Plant Cell Advance Publication. Published on December 15, 2015, doi:10.1105/tpc.15.00496
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Pipecolic acid orchestrates plant systemic acquired resistance and defense
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priming via salicylic acid-dependent and -independent pathways
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Friederike Bernsdorffa, Anne-Christin Döringa, Katrin Grunera, Stefan Schucka,
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Andrea Bräutigamb,c, Jürgen Zeiera,c,1
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a
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University, Universitätsstraße 1, D-40225 Düsseldorf, Germany
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b
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Universitätsstraße 1, D-40225 Düsseldorf, Germany
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c
Institute for Molecular Ecophysiology of Plants, Department of Biology, Heinrich Heine
Institute for Plant Biochemistry, Department of Biology, Heinrich Heine University,
Cluster of Excellence on Plant Sciences (CEPLAS)
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1
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The author responsible for distribution of materials integral to the findings presented in
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this article in accordance with the policy described in the Instructions for Authors
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(www.plantcell.org) is: Jürgen Zeier ([email protected]).
Address correspondence to [email protected].
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Running title: Pipecolic acid and salicylic acid in SAR
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One sentence summary: Systemic acquired resistance and the associated priming
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response are regulated by a pipecolic acid/FMO1 signaling module that controls both
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salicylic acid-dependent and independent defense pathways.
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Keywords: Arabidopsis, pipecolic acid, salicylic acid, systemic acquired resistance,
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defense priming, basal resistance, FLAVIN-DEPENDENT MONOOXYGENASE1,
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transcriptional reprogramming
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©2015 American Society of Plant Biologists. All Rights Reserved.
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ABSTRACT
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We investigated the relationships of the two immune-regulatory plant metabolites
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salicylic acid (SA) and pipecolic acid (Pip) in the establishment of plant systemic
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acquired resistance (SAR), SAR-associated defense priming, and basal immunity.
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Using SA-deficient sid2, Pip-deficient ald1, and sid2 ald1 plants deficient in both SA and
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Pip, we show that SA and Pip act both independently from each other and
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synergistically in Arabidopsis thaliana basal immunity to Pseudomonas syringae.
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Transcriptome analyses reveal that SAR establishment in Arabidopsis is characterized
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by a strong transcriptional response systemically induced in the foliage that prepares
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plants for future pathogen attack by pre-activating multiple stages of defense signaling,
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and that SA accumulation upon SAR activation leads to the down-regulation of
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photosynthesis and attenuated jasmonate-responses systemically within the plant.
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Whereas systemic Pip elevations are indispensable for SAR and necessary for virtually
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the whole transcriptional SAR response, a moderate but significant SA-independent
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component of SAR activation and SAR gene expression is revealed. During SAR, Pip
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orchestrates SA-dependent and SA-independent priming of pathogen responses in a
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FLAVIN-DEPENDENT-MONOOXYGENASE1
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conclude that a Pip/FMO1-signaling module acts as an indispensable switch for the
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activation of SAR and associated defense priming events, and that SA amplifies Pip-
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triggered responses to different degrees in the distal tissue of SAR-activated plants.
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2
(FMO1)-dependent
manner.
We
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INTRODUCTION
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In tissue inoculated by pathogenic microbes, plants are able to initiate a basal immune
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program that counteracts microbial infection. Plant basal resistance or pathogen-
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associated molecular pattern (PAMP)-triggered immunity (PTI) involves recognition of
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microbial structures by plant pattern recognition receptors, defense signal transduction,
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and transcriptional activation of defense-related gene expression (Boller and Felix,
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2009). Yet, PTI can be overcome by well-adapted pathogen isolates. Previous pathogen
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encounter, however, can render plants significantly more resistant to a future challenge.
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For instance, systemic acquired resistance (SAR), a state of heightened resistance of
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the entire plant foliage to a broad spectrum of biotrophic and hemibiotrophic
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phytopathogens, is induced by a localized leaf inoculation with avirulent or virulent
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microbial pathogens (Mishina and Zeier, 2007; Fu and Dong, 2013). Plants with
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activated SAR exhibit enhanced systemic expression of antimicrobial PR proteins and
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other augmented immune responses (Sticher et al., 1997). In addition, biologically
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induced SAR conditions plants to react more quickly and vigorously to subsequent
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pathogen attack (Jung et al. 2009; Návarová et al. 2012), a phenomenon also
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designated as defense priming (Conrath, 2011).
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The establishment of SAR is regulated by signal–active plant metabolites
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(Dempsey and Klessig, 2012; Shah and Zeier, 2013). From about 1980 onwards,
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multiple studies have provided evidence that the phenolic defense hormone salicylic
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acid (SA) plays a pivotal role in SAR (reviewed in Vlot et al., 2009). The pathogen-
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induced biosynthesis of SA in Arabidopsis thaliana proceeds via isochorismate
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synthase-mediated conversion of chorismate to isochorismate (Nawrath and Métraux,
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1999; Wildermuth et al., 2001). The Arabidopsis sid2-1 mutant, which is defective in
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ISOCHORISMATE SYNTHASE1 (ICS1), is unable to accumulate pathogen- and stress-
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inducible SA and is impaired in SAR activation (Nawrath and Métraux, 1999). Although
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SA was previously proposed as the mobile compound that travels from inoculated to
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distal leaves to induce SAR (Shulaev et al., 1995; Mölders et al., 1996), genetic studies
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support the notion that SA is not a SAR long-distance signal but that its isochorismate-
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derived de novo biosynthesis in systemic leaf tissue is required for proper SAR
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(Vernooij et al., 1994; Attaran et al., 2009). Since then, several other candidate long3
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distance signals have been suggested (reviewed in Shah and Zeier, 2013). A
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predominant portion of SA downstream responses is dependent on the transcriptional
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co-activator NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1),
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which has been identified, in addition to its paralogues NPR3 and NPR4, as a bona fide
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SA receptor (Wu et al., 2012; Fu et al., 2012). NPR1 acts as a central regulator of SAR
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(Fu and Dong, 2013).
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In addition to SA and NPR1, FLAVIN-DEPENDENT MONOOXYGENASE1
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(FMO1) is a critical component of biologically-induced SAR in Arabidopsis (Mishina and
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Zeier, 2006; Jing et al., 2011). The strongly pathogen-inducible FMO1 gene encodes a
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flavin monooxygenase that is activated in both locally inoculated and distal leaves
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(Bartsch et al., 2006; Koch et al., 2006; Mishina and Zeier, 2006). Notably, functional
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FMO1 is necessary for SA accumulation in the systemic, non-inoculated leaves but
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dispensable for SA production in inoculated tissue (Mishina and Zeier, 2006). A
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biochemical characteristic of distinct FMOs from plants, insects, and mammals is that
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they are able to oxidize amino- or sulphide groups within small metabolic substrates
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(Schlaich, 2007). We therefore previously hypothesized that an endogenously produced
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plant amine, amino acid, or S-containing metabolite might play a central function in SAR
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(Mishina and Zeier, 2006).
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This hypothesis was confirmed by identifying the non-protein amino acid
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pipecolic acid (Pip; homoproline) as a critical SAR regulator (Návarová et al., 2012).
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Alongside with SA, Pip accumulates to high amounts in Arabidopsis leaves inoculated
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with SAR-inducing Pseudomonas syringae bacteria as well as in leaves distant from
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initial inoculation. Very specifically, and in contrast to SA and many other accumulating
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metabolites, Pip is enriched in phloem exudates collected from inoculated leaves,
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indicating specific transport of Pip out of inoculated leaves (Návarová et al., 2012).
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Pipecolic acid is a plant natural product with widespread occurrence throughout the
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Angiosperms (Morrison, 1953; Zacharius et al., 1954), and its accumulation in leaves
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after inoculation with bacterial, fungal, or viral pathogens has been documented for rice,
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potato, tobacco, soybean, and Arabidopsis (Pálfi and Dészi, 1968; Návarová et al.,
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2012; Vogel-Adghough et al., 2013; Aliferis et al., 2014). In addition, Pip is
4
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overproduced in Arabidopsis autophagy mutants that exhibit stress-related phenotypes
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(Masclaux-Daubresse et al., 2014).
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Feeding studies with isotope-labelled Lys demonstrated that, like animals
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(Broquist, 1991), plants synthesize L-Pip from Lys (Fujioka and Sakurai, 1997), and
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strongly suggested that Lys to Pip conversion in plants involves both a classical
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aminotransferase reaction removing the α-amino group from Lys and a subsequent
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reductase activity (Gupta and Spenser, 1969; Zeier, 2013). An aminotransferase with
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strong substrate specificity for Lys is encoded by AGD2-LIKE DEFENSE RESPONSE
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PROTEIN1 (ALD1) (Song et al., 2004a). Similar to FMO1 (Mishina and Zeier, 2006),
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ALD1 has been identified as an essential SAR component that is up-regulated in both
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locally inoculated and distal leaf tissue (Song et al., 2004b). Our recent finding that ald1
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knockout-mutants are not able to biosynthesize and accumulate Pip after pathogen
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inoculation indicates that ALD1 is the aminotransferase required for the Lys-derived
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biosynthesis of Pip (Návarová et al., 2012). Moreover, the SAR defect of Pip-deficient
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ald1 can be rescued by exogenous supply of Pip in physiological doses, demonstrating
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that Pip accumulation is required for SAR activation (Návarová et al., 2012). Exogenous
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Pip is also sufficient to increase resistance to P. syringae in wild-type and ald1 plants to
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a similar degree as biological SAR (Návarová et al., 2012). Notably, Pip feeding is able
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neither to restore the SAR-defect of fmo1 nor to increase pathogen resistance in this
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mutant, indicating that Pip requires functional FMO1 to activate SAR (Návarová et al.,
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2012). Like for P. syringae-induced SAR, Pip accumulation and FMO1 are also integral
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parts of the systemic immune response induced by local oviposition of insect eggs in
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Arabidopsis (Hilfiker et al., 2014).
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The activation of SAR in non-infected distal tissue of pathogen-inoculated plants
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relies on the perception and amplification of endogenous plant signals (Shah and Zeier,
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2013). Our current working model implies that Pip is a central player of a feedback
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amplification
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establishment (Návarová et al., 2012; Zeier, 2013). Once established, SAR primes
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Arabidopsis plants for effective defense responses to future pathogen challenge. These
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responses include the accumulation of the phytoalexin camalexin and expression of a
mechanism
that
realizes
systemic
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SA
accumulation
and
SAR
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series of defense-related genes, including ALD1, FMO1, and PR1. Systemically
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elevated Pip during SAR is necessary and sufficient for the SAR-associated priming
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response (Návarová et al., 2012; Zeier, 2013).
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In the present study, we investigate the interplay of Pip and SA in Arabidopsis
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immune signaling and provide a detailed characterization of the systemic transcriptional
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response associated with SAR. Systemic transcriptional reprogramming upon localized
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P. syringae inoculation involves enhanced expression of microbial pattern recognition
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receptors, various defense signaling components, and specific transcription factor
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classes, as well as a massive down-regulation of photosynthetic and growth-related
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genes. We show that Pip orchestrates SAR and virtually the whole transcriptional SAR
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response via SA-dependent and, less prominently, SA-independent activation
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pathways. Our data indicate that activation of a primed state in distal leaves of locally-
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inoculated plants requires functional FMO1 downstream of Pip, and that SAR priming of
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a subset of genes proceeds in SA-deficient sid2 to a similar extent as in wild type. Our
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results therefore emphasize the significance of partially SA-independent signaling
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events during SAR establishment and the realization of SAR-associated defense
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priming. Moreover, they indicate that Pip and SA act both synergistically and
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independently from each other to mediate PR gene expression and plant basal
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resistance to P. syringae.
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RESULTS
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The SA and Pip defense pathways provide additive contributions to basal
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resistance
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To obtain deeper insights into the interplay between the immune signals Pip and SA in
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mediating Arabidopsis basal resistance, SAR establishment, and defense priming, we
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comparatively investigated resistance responses of the Col-0 wild type, Pip-deficient
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ald1 (Návarová et al., 2012), SA induction-deficient sid2-1 (sid2; Nawrath and Métraux,
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1999), and a sid2 ald1 double mutant unable to generate both SA and Pip after
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pathogen inoculation. Whereas ald1 represents a T-DNA knockout line for ALD1 (Song
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et al., 2004b; Návarová et al., 2012), sid2 was previously obtained by ethylmethane
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sulfonate-mutagenesis, and carries a single base-pair mutation in the ICS1 coding
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region that results in a premature stop codon and a full loss of ICS1 function (Nawrath
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and Métraux, 1999; Wildermuth et al., 2001). The sid2 ald1 double mutant was
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generated by screening progeny from a cross of the single mutants (Supplemental
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Figures 1 and 2).
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Upon leaf inoculation with the SAR-inducing bacterial strain Pseudomonas
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syringae pv. maculicola ES4326 (Psm) (Mishina and Zeier, 2007; Attaran et al., 2009;
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Jing et al., 2011), we observed strong increases of ALD1 and ICS1 transcript levels in
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wild-type Col-0 plants, increases of ICS1 but not ALD1 in ald1, and elevations of ALD1
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but not ICS1 in sid2. Moreover, sid2 ald1 lacked both basal and pathogen-induced
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expression of ALD1 and ICS1 (Fig. 1A). On the metabolite level, Psm attack triggered a
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strong accumulation of both Pip and SA in inoculated (1°) leaves and in distal (2°), non-
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inoculated leaves of Col-0 plants. By contrast, sid2 ald1 produced neither Pip nor SA
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after pathogen inoculation, and contained only faint basal levels of the two immune-
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regulatory metabolites (Fig. 1B, C). Consistent with our previous analyses (Návarová et
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al., 2012), ald1 completely lacked pathogen-induced Pip accumulation but was able to
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activate SA production in inoculated leaves, whereas sid2 showed a reciprocal
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accumulation pattern (Fig. 1B, 1C).
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To assess basal resistance to bacterial attack, we compared the growth of the
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virulent Psm strain in leaves of Col-0, ald1, sid2, and sid2 ald1 3 days after inoculation
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(DAI). Both ald1 and sid2 allowed higher bacterial multiplication than the wild type (Fig.
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2A), confirming the previously suggested requirements for Pip and SA in the full basal
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immunity program at inoculation sites (Nawrath and Métraux, 1999; Návarová et al.,
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2012). The sid2 mutant permitted significantly higher bacterial multiplication than ald1,
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indicating that the relative contribution of SA to basal resistance is higher than that of
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Pip. Notably, leaf-inoculated sid2 ald1 showed the weakest resistance phenotype of all
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investigated lines and allowed a significantly higher bacterial multiplication than Col-0,
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ald1, and sid2 (Fig. 2A). This indicates that SA and Pip provide additive contributions to
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Arabidopsis basal immunity against P. syringae.
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Pip regulates SAR via SA-dependent and SA-independent activation pathways
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2° leaves of locally inoculated sid2 plants accumulated Pip to a moderate but
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significant extent, suggesting that pathogen-triggered systemic responses are not fully
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suppressed in sid2 (Fig. 1B). On the contrary, ald1 was unable to elevate SA in 2°
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leaves, corroborating our previous results showing the necessity of Pip for the activation
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of SA biosynthesis and concomitant SA accumulation in distal leaves (Fig. 1C,
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Návarová et al., 2012). To directly examine the SAR response in the lines under
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investigation, we inoculated plants with Psm in lower, 1° leaves to induce SAR or
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performed mock treatments with 10 mM MgCl2 to generate appropriate non-induced
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control plants, and then challenge-inoculated upper, 2° leaves of both pathogen-
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inoculated and mock-treated plants with Psm two days after the primary treatment.
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Bacterial growth in upper leaves was assessed another three days later (Mishina and
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Zeier, 2007; Attaran et al., 2009; Jing et al., 2011; Návarová et al., 2012). In these
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assays, wild-type Col-0 plants exhibited a strong SAR response and the bacterial
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multiplication in challenge-infected leaves was generally attenuated by 95 to 98% as a
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consequence of SAR induction (Fig. 2B). The Pip-deficient ald1 mutant was not able to
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activate any SAR upon attempted induction (Song et al., 2004b; Návarová et al., 2012),
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confirming the previously reported necessity of Pip accumulation in SAR establishment
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(Návarová et al., 2012). Remarkably, the SA-deficient sid2 mutant was able to
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significantly induce resistance upon localized Psm inoculation in distal leaves (Fig. 2B).
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Although the observed SAR response in sid2 was small compared to wild-type SAR (a
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50 to 80% reduction of bacterial growth), the effect was reproducible between
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experiments and occurred, besides in sid2, in sid1 (Fig. 2C), another Arabidopsis
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mutant unable to activate stress-induced SA biosynthesis (Nawrath and Métraux, 1999),
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and in an ics1 ics2 double mutant (Supplemental Figure 3), which is not only blocked in
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induced SA production but also exhibits strongly diminished basal SA levels (Garcion et
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al., 2008). Moreover, the hypersensitive response (HR)-inducing Psm avrRpm1 strain
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triggered partial SAR activation in sid2 in a similar manner than the compatible Psm
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strain (Fig. 2C). These results show that a moderate SAR response in plants can be
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triggered independently of inducible SA biosynthesis but not independently of Pip
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228
biosynthesis. However, SA accumulation upon pathogen encounter is required to
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realize a full SAR response. The residual, SA-independent SAR effect is absent in the
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sid2 ald1 double mutant, indicating that activation of this pathway and of the
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predominant, SA-dependent SAR pathway do both require Pip (Fig. 2B). In sum, these
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data strongly suggest that Pip is a central regulatory metabolite for SAR that controls
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both SA-dependent and SA-independent SAR activation pathways.
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Transcriptional reprogramming in distal leaves of SAR-activated plants:
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increased
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photosynthesis, general metabolism and growth
readiness
for
pathogen
defense
coupled
with
decreased
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SAR establishment following 1° leaf inoculation involves increased expression of
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a whole battery of defense-related genes in the distal leaves (Ward et al., 1991; Gruner
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et al., 2013). To assess the contribution of Pip and SA to SAR on the transcriptional
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level, the transcriptional SAR response of Col-0 was characterized at the whole-genome
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level and compared to the responses in sid2 and ald1. Compatible Psm or HR-inducing
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Psm avrRpm1 trigger SAR in Col-0 plants between day 1 and day 2, and the full
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resistance response is apparent at 2 DAI (Mishina et al., 2008; Návarová et al., 2012).
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We thus determined the transcriptional changes that occur in 2° leaves two days after
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Psm-treatment of 1° leaves compared to 1°-mock treatment by Illumina TruSeq™ RNA
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sequencing analyses. A first experimental set consisted of three independent, replicate
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SAR experiments (experiments 1 to 3) with Col-0 and sid2 plants, and a second
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analogous set involved Col-0 and ald1 (experiments 4 to 6). Principle component
250
analysis (PCA) identified Psm-treatment variation as the major variable between the
251
samples, accounting for 58.0 % of the variation. The second variable was experiment
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variation between the first and second experimental sets and accounted for 15.7 % of
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the variation, indicating that experimental variation was small compared to treatment
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variation. To be conservative, the 6 Col-0 replicates were combined for analysis. The
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PCA also showed that in Col-0, the distance between mock- and Psm-treatment
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samples was widest. The distance between sid2 samples was small and the distance
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between ald1 samples was virtually non-existent (Supplemental Figure 4).
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A threshold cut-off of expression values (reads per million) was defined that
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excluded genes with very low expression levels from the RNA-seq data set, i.e. genes
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that did not reach expression values of at least 5 in any of the mock- or Psm-samples.
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This reduced the set of 28496 totally RNA-seq covered genes to a set of 15239
262
expressed genes. To determine statistically significant changes in gene expression of
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Psm- versus mock-treatments for Col-0, sid2, and ald1, a false discovery rate (FDR) of
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0.01 was assumed (Benjamini and Hochberg, 1995). Among the 15239 investigated
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genes, 3413 were up-regulated (designated as SAR+ genes), and 2893 were down-
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regulated (SAR- genes) in a statistically significant manner in the Col-0 wild-type (Fig.
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3A; Supplemental Data Set 1). To quantitatively assess the transcriptional changes
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between the SAR-induced and mock-control state, we calculated log2-transformed
269
ratios of the mean of expression values for Psm and mock samples (P/M-fold changes).
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The log2 P/M-fold changes for Col-0 averaged over all the SAR+ and SAR- genes were
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2.05 and -1.57, respectively (Fig. 3B).
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To identify and illustrate physiological and metabolic processes altered in Col-0
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plants upon SAR establishment, we tested whether the up- and down-regulated SAR
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genes are enriched in MapMan bins or particular Arabidopsis gene families (Thimm et
275
al., 2004; http://www.arabidopsis.org/). Strikingly, 86 % of the investigated genes
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annotated for an involvement in photosynthesis were significantly down-regulated upon
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SAR induction in Col-0 (Fig. 4A). Similarly, genes belonging to the MapMan categories
278
photosynthetic
279
biosynthesis were predominantly down-regulated (Fig. 4A, Supplemental Figures 5 to
280
7). Moreover, genes involved in starch metabolism and genes of the category major
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CHO metabolism were strongly overrepresented in the SAR- gene group (Fig. 4A, B).
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To a lesser extent, this was also valid for genes of the categories lipid metabolism,
283
amino acid metabolism, and secondary metabolism (Fig. 4B). In addition, genes of the
284
MapMan category cell wall were enriched among SAR- genes (Fig. 4B). A closer look at
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the family level revealed that many genes coding for proteins involved in cell wall
286
modification and growth (fasciclin-like arabinogalactan proteins, expansins, xyloglucan
287
endotransglucosylase/hydrolases) as well as genes associated with wax and cutin
288
biosynthesis were strongly down-regulated during SAR (Fig. 4C). This suggests that in
light
reactions,
Calvin
cycle,
10
photorespiration
and
tetrapyrrole
289
the distal leaves of SAR-activated plants, photosynthesis, several primary and
290
secondary metabolic pathways, and growth processes are reduced compared to
291
respective leaves of control plants. Gene ontology (GO) term enrichment analysis
292
confirmed the reduction in growth-related processes (Supplemental Data Set 2).
293
The massive down-regulation of photosynthesis-associated genes upon SAR
294
induction in Col-0 prompted us to comparatively investigate the photosynthetic rates of
295
2° leaves of plants infiltrated in 1° leaves two days earlier with Psm- or mock-solution.
296
We measured the CO2-uptake of individual leaves using infrared gas analysis (IRGA) to
297
determine the maximum rates of photosynthetic carbon assimilation (von Caemmerer
298
and Farquhar, 1981). Upon SAR induction in Col-0, the rate of CO2 uptake of 2° leaves
299
significantly dropped from about 8 to 4 µmol m-2 s-1, indicating a marked decrease in
300
photosynthetic rates (Fig. 5A). Our data thus reveal an attenuation of photosynthesis in
301
the non-inoculated distal leaves of SAR-induced wild-type plants both at the
302
transcriptional and the physiological level. Infrared gas analysis also showed decreased
303
water loss from 2° leaves of SAR-induced Col-0 plants, suggesting a significant decline
304
of leaf transpiration when SAR is established (Fig. 5B). Decreased stomatal apertures
305
may thus account for the attenuation of photosynthesis in SAR-induced plants.
306
Analyses of the transcriptional SAR response also revealed that the categories
307
biotic stress and signaling were over-represented among the SAR+ genes (Fig. 4D). GO
308
term enrichment analysis of the SAR+ genes specified the biotic stress and signaling
309
categories by identifying regulation of the hypersensitive response, SAR, and SA
310
signaling and their respective parent terms as enriched processes. In addition, N-
311
terminal protein myristoylation, Golgi-based protein targeting to membranes, and the
312
endoplasmic reticulum unfolded protein response as well as their respective parent
313
terms were enriched (Supplemental Data Set 3). Moreover, gene families typically
314
involved in the perception of pathogen-derived elicitors, i.e. genes coding for nucleotide
315
binding site (NBS)-containing resistance proteins (Tan et al., 2007), receptor-like protein
316
kinases (RLK, Shiu and Bleecker, 2001), and receptor-like proteins (Fritz-Laylin et al.,
317
2005) were markedly enriched in the SAR+ gene group (Fig. 4E). Notably, a preferential
318
accumulation of members representing specific subfamilies of the large RLK family in
319
the SAR+ group was apparent. For example, about 70 % of cysteine-rich protein kinases
11
320
in the investigated gene set were consistently up-regulated upon SAR induction
321
(Supplemental Figure 8A). In addition, defense signaling components such as mitogen-
322
activated protein kinase kinases (MAPKK), calcium-dependent protein kinases, EF-
323
hand containing proteins, and calmodulin-binding proteins were overrepresented among
324
the SAR+ genes (Fig. 4F, G). Among transcription factor families, a strong
325
overrepresentation of WRKY-, and NAC-type transcription factor genes was observed in
326
the SAR+ gene group, whereas genes for transcription factor types such as MYB, bHLH
327
or bZIP were not enriched (Fig. 4H). Finally, compared to other major types of enzyme
328
classes, a prominent overrepresentation of glutathione-S-transferases among the SAR+
329
genes was discernable (Fig. 4I). Other, less prominent gene classes that were strongly
330
overrepresented in the SAR+ group involved senescence-associated genes as well as
331
genes coding for stomatin/prohibitin/flotillin/HflK/C (SPFH) domain-containing, FAD
332
berberine-type, VQ motif-containing proteins, and plant U-box proteins (PUB)
333
(Supplemental Figure 8B). By contrast, the general expression patterns of genes from
334
MapMan categories such as development, cell, transport, monolignol biosynthesis or
335
class III peroxidase genes were hardly or not at all changed upon SAR induction
336
(Supplemental Figure 8C).
337
Overall, these characteristics suggest that SAR-activated plants prepare
338
themselves for future pathogen attack by up-regulating genes involved at different
339
stages of defense signaling, such as elicitor perception, signal transduction, and
340
transcriptional gene activation, and by down-regulating photosynthesis and growth-
341
associated processes.
342
343
The transcriptional SAR response involves a subset of genes whose systemic
344
expression is partially SA-independent
345
The transcriptional SAR response in the mutants was compared qualitatively and
346
quantitatively to the wild-type response (Fig. 3). According to the statistical RNA-seq
347
analysis, the transcript levels of 2672 out of the 3413 genes that were systemically up-
348
regulated in the Col-0 wild-type following SAR induction were not elevated in sid2 in a
349
statistically significant manner (Fig. 3A). Therefore, a predominant part of the
12
350
transcriptional SAR response is SA-dependent. We created a list of all the SAR+ genes
351
from this group and sorted them according to their mean P/M-fold change in sid2 in
352
ascending order. Table 1 depicts the top 15 SAR+ genes from this list. Among the tightly
353
SA-regulated SAR+ genes are PATHOGENESIS-RELATED GENE1 (PR1), a classical
354
marker gene for an activated SA signaling pathway and SAR (Sticher et al., 1997),
355
ACIREDUCTONE DIOXYGENASE3 (ARD3), and GLUTAREDOXIN 13 (GRXS13).
356
The quantitative RNA-seq assessment indicated that the median log2 P/M-fold
357
change over all the SAR+ genes for sid2 accounted for 0.95 (Fig. 3B). Although lower
358
than the respective Col-0 value of 2.05, this value was markedly higher than the values
359
calculated from different sets of randomly chosen genes from the total Arabidopsis
360
genome (0.23 to 0.28). Therefore, sid2 exhibits an attenuated but still detectable
361
transcriptional response to Psm in tissue distal from inoculation. Qualitatively, 845
362
genes were significantly up-regulated in 2° leaves of sid2 upon 1°-leaf inoculation, and
363
741 of them were induced in a significant manner also in the Col-0 wild-type (Fig. 3A).
364
We sorted the SAR+ genes within this group according to their mean P/M-fold change
365
values in sid2 in descending order. Table 2 lists 15 prominently expressed genes with
366
strong up-regulation in sid2.
367
A common feature of these genes is that their P/M-fold changes (their
368
inducibility) in sid2 are similar or even higher than in the wild-type, but that their
369
absolute expression in 2° tissue of mock- and pathogen-treated sid2 plants is markedly
370
lower than in Col-0. This indicates that SA amplifies their expression under both
371
inducing and basal conditions (Table 2; Supplemental Data Set 1; Supplemental Figure
372
4), and we have therefore designated these genes as partially SA-independent.
373
Strikingly, ALD1 and FMO1 rank among the most strongly induced genes in the
374
systemic tissue of sid2, demonstrating that the transcriptional activation of Pip
375
biosynthesis and downstream signaling can be activated during SAR in a manner that is
376
partially SA-independent. Other paradigm examples of SAR+ genes that can be strongly
377
up-regulated in the absence of elevated SA are TYROSINE AMINOTRANSFERASE3
378
(TAT3), GLUTATHIONE S-TRANSFERASE22 (GST22), PHYTOALEXIN-DEFICIENT3
379
(PAD3), and SENESCENCE-ASSOCIATED GENE13 (SAG13) (Table 2). For the top
380
members of the gene list, the high inducibility in sid2 resulted in expression values for
13
381
Psm-treated sid2 samples that markedly exceeded those of mock-treated Col-0. Genes
382
from the middle or bottom part of the gene list, however, generally showed a lower
383
inducibility in sid2 than in Col-0, and the values from Psm-inoculated sid2 only
384
moderately exceeded the Col-0 mock-values (e.g. PR5, AAC3, P4H5) or even stayed
385
below (e.g. FRK1) (Table 2). Therefore, the absolute expression of the genes from the
386
bottom part of the gene list such as FRK1 still very much depends on SA, although a
387
slight (but statistically significant) up-regulation exists in sid2.
388
Together, these expression analyses indicate that the extent of systemic up-
389
regulation for virtually all SAR+ genes is positively regulated by SA. For the majority of
390
genes, this SA-mediated amplification is essential for a significant up-regulation (SA-
391
dependent SAR+ genes, Table 1) or a noticeable induction well-above basal wild-type
392
levels (bottom part of list of partly SA-independent genes, as exemplified by FRK1 in
393
Table 2). However, the dependency on SA is lower for several hundred SAR+ genes
394
which therefore exhibit a marked systemic up-regulation in sid2 (major part of the 741
395
partly SA-independent genes, as exemplified by the top 15 genes of Table 2). Notably,
396
the SAR regulatory and pipecolic acid pathway genes ALD1 and FMO1 belong to the
397
SAR+ genes with a pronounced SA-independent component of induction.
398
399
An activated SA pathway upon SAR induction suppresses systemic jasmonate
400
responses
401
The RNA-seq results show that 104 of the 845 genes systemically induced in sid2 were
402
not up-regulated or even significantly down-regulated in Col-0 upon SAR activation (Fig.
403
3A). We listed these genes according to their Col-0 mean P/M-fold change values in
404
ascending order, and Table 3 exemplifies 15 of these specifically sid2 up-regulated
405
genes. Several jasmonate-responsive genes such as VEGETATIVE STORAGE
406
PROTEIN2 (VSP2) or BENZOIC ACID/SALICYLIC ACID METHYLTRANSFERASE1
407
(BSMT1) belong to this group. We therefore tested whether this gene category would be
408
actually enriched in jasmonic acid (JA)-responsive genes. A list of JA-responsive genes
409
was taken from microarray data of Goda et al. (2008), studying the response of
410
Arabidopsis to methyl jasmonate-treatment. According to this list, the examined group of
14
411
15239 Arabidopsis genes contained 3.2 % of JA-responsive genes, and the genes
412
systemically up-regulated in Col-0 (SAR+ genes) showed a similar percentage of JA-
413
regulated genes (3.7 %; Table 4). By comparison, 12.8 % of the genes systemically up-
414
regulated in sid2 were JA-responsive, and the subgroup thereof containing only genes
415
that were not up-regulated in Col-0 showed by far the highest enrichment (48.1 % JA-
416
responsive genes). These findings show that a 1°-leaf-inoculation with Psm triggers a
417
significantly higher expression of JA-responsive genes in 2° leaves of sid2 plants
418
compared to Col-0 plants and indicate that an activated SA pathway in the SAR-induced
419
wild type suppresses pathogen-inducible JA responses in systemic tissue.
420
421
Pipecolic
422
reprogramming response associated with SAR
423
Col-0 plants responded to the 1° Psm-inoculation with the up-regulation of 3413 and the
424
down-regulation of 2893 genes in 2° leaves. Strikingly, for the Pip-deficient ald1 plants,
425
only two genes, CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASE6 (CRK6) and
426
PLANT CADMIUM RESISTANCE1 (PCR1), were systemically up-regulated in a
427
statistically significant manner, and not a single gene was significantly down-regulated
428
(Fig. 3A; Table 5). Moreover, albeit the statistical analyses identified CRK6 and PCR1
429
as up-regulated in ald1, their absolute expression levels after Psm-treatment were low
430
and did not even exceed the values of the Col-0 mock-control samples (Table 5).
431
Therefore, the strong transcriptional reprogramming observed in 2° leaves of Col-0
432
plants after 1°-leaf inoculation is essentially absent in ald1. This indicates that the
433
accumulation of Pip upon pathogen-inoculation is necessary for virtually the entire
434
transcriptional SAR response. This also becomes evident when the MapMan heat maps
435
of central metabolism for Col-0 and ald1 upon (attempted) SAR induction are compared
436
(Supplemental Figures 5 and 7). Moreover, the mean P/M-fold change averaged over
437
all the SAR+ genes for ald1 accounted for a value of 0.28, which was low compared with
438
the Col-0 (2.05) or sid2 (0.95) values and in the range of the values for groups of
439
randomly chosen genes (Fig. 3B).
acid
is
a
central
regulator
15
of
the
systemic
transcriptional
440
The PCA showed that the transcriptome differences between the mock-samples
441
of Col-0 and those of ald1 were lower than the differences of mock-values between Col-
442
0 and sid2 (Supplemental Figure 4). This might indicate, as deduced before from the
443
bacterial growth data (Fig. 2A), that the contribution of Pip to basal resistance against P.
444
syringae is lower than the contribution of SA. In addition, the SAR-associated down-
445
regulation of genes was not only blocked in ald1 but also severely compromised in sid2
446
(Figs. 3A and B; Supplemental Figures 5 to 7). The RNA-seq results indicate that only
447
17 genes were significantly down-regulated in sid2, whereas 2893 genes were
448
repressed in Col-0 (Fig. 3A). Therefore, a lack of induction of SA biosynthesis in plants
449
seems to affect the pathogen-induced systemic gene repression to a broader extent
450
than gene activation. Since the systemic down-regulation of photosynthetic genes is
451
one hallmark of the transcriptional SAR response in the wild-type (Fig. 4A), we expected
452
that the observed induced systemic attenuation of photosynthetic rates in Col-0 would
453
be severely affected in ald1 and sid2. IRGA analyses confirmed this assumption, since
454
1° leaf-inoculation changed neither the photosynthetic rates nor the stomatal
455
conductance in ald1, sid2 or sid2 ald1, indicating that both Pip and SA are required to
456
mediate these responses (Fig. 5A, B).
457
458
Pipecolic
459
responses upon SAR activation
460
We have previously shown that the induction of SAR conditions Arabidopsis for timely
461
and effective defense gene activation, SA biosynthesis, and camalexin accumulation.
462
This state of defense priming becomes apparent upon a challenge infection of
463
previously uninfected 2° leaves. Pip is a critical mediator of this SAR-associated priming
464
response, because Pip-deficient ald1 plants completely lack this phenomenon.
465
Moreover, exogenously applied Pip is sufficient to promote both Col-0 and ald1 plants
466
into a primed, SAR-like state (Návarová et al., 2012). To investigate a possible interplay
467
between Pip and SA in the activation of SAR-associated defense conditioning, we
468
directly compared the abilities of Col-0, ald1, sid2, and sid2 ald1 plants to realize
469
biologically-induced defense priming.
acid
orchestrates
SA-dependent
16
and
SA-independent
priming
470
For this purpose, the plants were infiltrated with Psm or mock solution in their
471
lower (1°) leaves and two days later, the distal (2°) leaves were challenged with Psm or
472
mock-infiltrated obtaining four combinations: (1°/2°) mock/mock, Psm/mock, mock/Psm,
473
and Psm/Psm (Supplemental Figure 9A). The magnitude of defense in the challenged
474
2° leaves was determined for the four combinations at 10 HAI (Návarová et al., 2012).
475
We defined a particular defense response as primed if the differences between the
476
responses of SAR-induced plants to 2°-Psm and 2°-mock-treatments (Psm/Psm –
477
Psm/mock), respectively, were significantly larger than the same differences in non-
478
induced plants (mock/Psm – mock/mock) (Supplemental Figure 9B). Moreover, to
479
estimate quantitative differences in the strength of priming between genotypes with
480
activated priming, we calculated the prgain (response gain due to priming)-value (see
481
Supplemental Figure 9C for details).
482
We first monitored the expression of ALD1, FMO1, and SAG13 (partially SA-
483
independent SAR+ genes; Table 2) and of GRXS13, ARD3, and PR1 (SA-dependent
484
SAR+ genes; Table 1), as defense outputs of the SAR priming assay. SAR induction
485
significantly primed Col-0 wild-type plants for enhanced expression of all these genes
486
(Fig. 6A; Supplemental Figure 10A). This conditioning of gene expression was
487
completely absent in both ald1 and sid2 ald1 for all the examined genes (Fig. 6A;
488
Supplemental Figure 10A), corroborating the previously identified central role for Pip in
489
the activation of SAR-associated defense priming (Návarová et al., 2012). For sid2
490
plants, by contrast, the outcome of the priming assay depended on the nature of the
491
investigated response. FMO1 and ALD1 expression were primed in sid2 to a higher or
492
similar extent, respectively, than in Col-0. The expression of SAG13 was also
493
significantly primed in sid2 but this response was markedly lower than in Col-0.
494
Moreover, only a weak priming effect was observed for GRXS13 expression, and
495
priming for both ARD3 and PR1 expression was completely abolished in sid2 (Fig. 6A;
496
Supplemental Figure 10A). Therefore, SAR induction primes sid2 plants for enhanced
497
expression of three members of the partially SA-independent cluster of SAR+ genes,
498
whereas priming is weak or fully absent with respect to expression of the three SA-
499
dependent SAR+ genes. In addition to gene expression, we measured priming at the
500
metabolite level. As previously shown (Návarová et al., 2012), we found that an
17
501
activated SAR state primes Col-0 plants for enhanced camalexin and SA biosynthesis
502
upon pathogen inoculation in a Pip-dependent manner (Fig. 7A; Supplemental Figure
503
11A). Priming of camalexin accumulation was also absent in sid2, indicating that both
504
Pip and SA are required for conditioning of camalexin biosynthesis during biological
505
SAR (Fig. 7A; Supplemental Figure 11A).
506
Together, these results indicate that Pip is able to prime plants for enhanced
507
activation of a subset of defense responses such as ALD1 and FMO1 expression during
508
biologically-induced SAR independently of SA. For the priming of a second category of
509
defense responses that involve ARD3 expression, PR1 expression, and camalexin
510
accumulation, however, both Pip and SA are necessary. Notably, our data suggest
511
overlapping regulatory principles of SAR activation and the realization of defense
512
priming in challenge-infected plants: priming of expression of partially SA-independent
513
SAR+ genes can be achieved independently of SA, whereas priming of SA-dependent
514
SAR+ genes requires SA (Tables 1 and 2; Fig. 6A; Supplemental Figure 10A).
515
Arabidopsis plants exogenously supplied with 10 µmol Pip over the root system
516
accumulate Pip in leaves to similar levels as 2° leaves of plants exhibiting P. syringae-
517
induced SAR (Návarová et al., 2012). Pip applied in this way is sufficient to enhance
518
resistance to P. syringae and induces defense priming to a similar extent as biological
519
SAR (Návarová et al., 2012). To examine the role of SA in Pip-induced defense priming,
520
we fed Col-0, sid2, ald1, and sid2 ald1 plants with 10 ml of 1 mM (= 10 µmol) Pip,
521
challenged leaves with Psm one day later, and compared their defense responses 10 h
522
after the challenge-infection with those of unfed plants (supplied with 10 ml H2O).
523
Similar to the SAR priming assay, we distinguished four treatments (soil/leaf): a control
524
situation (H2O/mock), Pip-treatment alone (Pip/mock), pathogen challenge alone
525
(H2O/Psm), and Pip-treatment with subsequent pathogen challenge (Pip/Psm), and
526
defined defense priming by analogous criteria as for the SAR priming assay (Pip/Psm –
527
Pip/mock > H2O/Psm – H2O/mock; Supplemental Figure 9). Exogenously supplied Pip
528
markedly intensified the Psm-triggered expression of FMO1 and ALD1 in Col-0, and this
529
priming response was even more pronounced in sid2 (Fig. 8A; Supplemental Figure
530
12A). Pip feeding also primed Col-0 plants for enhanced PR1 expression during a Psm
531
challenge infection, and here, the priming effect was almost fully suppressed in sid2
18
532
(Fig. 8A; Supplemental Figure 12A). Pip-induced priming for camalexin production was
533
also stronger in Col-0 than in sid2 (Fig. 8B; Supplemental Figure 12B). These results
534
overlap with those of the SAR priming assay, and substantiate our conclusion that Pip
535
regulates priming for certain responses in an SA-independent manner, whereas it
536
requires SA to mediate priming of other responses. The responses triggered by
537
exogenous Pip were generally somewhat lower in ald1 or sid2 ald1 than in Col-0 plants,
538
indicating that endogenous Pip also amplifies the priming effects in these assays (Fig.
539
8A, B; Supplemental Figure 12A, B).
540
541
Pipecolic
542
DEPENDENT-MONOOXYGENASE1
543
FMO1 is an essential component of biologically-induced SAR (Mishina and Zeier,
544
2006). The finding that fmo1 mutant plants are unable to increase resistance upon Pip
545
treatment indicates that FMO1 acts downstream of Pip in SAR signal transduction
546
(Návarová et al. 2012). To investigate the role of FMO1 in defense conditioning, we
547
subjected fmo1 plants to the priming assays described above. Like ald1, fmo1 plants
548
were unable to intensify expression of partially SA-dependent or SA-independent SAR+
549
genes (Fig. 6B; Supplemental Figure 10B), and to potentiate camalexin and SA
550
production in challenge-infected 2° leaves upon a previous 1° pathogen inoculation (Fig.
551
7B; Supplemental Figure 11B). In contrast to ald1, however, fmo1 was not capable of
552
activating defense priming upon Pip feeding, because challenge-infected leaves of Pip-
553
supplied plants were not or only very faintly able to intensify ALD1 expression, PR1
554
expression (Fig. 8C; Supplemental Figure 12C), camalexin accumulation (Fig. 8D;
555
Supplemental Figure 12D), and SA biosynthesis (Supplemental Figure 13). This
556
indicates that FMO1 is a critical mediator of Pip-activated conditioning events that
557
determine SAR-associated defense priming.
acid
mediates
SAR-associated
defense
priming
via
FLAVIN-
558
559
Pip and SA act synergistically and independently from each other to induce PR
560
gene expression and disease resistance
19
561
Exogenous SA is sufficient to induce expression of a set of defense-related genes, and
562
to confer enhanced plant resistance to different hemibiotrophic and biotrophic
563
pathogens (Delaney et al., 1995; Sticher et al., 1997; Thibaud-Nissen et al., 2006). To
564
examine a possible interplay of Pip and SA in the regulation of plant immune responses,
565
we watered plants with 10 µmol Pip via the root system, subsequently infiltrated 0.5 mM
566
SA into leaves, and determined the transcript levels of the classical SA-inducible gene
567
PR1 4 hours after SA treatment. Single Pip and SA applications as well as a control
568
treatment were included, so that, as for the priming assays described above, four cases
569
could be distinguished and priming assessed in an analogous manner (Pip/SA –
570
Pip/mock > H2O/SA – H2O/mock; Supplemental Figure 9). SA alone induced strong
571
expression of PR1 in Col-0, ald1, sid2, and sid2 ald1 plants, indicating that elevated SA
572
levels are sufficient to trigger PR1 expression in the absence of Pip. However, this
573
response was markedly fortified in all four genotypes when plants had been pre-treated
574
with Pip, indicating synergism between SA and Pip in the induction of PR1 (Fig. 9A;
575
Supplemental Figure 14A). Further, the response to SA alone was higher in the Col-0
576
wild-type than in the other lines, suggesting that the capacity of endogenously
577
synthesizing Pip or SA positively affects the induction of PR1 by exogenous SA. Pip
578
treatment alone caused increased expression of PR1 as well. This induction was almost
579
absent in sid2 and in sid2 ald1, indicating that Pip-induced PR1 expression depends on
580
an intact SA biosynthetic pathway (Fig. 9A; Supplemental Figure 14A).
581
In addition to functional FMO1, intact PAD4 and NPR1 genes are required for a
582
strong resistance induction by exogenous Pip (Návarová et al., 2012). To get deeper
583
mechanistic information about the synergistic interplay of Pip and SA in PR1 induction,
584
we examined the behavior of fmo1, pad4, and npr1 mutant plants in our assay. SA
585
alone triggered a strong, wild-type-like expression of PR1 in fmo1. However, in contrast
586
to the wild type, we observed neither significantly increased PR1 expression upon Pip-
587
treatment alone nor the Pip-triggered intensification of SA-induced PR1 expression in
588
fmo1 (Fig. 9B; Supplemental Figure 14B). This indicates that FMO1 acts downstream of
589
Pip but upstream of SA in inducible PR1 expression. Moreover, FMO1 is required for
590
the intensification of SA-induced PR1 expression by Pip. Similar to FMO1, PAD4 is not
591
essential for SA-induced, but required for Pip-induced, PR1 expression, indicating that
20
592
PAD4 is also positioned between Pip and SA in the signaling pathway leading to PR1
593
induction. In contrast to fmo1, however, pad4 was not impaired in the Pip-mediated
594
intensification of PR1 expression, suggesting that PAD4 is not involved in the
595
synergistic interplay between SA and Pip (Fig. 9B; Supplemental Figure 14B). Finally,
596
the npr1 mutant did not show discernible expression of PR1 after any of the treatments,
597
indicating that NPR1 functions downstream of both Pip and SA in the induction of PR1
598
(Fig. 9B; Supplemental Figure 14B).
599
Exogenous Pip treatment results in a significant resistance induction in Col-0,
600
ald1, and sid2 but not in fmo1 plants (Návarová et al., 2012). Complementarily, we now
601
tested the abilities of Col-0, ald1, fmo1, and sid2 to augment basal resistance to Psm in
602
response to exogenous SA (Fig. 9C). SA treatment strongly increased resistance to
603
Psm in all the genotypes. In fact, the degree of resistance induction was even higher in
604
ald1 and fmo1 than in Col-0, since bacterial growth was attenuated by exogenous SA to
605
93-95% in the mutants but only to 88% in the wild-type (Fig. 9C). This indicates that
606
exogenous SA can induce both PR gene expression (Fig. 9A; Supplemental Figure
607
14A) and disease resistance (Fig. 9C) past Pip/FMO1-signaling and points to a
608
redundant mode of action of Pip and SA. Nevertheless, the above-described
609
amplification of SA signaling by Pip/FMO1 (Fig. 9A; Supplemental Figure 14A) was still
610
apparent in this resistance assay, because exogenous SA restricted bacterial growth to
611
lower absolute numbers in Col-0 than in ald1 or fmo1 (Fig. 9C). Yet more strikingly, both
612
Pip and SA pre-treatments failed to reduce bacterial multiplication in sid2 to the same
613
absolute values as in the wild-type (Fig. 9C, Návarová et al., 2012), indicating the
614
necessity for endogenous SA production for full resistance induction. Together, the
615
response patterns observed here for exogenous SA and by Návarová et al. (2012) for
616
exogenous Pip treatments place FMO1 downstream of Pip but upstream of SA in
617
resistance induction. Exogenous SA can trigger a strong but not a complete immune
618
response without functional Pip signaling. Reciprocally, Pip can induce a marked but
619
not a full resistance response independently from SA biosynthesis. Together, our
620
findings show that Pip and SA exhibit synergistic, independent, and redundant modes of
621
action in plant immunity.
622
21
623
DISCUSSION
624
The current study provides insights into the interplay of two key SAR regulatory plant
625
metabolites, the phenolic SA and the non-protein amino acid Pip (Nawrath and Métraux,
626
1999; Wildermuth et al., 2001; Návarová et al., 2012), in SAR establishment, SAR-
627
associated defense priming, and basal plant immunity. Key Pip and SA biosynthetic and
628
signaling genes exhibit strong transcriptional activation upon SAR induction throughout
629
the plant (Fig. 1A; Song et al., 2004b; Mishina and Zeier, 2006; Attaran et al., 2009;
630
Návarová et al., 2012), and both metabolites accumulate systemically in the foliage of
631
Arabidopsis plants locally leaf-inoculated with P. syringae, whereby the initial rise of Pip
632
in the systemic tissue timely precedes the increase of SA (Návarová et al., 2012).
633
Similar to many other studies (Nawrath and Métraux, 1999; Wildermuth et al., 2001; Vlot
634
et al., 2009), we assessed here resistance responses of the SA-induction-deficient sid2
635
mutant, which is defective in the pathogen-inducible SA biosynthesis gene ICS1, to
636
investigate the function of SA in basal immunity and SAR. By analogy, we used ald1
637
plants defective in Pip accumulation to deduce the immune responses that are
638
regulated by Pip. Taken together, isotope-labelling, biochemical, and metabolite studies
639
strongly suggest that Pip is derived from Lys by a two-step mechanism, and that ALD1
640
catalyzes a first aminotransferase step therein (Gupta and Spenser, 1969; Song et al.,
641
2004a; Návarová et al., 2012; Zeier, 2013). The direct involvement of ALD1 in Pip
642
biosynthesis and the fact that exogenous Pip can complement ald1 resistance defects
643
(Návarová et al., 2012) suggest that, just as sid2 is useful for SA-related research, ald1
644
is a suitable genetic tool to assess the function of Pip in plants. Given the above-
645
mentioned lines of evidence, the possibilities that metabolites other than SA and Pip (or
646
direct derivatives thereof) substantially contribute to the sid2 and ald1 phenotypes,
647
respectively, are unlikely in our opinion. Whereas the recent identification of the bona
648
fide SA receptors NPR1, NPR3, and NPR4 (Wu et al., 2012; Fu et al., 2012) has
649
validated that free SA is an active signal, it is not clear yet whether unmodified Pip or a
650
direct, possibly FMO1-generated Pip-derivative is signalling-active (see further
651
discussion below regarding FMO1; Zeier, 2013). In addition to the respective single
652
mutants, we have generated a Pip- and SA-deficient sid2 ald1 double mutant as one of
22
653
the tools to study the interplay between Pip and SA in basal and systemic immunity
654
(Supplemental Figures 1 and 2).
655
We showed that SAR establishment in Arabidopsis is characterized by a strong
656
transcriptional reprogramming of the systemic leaf tissue that involves robust activation
657
of more than 3400 SAR+ genes and suppression of nearly 2900 SAR- genes. Along with
658
systemic resistance induction (Fig. 2B; Song et al., 2004b; Návarová et al., 2012), this
659
massive transcriptional SAR response is virtually absent in ald1, indicating that
660
transcriptional reprogramming during SAR depends on the ability of plants to
661
biosynthesize Pip after pathogen inoculation (Fig. 1B, Fig. 3A, B; Supplemental Figures
662
5 and 7; Návarová et al., 2012). Since knockout of ALD1 alone is sufficient to fully
663
abrogate the transcriptional reprogramming in distal leaves, we consider it unlikely that
664
analogous RNA-seq analyses with the sid2 ald1 double mutant, which we have not
665
performed in this study, would provide mechanistic information about the transcriptional
666
SAR response beyond that acquired for ald1, although we cannot fully rule out this
667
possibility. However, in addition to ALD1, SAR and the transcriptional response
668
associated with SAR fully require intact FMO1 (Mishina and Zeier, 2006; Gruner et al.,
669
2013). Further, elevation of Pip levels by exogenous application induces a SAR-like
670
resistance response in a FMO1-dependent manner (Návarová et al., 2012). These
671
findings indicate that the establishment of biological SAR and the associated
672
transcriptional reprogramming of systemic leaf tissue are regulated by a master module
673
that involves accumulating Pip and its downstream-acting component FMO1 (Fig. 10A).
674
Whereas the critical function of Pip for SAR has been recognized only recently
675
(Návarová et al., 2012), it has been known for more than two decades that SA functions
676
as another key SAR player (Vernooij et al., 1994; Nawrath and Métraux, 1999). The
677
current study confirms this assessment and shows that the extent of SAR and of the
678
transcriptional response in systemic tissue is strongly attenuated in sid2 plants (Fig. 2B,
679
C; Fig. 3A, B). Our findings indicate that the degree of up-regulation of the vast majority
680
of the SAR+ genes in systemic tissue is positively influenced by SA (Tables 1 and 2).
681
However, our study adds an important aspect to the current viewpoint of SAR
682
regulation. We show that in the absence of induced SA production, a moderate SAR
683
and a partial transcriptional response in systemic tissue occurs upon primary bacterial
23
684
inoculation (Figs. 2B, 2C, 3A, 3B). This response not only becomes apparent in the
685
ICS1-defective sid2 plants, but also in another SA-induction-deficient mutant, sid1,
686
which bears a defect in the multidrug and toxin extrusion-like transporter ENHANCED
687
DISEASE SUSCEPTIBILITY5 (EDS5) (Fig. 2C). EDS5 is required for the export of
688
chloroplast-synthesized SA from this organelle, and SA accumulation in sid1/eds5 is
689
supposedly inhibited by auto-inhibitory feedback (Serrano et al., 2013). Further, the
690
partial SAR response is established in ics1 ics2, in which both isochorismate synthase
691
isoforms of Col-0 are inactive (Supplemental Figure 3). The ics1 ics2 double mutant not
692
only is impaired in stress-inducible SA biosynthesis but also displays strongly reduced
693
basal SA levels (Garcion et al., 2008). Therefore, our study reveals the existence of an
694
SA-independent signaling pathway that is able to activate a comparatively small but
695
significant SAR immune response in Arabidopsis. We show that this pathway is induced
696
upon inoculation with two inherently different bacterial pathogen types, compatible
697
(virulent) P. syringae pv. maculicola ES4326 (Psm), and incompatible (avirulent) Psm
698
avrRpm1, which, in contrast to Psm, causes a hypersensitive cell death response in
699
Col-0 leaves (Bisgrove et al., 1994). SAR assays conducted with the sid2 ald1 double
700
mutant indicate that the major, SA-dependent SAR activation pathway and the minor,
701
SA-independent activation pathway are both regulated by Pip (Fig. 2B). Therefore, the
702
Pip/FMO1 module is able to switch on a moderate but clearly discernable SAR
703
response in the absence of inducible SA biosynthesis (Fig. 10B).
704
From our expression analyses we deduce that, after accumulating in systemic
705
leaf tissue in a Pip/FMO1-dependent manner (Song et al., 2004b; Mishina and Zeier,
706
2006; Návarová et al., 2012), SA amplifies the expression of different SAR+ genes to
707
varying degrees (Tables 1 and 2). Systemic expression of the predominant portion of
708
the SAR+ genes shows a strict SA dependency, which signifies that these genes are not
709
up-regulated in a statistically significant manner in distal leaves of sid2 (Fig. 3A; Table
710
1). Paradigm examples for these strictly SA-dependent SAR+ genes are PR1, a classical
711
marker for the SA signaling pathway in plants and SAR establishment (Sticher et al.,
712
1997), ARD3, an Arabidopsis gene with sequence similarity to a rice acireductone
713
dioxygenase involved in the Yang cycle (Sauter et al., 2005), and GRXS13, encoding a
714
glutaredoxin facilitating infection of Arabidopsis with the fungal necrotroph Botrytis
24
715
cinerea (La Camera et al., 2011). About one quarter of SAR+ genes, however, were
716
significantly up-regulated in the systemic leaf tissue of sid2 upon 1°-inoculation and
717
showed higher absolute expression values in Psm-inoculated sid2 mutants than mock-
718
treated Col-0 plants (Fig. 3A; Table 2). These genes require SA for their full expression
719
in systemic tissue but can be induced to different degrees without SA elevation.
720
Notably, the two SAR regulatory and Pip pathway genes ALD1 and FMO1 rank among
721
the SAR+ genes with the strongest SA-independent expression characteristics (Table
722
2). This suggests that the Pip signaling pathway can function separately from SA to
723
switch on a subset of systemic resistance responses to a certain level. Nevertheless, for
724
a full and strong SAR response, accumulation of SA is required to potentiate these
725
partially SA-independent responses that include Pip production and signaling, and to
726
turn on other, strictly SA-dependent responses such as PR1 expression (Fig. 10A, B).
727
Functional ICS1 is not a sufficient prerequisite for pathogen-induced SAR, as
728
exemplified by the full SAR defects of ald1 and fmo1 (Fig. 2B; Song et al., 2004b;
729
Mishina and Zeier, 2006; Návarová et al., 2012). This is the case because Pip
730
biosynthesis and signal transduction via FMO1 are required to switch on virtually every
731
response in distal leaves of locally inoculated plants, including the expression of ICS1,
732
SA biosynthesis, and SA downstream responses (Fig. 1B, C; Fig. 3B; Mishina and
733
Zeier, 2006; Gruner et al., 2013; Fig. 10A-C). However, once SA levels elevate in
734
leaves, significant SA responses can be activated in the absence of a functional
735
Pip/FMO1 module. This is experimentally revealed in ald1 and fmo1 plants exogenously
736
supplied with SA, because these plants exhibit markedly enhanced expression of PR-1
737
and elevated resistance to Psm (Fig. 9). This aspect is relevant for defense processes
738
in inoculated leaves because here, in contrast to distal leaves, pathogen-induced ICS1-
739
expression and SA accumulation are still active in ald1 and fmo1 (Figs. 1A, C; Song et
740
al., 2004b; Mishina and Zeier, 2006; Bartsch et al., 2006). Our findings that SA-deficient
741
sid2 plants exhibit a more pronounced defect in local resistance to Psm than ald1 or
742
fmo1 (Fig. 2A; Fig. 9C) indicate that the contribution of the SA pathway to basal
743
immunity exceeds that of the Pip/FMO1-pathway. A largely Pip/FMO1-independent
744
activation of the SA defense pathway at pathogen-inoculation sites might explain why
745
defects in Pip biosynthesis and FMO1 result in comparatively moderate decreases of
25
746
basal immunity. In fact, the importance of FMO1 for local plant immune responses is
747
variable and depends on the nature of the attacking pathogen, with particular
748
significance of FMO1 for local immunity to pathogens that activate EDS1-dependent
749
and SA-independent branches of plant defense (Bartsch et al., 2006; Koch et al., 2006).
750
Interestingly, EDS1 and its interacting partner PAD4, both master regulators of basal
751
immunity (Feys et al., 2001), have also been identified as necessary SAR components
752
(Mishina and Zeier, 2006; Jing et al., 2011; Rietz et al., 2011; Breitenbach et al., 2014).
753
PAD4 positively regulates the transcription of a whole battery of defense genes
754
including the SAR-relevant genes ALD1, FMO1, and ICS1 (Zhou et al., 1998; Jirage et
755
al., 1999; Song et al., 2004b; Bartsch et al., 2006). Therefore, overlapping regulatory
756
principles of basal immunity and SAR exist. However, the relevance of the Pip/FMO1
757
module for SA pathway activation seems to be a main distinguishing feature of the
758
resistance modes at sites of Psm attack and in distal tissue (SAR). Whereas the
759
Pip/FMO1 module possesses an obligate switch function for SAR activation, it seems to
760
play a supportive but less rigorous role for resistance induction at inoculation sites.
761
Notably, our resistance assays with sid2 ald1 reveal that the SA and Pip defense
762
pathways provide additive, independent contributions to Arabidopsis basal resistance
763
towards Psm infection (Fig. 2A). This is further corroborated by the findings that
764
exogenous Pip is able to enhance resistance of sid2 to Psm (Návarová et al., 2012),
765
and that exogenous SA increases resistance of ald1 to Psm (Fig. 9C). Moreover,
766
Bartsch et al. (2006) have shown that a sid2 fmo1 double mutant is more susceptible to
767
the Hyaloperonospora arabidopsidis (Hpa) isolate Cala2, an avirulent oomycete
768
pathogen that is recognized by the RPP2 resistance protein in the Arabidopsis
769
accession Col-0, than either sid2 or fmo1 single mutants. This indicates additive
770
contributions of the SA- and Pip/FMO1-pathways also in resistance gene-mediated
771
responses.
772
However, our data also show a synergistic relationship between Pip and SA
773
signaling on several levels. First, application of Pip intensifies PR1 expression induced
774
by exogenous SA in a FMO1-dependent manner (Fig. 9A, B; Supplemental Figure 14A,
775
B; Fig. 10D). This enhancement, which also takes place in SA-deficient sid2 (Fig. 9A),
776
indicates that the Pip/FMO1 module amplifies SA downstream signaling events.
26
777
Second, elevated Pip levels trigger PR1 expression via the induction of SA biosynthesis
778
(Fig. 10E), as indicated by both the shortcoming of sid2 to induce PR1 upon exogenous
779
Pip feeding (Fig. 9A) and the ability of exogenous Pip to moderately induce SA
780
accumulation in Arabidopsis (Návarová et al., 2012). Pip therefore acts both upstream
781
of SA to trigger PR1 expression and downstream of SA to fortify SA-induced PR1
782
expression (Fig. 10D, E). The SA-mediated, PR1-inducing action of Pip as well as its
783
function as an SA signal intensifier share overlapping mechanisms because both
784
functions require functional FMO1 and operate upstream of NPR1. However, both
785
operation modes of Pip also differ mechanistically because the PR1-inducing but not the
786
intensifying mode requires PAD4 (Fig. 9B; Fig. 10D, E). Complementary to these
787
positive effects of Pip on SA biosynthesis and signaling, SA feeding alone is sufficient to
788
trigger small but significant elevations of ALD1 expression and Pip levels (Návarová et
789
al., 2012). These findings exemplify that, in addition to their above-described
790
independent modes of action, the Pip and SA pathways mutually reinforce each other in
791
the regulation of plant immunity and SAR.
792
Cross-talk between different stress-related plant signals is well-established
793
(Derksen et al, 2013). For example, synergism between JA and ethylene (ET) signaling
794
ensures effective induction of plant defenses directed against necrotrophic pathogen
795
attack (Memelink, 2009). Moreover, the JA/ET- and SA-signaling pathways negatively
796
influence each other to prioritize specific defense outputs in plant-pathogen interactions
797
(Koornneef and Pieterse, 2008; Zander et al., 2014). JA biosynthesis and signaling is
798
strongly induced by P. syringae infection in inoculated leaves. However, activation of
799
the JA pathway is variable and strongly correlates with necrotic tissue disruption but not
800
with systemic responses or SAR (Mishina and Zeier, 2007; Zoeller et al., 2012). With
801
the inoculation conditions to induce SAR used in this study, JA biosynthesis and
802
expression of JA-responsive genes are not elevated in systemic tissue of Col-0 wild-
803
type plants (Table 4; Gruner et al., 2013). Although a considerable systemic JA
804
response in Arabidopsis early after inoculation with high titers of avirulent P. syringae
805
has been detected and associated with SAR (Truman et al., 2007), genetic analyses
806
argue against a role of the JA pathway in Arabidopsis SAR establishment (Chaturvedi et
807
al., 2008; Attaran et al., 2009). Notably, our comparative RNA-seq analyses reveal that
27
808
distal leaves of locally inoculated sid2 plants express JA-responsive genes to a
809
significantly higher extent than Col-0 plants. In particular, the genes that were up-
810
regulated in sid2 but not changed or down-regulated in Col-0 are markedly enriched in
811
JA-inducible genes (Table 4). This indicates that the accumulation of SA during SAR in
812
wild-type plants suppresses systemic JA responses and directly illustrates the
813
occurrence of SA/JA antagonism within SAR. The apparent prevalence of SA over JA
814
signaling in SAR-induced plants might explain why SAR provides protection particularly
815
against pathogens with a (hemi)biotrophic lifestyle (Sticher et al., 1997). Potential cross-
816
talk of Pip with plant stress signals and hormones other than SA is not yet established.
817
However, a recent study reports that treatment of Arabidopsis seedlings with methyl
818
jasmonate decreases ALD1 expression and Pip levels in roots, indicating negative
819
influences of JA on Pip biosynthesis (Yan et al. 2014).
820
SAR can condition plants to induce defense responses more quickly and
821
efficiently after pathogen challenge (Jung et al., 2009; Návarová et al., 2012), a
822
phenomenon designated as defense priming (Conrath, 2011). Within our experimental
823
setup, we have defined a particular defense response as primed, if the difference
824
between the pathogen- and mock-response of SAR-activated (or Pip-stimulated) plants
825
is significantly larger than the same difference of non-activated plants (Supplemental
826
Figure 9B), resulting in the strongest absolute response level in pathogen-challenged,
827
SAR-activated (or Pip-stimulated) plants (Figs. 6 to 8; Supplemental Figures 10 to 12).
828
This does not mean that the pathogen-triggered fold-change of the response in SAR-
829
(Pip-) activated plants is larger than the respective fold-change in control plants. For
830
example, in the SAR-priming assay shown in Fig. 6A (Supplemental Figure 10A), the
831
quotient between the averaged 1°-Psm/2°-Psm- to the averaged 1°-Psm/2°-mock-
832
expression value for GRXS13 is 5.2 for the Col-0 wildtype, whereas the quotient
833
between the 1°-mock/2°-Psm- to the 1°-mock/2°-mock-value is 5.7. Therefore, an
834
important determinant of the response gain due to priming is the marked increase of the
835
response level caused by the inducing stimulus alone (SAR activation, Pip-stimulation).
836
A further increase of a strongly pre-elevated response level due to 2° challenge will
837
ultimately lead to a much stronger absolute response level than a similar increase from
838
a very small level present in non-induced plants. This mechanistic aspect of priming is
28
839
consistent with a general model of the priming phenomenon proposed by Bruce and
840
colleagues for two subsequent stress exposures (Bruce et al., 2007).
841
Between independent SAR- or Pip-induction experiments, the observed priming
842
patterns in wild-type plants are reproducible and can be tested with our proposed
843
algorithm (Supplemental Figure 9B, C). However, absolute or relative measurement
844
values resulting from one of the four treatment cases (e.g. mock/mock, mock/Psm,
845
Psm/mock or Psm/Psm) might quantitatively vary between experiments (for example,
846
compare Fig. 6A vs. Fig. 6B, Fig. 7A vs. Fig. 7B, or Figs. 8A/B vs. Figs. 8C/D). In this
847
context, we performed linear model-based analyses of the SAR-associated priming
848
responses (Figs. 6 and 7), the Pip-induced priming responses (Fig. 8), and the Pip-
849
mediated intensification of SA-inducible PR1 expression (Fig. 9A, B) in the Col-0 wild-
850
type using combined data of three independent experiments to estimate treatment and
851
experimental effect terms (Supplemental Tables 1, 2, and 3 ). In doing so, we assumed
852
that priming in our assays corresponds to the interaction of a pre-treatment (treatment
853
1) on the response of a second treatment (treatment 2). These analyses confirm that,
854
although experimental variation significantly influences the individual treatment
855
responses and the interaction term (priming) for most measured values, the individual
856
treatment responses and the priming response are stable over different experiments
857
(Supplemental Tables 1, 2, and 3).
858
Besides microbial encounters, adverse abiotic conditions can prime plants for
859
enhanced pathogen responses (Singh et al., 2014). For more than two decades, SA has
860
been attributed an important role in defense priming. For instance, in leaf tissue directly
861
adjacent to pathogen attack, SA has been implicated in the potentiation of expression of
862
defense genes that are not directly SA responsive (Mur et al., 1996). Further,
863
exogenous SA or pre-treatment with chemicals such as 2,6-dichloroisonicotinic acid or
864
S-methyl-1,2,3-benzothiadiazole-7-carbothioate
865
designated as SA analogues, are able to prime cultured plant cells or intact plants for
866
enhanced defense responses (Kauss et al., 1992; Thulke and Conrath, 1998), and
867
BTH-induced priming proceeds via the SA downstream regulator NPR1 (Kohler et al.,
868
2002). Moreover, treatments with chemicals that inhibit SA glycosylation and thereby
869
endogenously elevate free SA levels lead to priming responses in Arabidopsis
29
(BTH),
which
are
often
vaguely
870
(Noutoshi et al., 2012), and defense priming triggered by exogenous β-amino butyric
871
acid or the bacterial quorum sensing compound N-acyl-homoserine lactone requires an
872
intact SA signaling pathway (Zimmerli et al., 2000; Schenk et al., 2014). Our study
873
reveals a differentiated role of SA in the defense priming responses associated with
874
SAR: SA is required to prime plants for enhanced activation of certain defense
875
responses, but is dispensable for the potentiation of others. On the one hand, genes
876
such as PR1, ARD3 and GRXS13, which we had classified as SA dependent according
877
to their expression characteristics in systemic tissue upon SAR induction, also require
878
SA accumulation for augmented expression during challenge infection (Fig. 6A). On the
879
other hand, the expression of the partially SA-independent SAR genes ALD1, FMO1,
880
and SAG13 is potentiated in the SAR priming assays to a similar extent in Col-0 and
881
sid2, and a primary inoculation thus primes plants for enhanced expression of these
882
genes in an SA-independent manner in systemic tissue (Fig. 6A).
883
Our previous work has identified Pip as a critical regulator of SAR-associated
884
defense priming in Arabidopsis (Návarová et al., 2012). Exogenous Pip also increases
885
resistance of tobacco plants to P. syringae pv. tabaci and primes plants for early SA
886
accumulation, indicating a conserved role of Pip in plant defense priming (Vogel-
887
Adghough et al., 2013). The current study shows that Pip elevations in leaves are both
888
required and sufficient for the priming of both SA-dependent and SA-independent SAR
889
responses (Figs. 6 to 8; Supplemental Figures 10 to 12). Moreover, our results provide
890
evidence that, in addition to Pip, functional FMO1 is essential for the SAR-associated
891
priming phenomenon (Fig. 6B). In fact, FMO1 acts downstream of Pip in the activation
892
of defense priming because the priming responses induced by exogenous Pip are
893
virtually blocked in fmo1 (Fig. 8B; Supplemental Figure 13). This finding is consistent
894
with the critical role of FMO1 for SAR and resistance induction by Pip (Mishina and
895
Zeier, 2006; Návarová et al., 2012; Gruner et al., 2013). Future work should focus on
896
the biochemical function of the flavin monooxygenase FMO1. A conceivable scenario is
897
that FMO1 catalyzes the oxidation of Pip or a Pip-derived metabolite for defense signal
898
transduction (Zeier, 2013).
30
899
The existence of SA-independent defense priming responses associated with
900
SAR parallel recent findings on silicon-induced resistance in Arabidopsis, in which
901
increased silicon absorption primed plants for effective responses also in a sid2
902
background (Vivancos et al., 2014). Moreover, SA-independent SAR activation, which is
903
moderate for Arabidopsis (Fig. 2B, C), might be more pronounced in monocots, as
904
activation of systemic immunity in barley by leaf inoculation with bacterial pathogens
905
has been shown to proceed in an NPR1-independent manner (Dey et al., 2014). Which
906
molecular components besides Pip and FMO1 could be involved in the SA-independent
907
signaling pathway leading to partial SAR and defense priming in Arabidopsis? Recent
908
data point to a role of MAPK signaling cascades in the activation of SA-independent
909
resistance responses and SAR. On one hand, Tsuda and colleagues have shown that a
910
sustained activation of mitogen activated protein kinase 3 (MPK3) and MPK6 in leaves
911
of transgenic Arabidopsis plants results in the induction of PR1 and other SA-
912
responsive genes in sid2 and npr1, indicating that strong activation of MAPK signaling
913
can induce defense gene expression independently of a functional SA pathway (Tsuda
914
et al., 2013). PR1 induction in systemic leaf tissue of P. syringae-inoculated plants,
915
however, is strictly SA dependent (Table 1), indicating differences between the SA-
916
independent branch of biological SAR and the SA-independent signaling pathway
917
activated by sustained MAPK expression (Tsuda et al., 2013). Nevertheless, Beckers et
918
al. (2009) have reported that priming of abiotic stress responses and SAR are
919
compromised in a mpk3 mutant and attenuated in mpk6, indicating a role for MAPK3/6
920
signaling in SAR. In our SAR analyses, several MAPK, MAPKK, and MAPKKK genes
921
are significantly up-regulated upon SAR induction (Fig. 4F), including MPK3 and MPK6.
922
In sid2, however, systemic up-regulation of MAPK cascade genes is only weakly
923
induced, suggesting that transcriptional activation of these genes is largely SA
924
dependent (Supplemental Figure 15). Future experiments should further dissect the role
925
of MAPK signaling in SAR and, in particular, clarify its relationship to the Pip signaling
926
pathway.
927
Our RNA-seq data suggest that SAR prepares plants for future pathogen attack
928
by the transcriptional pre-activation of different, consecutive stages of defense
929
signaling. First, a widespread up-regulation of genes putatively involved in pathogen
31
930
recognition and early signal transduction occurs upon SAR establishment. For example,
931
many Toll-interleukin-1 receptor-like domain (TIR)-nucleotide binding site (NBS)-leucine
932
rich repeat (LRR) or coiled coil (CC)-NBS-LRR-type resistance proteins are up-
933
regulated upon SAR activation (Fig. 4E; Supplemental Figure 15). These include
934
VICTR, which encodes a TIR-NBS-LRR protein that associates with EDS1 and PAD4 in
935
protein complexes and is required for small molecule-mediated cross-talk between
936
abscisic acid and immune signaling (Kim et al., 2012). Further, genes coding for
937
receptor-like proteins and members of specific receptor-like kinase families prominently
938
occur in the SAR+ gene group (Fig. 4E). For instance, cysteine-rich protein kinase
939
(CRK) genes are widely up-regulated upon SAR establishment. Dexamethasone-
940
inducible expression of CRK13 in Arabidopsis, one of the most strongly up-regulated
941
CRK genes during SAR (Supplemental Figure 15), resulted in increased SA-mediated
942
resistance to P. syringae (Acharya et al., 2007). In addition, another SAR+ gene, the
943
RLK IMPAIRED OOMYCETE SUSCEPTIBILITY1 (IOS1), has been shown to regulate
944
priming of early PTI responses such as cell wall callose deposition (Chen et al., 2014).
945
These results suggest that the increased, broad-spectrum expression of resistance
946
proteins and pattern recognition receptors during SAR is an integral part of systemic
947
resistance activation and defense priming. Moreover, the well-characterized pattern
948
recognition receptor genes EFR, BAK1, and CERK1, but not FLS2 (Zipfel, 2014) are
949
among the SAR+ genes (Supplemental Figure 15). SA has been shown to dynamically
950
regulate the levels of FLS2 and BAK1 and to promote their up-regulation at later
951
signaling stages (Tateda et al., 2014). Consistent with the notion that SA positively
952
regulates microbial pattern receptors, sid2 shows strongly reduced up-regulation of
953
these genes. However, a marked SA-independent (but Pip-dependent) component of
954
receptor up-regulation is discernable, as exemplified by increased systemic expression
955
of several CRK genes in sid2 (Supplemental Figure 15).
956
Besides the above-discussed MAPK members, several other gene types
957
involved in signaling steps downstream of pathogen perception are broadly activated in
958
systemic tissue of SAR-induced plants. These include genes coding for Ca2+-signaling-
959
related proteins such as calcium-dependent protein kinases (CDPK), EF-hand
960
containing proteins, and calmodulin-binding proteins (Fig. 4G). Among those is CPK5
32
961
(Supplemental Figure 15), an Arabidopsis CDPK that phosphorylates the NADPH
962
oxidase isoform RbohD (Respiratory burst oxidase homolog D). CPK5 is required for
963
the rapid leaf-to-leaf propagation of defense responses upon PAMP perception, and
964
reactive oxygen species (ROS)-mediated cell-to-cell communication involving CPK5
965
and RbohD has been suggested as a basis for long-distance defense signal
966
propagation (Dubiella et al., 2013). However, a direct function of CPK5 in pathogen-
967
induced SAR has not yet been established.
968
Many immune-related signal transduction pathways culminate in the activation of
969
transcription factors and cofactors in the nucleus and thereby trigger enhanced
970
expression of defense-related genes. The transcriptional coactivator NPR1 acts as a
971
central transducer of SA responses and is essential for biological SAR as well as for
972
SAR-related transcriptional responses (Fu and Dong, 2013). For example, NPR1 is
973
required for the expression of about 98.5 % of BTH-inducible genes (Wang et al., 2006).
974
It is likely that NPR1 also largely regulates, besides the SA-dependent SAR activation
975
pathway, also the here-described Pip-dependent and partially SA-independent branch
976
of SAR induction, because exogenous Pip induces a much weaker resistance response
977
in npr1 than in sid2 (Návarová et al., 2012). NPR1 interacts with different members of
978
the TGA subfamily of bZIP transcription factors to regulate PR gene expression (Fu and
979
Dong, 2013). NPR1, its paralog NPR3, and three TGA genes (TGA5, TGA3, and TGA1)
980
are SAR+ genes showing a moderate but significant degree of up-regulation upon SAR
981
induction (Supplemental Figure 15).
982
Remarkably, more than one third of the WRKY family of transcription factors
983
belong to the SAR+ group, suggesting that WRKY factors play a major role in the SAR
984
transcriptional response (Fig. 4H). For many WRKYs, positive or negative regulatory
985
roles in distinct plant immune responses have been established (Pandey and Somssich,
986
2009). Among the SAR+ genes are several WRKY factor genes whose BTH-induced
987
expression depend on NPR1 (Supplemental Figure 15; Wang et al., 2006). WRKY18,
988
for example, controls about one fourth of BTH-induced and NPR1-regulated genes in
989
Arabidopsis, indicating that individual WRKY factors regulate expression of subgroups
990
of the SAR transcriptome (Wang et al., 2006). Interestingly, the marked up-regulation of
991
WRKY genes correlates with an enrichment of VQ motif-containing genes in the SAR+
33
992
group (Supplemental Figure 8B). VQ motif-containing proteins have been shown to
993
interact with WRKY proteins and appear to be part of transcriptional-regulatory protein
994
complexes in plant immunity (Cheng et al., 2012). Besides WRKYs and TGAs, NAC
995
transcription factors are overrepresented in the SAR+ gene group, whereas other
996
classes are not (Fig. 4H). This suggests a dominant function of particular transcription
997
factor families in mediating the SAR transcriptional response. Sequential waves of
998
specific transcription factor activities have been suggested to orchestrate plant immunity
999
(Moore et al., 2011), and this might be particularly true for SAR activation. For the
1000
initiation of this transcriptional program leading to SAR, the Pip/FMO1 module and the
1001
transcriptional co-regulator NPR1 are indispensable.
1002
Our results not only illustrate that SAR equips plants with an increased defense
1003
capacity at different signaling levels, but also show that the state of increased pathogen
1004
resistance is associated with a diminished photosynthetic rate systemically in the foliage
1005
and strongly decreased expression of photosynthesis-related genes (Fig. 4A; Fig. 5A;
1006
Supplemental Figure 5 and Supplemental Data Set 2). Moreover, the transcriptional
1007
SAR response illustrates an overall decline of several anabolic pathways, secondary
1008
metabolism, cell wall remodeling, and wax and cutin biosynthesis (Fig. 4A-C;
1009
Supplemental Data Set 2), suggesting that SAR is associated with reduced plant
1010
growth. The induced decline in photosynthetic rates is completely absent in both ald1
1011
and sid2 (Fig. 5A), indicating that the Pip-dependent accumulation of SA attenuates
1012
photosynthesis systemically in the plant. This interpretation is consistent with the
1013
observation that plants exhibiting constitutively high SA levels show decreased
1014
efficiency of several photosynthetic parameters (Mateo et al., 2006). Similarly,
1015
enhanced SA levels during SAR appear to trigger growth alleviation, because
1016
transcriptional gene down-regulation is strongly suppressed in sid2 (Fig. 5A), and plants
1017
with high constitutive SA levels exhibit retarded growth (Bowling et al., 1997; Mauch et
1018
al., 2001). The decrease in photosynthesis (and growth) might be a consequence of
1019
reduced stomatal conductance in SAR-activated plants, as the decreases in
1020
transpiration rates indicate (Fig. 5B). This would in turn limit CO2 uptake and carbon
1021
fixation. Since the transpiration rates do not systemically decrease in ald1 and sid2 (Fig.
1022
5B), and exogenous SA is able to induce stomatal closure in Arabidopsis (Zeng et al.,
34
1023
2011), accumulating SA during SAR also likely accounts for this phenomenon. A
1024
closure of stomata represents an integral part of the PAMP-induced immune response
1025
of plants to bacterial pathogen entry (Melotto et al., 2006), and our data suggest that the
1026
stomatal defense response is also pre-activated systemically in the foliage upon
1027
localized pathogen encounter.
1028
In
conclusion,
Arabidopsis
SAR
and
the
associated
defense
priming
1029
phenomenon are master-regulated by Pip accumulation and the action of FMO1
1030
downstream of Pip. The Pip/FMO1 switch module activates SA-dependent and SA-
1031
independent responses that both contribute to SAR establishment and the execution of
1032
defense priming. SA amplifies Pip/FMO1-dependent responses to different degrees and
1033
thereby ensures strong SAR and priming reactions. This work further indicates that Pip
1034
and SA cooperatively interact but can also function independently from each other in the
1035
induction of plant immune responses.
1036
1037
METHODS
1038
1039
Plant material and cultivation
1040
Arabidopsis thaliana L. Heynh. plants were grown individually in pots containing a
1041
mixture of soil (Klasmann-Deilmann, Substrat BP3), vermiculite and sand (8:1:1) in a
1042
controlled cultivation chamber with a 10-h day (9 AM to 7 PM; photon flux density 100
1043
µmol m-2 s-1) / 14-h night cycle and a relative humidity of 70 %. Day and night
1044
temperatures were set to 21°C and 18°C, respectively. Experiments were performed
1045
with 5- to 6-week-old, naïve plants exhibiting a uniform appearance.
1046
The ald1 and fmo1 mutants represent the SALK lines SALK_007673 and
1047
SALK_026163, respectively (Mishina and Zeier, 2006; Návarová et al., 2012). Further,
1048
sid2-1 (sid2, ics1; Nawrath and Métraux, 1999), ics1 ics2 (Garcion et al., 2008), npr1-2
1049
(npr1, NASC ID: N3801), and pad4-1 (pad4; Jirage et al., 1999) were used in this study.
1050
All mutant lines are in the Col-0 background.
35
1051
The sid2-1 ald1 (sid2 ald1) double mutant was generated by crossing of the sid2-
1052
1 (female parent) and ald1 (male parent) single mutants. From every fertilized silique,
1053
the F1 seeds were collected individually and dried before planting. About 110 sid2 ald1
1054
candidate plants were analyzed in the F2 generation as outlined in Supplemental
1055
Figures 1 and 2. One positive F2 candidate (sid2 ald1 #6) was self-pollinated and used
1056
for experiments. The position of the EMS-generated mutation in sid2-1 results in a stop
1057
codon (TAA) at residue 449 instead of a glutamine (Wildermuth et al., 2001). To identify
1058
the EMS-generated mutation, site specific primers were designed and the specific
1059
annealing temperature of 64°C determined in a gradient PCR. To identify the
1060
homozygous T-DNA insertion of ald1 by PCR, the method described by Alonso et al.
1061
(2003) was applied, using gene-specific primers (Supplemental Table 4). Seeds of ics1
1062
ics2 were sterilized and germinated on full MS medium containing 2% sucrose (pH 5.7)
1063
before the seedlings were transferred to soil (Garcion et al., 2008).
1064
1065
Cultivation of bacteria
1066
Pseudomonas syringae pv. maculicola strain ES4326 (Psm) and Psm harboring the
1067
avrRpm1 avirulence gene (Psm avrRpm1) were cultivated at 28°C in King’s B medium
1068
to which appropriate antibiotics were added (Zeier et al., 2004). Overnight log phase
1069
cultures were diluted to different final optical densities at 600 nm (OD600) for leaf
1070
inoculations. As experimentally determined, OD600 = 1 corresponds to 3.2*108 colony
1071
forming units (cfu) per ml.
1072
1073
SAR experiments, defense priming, and plant basal resistance
1074
To activate SAR, plants were infiltrated between 10 AM and 12 AM into three lower (1°)
1075
leaves with bacterial suspensions of OD600 = 0.005. Infiltration with 10 mM MgCl2
1076
served as the mock-control treatment. Upper (2°) leaves were harvested 48 h after the
1077
primary treatment for the determination of systemic gene expression and metabolite
1078
contents, as well as for RNA-seq analyses. For systemic resistance assays, 2° leaves
1079
were inoculated with Psm (OD600 = 0.001) two days after the 1° treatment. The number
36
1080
of Psm bacteria in 2° leaves was assessed another three days later as described in
1081
Návarová et al. (2012). SAR-associated defense priming was assessed by challenging
1082
2° leaves with either Psm (OD600 = 0.005) or infiltrating 10 mM MgCl2 as a mock-control
1083
two days after the 1°-inducing or 1°-mock treatments. 2° leaves were collected 10 h
1084
later for analyses (Supplemental Figure 9). For the determination of basal resistance,
1085
suspensions of Psm (OD600 = 0.001) were infiltrated into three full-grown leaves of naive
1086
plants. Bacterial growth was assessed three days later (Návarová et al., 2012).
1087
1088
Exogenous treatments with Pip and/or SA
1089
Exogenous Pip was applied to plants by pipetting 10 ml of a 1 mM (10 µmol)
1090
D,L-pipecolic acid solution (S47167; Sigma-Aldrich) onto the soil substrate of
1091
individually cultivated plants. Control plants were supplemented with 10 ml of H2O
1092
instead. Pip-feeding in this way served as the inducing treatment for Pip-priming
1093
assays. The bacterial challenge was performed one day after Pip application as
1094
described for the SAR priming assay. To determine synergism between Pip and SA, SA
1095
was infiltrated into leaves in a concentration of 0.5 mM one day after Pip-feeding, and
1096
H2O-infiltration served as a mock control. Leaf samples were collected 4 h after
1097
treatment.
1098
1099
Metabolite determination
1100
Determination of pipecolic acid levels in leaves was performed by a protocol detailed in
1101
Návarová et al. (2012), using GC/MS-based analysis following propyl chloroformate
1102
derivatisation. Free SA, conjugated SA and camalexin were determined by a method
1103
based on vapour-phase extraction and GC/MS analysis of metabolites as described by
1104
Attaran et al. (2009) and Návarová et al. (2012).
1105
1106
Quantitative real-time PCR analysis
1107
Quantitative real-time PCR (qPCR) analysis to assess gene expression in leaves was
1108
performed as detailed in Návarová et al. (2012) with minor modifications. In brief, 1 µg
37
1109
total RNA extracted with PeqGOLD RNAPure reagent (PeqLab, Erlangen, Germany)
1110
was used for cDNA synthesis using the RevertAid RT Reverse Transcription kit from
1111
Thermo Fisher Scientific Inc. (Waltham, USA). Transcript levels were measured based
1112
on SYBR Green technology using the GoTaq qPCR master mix of Promega (Promega
1113
Corporation, Madison, USA) according to the manufacturer´s instructions. Data was
1114
analyzed using the ΔΔCT method. Primers used for qPCR analyses are given in
1115
Supplemental Table 4.
1116
1117
Assessment of photosynthesis and transpiration rates by gas exchange
1118
Maximum photosynthesis rates were determined by CO2 uptake (µmol CO2 m−2 s−1)
1119
measurements, and stomatal conductance was assessed via the determination of
1120
transpiration rates (mmol H2O m−2 s−1) in intact leaves at a photosynthetic photon flux
1121
density of 1000 µmol photons m−2 s−1 using a LI-6400XT Portable Photosynthesis
1122
System (LI-COR Environmental, Lincoln, Nebraska, USA). Before recording the
1123
maximum photosynthetic rates, leaves were incubated for about 20 minutes in the
1124
measurement chamber until photosynthetic rates were stable. The temperature in the
1125
measuring cell was set to 22–23°C and the relative humidity was kept constant at 40 %
1126
during measurements.
1127
1128
Reproducibility of experiments and statistical analyses
1129
The presented results are generally derived from one experimental dataset, consisting
1130
of at least three biological replicates per treatment and genotype for metabolite and
1131
qPCR analyses, of seven biological replicates for bacterial growth assays, and of four
1132
biological replicates for IRGA analyses. For qPCR analyses, values for biological
1133
replicates were calculated as the mean of two technical replicates. The presented
1134
results were similar in three independent experiments. For the statistical evaluation of
1135
the qPCR and metabolite data by Student’s t test, log10-transformed relative expression
1136
and metabolite content values were analyzed. Bacterial growth data were analyzed by
1137
Student’s t test without prior log-transformation.
38
1138
Assessment of the primed state was performed as described in Supplemental
1139
Figure 9 by comparing permutated differences of measured values by a two-sided
1140
Mann-Whitney-U test (α = 0.005). To assess treatment effects, interaction terms
1141
between the two consecutive treatments, and between-experiment variation in the SAR-
1142
associated priming assays, the Pip-induced priming assays, and the Pip/SA-co-
1143
application assays, we assumed a linear model and performed analyses of variance
1144
(ANOVA) (Brady et al., 2015) with type II-sum of squares on Col-0 response data from
1145
three independent experiments using the R statistical package (https://www.r-
1146
project.org/) and the command:
1147
Anova(lm(Phenotype~Treatment1+Treatment2+Experiment+Treatment1*Treatment2+T
1148
reatment1*Experiment+Treatment2*Experiment+Treatment1*Treatment2*Experiment,
1149
data=object1),type=2) (Supplemental Tables 1, 2, and 3).
1150
The statistical analysis of the RNA-seq data is described below.
1151
1152
Genome-wide transcriptional analyses of the SAR state
1153
Three biologically independent, replicate SAR induction experiments were performed as
1154
described in the paragraph “SAR experiments” with Col-0 and sid2 plants (experimental
1155
set 1), and three other biologically independent experiments with Col-0 and ald1 plants
1156
(experimental set 2). In each SAR experiment, at least 6 upper (2°) leaves from 6
1157
different plants pre-treated in 1° leaves with Psm (MgCl2) were pooled for one biological
1158
Psm- (mock-control) replicate. In this way, 3 biologically independent, replicate samples
1159
per treatment and plant genotype were obtained within each SAR set.
1160
RNA was extracted from leaf sample replicates with the Plant RNeasy extraction
1161
kit (Qiagen, Germany), and treated on-column (Qiagen, Germany) and in solution with
1162
RNA-free DNAse (New England Biolabs, MA, USA). RNA integrity, sequencing library
1163
quality and fragment size were checked on a 2100 Bioanalyzer (Agilent, CA, USA).
1164
Libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina, San
1165
Diego, CA) and library quantification was performed with a Qubit 2.0 (Invitrogen,
1166
Germany). Leaf samples were multiplexed 12 libraries per lane, yielding approximately
39
1167
150 million reads per lane. All libraries were sequenced on the HISEQ2500 Illumina
1168
platform (San Diego, CA). Libraries for the not stranded RNA-seq experiments were
1169
sequenced in the single end (SE) mode with length of 50 or 100 nucleotides.
1170
Reads were de-multiplexed and mapped to the Arabidopsis thaliana genome by
1171
CLC bio Genomics Workbench© with default parameters (alignment over at least 80 %
1172
of the length of the read, up to three mismatches allowed) without alternative transcript
1173
detection. Differences in change ratios between mock and Psm-treatment for the two
1174
independent Col-0 experimental sets were determined using a linear model framework
1175
[lm(log2(rpm) ~ experiment*treatment)] resulting in no statistically different change
1176
ratios. The data for the two experimental sets were then combined for analyses.
1177
Differential expression between mock-treated and Psm-treated samples was calculated
1178
as a pairwise using edgeRs classic method on raw read counts as implemented in
1179
Bioconductor (Robinson et al., 2010). Genes are considered significantly differentially
1180
expressed if the FDR is below 0.01 (Benjamini and Hochberg, 1995). Reads per million
1181
(rpm) are reported for all transcripts. Rpm were averaged for the replicates of each
1182
condition and the log2 of fold-changes between Psm- and mock values (= P/M-fold
1183
changes) were calculated by formally adding one read to all rpm values to account for
1184
transcripts with no expression detected in one or more samples before fold-change
1185
calculation and logarithmic transformation (Bräutigam et al., 2011). This allowed us to
1186
keep genes with very low mock-expression values but marked Psm-triggered
1187
expression in the data sets without significantly changing the actual logarithmized P/M-
1188
ratios. Differences in change ratios between Col-0 and sid2 for all genes without 0
1189
counts in any experiment were determined using a linear model framework
1190
[lm(log2(rpm) ~ genotype*treatment)] with p-values corrected (Benjamini and Hochberg,
1191
1995). The complete RNA-seq data of the SAR experiments are provided in
1192
Supplemental Data Set 1. Arabidopsis transcripts are annotated with descriptions from
1193
TAIR10 (Swarbreck et al., 2008) and functional annotations from MapMan (Thimm et
1194
al., 2004). Overlapping and exclusive membership in the significantly changed genes
1195
were calculated with Linux functions. To determine the percentages of SAR genes in
1196
specific gene categories or families (http://www.arabidopsis.org/), selected gene sets
1197
[major MapMan categories (Thimm et al., 2004), categories with a presumed role in
40
1198
defense signaling, and categories with an evident enrichment in SAR+ and SAR- genes
1199
based on careful inspection by eye of the whole data set] were aligned to the RNA-seq
1200
data set using the Excel® macro FIRe (Garcion et al., 2006). Fisher’s exact test (P <
1201
0.01) corrected according to Benjamini and Hochberg (1995) was used to determine
1202
whether gene categories were significantly enriched or depleted in SAR+ and SAR-
1203
genes. GO term enrichment was analyzed and visualized using the GOrilla tool using
1204
the target and background genelist method (Eden et al., 2009).
1205
1206
Accession Numbers
1207
RNA-seq data were deposited in the ArrayExpress database under the accession
1208
XXXXX. Sequence data from this article for the major genes discussed can be found in
1209
the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following
1210
accession numbers: ALD1 (At2g13810), FMO1 (At1g19250), ICS1 (At1g74710), PR-1
1211
(At2g14610), PAD4 (At3g52430), NPR1 (At1g64280), ARD3 (At2g26400), SAG13
1212
(At2g29350), GRXS13 (At1g03850).
1213
1214
Supplemental Data
1215
The following materials are available in the version of this article.
1216
Supplemental Figure 1. Identification of the sid2 ald1 (sid2-1 ald1) double mutant.
1217
Supplemental Figure 2. Characterization of the sid2 ald1 double mutant.
1218
Supplemental Figure 3. Systemic acquired resistance assay with Col-0 and ics1 ics2
1219
plants.
1220
Supplemental Figure 4. Principle component analysis of the normalized transcriptome
1221
data.
1222
Supplemental Figure 5. MapMan visualization: the transcriptional SAR response in
1223
Col-0.
1224
Supplemental Figure 6. MapMan visualization: a diminished transcriptional SAR
1225
response exists in sid2.
41
1226
Supplemental Figure 7. MapMan visualization – the transcriptional SAR response is
1227
absent in ald1.
1228
Supplemental Figure 8. Percentage of SAR+ and SAR- genes in additional gene
1229
classes/families.
1230
Supplemental Figure 9. SAR-associated defense priming – assay and definition.
1231
Supplemental Figure 10. Graphs of Figure 6 with a linear scale for the y-axes instead of
1232
a log-scale.
1233
Supplemental Figure 11. Graphs of Figure 7 with a linear scale for the y-axes instead of
1234
a log-scale.
1235
Supplemental Figure 12. Graphs of Figure 8 with a linear scale for the y-axes instead of
1236
a log-scale.
1237
Supplemental Figure 13. Pip-induced priming of salicylic acid biosynthesis requires
1238
functional FMO1.
1239
Supplemental Figure 14. Graphs of Figures 9A and 9B with a linear scale for the y-axes
1240
instead of a log-scale.
1241
Supplemental Figure 15. The transcriptional SAR response: activation of multiple stages
1242
of defense signaling. Expression values of selected genes.
1243
Supplemental Table 1. Linear model-based analysis of the SAR-associated priming
1244
response in Col-0 plants to estimate treatment and experimental effect terms.
1245
Supplemental Table 2. Linear model-based analysis of the pipecolic acid-induced
1246
priming response in Col-0 plants to estimate treatment and experimental effect terms.
1247
Supplemental Table 3. Linear model-based analysis of the amplification of salicylic acid-
1248
induced PR1 expression by pipecolic acid in Col-0 plants to estimate treatment and
1249
experimental effect terms.
1250
Supplemental Table 4. List of primers used in this study.
1251
Supplemental Data Set 1. Complete RNA-seq data of the SAR experiments with Col-0,
1252
sid2, and ald1.
42
1253
Supplemental Data Set 2. GO term enrichment analysis of the SAR- gene group.
1254
Supplemental Data Set 3. GO term enrichment analysis of the SAR+ gene group.
1255
1256
ACKNOWLEDGMENTS
1257
This work was supported by the German Research Foundation (DFG grant ZE467/6-1
1258
and Graduate program IRTG 1525). We are indebted to Anna Busch for excellent
1259
technical assistance, Wolfgang Kaisers (Center for Bioinformatics and Biostatistics,
1260
HHU Düsseldorf), Markus Kollmann and Martin Lercher for support on statistical issues,
1261
and Jean-Pierre Métraux for kindly providing ics1 ics2 seeds. We thank Michael
1262
Hartmann and Philippe Reymond for critically reading the manuscript.
1263
1264
AUTHOR CONTRIBUTIONS
1265
F.B., A.-C.D., K.G., and St.S. performed the experiments. A.B., F.B., and J.Z.
1266
contributed to RNA-seq analyses and evaluation of expression data. F.B. and A.B.
1267
assisted J.Z. in manuscript preparation. J.Z. designed the research and wrote the
1268
manuscript.
43
1269
Tables
1270
Table 1. SAR+ genes tightly regulated by SA (SA-dependent SAR+ genes).
1271
RNA-seq analyses identified 2672 SAR+ genes with significant Psm-induced up-regulation in the Col-0 wild-type and no significant
1272
induction in sid2. RNA samples originate from distal leaves of Psm (P)-inoculated and mock (M)-treated Col-0 and sid2 plants at 48
1273
HAI. Mean log2-transformed P/M-ratios (fold-changes) are depicted, and asterisks indicate significant changes between P- and M-
1274
treatments (FDR < 0.01). The 2672 genes were listed in ascending order according to their sid2 P/M ratios. The top 15 SAR+ genes
1275
from this category (i.e. those with highest P/M-fold changes in Col-0) are shown. P values for differences in log2-fold-changes in
1276
Col-0 and sid2, determined by using a linear model framework [lm(log2(rpm) ~ genotype*treatment)], are given. Number values
1277
indicate significant differences (P < 0.05).
1278
Mean Expression Value
Fold-change
(log2)
P value
log2 (P/M)
AGI Code
Name
Gene Name / Description
Col-0
M
Col-0
P
sid2
M
sid2
P
Col-0
P/M
sid2
P/M
Col-0 vs
sid2
At2g26400
ARD3
ACIREDUCTONE DIOXYGENASE 3
17.5
1395.8
2.2
2.7
6.2*
0.2
0.0115#
At2g14620
XTH10
xyloglucan endotransglucosylase/hydrolase 10
3.1
156.4
0.9
1.6
5.3*
0.4
0.0125#
At2g14610
PR1
PATHOGENESIS-RELATED PROTEIN 1
98.7
3801.4
6.0
10.3
5.3*
0.7
0.0243#
At3g22910
ACA13
putative calcium-transporting ATPase 13
5.6
247.0
1.6
3.1
5.2*
0.6
0.0160#
At3g28510
-
AAA-type ATPase family protein
11.0
411.5
1.8
2.7
5.1*
0.4
0.0271#
At3g13950
-
unknown protein
5.4
173.3
5.3
18.6
4.8*
1.6
0.0357#
At4g10860
-
unknown protein
1.6
57.2
0.1
0.3
4.5*
0.2
0.0634
At3g53150
UGT73D1
UDP-GLUCOSYLTRANSFERASE 73D1
1.4
49.9
0.1
0.6
4.4*
0.6
0.1793
At4g35180
LHT7
LYS/HIS TRANSPORTER 7
4.2
105.9
0.7
1.9
4.4*
0.7
0.0959
At1g03850
GRXS13
GLUTAREDOXIN 13
7.8
175.8
2.7
5.2
4.4*
0.7
0.0138#
At4g01870
-
tolB protein-related
8.6
196.5
3.5
17.2
4.4*
2.0
0.0716
At1g65610
GH9A2
GLYCOSYL HYDROLASE 9A2
1.6
51.6
0.6
2.2
4.4*
1.0
0.0274#
At3g61190
BAP1
BON ASSOCIATION PROTEIN 1
2.1
58.6
1.2
5.8
4.2*
1.6
0.1060
At4g37530
PER51
peroxidase superfamily protein
12.4
241.7
8.0
20.6
4.2*
1.3
0.0132#
At5g18270
ANAC087
NAC DOMAIN CONTAINING PROTEIN 87
0.8
31.3
0.3
1.3
4.2*
0.8
0.0191#
1279
1280
1281
1282
45
1283
Table 2. Partially SA-independent SAR+ genes.
1284
SAR+ genes with significant Psm-induced up-regulation in distal leaves of both Col-0 and sid2. The subgroup of SAR+ genes (741
1285
genes in total) was listed in descending order according to their sid2 P/M ratios. Asterisks indicate significant changes (FDR <
1286
0.01). The 15 genes with the highest P/M-fold change in sid2 and Col-0 log2P/M values > 4.0 are depicted. Four selected genes
1287
from lower parts of the gene list are also shown. The position of each gene in the full SAR+ gene list is indicated in the first column.
1288
Note that the Pip-pathway genes ALD1 and FMO1 occur at the top of the list. P values for differences in log2-fold-changes in Col-0
1289
and sid2 are given. Number values indicate significant differences (P < 0.05).
1290
Mean Expression Value
Col-0
P/M
sid2
P/M
Col-0 vs
sid2
55.6 2089.5 11.1 1302.5
5.2*
6.8*
0.8032
AGD2-LIKE DEFENSE RESPONSE PROTEIN 1
12.1
378.6
0.8
140.2
4.9*
6.3*
0.1956
FMO1
FLAVIN-DEPENDENT MONOOXYGENASE 1
3.9
173.2
0.4
84.3
5.2*
6.0*
0.6120
At2g43570
CHI
putative chitinase
45.7 1523.2
3.7
286.4
5.2*
5.9*
0.7260
6
At2g29460
GST22
GLUTATHIONE S-TRANSFERASE 22
5.4
274.4
1.1
112.2
5.6*
5.8*
0.9231
7
At3g09940
MDAR3
MONODEHYDROASCORBATE REDUCTASE 3
4.9
126.6
0.8
93.8
4.8*
5.7*
0.8622
9
At1g02930
GSTF6
GLUTATHIONE S-TRANSFERASE 6
115.7 2530.5 14.7
596.6
4.1*
5.3*
0.4248
10
At1g33960
AIG1
AVRRPT2-INDUCED GENE 1
54.1 2285.9
7.6
325.5
5.4*
5.3*
0.7475
11
At3g57260
PR2
PATHOGENESIS-RELATED PROTEIN 2
179.9 3193.3 15.9
634.4
4.3*
5.2*
0.8218
12
At3g22600
LTPG5
GPI-ANCHORED LIPID TRANSFER PROTEIN 5
15.6
81.1
5.8*
5.2*
0.8393
Pos.
AGI Code
Name
Gene Name / Description
1
At2g24850
TAT3
TYROSINE AMINOTRANSFERASE 3
3
At2g13810
ALD1
4
At1g19250
5
46
Col-0
M
Col-0
P
Fold-change P value
(log2)
log2 (P/M)
775.0
sid2
M
1.2
sid2
P
13
At3g26830
12.1
421.8
0.9
67.5
5.3*
5.2*
0.8512
14
At2g38240
0.2
20.0
0.2
41.4
4.8*
5.1*
0.7480
15
At2g29350
SAG13
SENESCENCE-ASSOCIATED GENE 13
27.4 1135.6
4.0
165.1
5.8*
5.0*
0.5042
16
At1g57630
-
Toll-Interleukin-Resistance domain family protein
6.2
255.9
0.9
55.9
5.3*
4.9*
0.9346
17
At2g04450
NUDT6
nudix hydrolase homolog 6
35.7
623.0
2.7
102.0
4.1*
4.8*
0.9346
120
At1g75040
PR5
PATHOGENESIS-RELATED PROTEIN 5
274.8 5349.1 65.7
539.2
4.3*
3.0*
0.1159
350
At4g28390
AAC3
ADP/ATP CARRIER 3
15.7
165.0
5.9
25.6
3.3*
2.0*
0.0473#
510
At2g17720
P4H5
PROLYL 4-HYDROXYLASE 5
28.2
188.0
18.5
58.3
2.7*
1.6*
0.0882
655
At2g19190
FRK1
FLG22-INDUCED RECEPTOR-LIKE KINASE 1
2.9
70.2
0.2
1.7
4.2*
1.2*
0.3649
PAD3
PHYTOALEXIN DEFICIENT 3
DOXC46 2–oxoglutarate-dependent dioxygenase superfamily
1291
47
1292
Table 3. Genes induced in systemic tissue of sid2 but not in Col-0.
1293
104 genes were identified with significant Psm-induced up-regulation in distal leaves of sid2 but not Col-0 plants (Fig. 3A). The
1294
genes were listed in descending order according to their sid2 P/M ratios. The 10 genes with highest P/M ratios in sid2 and all genes
1295
significantly down-regulated in Col-0 (SAR- genes) from this group are depicted. The position of each gene in the list is indicated.
1296
Asterisks indicate significant changes between Psm- and mock-treatments (FDR < 0.01).
1297
Mean Expression Value
Fold-change
(log2)
Pos.
AGI Code
Name
Gene Name / Description
Col-0
M
Col-0
P
sid2
M
sid2
P
Col-0
P/M
sid2
P/M
1
At5g24770
VSP2
VEGETATIVE STORAGE PROTEIN 2
2.9
7.6
10.4
1108.4
1.1
6.6*
2
At5g24780
VSP1
VEGETATIVE STORAGE PROTEIN 1
0.8
1.9
1.8
260.4
0.7
6.5*
3
At4g16590
CSLA01
CELLULOSE SYNTHASE-LIKE A01
0.8
1.2
1.6
133.2
0.3
5.7*
4
At1g76790
IGMT5
INDOLE GLUCOSINOLATE OMETHYLTRANSFERASE 5
0.6
0.4
1.8
116.5
-0.2
5.4*
5
At4g17470
-
α/β-hydrolases superfamily protein
1.1
2.6
2.7
148.8
0.8
5.3*
6
At2g24210
TPS10
TERPENE SYNTHASE 10
0.1
0.5
0.5
54.8
0.4
5.3
7
At3g11480
BSMT1
BENZOIC ACID/SALICYLIC ACID
METHYLTRANSFERASE
0.1
0.6
0.1
40.9
0.6
5.2*
8
At1g51780
ILL5
IAA-LEUCINE RESISTANT (ILR)-LIKE 5
0.0
0.4
0.1
27.8
0.5
4.8*
9
At4g13410
CSLA15
CELLULOSE SYNTHASE LIKE A15
3.8
1.0
2.6
95.5
-1.3
4.7*
10
At1g24070
CSLA10
CELLULOSE SYNTHASE-LIKE A10
1.3
1.0
1.4
55.0
-0.2
4.5*
48
11
At3g28220
-
TRAF-like family protein
26.3
6.5
31.2
686.5
-1.9*
4.4*
32
At3g28290
-
sequence similarity to integrins
2.4
0.5
1.9
28.9
-1.2*
3.4*
33
At3g28300
-
sequence similarity to integrins
1.9
0.5
2.0
29.3
-0.9*
3.3*
41
At1g52000
-
mannose-binding lectin superfamily
28.5
6.6
33.4
306.8
-2.0*
3.2*
70
At2g43550
-
scorpion toxin-like knottin superfamily
11.8
2.1
30.5
146.1
-2.0*
2.2*
91
At5g02940
-
protein of unknown function
109.2
32.0
148.3
464.3
-1.7*
1.6*
1298
1299
49
1300
1301
Table 4. Genes systemically induced in sid2 are strongly enriched in JA-responsive genes.
1302
Percentage of JA-responsive genes in the total number of RNA-seq-analyzed genes and in different gene categories (as illustrated
1303
in the left Venn diagram of Fig. 3A).
1304
Gene category
Number of genes
% JA-inducible genesb
P valuec
Whole gene seta
15239
3.2
-
Col-0 up (SAR )
3413
3.7
0.068
sid2 up (total)
845
12.8
9*10-38
sid2 up / Col-0 not upd
104
48.1
4*10-44
+
1305
a
after threshold cut-off
1306
b
Percentage of genes determined as JA-inducible by Goda et al. (2008)
1307
c
by Fisher’s exact test; indicates significance of enrichment
1308
d
genes up-regulated in sid2 but not in Col-0
1309
1310
50
1311
Table 5. The transcriptional SAR response is virtually absent in ald1.
1312
SAR genes with significant Psm-induced transcript changes in distal leaves of ald1 (FDR < 0.01). From the whole gene set, only
1313
two genes were significantly up-regulated in ald1. Moreover, the Psm-induced expression values of these genes in ald1 did not
1314
exceed the basal expression values in Col-0.
1315
Mean Expression Value
Fold-change
(log2)
AGI Code
Name
Gene Name / Description
Col-0
M
Col-0
P
ald1
M
ald1
P
Col-0
P/M
ald1
P/M
At4g23140
CRK6
CYSTEINE-RICH RECEPTOR-LIKE KINASE 6
52.4
636.3
5.3
26.1
3.6*
2.1*
At1g14880
PCR1
PLANT CADMIUM RESISTANCE 1
270.6 5673.3
7.2
29.0
4.4*
1.9*
1316
1317
51
1318
REFERENCES
1319
1320
Acharya, B.R., Raina, S., Maqbool, S.B., Jagadeeswaran, G., Mosher, S.L., Appel,
1321
H.M., Schultz, J.C., Klessig, D.F., and Raina, R. (2007). Overexpression of CRK13,
1322
an Arabidopsis cysteine-rich receptor-like kinase, results in enhanced resistance to
1323
Pseudomonas syringae. Plant J 50: 488-499.
1324
1325
1326
1327
Aliferis, K.A., Faubert, D., and Jabaji, S. (2014). A metabolic profiling strategy for the
dissection of plant defense against fungal pathogens. PLoS One 9: e111930.
Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis
thaliana. Science 301: 653-657.
1328
Attaran, E., Zeier, T. E., Griebel, T., and Zeier, J. (2009). Methyl salicylate production
1329
and jasmonate signaling are not essential for systemic acquired resistance in
1330
Arabidopsis. Plant Cell 21: 954-971.
1331
Bartsch, M., Gobbato, E., Bednarek, P., Debey, S., Schultze, J.L., Bautor, J., and
1332
Parker,
1333
SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by
1334
the monoxygenase FMO1 and the nudix hydrolase NUDT7. Plant Cell 18: 1038-
1335
1051.
J.E.
(2006).
Salicylic
acid-independent
ENHANCED
DISEASE
1336
Beckers, G. J., Jaskiewicz, M., Liu, Y., Underwood, W.R., He, S.Y., Zhang, S., and
1337
Conrath, U. (2009). Mitogen-activated protein kinases 3 and 6 are required for full
1338
priming of stress responses in Arabidopsis thaliana. Plant Cell 21: 944-953.
1339
Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate: a practical
1340
and powerful approach to multiple testing. J Roy Statist Soc Ser B (Methodological)
1341
57: 289-300.
1342
Bisgrove, S.R., Simonich, M.T., Smith, N.M., Sattler, A., and Innes, R.W. (1994). A
1343
disease resistance gene in Arabidopsis with specificity for two different pathogen
1344
avirulence genes. Plant Cell 6: 927-933.
1345
Bowling, S.A., Clarke, J.D., Liu, Y., Klessig, D.F., and Dong, X. (1997). The cpr5 mutant
1346
of Arabidopsis expresses both NPR1-dependent and NPR1-independent resistance.
1347
Plant Cell 9: 1573-1584.
1348
Boller, T., and Felix, G. (2009). A renaissance of elicitors: perception of microbe-
1349
associated molecular patterns and danger signals by pattern-recognition receptors.
1350
Annu Rev Plant Biol 60: 379-406.
1351
Brady, S.M., Burow, M., Busch, W., Carlborg, Ö., Denby, K.J., Glazebrook, J., Hamilton,
1352
E.S., Harmer, S.L., Haswell, E.S., Maloof, J.N., Springer, N.M., and Kliebenstein,
1353
D.J. (2015). Reassess the t Test: Interact with All Your Data via ANOVA. Plant Cell
1354
27: 2088-2094.
1355
Bräutigam, A., Kajala, K., Wullenweber, J., Sommer, M., Gagneul, D., Weber, K.L.,
1356
Carr, K.M., Gowik, U., Mass, J., Lercher, M.J., Westhoff, P., Hibberd, J.M., and
1357
Weber, A.P. (2011). An mRNA blueprint for C4 photosynthesis derived from
1358
comparative transcriptomics of closely related C3 and C4 species. Plant Physiol
1359
155: 142-156.
1360
Breitenbach, H.H., Wenig, M., Wittek, F., Jordá, L., Maldonado-Alconada, A.M.,
1361
Sarioglu, H., Colby, T., Knappe, C., Bichlmeier, M., Pabst, E., Mackey, D., Parker,
1362
J.E., and Vlot A.C. (2014). Contrasting roles of the apoplastic aspartyl protease
1363
APOPLASTIC, ENHANCED DISEASE SUSCEPTIBILITY1-DEPENDENT1 and
1364
LEGUME LECTIN-LIKE PROTEIN1 in Arabidopsis systemic acquired resistance.
1365
Plant Physiol 165: 791-809.
1366
1367
1368
1369
Broquist, H.P. (1991). Lysine-pipecolic acid metabolic relationships in microbes and
mammals. Annu Rev Nutr 11: 435-448.
Bruce T.J., Matthes, M.C., Napier, J.A., and Pickett. J.A. (2007). Stressful “memories” of
plants: evidence for possible mechanisms. Plant Sci 173: 603-608.
1370
Chaturvedi, R., Krothapalli, K., Makandar, R., Nandi, A., Sparks, A.A., Roth, M.R., Welti,
1371
R., and Shah, J. (2008). Plastid omega 3-fatty acid desaturase-dependent
1372
accumulation of a systemic acquired resistance inducing activity in petiole exudates
1373
of Arabidopsis thaliana is independent of jasmonic acid. Plant J 54: 106-117.
53
1374
Chen, C.W., Panzeri, D., Yeh, Y.H., Kadota, Y., Huang, P.Y., Tao, C.N., Roux, M.,
1375
Chien. S.C., Chin, T.C., Chu, P.W., Zipfel, C., and Zimmerli, L. (2014). The
1376
Arabidopsis malectin-like leucine-rich repeat receptor-like kinase IOS1 associates
1377
with the pattern recognition receptors FLS2 and EFR and is critical for priming of
1378
pattern-triggered immunity. Plant Cell 26: 3201-3219.
1379
Cheng, Y., Zhou, Y., Yang, Y., Chi, Y.J., Zhou, J., Chen, J.Y., Wang, F., Fan, B., Shi,
1380
K., Zhou, Y.H., Yu, J.Q., and Chen, Z. (2012). Structural and functional analysis of
1381
VQ motif-containing proteins in Arabidopsis as interacting proteins of WRKY
1382
transcription factors. Plant Physiol 159: 810-825.
1383
1384
Conrath, U. (2011). Molecular aspects of defence priming. Trends Plant Sci 16: 524531.
1385
Delaney, T.P., Friedrich, L., and Ryals, J.A. (1995). Arabidopsis signal transduction
1386
mutant defective in chemically and biologically induced disease resistance. Proc Natl
1387
Acad Sci USA 91: 8955–8959.
1388
1389
1390
1391
Dempsey, D. A., and Klessig, D. F. (2012). SOS - too many signals for systemic
acquired resistance? Trends Plant Sci 17: 538-545.
Derksen, H., Rampitsch, C., and Daayf, F. (2013). Signaling cross-talk in plant disease
resistance. Plant Sci 207: 79-87.
1392
Dey, S., Wenig, M., Langen, G., Sharma, S., Kugler, K.G., Knappe, C., Hause, B.,
1393
Bichlmeier, M., Babaeizad, V., Imani, J., Janzik, I., Stempfl, T., Hückelhoven, R.,
1394
Kogel, K.H., Mayer, K.F., and Vlot, A.C. (2014). Bacteria-triggered systemic
1395
immunity in barley is associated with WRKY and ETHYLENE RESPONSIVE
1396
FACTORs but not with salicylic acid. Plant Physiol 166: 2133-2151.
1397
Dubiella, U., Seybold, H., Durian, G., Komander, E., Lassig, R., Witte, C.P., Schulze,
1398
W.X., and Romeis, T. (2013). Calcium-dependent protein kinase/NADPH oxidase
1399
activation circuit is required for rapid defense signal propagation. Proc Natl Acad Sci
1400
USA 110: 8744–8749.
54
1401
Eden, E., Navon, R., Steinfeld, I., Lipson, D., and Yakhini, Z. (2009). GOrilla: A tool for
1402
discovery and visualization of enriched GO terms in ranked gene lists. BMC
1403
Bioinformatics 10: 48.
1404
Feys, B.J., Moisan, L.J., Newman, M.A., and Parker, J.E. (2001). Direct interaction
1405
between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4.
1406
EMBO J. 20: 5400-5411.
1407
Fritz-Laylin, L. K., Krishnamurthy, N., Tör, M., Sjölander, K. V., and Jones, J. D. G.
1408
(2005). Phylogenomic analysis of the receptor-Like proteins of rice and Arabidopsis.
1409
Plant Physiol 138: 611-623.
1410
Fu, Z. Q., Yan, S., Saleh, A., Wang, W., Ruble, J., Oka, N., Mohan, R., Spoel, S. H.,
1411
Tada, Y., Zheng, N., and Dong, X. (2012). NPR3 and NPR4 are receptors for the
1412
immune signal salicylic acid in plants. Nature 486: 228-232.
1413
1414
Fu, Z.Q., and Dong, X. (2013). Systemic acquired resistance: turning local infection into
global defense. Annu Rev Plant Biol. 64: 839-863.
1415
Fujioka, S., Sakurai, A., Yamaguchi, I., Murofushi, N., Takahashi, N., Kaihara, S., and
1416
Takimoto, A. (1987). Isolation and identification of L-pipecolic acid and nicotinamide
1417
as flower-inducing substances in Lemna. Plant Cell Physiol. 28: 995-1003.
1418
Garcion, C., Baltensperger, R., Fournier, T., Pasquier, J., Schnetzer, M.A., Gabriel, J.P.,
1419
and Métraux, J.-P. (2006). FiRe and microarrays: a fast answer to burning
1420
questions. Trends Plant Sci 11: 320-322.
1421
Garcion, C., Lohmann, A., Lamodière, E., Catinot, J., Buchala, A., Doermann, P., and
1422
Métraux,
1423
ISOCHORISMATE SYNTHASE2 gene of Arabidopsis. Plant Physiol 147: 1279-
1424
1287.
J.P.
(2008).
Characterization
and
biological
function
of
the
1425
Goda, H. et al. (2008). The AtGenExpress hormone and chemical treatment data set:
1426
experimental design, data evaluation, model data analysis and data access. Plant J
1427
55: 526-542.
1428
1429
Gruner, K., Griebel, T., Návarová, H., Attaran, E., and Zeier, J. (2013). Reprogramming
of plants during systemic acquired resistance. Front Plant Sci 4: 252.
55
1430
Gupta, R. N., and Spenser, I. D. (1969). Biosynthesis of the piperidine nucleus. The
1431
mode of incorporation of lysine into pipecolic acid and into piperidine alkaloids. J Biol
1432
Chem 244: 88-94.
1433
Hilfiker, O., Groux, R., Bruessow, F., Kiefer, K., Zeier, J., and Reymond, P. (2014).
1434
Insect eggs induce a systemic acquired resistance in Arabidopsis. Plant J 80: 1085-
1435
1094.
1436
Jing, B., Xu, S., Xu, M., Li, Y., Li, S., Ding, J., and Zhang, Y. (2011). Brush and spray: a
1437
high-throughput systemic acquired resistance assay suitable for large-scale genetic
1438
screening. Plant Physiol 157: 973-980.
1439
Jirage, D., Tootle, T.L., Reuber, T.L., Frost, L.N., Feys, B.J., Parker J.E., Ausubel, F.M.,
1440
and Glazebrook, J. (1999). Arabidopsis thaliana PAD4 encodes a lipase-like gene
1441
that is important for salicylic acid signaling. Proc Natl Acad Sci USA 96: 13583-
1442
13588.
1443
1444
Jung, H.W., Tschaplinski, T.J., Wang, L., Glazebrook, J., and Greenberg, J.T. (2009).
Priming in systemic plant immunity. Science 324: 89-91.
1445
Kauss, H., Theisinger-Hinkel, E., Mindermann, R., and Conrath, U. (1992).
1446
Dichloroisonicotinic and salicylic acid, inducers of systemic acquired resistance,
1447
enhance fungal elicitor responses in parsley cells. Plant J 2: 655–660
1448
Kim, T.H., Kunz, H.H., Bhattacharjee, S., Hauser, F., Park, J., Engineer, C., Liu, A., Ha,
1449
T., Parker, J.E., Gassmann, W., and Schroeder, J.I. (2012). Natural variation in
1450
small molecule-induced TIR-NB-LRR signaling induces root growth arrest via EDS1-
1451
and PAD4-complexed R protein VICTR in Arabidopsis. Plant Cell 24: 5177-5192.
1452
Koch, M., Vorwerk, S., Masur, C., Sharifi-Sirchi, G., Olivieri, N., and Schlaich, N.L.
1453
(2006). A role for a flavin-containing monooxygenase in resistance against microbial
1454
pathogens in Arabidopsis. Plant J 47: 629-639.
1455
Kohler, A., Schwindling, S., and Conrath, U. (2002). Benzothiadiazole-induced priming
1456
for potentiated responses to pathogen infection, wounding, and infiltration of water
1457
into leaves requires the NPR1/NIM1 gene in Arabidopsis. Plant Physiol 128: 1046-
1458
1056.
56
1459
1460
Koornneef, A., and Pieterse, C.M.J. (2008). Cross talk in defense signaling. Plant
Physiol 146: 839-844.
1461
La Camera, S., L'Haridon, F., Astier, J., Zander, M., Abou-Mansour, E., Page, G.,
1462
Thurow, C., Wendehenne, D., Gatz, C., Métraux, J.-P., and Lamotte, O. (2011). The
1463
glutaredoxin ATGRXS13 is required to facilitate Botrytis cinerea infection of
1464
Arabidopsis thaliana plants. Plant J 68: 507-519.
1465
Masclaux-Daubresse, C., Clément, G., Anne, P., Routaboul, J.M., Guiboileau, A.,
1466
Soulay, F., Shirasu, K., and Yoshimoto K. (2014). Stitching together the multiple
1467
dimensions of autophagy using metabolomics and transcriptomics reveals impacts
1468
on metabolism, development, and plant responses to the environment in
1469
Arabidopsis. Plant Cell 26: 1857-1877.
1470
Mateo, A., Funck, D., Mühlenbock, P., Kular, B., Mullineaux, P.M., and Karpinski, S.
1471
(2006). Controlled levels of salicylic acid are required for optimal photosynthesis and
1472
redox homeostasis. J Exp Bot 57: 1795-1807.
1473
Mauch, F., Mauch-Mani, B., Gaille, C., Kull, B., Haas, D., and Reimmann, C. (2001).
1474
Manipulation of salicylate content in Arabidopsis thaliana by the expression of an
1475
engineered bacterial salicylate synthase. Plant J 25: 67-77.
1476
Melotto, M., Underwood, W., Koczan, J., Nomura, K., and He, S.Y. (2006). Plant
1477
stomata function in innate immunity against bacterial invasion. Cell 126: 969-980.
1478
Memelink, J. (2009). Regulation of gene expression by jasmonate hormones.
1479
Phytochem 70: 1560-1570.
1480
Mishina, T.E., and Zeier, J. (2006). The Arabidopsis flavin-dependent monooxygenase
1481
FMO1 is an essential component of biologically induced systemic acquired
1482
resistance. Plant Physiol 141: 1666-1675.
1483
Mishina, T.E., and Zeier, J. (2007). Pathogen-associated molecular pattern recognition
1484
rather than development of tissue necrosis contributes to bacterial induction of
1485
systemic acquired resistance in Arabidopsis. Plant J 50: 500-513.
1486
Mishina, T.E., Griebel, T., Geuecke, M., Attaran, E., and Zeier, J. (2008). New insights
1487
into the molecular events underlying systemic acquired resistance. Paper 81 in: M
57
1488
Lorito, SL Woo, F Scala, eds, Biology of Plant-Microbe Interactions, Vol 6,
1489
International Society for Molecular Plant-Microbe Interactions, St. Paul, MN.
1490
1491
1492
1493
1494
1495
Mölders, W., Buchala, A., and Métraux J.-P. (1996). Transport of salicylic acid in
tobacco necrosis virus-infected cucumber plants. Plant Physiol. 112: 787-792.
Moore, J.W., Loake, G.J., and Spoel, S.H. (2011). Transcription dynamics in plant
immunity. Plant Cell 23: 2809-2820.
Morrison, R.I. (1953). The isolation of L-pipecolinic acid from Trifolium repens. Biochem
J. 53: 474-478.
1496
Mur, L.A.J., Grant, N., Warner, S.A.J., Sugars, J.M., White, R.F., and Draper, J. (1996).
1497
Salicylic acid potentiates defence gene expression in tissue exhibiting acquired
1498
resistance to pathogen attack. Plant J 9: 550-571.
1499
Návarová, H., Bernsdorff, F., Döring, A.-C., and Zeier, J. (2012). Pipecolic acid, an
1500
endogenous mediator of defense amplification and priming, is a critical regulator of
1501
inducible plant immunity. Plant Cell 24: 5123-5141.
1502
Nawrath, C., and Métraux, J.-P. (1999). Salicylic acid induction-deficient mutants of
1503
Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after
1504
pathogen inoculation. Plant Cell 11: 1393-1404.
1505
Noutoshi, Y., Okazaki, M., Kida, T., Nishina, Y., Morishita, Y., Ogawa, T., Suzuki, H.,
1506
Shibata, D., Jikumaru, Y., Hanada, A., Kamiya, Y., and Shirasu, K. (2012). Novel
1507
plant immune-priming compounds identified via high-throughput chemical screening
1508
target salicylic acid glucosyltransferases in Arabidopsis. Plant Cell 24: 3795-3804.
1509
Pálfi, G., and Dézsi, L. (1968). Pipecolic acid as an indicator of abnormal protein
1510
1511
1512
metabolism in diseased plants. Plant Soil 29: 285-291.
Pandey, S.P., and Somssich, I.E. (2009). The role of WRKY transcription factors in
plant immunity. Plant Physiol 150: 1648-1655.
1513
Rietz, S., Stamm, A., Malonek, S., Wagner, S., Becker, D., Medina-Escobar, N., Vlot,
1514
A.C., Feys, B.J., Niefind, K., and Parker, J.E. (2011). Different roles of Enhanced
58
1515
Disease Susceptibility1 (EDS1) bound to and dissociated from Phytoalexin
1516
Deficient4 (PAD4) in Arabidopsis immunity. New Phytol 191: 107-119.
1517
Robinson, M.D., McCarthy, D.J. and Smyth, G.K. (2010). edgeR: a Bioconductor
1518
package for differential expression analysis of digital gene expression data.
1519
Bioinformatics 26: 139-140.
1520
Sauter, M., Lorbiecke, R., Ouyang, B., Pochapsky, T.C., and Rzewuski, G. (2005). The
1521
immediate-early ethylene response gene OsARD1 encodes an acireductone
1522
dioxygenase involved in recycling of the ethylene precursor S-adenosylmethionine.
1523
Plant J 44: 718-729.
1524
Schenk, S.T., Hernandez-Reyes, C., Samans, B., Stein, E., Neumann, C., Schikora, M.,
1525
Reichelt, M., Mithöfer, A, Becker, A., Kogel, K.H., and Schikora, A. (2014). N-Acyl-
1526
Homoserine lactone primes plants for cell wall reinforcement and induces resistance
1527
to bacterial pathogens via the salicylic acid/oxylipin pathway. Plant Cell 26: 2708–
1528
2723.
1529
1530
Schlaich, N. L. (2007). Flavin-containing monooxygenases in plants: looking beyond
detox. Trends Plant Sci. 12: 412-418.
1531
Serrano, M., Wang, B., Aryal, B., Garcion, C., Abou-Mansour, E., Heck, S., Geisler, M.,
1532
Mauch, F., Nawrath, C., and Métraux, J.-P. (2013). Export of salicylic acid from the
1533
chloroplast requires the multidrug and toxin extrusion-like transporter EDS5. Plant
1534
Physiol 162: 1815-1821.
1535
1536
Shah, J., and Zeier, J. (2013). Long-distance communication and signal amplification in
systemic acquired resistance. Front Plant Sci 4: 30.
1537
Shiu, S.H., and Bleecker, A.B. (2001). Receptor-like kinases from Arabidopsis form a
1538
monophyletic gene family related to animal receptor kinases. Proc Natl Acad Sci
1539
USA 98: 10763–10768.
1540
1541
Shulaev, V., Leon, J., and Raskin, I. (1995). Is salicylic acid a translocated signal of
systemic acquired resistance in tobacco? Plant Cell 7: 1691-1701.
59
1542
Singh, P., Yekondi, S., Chen, P.W., Tsai, C.H., Yu, C.W., Wu, K., and Zimmerli, L.
1543
(2014). Environmental history modulates Arabidopsis pattern-triggered immunity in a
1544
HISTONE ACETYLTRANSFERASE1-dependent manner. Plant Cell 26: 2676-2688.
1545
Song, J.T., Lu, H., and Greenberg, J.T. (2004a). Divergent roles in Arabidopsis thaliana
1546
development and defense of two homologous genes, aberrant growth and death2
1547
and
1548
aminotransferases. Plant Cell 16: 353-366.
1549
1550
1551
1552
1553
1554
AGD2-LIKE
DEFENSE
RESPONSE
PROTEIN1,
encoding
novel
Song, J.T., Lu, H., McDowell, J.M., and Greenberg, J.T. (2004b). A key role for ALD1 in
activation of local and systemic defenses in Arabidopsis. Plant J 40: 200-212.
Sticher, L., Mauch-Mani, B., and Métraux, J.P. (1997). Systemic acquired resistance.
Annu Rev Phytopathol 35: 235-270.
Swarbreck, D., et al. (2008). The Arabidopsis Information Resource (TAIR): gene
structure and function annotation. Nucleic Acids Res 36: D1009-D1014.
1555
Tan, X., Meyers, B., Kozik, A., West, M., Morgante, M., St Clair, D., Bent, A., and
1556
Michelmore, R. (2007). Global expression analysis of nucleotide binding site-leucine
1557
rich repeat-encoding and related genes in Arabidopsis. BMC Plant Biol. 7: 56.
1558
Tateda, C., Zhang, Z., Shrestha, J., Jelenska, J., Chinchilla, D., and Greenberg, J.T.
1559
(2014). Salicylic acid regulates Arabidopsis microbial pattern receptor kinase levels
1560
and signaling. Plant Cell 26: 4171-4187.
1561
Thibaud-Nissen, F., Wu, H., Richmond, T., Redman, J. C., Johnson, C., Green, R.,
1562
Arias, J., and Town, C. D. (2006). Development of Arabidopsis whole-genome
1563
microarrays and their application to the discovery of binding sites for the TGA2
1564
transcription factor in salicylic acid-treated plants. Plant J 47: 152-162.
1565
Thimm, O., Blaesing, O., Gibon, Y., Nagel, A., Meyer, S., Krüger, P., Selbig, J., Müller,
1566
L.A., Rhee, S.Y., and Stitt, M. (2004). MAPMAN: a user-driven tool to display
1567
genomics data sets onto diagrams of metabolic pathways and other biological
1568
processes. Plant J 37: 914-939.
1569
1570
Thulke, O.U., and Conrath, U. (1998). Salicylic acid has a dual role in the activation of
defense-related genes in parsley. Plant J 14: 35-42.
60
1571
Truman, W., Bennett, M.H., Kubigsteltig, I., Turnbull, C., and Grant, M. (2007).
1572
Arabidopsis systemic immunity uses conserved defense signaling pathways and is
1573
mediated by jasmonates. Proc Natl Acad Sci USA 104: 1075-1080.
1574
Tsuda, K., Mine, A., Bethke, G., Igarashi, D., Botanga, C.J., Tsuda, Y., Glazebrook, J.,
1575
Sato, M., and Katagiri, F. (2013). Dual regulation of gene expression mediated by
1576
extended MAPK activation and salicylic acid contributes to robust innate immunity in
1577
Arabidopsis thaliana. PLoS Genet 9: e1004015.
1578
Vernooij, B., Friedrich, L., Morse, A., Reist, R., Kolditz-Jawhar, R., Ward, E., Uknes, S.,
1579
Kessmann, H., and Ryals, J. (1994). Salicylic acid is not the translocated signal
1580
responsible for inducing systemic acquired resistance but is required in signal
1581
transduction. Plant Cell 6: 959-965.
1582
Vivancos, J., Labbé, C., Menzies, J.G., and Bélanger, R.R. (2014). Silicon-mediated
1583
resistance of Arabidopsis against powdery mildew involves mechanisms other than
1584
the salicylic acid (SA)-dependent defence pathway. PMID: 25346281.
1585
1586
Vlot, A.C., Dempsey, D.A., and Klessig, D.F. (2009). Salicylic Acid, a multifaceted
hormone to combat disease. Annu Rev Phytopathol 47: 177-206.
1587
Vogel-Adghough, D., Stahl, E., Návarová, H., and Zeier, J. (2013). Pipecolic acid
1588
enhances resistance to bacterial infection and primes salicylic acid and nicotine
1589
accumulation in tobacco. Plant Sig Behav 8: e26366.
1590
von Caemmerer S., and Farquhar, G.D. (1981). Some relationships between the
1591
biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 376-
1592
387.
1593
Wang, D., Amornsiripanitch, N., and Dong, X. (2006). A genomic approach to identify
1594
regulatory nodes in the transcriptional network of systemic acquired resistance in
1595
plants. PLoS Pathog 2: e123.
1596
Ward, E.R., Uknes, S.J., Williams, S.C., Dincher, S.S., Wiederhold, D.L., Alexander,
1597
D.C., Ahl-Goy, P., Métraux, J.P., and Ryals, J.A. (1991). Coordinate gene activity in
1598
response to agents that induce systemic acquired resistance. Plant Cell 3: 1085-
1599
1094.
61
1600
Wildermuth, M. C., Dewdney, J., Wu, G., and Ausubel, F. M. (2001). Isochorismate
1601
synthase is required to synthesize salicylic acid for plant defence. Nature 414: 562-
1602
565.
1603
Wu, Y., Zhang, D., Chu, J. Y., Boyle, P., Wang, Y., Brindle, I. D., De Luca, V., and
1604
Després, C. (2012). The Arabidopsis NPR1 protein is a receptor for the plant
1605
defense hormone salicylic acid. Cell Reports 1: 639-647.
1606
Yan, H., Yoo, M.J., Koh, J., Liu, L., Chen, Y., Acikgoz, D., Wang, Q., and Chen, S.
1607
(2014). Molecular reprogramming of Arabidopsis in response to perturbation of
1608
jasmonate signaling. J Proteome Res 13: 5751-5766.
1609
Zacharius, R. M., Thompson, J. F., and Steward, F. C. (1954). The detection, isolation
1610
and identification of L(-)-pipecolic acid in the non-protein fraction of beans
1611
(Phaseolus vulgaris). J Am Chem Soc 76: 2908-2912.
1612
Zander, M., Thurow, C., and Gatz, C. (2014). TGA transcription factors activate the
1613
salicylic acid-suppressible branch of the ethylene-induced defense program by
1614
regulating ORA59 expression. Plant Physiol 165: 1671-1683.
1615
Zeier, J., Pink, B., Mueller, M.J., and Berger, S. (2004). Light conditions influence
1616
specific defence responses in incompatible plant-pathogen interactions: uncoupling
1617
systemic resistance from salicylic acid and PR-1 accumulation. Planta 219: 673-683.
1618
Zeier, J. (2013). New insights into the regulation of plant immunity by amino acid
1619
metabolic pathways. Plant Cell Environ 36: 2085–2103.
1620
Zeng, W., Brutus, A., Kremer, J.M., Withers, J.C., Gao, X., Jones, D., and He, S.Y.
1621
(2011). A genetic screen reveals Arabidopsis stomatal and/or apoplastic defenses
1622
against Pseudomonas syringae pv. tomato DC3000. PLoS Pathog 7: e1002291.
1623
Zhou N., Tootle T.L., Tsui F., Klessig D.F., and Glazebrook J. (1998). PAD4 functions
1624
upstream from salicylic acid to control defence responses in Arabidopsis. Plant Cell
1625
10: 1021-1030.
1626
Zimmerli, L., Jakab, G., Métraux, J.-P., and Mauch-Mani, B. (2000). Potentiation of
1627
pathogen-specific defense mechanisms in Arabidopsis by β-aminobutyric acid. Proc
1628
Natl Acad Sci USA 97: 12920-12925.
62
1629
Zipfel, C. (2014). Plant pattern-recognition receptors. Trends Immunol 35: 345-351.
1630
Zoeller, M., Stingl, N., Krischke, M., Fekete, A., Waller, F., Berger, S., and Mueller M.J.
1631
(2012). Lipid profiling of the Arabidopsis hypersensitive response reveals specific
1632
lipid peroxidation and fragmentation processes: biogenesis of pimelic and azelaic
1633
acid. Plant Physiol 160: 365-378.
1634
63
A
ALD1
1000
100
MgCl2
ICS1
10
1
0
0.1
0
0.01
Col-0
inoculated (1°) leaves
24 HAI
***
Col-0
ald1
sid2
ald1
sid2
C
5
Col-0
ald1
sid2
sid2
ald1
2
1
24 HAI
***
***
Col-0
ald1
sid2
sid2
ald1
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
***
0.02 ± 0.01
0.02 ± 0.01
0.5
3
0
Col-0
inoculated (1°) leaves
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
**
4
1.0
0
Psm
6
0.09 ± 0.03
0.65 ± 0.10
40
0.02 ± 0.003
0.02 ± 0.001
8
0.03 ± 0.004
0.03 ± 0.003
60
50
MgCl2
7
80
70
12
10
0
8
0.07 ± 0.06
0.09 ± 0.05
16
14
0.5
***
48 HAI
***
***
1.0
distal (2°) leaves, 48 HAI
0.08 ± 0.06
0.10 ± 0.04
***
sid2
ald1
sid2
ald1
0.03 ± 0.01
0.04 ± 0.01
B
0.001
0
sid2
ald1
sid2
0.16 ± 0.07
ald1
Col-0
Pip (µg g-1 FW)
1
0
0.1
0.01
0
SA (µg g-1 FW)
10
relative expression
relative expression
Psm
100
ald1
sid2
ald1
sid2
distal (2°) leaves, 48 HAI
48 HAI
***
2.0
***
MgCl2
Psm
1.5
1.0
0.5
0.0
Col-0
ald1
sid2
sid2
ald1
Col-0
ald1
sid2
sid2
ald1
Fig. 1
Figure 1. Pipecolic acid and salicylic acid biosynthesis in local and systemic tissue of wild-type Col-0,
ald1, sid2, and sid2 ald1 plants inoculated with SAR-inducing P. syringae pv. maculicola (Psm).
(A) Expression of ALD1 (left) and ICS1 (right) in Psm-inoculated leaves at 24 hours after inoculation
(HAI). Infiltration with 10 mM MgCl2 served as a mock-control treatment. Transcript levels were
assessed by quantitative real-time PCR (qPCR) analysis, and expressed relative to the Col-0 mockcontrol value. Data represent the mean ± SD of three biological replicate leaf samples from different
plants. Each biological replicate consists of two leaves from one plant. Expression values for each
biological replicate represent the mean of two technical replicates.
(B) and (C) Accumulation of Pip (B) and free SA (C) in Psm-inoculated (1°) leaves at 24 HAI and 48 HAI
(left) and in distal, non-inoculated (2°) leaves (right) at 48 HAI. Data represent the mean ± SD of at least
three biological replicate leaf samples from different plants. Each biological replicate consists of 6 leaves
from two plants. Asterisks denote statistically significant differences between Psm- and MgCl2-samples
(***: P < 0.001; **: P < 0.01; two-tailed t test). Numerical values for samples with very low metabolite
contents are given above the respective bars.
A
***
Psm (cfu cm-2 * 10-4)
Bacterial numbers
**
***
10000
###
###
1000
###
100
10
Col-0
B
ald1
sid2
ald1
sid2
ns
10000
***
ns
***
Psm (cfu cm-2 * 10-4)
Bacterial numbers, 2° leaves
1° : MgCl2
1° : Psm
1000
100
10
1
Col-0
C
sid2
sid2 ald1
1° : MgCl2
1° : Psm
1° : Psm avrRpm1
***
Psm (cfu cm-2 * 10-4)
Bacterial numbers, 2° leaves
10000
ald1
***
*** ***
1000
100
*** ***
10
1
Col-0
sid2
sid1
(eds5)
Fig. 2
Figure 2. Analyses of basal resistance to Psm and systemic acquired resistance in Pip- and/or SAdeficient mutant plants.
(A) Basal resistance of Col-0, ald1, sid2, and sid2 ald1 plants to Psm. Three leaves per plant were
inoculated with a suspension of Psm (OD600 = 0.001), and bacterial numbers quantified three days
later. Bars represent mean values (± SD) of colony forming units (cfu) per square centimetre from at
least seven biological replicate samples (n) derived from different plants. Each biological replicate
consists of 3 leaf discs harvested from different leaves of one plant. Number signs denote statistically
significant differences from the Col-0 wild-type value (#: P < 0.05, ##: P < 0.01, ###: P < 0.001; two-tailed
t-test). Asterisks designate statistically significant differences between indicated samples. To test
whether the effects of ald1 and sid2 on bacterial proliferation are additive or synergistic, a linear model
was used (log10 bacterial count ~ ald1*sid2). No significant interaction of ald1*sid2 was detected both
according to the F-test and according to Akaikes information criterion (p=0.0508, AICsynergistic = -120
AICadditive = -122.7), hence the effect is additive.
(B) Systemic acquired resistance assay with Col-0, ald1, sid2, and sid2 ald1 plants. Lower (1°) leaves
were infiltrated with either 10 mM MgCl2 or Psm (OD600 = 0.005) to induce SAR, and two days later,
three upper leaves (2°) were challenge-infected with Psm (OD600 = 0.001). Bacterial growth in upper
leaves was assessed 3 days post 2° leaf inoculation (n ≥ 7; as described in 2A). Asterisks denote
statistically significant differences between Psm-pre-treated and mock-control samples (***: P < 0.001;
ns: not significant, two-tailed t test).
(C) Biological SAR induction upon 1° leaf inoculation with compatible Psm and incompatible Psm
avrRpm1 in Col-0, sid2, and sid1 plants (n ≥ 7; as described in 2A and 2B). Asterisks denote
statistically significant differences between Psm or Psm avrRpm1 (OD600 = 0.005) pre-treated and
mock-control samples (***: P < 0.001; two-tailed t test).
up-regulated genes
down-regulated genes
A
sid2
739
104
17
2
2876
2672
ald1
Col-0
8
6
6
4
4
2
0
log2 fold-change
log2 fold-change
B
2
0
-2
-2
-4
-4
-6
Figure 3. Transcriptional SAR response in distal leaves of plants inoculated in primary leaves with Psm
(OD600 = 0.005) at 48 HAI. 6 independent SAR assays for Col-0, and 3 independent SAR experiments
for both sid2 and ald1 were performed. Gene expression was analysed by RNA-seq analyses of the
resulting replicate samples for Psm- and mock-treatments at the whole-genome level.
(A) Venn diagram depicting numbers of differentially regulated genes between Psm- and mocktreatments of Col-0 (black), sid2 (red) and ald1 (blue) [false discovery rate (FDR) < 0.01]. Overlap of
genes is indicated. Left: significantly up-regulated genes (the Col-0 genes correspond to the SAR+
genes). Right: significantly down-regulated genes (the Col-0 genes correspond to the SAR- genes).
Note that only two genes are differentially regulated in ald1.
(B) Distribution of Psm (P) / Mock (M)-fold changes of SAR genes in Col-0, sid2, and ald1. Box plots
depict log2-transformed P/M-fold changes. The distribution of log2 P/M-fold changes for three sets of
randomly-selected genes (left: 3413 genes, right: 2893 genes) is included (random a, b, c). Left: SAR+
genes. Right: SAR- genes.
Fig. 3
% SAR- genes
% SAR+ genes
A
all (N = 15239)
photosynthesis (155)
tetrapyrrole synthesis (40)
starch metabolism (48)
mitochondrial electron transport (98)
*
*
*
*
*
80
60
40
20
0
20
%
D
B
all (15239)
major CHO metabolism (76)
minor CHO metabolism (91)
glycolysis (58)
OPP pathway (24)
citric acid cycle (61)
co-factor metabolism (70)
lipid metabolism (289)
20
amino acid metabolism (215)
N-metabolism (23)
E
C1-metabolism (29)
secondary metabolism (238)
cell wall (261)
*
*
*
*
*
40
20
0
20
40
*
*
0
*
*
*
40
20
0
%
*
*
*
all (15239)
f.-AGP (12)
expansin (14)
XTH (18)
Ces/Csl (26)
wax/cutin (29)
*
60
40
*
40
80
20
%
C
100
all (15239)
stress (274)
* biotic stress (268)
signalling (830)
redox (162)
hormone metabolism (287)
fermentation (9)
20
20
0
20
40
60
F
*
*
40
20
0
20
40
60
*
*
*
40
20
Fig. 4
*
0
20
40
60
80 %
all (15239)
MAPK (17)
MAPKK (6)
MAPKKK (46)
80 %
40 %
G
H
all (15239)
NBS (118)
RLK (355)
RLP (53)
all (15239)
WRKY (42)
NAC (42)
TGA (7)
other bZIP (46)
AP2-EREBP (60)
zinc finger (150)
GRAS (25)
bHLH (60)
MYB (51)
HB (44)
MADS (15)
80 %
*
*
*
*
20
*
*
*
*
0
20
40
60 %
I
*
*
40
20
0
20
all (15239)
CDPK (24)
EF-hand (126)
calmodulin (80)
PP2C (55)
GLR (15)
PLD (10)
40
all (15239)
GST (38)
UGT (64)
GH (171)
CYP (104)
60
%
Figure 4. Percentage of SAR+ and SAR- genes in defined gene groups representing MapMan
metabolic pathways, functional categories, or Arabidopsis gene families (http://www.arabidopsis.org).
Dashed vertical lines illustrate the percentage of SAR+ and SAR- genes in the whole, RNA-seqcovered transcriptome (28496 genes) after threshold cut-off (15239 genes). The number of genes in
each category is given in brackets. Asterisks on the bars indicate significant enrichment or depletion of
gene categories in SAR+ (right) and SAR- (left) genes (Fisher’s exact test, P < 0.01).
(A), (B), and (D) MapMan metabolic pathways and functional categories.
(C) Gene families involved in cell wall remodelling and wax/cutin biosynthesis (f.-AGP: fasciclin-like
arabinogalactan proteins; XTH: xyloglucan endotrans-glucosylase/ hydrolases; Ces/Csl: cellulose
synthase/cellulose synthase-like).
(E) Gene families involved in the perception of microbial structures and early defense signal
transduction (NBS: nucleotide binding site-containing resistance proteins; RLK: receptor-like protein
kinases; RLP: receptor-like proteins).
(F) Mitogen-activated protein (MAP) kinase cascade members (MAPK: MAP kinases, MAPKK: MAP
kinase kinases; MAPKKK: MAP kinase kinase kinases).
(G) Other gene categories involved in defense signaling (CDPK: calcium-dependent protein kinases;
EF-hand: EF-hand containing proteins; calmodulin: calmodulin-binding proteins; GLR: glutamate
receptor-like family; PLD: phospholipase D family).
(H) Main transcription factor families (WRKY: WRKY domain family; NAC: NAM-ATAF1,2-CUC2
transcription factors; TGA: TGACG motif-binding factor; bZIP: basic leucine zipper; AP2-EREBP:
APETALA2 and ethylene-responsive element binding proteins; zinc finger: zinc finger superfamily;
GRAS: GRAS family; bHLH: basic helix-loop-helix; MYB: MYB family; HB: homeobox-leucine zipper;
MADS: MADS-box).
(I) Genes for different enzyme classes (GST: glutathione-S-transferases; UGT: UDP-dependent
glycosyltransferases; GH: Glycosyl hydrolases; CYP: cytochrome P450 superfamily).
Photosynthesis rate of 2° leaves
(µmol CO2 m-2 s-1), 48 HAI
A
12
1° : MgCl2
**
1° : Psm
10
8
6
4
2
0
Col-0
ald1
sid2
sid2
ald1
B
1° : MgCl2
Transpiration rate of 2° leaves
(mmol H2O m-2 s-1), 48 HAI
5.0
4.5
1° : Psm
**
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Col-0
ald1
sid2
sid2
ald1
Figure 5. Photosynthesis and transpiration rates in 2° leaves of 1°-leaf-inoculated Col-0, ald1, sid2, and
sid2 ald1 plants.
(A) CO2-uptake rates as a measure of photosynthetic capacity at 48 h after Psm inoculation or MgCl2infiltration measured in distal, untreated leaves of Col-0, sid2, ald1 and sid2 ald1 plants, as determined
by Infrared Gas Analyses (IRGA). Data represent the mean ± SD of four biological replicates (CO2
uptake rate of four distal leaves from different plants). Asterisks denote statistically significant
differences between Psm-treated and mock-control plants (**: P < 0.01; two-tailed t test).
(B) Rates of transpiration (water loss) in distal leaves, determined as described above by IRGA.
Fig. 5
0.1
0
P (52)
sid2
P (57)
sid2 ald1
ALD1
1
0
0.1
Col-0
ald1
P (24)
sid2
P (11)
sid2 ald1
SAG13
10
1
0.1
0
20
10
Col-0
ald1
sid2
P (7.1)
GRXS13
P (0.9)
0
0.1
100
Col-0
ald1
sid2
P (62)
10
1
0
0.1
0.01
0
0.001
0
70
ald1
sid2
1
0.1
0
0.01
0
300
100
P (26)
10
1
0
0.1
Col-0
ald1
sid2
sid2 ald1
Col-0
ald1
P (130)
fmo1
ALD1
10
1
0.1
0
0.01
0
600
Col-0
ald1
P (253)
fmo1
SAG13
100
10
1
100
Col-0
ald1
P (41)
fmo1
GRXS13
10
1
200
100
Col-0
ald1
P (111)
fmo1
ARD3
10
1
sid2 ald1
PR1
0
0.01
10
0.1
0
Col-0
FMO1
100
0.1
0
sid2 ald1
ARD3
1000
0.1
0
sid2 ald1
1
0
0.01
relative expression
ald1
relative expression
relative expression
Col-0
10
60
P (703)
relative expression
relative expression
100
B
relative expression
1
0.01
0
relative expression
FMO1
10
0
0.01
relative expression
1° Psm / 2° Psm
100
0
0.01
relative expression
P (411)
P (215)
1° MgCl2 / 2° Psm
relative expression
500
1° Psm / 2° MgCl2
relative expression
relative expression
A
1° MgCl2 / 2° MgCl2
300
100
Col-0
ald1
P (51)
fmo1
PR1
10
1
0.1
0
Col-0
ald1
fmo1
Fig. 6
Figure 6. SAR-associated priming of defense-related gene expression fully depends on a functional
Pip/FMO1-module but is only partially SA-dependent.
(A) SAR priming assays for Col-0, ald1, sid2, and sid2 ald1 plants.
(B) SAR priming assays for Col-0, ald1, and fmo1 plants (independent experiment).
The priming assay consisted of an inductive Psm-inoculation or mock (MgCl2)-treatment of 1° leaves,
followed by a Psm-challenge or mock-treatment of 2° leaves 48 h later. Gene expression in 2° leaves
was assessed 10 h after the second treatment (Supplemental Figure 9A). A particular defense
response was defined as primed if the differences between the (1°-Psm / 2°-Psm)- and the (1°-Psm /
2°-MgCl2)-values were significantly larger than the differences between the (1°-MgCl2 / 2°-Psm)- and
the (1°-MgCl2/ 2°-MgCl2)-values (two-sided Mann-Whitney U test, a = 0.005) (Supplemental Figure
9B). A P above the bars for a particular genotype indicates priming. Expression of three partially SAindependent SAR+ genes (FMO1, ALD1, SAG13; Table 1) and three SA-dependent SAR+ genes
(GRXS13, ARD3, PR1) were monitored. Transcript levels were assessed by quantitative real-time
PCR analysis and are given as means ± SD of three biological replicates. Each biological replicate
involves two technical replicates. The transcript levels are expressed relative to the respective Col-0
mock-control value. Note that the graphs use a base 10 logarithmic scale for the y-axes to ensure
recognizability of both high and low values. Graphs with a linear scale for the y-axes, which more
clearly illustrate differences between challenge-infected 1°-mock-treated and 1°-Psm-induced plants,
are depicted in Supplemental Figure 10. As a measure of the gain of a response due to priming, we
calculated the prgain (response gain due to priming) for each genotype with activated priming
according to the formula given in Supplemental Figure 9C. Prgain values are given in brackets behind
the priming indicator P and allow estimates about quantitative differences of the strength of priming
between genotypes. The higher the prgain value, the stronger the priming. The data sets depicted in
(A) and (B) are derived from independent experiments.
1° MgCl2 / 2° MgCl2
1° Psm / 2° MgCl2
1° MgCl2 / 2° Psm
1° Psm / 2° Psm
A
B
camalexin (µg g-1 FW)
1
0.5
0.10
0.05
0.010
20
total SA (µg g-1 FW)
P (104)
Col-0
ald1
sid2
P (15)
P (94)
1
0.5
0
0.1
20
Col-0
ald1
fmo1
ald1
fmo1
P (14)
10
10
5
1
0.5
0
0.1
20
10
5
0.01
0
sid2 ald1
total SA (µg g-1 FW)
camalexin (µg g-1 FW)
5
Col-0
ald1
sid2
sid2 ald1
5
1
0.5
0
0.1
Col-0
Figure 7. SAR-associated priming of camalexin and SA biosyntheses requires a functional Pip/FMO1module, and SAR priming of camalexin production is SA-dependent.
(A) SAR priming assays for Col-0, ald1, sid2, and sid2 ald1 plants. Camalexin levels and total SA levels
were determined as defense outputs. Values represent the mean ± SD of three biological replicates
from different plants. Each biological replicate consists of six leaves from two plants. A P above the
bars for a particular genotype indicates priming in this genotype. The prgain-values are given in
brackets. Details of the priming assessments are described in the legends of Fig. 6 and Supplemental
Figure 9.
(B) SAR priming assays for camalexin and total SA production in Col-0, ald1, and fmo1 plants, as
described in (A).
Note that the graphs use a logarithmic scale for the y-axes. The same graphs with linear scaling are
depicted in Supplemental Figure 11. The data sets depicted in (A) and (B) originate from independent
experiments.
Fig. 7
A
P (1041)
P (136)
P (138)
100
H2O / mock
Pip / mock
H2O / Psm
Pip / Psm
10
1
0.1
0
0.01
0
300
100
relative expression
P 353)
Col-0
ald1
sid2
sid2 ald1
C
P (146)
P (74)
50
ALD1
relative expression
relative expression
1000
FMO1
10
1
0
0.1
0
0.01
50
ald1
P (18)
P (13)
sid2
1
sid2 ald1
Col-0
fmo1
P (8.8)
20
PR1
PR1
10
1
P (0.2)
relative expression
10
relative expression
10
0
0.1
Col-0
ALD1
P (25)
P (0.2)
0
0.1
0.01
0
Col-0
ald1
sid2
0.1
0
sid2 ald1
B
1
Col-0
fmo1
D
P (9.1)
P (6.7)
P (73)
5
P (12)
camalexin (µg g-1 FW)
camalexin (µg g-1 FW)
5
P (21)
1
1.0
0.5
0.1
1
1.0
0.5
P (1.1)
0.1
0.01
0.0
0.01
0.0
Col-0
ald1
sid2
sid2 ald1
Col-0
fmo1
Fig. 8
Figure 8. Exogenous Pip confers defense priming in a FMO1-dependent and partially SA-independent
manner.
(A) Pip-induced priming of gene expression (FMO1, ALD1, PR1) in Col-0, ald1, sid2, and sid2 ald1
plants, as determined by qPCR analysis. Plants were supplied with 10 ml of 1 mM Pip ( dose of 10
µmol) or with 10 ml of H2O (control treatment) via the root system, and leaves challenge-inoculated
with Psm or mock-infiltrated one day later. Defense responses in leaves were determined 10 h after
the challenge treatment. Values represent the mean ± SD of three biological replicates from different
plants. Each biological replicate consists of two leaves from one plant and involves two technical
replicates. A P above the bars for a particular genotype indicates defense priming in this genotype, as
assessed in analogy to SAR priming. The prgain-values are given in brackets (see legend to Fig. 6 and
Supplemental Figure 9).
(B) Pip-induced priming for camalexin production in Col-0, ald1, sid2, and sid2 ald1 plants. Values
represent the mean ± SD of three biological replicates from different plants. Each biological replicate
consists of six leaves from two plants.
(C) Pip-induced priming assays in Col-0 and fmo1 plants, monitoring ALD1 and PR1 expression.
Sampling as outlined in (A). The data sets depicted in (A) and (C) originate from independent
experiments.
(D) Pip-induced priming assays in Col-0 and fmo1 plants, monitoring camalexin accumulation.
Sampling as outlined in (B). The data sets depicted in (B) and (D) originate from independent
experiments.
Note that the graphs use a logarithmic scale for the y-axes. The same graphs with linear scaling are
depicted in Supplemental Figure 12.
A
P (892)
P (1112)
P (580)
P (613)
relative PR1 expression
1000
100
10
1
0
0.1
H2O / mock
0.01
0
Col-0
B
ald1
sid2
H2O / SA
sid2 ald1
Pip / mock
Pip / SA
P (7583)
P (7315)
relative PR1 expression
10000
1000
100
10
1
0.10
0.010
Col-0
fmo1
pad4
C
npr1
***
H2O
###
**
Psm (cfu cm-2 * 10-4)
Bacterial numbers
1000
##
***
SA
##
###
**
100
###
###
10
1
Col-0
ald1
fmo1
sid2
Fig. 9
Figure 9. Pipecolic acid amplifies SA-induced PR1 expression in a FMO1-dependent manner and
exogenous SA enhances basal resistance in ald1, fmo1, and sid2.
(A) and (B) Plants were pre-treated with Pip (or H2O) as described in the legend to Fig. 8, and one day
later 0.5 mM SA (SA) or water (mock) was infiltrated into leaves. PR1 expression in leaves was
monitored 4 h after the SA (mock)-treatment by qPCR analysis. Values represent the mean ± SD of
three biological replicates from different plants. Each biological replicate consists of two leaves from
one plant and involves two technical replicates. PR1 transcript levels are expressed relative to the
H2O/mock value of Col-0. (A) Col-0, ald1, sid2, and sid2ald1. (B) Col-0, fmo1, pad4, and npr1. The data
sets depicted in (A) and (B) originate from independent experiments. A P above the bars for a particular
genotype indicates priming of SA responses in this genotype, as assessed in analogy to SAR priming.
The prgain values are given in brackets (see legend to Fig. 6 and Supplemental Figure 9). Note that the
graphs use a logarithmic scale for the y-axes. The same graphs with linear scaling are depicted in
Supplemental Figure 14.
(C) Basal resistance to Psm infection of Col-0, ald1, fmo1, and sid2 plants is enhanced by exogenous
SA. Three leaves per plant were pre-infiltrated with 0.5 mM SA or H2O, and 4 h later, the same leaves
were challenged with Psm (OD600 = 0.001). Bacterial growth was assayed 3 days later as outlined in the
legend to Fig. 2A. Asterisks denote statistically significant differences between SA- and H2O-pre-treated
plants (**: P < 0.01, ***: P < 0.001; two-tailed t-test). Number signs above bars of mutant values denote
statistically significant differences from the respective Col-0 wild-type value (##: P < 0.01, ###: P < 0.001;
two-tailed t-test).
A
Col-0
2° responses
ALD1, FMO1
inducing
stimuli
(from
1° leaves)
ALD1
Pip
FMO1
SAG13
ICS1
SAR
GRXS13
SA
PR1, ARD3
switch on
amplifier on
(signal intensifier)
B
priming
priming
priming
sid2
2° responses
ALD1, FMO1
inducing
stimuli
(from
1° leaves)
ALD1
Pip
FMO1
SA
switch on
C
SAG13
ICS1
partial
SAR
amplifier off
ald1
no 2° response
inducing
stimuli
(from
1° leaves)
ALD1
Pip
ICS1
FMO1
no SAR
SA
switch off
no defense priming
amplifier inactive
D
SA
Pip
FMO1
+
NPR1
E
Pip
FMO1/
PAD4
ICS1
PR1
SA
NPR1
PR1
Fig. 10
Figure 10. Summary of the roles of pipecolic acid, FMO1, and salicylic acid in Arabidopsis SAR and
associated defense priming, and modes of the synergistic interplay between Pip and SA.
(A) to (C) Regulation of the SAR transcriptional response, SAR establishment, and SAR-associated
defense priming by Pip, FMO1, and SA. (A) Situation in wild-type plants. The full SAR and priming
responses are established by elevated levels of ALD1-generated Pip, the action of FMO1 downstream
of Pip, and ICS1-synthesized SA in 2° leaf tissue. The Pip/FMO1-module acts as an indispensable
switch for SAR activation, and SA amplifies Pip-dependent responses to different degrees. (B)
Situation in sid2 mutant plants. In the absence of functional ICS1 and elevated SA, the Pip/FMO1module is sufficient to induce a set of partially SA-independent responses to a certain level and trigger
a moderate SAR response. Notably, an intact Pip/FMO1-module is also capable to prime plants for
enhanced activation of partially SA-independent responses in the absence of SA elevations (bent red
arrow). (C) Situation in ald1 and fmo1 mutant plants. Functional ICS1 alone is not sufficient for SAR
activation. Without Pip elevations or functional FMO1, SA biosynthesis is not activated. Failure of both
Pip and SA elevations prevent the establishment of a primed state. Without the Pip/FMO1-module,
inducing stimuli from 1° leaves are either not transduced into a meaningful response in 2° leaves or
too weak to induce a noticeable response.
(D) and (E) Two modes of synergistic interplay between the immune signals Pip and SA. (D) Elevated
Pip levels amplify SA-induced PR1 expression. This amplification response is mediated by FMO1 but
does not depend on PAD4. (E) Elevated Pip levels induce PR1 expression via ICS1-triggered SA
production. This signaling mode of Pip depends on both FMO1 and PAD4.
Pipecolic acid orchestrates plant systemic acquired resistance and defense priming via salicylic
acid-dependent and independent pathways
Jürgen Zeier, Friederike Bernsdorff, Anne-Christin Doering, Katrin Gruner, Andrea Bräutigam and Stefan
Schuck
Plant Cell; originally published online December 15, 2015;
DOI 10.1105/tpc.15.00496
This information is current as of June 17, 2017
Supplemental Data
/content/suppl/2015/12/15/tpc.15.00496.DC1.html
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