Plant Physiology Preview. Published on February 3, 2014, as DOI:10.1104/pp.113.232090
Du et al. miR159 and CMV symptoms
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Running head: miR159 and CMV symptoms
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Address correspondence to
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Z. Du
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College of Life Sciences, Zhejiang Sci-Tech University, No. 2 Street, Xiasha, Hangzhou, Zhejiang,
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310018, P.R. China. [email protected], +86-13958122256
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or
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J.P. Carr
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Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA,
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U.K.
[email protected], +44-1223 333900
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Research area: Genes, Development and Evolution; Host-Virus Interaction
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Copyright 2014 by the American Society of Plant Biologists
Du et al. miR159 and CMV symptoms
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Using a viral vector to reveal the role of miR159 in disease symptom induction
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by a severe strain of cucumber mosaic virus
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One-sentence summary: A vector based on a mild strain of cucumber mosaic virus carrying a
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microRNA decoy sequence uncovered the relevance of miR159 to disease symptoms induced by a
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severe CMV strain
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Zhiyou Du1, 2*, Aizhong Chen1, Wenhu Chen1, Jack H. Westwood2, David C. Baulcombe2, John P.
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Carr2*
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1
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, 310018, China
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2
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA,
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U.K.
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* Corresponding authors: Z. Du and J. P. Carr
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Du et al. miR159 and CMV symptoms
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Footnotes:
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This work was supported by the National Natural Science Foundation of China (30800043,
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31170141), a Marie Curie International Incoming Fellowship (PIIF-GA-2009-236443) and a 521
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Talents Development Project grant (11610032521303) to Z.D, and awards by the Leverhulme Trust
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(F/09741/F, RPG-2012-667) and UK Biotechnology and Biological Sciences Research Council
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(BB/D014376/1, BB/J011762/1) to J.P.C.
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Address correspondence to [email protected] or [email protected].
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Du et al. miR159 and CMV symptoms
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ABSTRACT
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In transgenic Arabidopsis, expression of the cucumber mosaic virus (CMV) 2b silencing suppressor
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protein from the severe subgroup IA strain Fny disrupted microRNA-regulated development but
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orthologs from mild subgroup II strains (Q and LS) did not, explaining strain-specific differences in
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symptom severity.
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viral symptoms.
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and that Fny2b altered miR159ab-regulated transcript levels suggested a role for miR159ab in
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elicitation of severe symptoms by Fny-CMV.
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plants expressing an artificial microRNA as a proof-of-concept, we developed a LS-CMV-based
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vector to express sequences mimicking microRNA targets.
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sequence using LS-CMV depleted miR159 and induced symptoms resembling those of Fny-CMV.
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Suppression of Fny-CMV-induced symptoms in plants harboring mutant alleles for the miR159ab
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targets MYB33 and MYB65 confirmed the importance of this microRNA in pathogenesis. The
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work demonstrates the utility of a viral vector to express microRNA target mimics to facilitate
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functional studies of microRNAs in plants.
However, it is unknown which microRNA(s) affected by Fny2b critically affect
Observations that Fny2b-transgenic plants phenocopy mir159ab mutant plants
Using restoration of normal phenotype in transgenic
Expressing a miR159 target mimic
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Du et al. miR159 and CMV symptoms
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INTRODUCTION
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RNA silencing is a set of mechanisms by which small RNA molecules, including microRNAs
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(miRNA) and short-interfering RNAs (siRNA), guide RNA-induced silencing complexes (RISC) to
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degrade transcripts, or in some cases hinder their translation, in a sequence-specific manner
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(Chapman and Carrington, 2007). Ranging in size from 19 to 24-nucleotides (nt), plant miRNAs
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are encoded by nuclear genes that are transcribed to produce non-coding primary transcripts called
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pri-miRNAs, which are processed into dsRNAs with imperfect stem–loop structures
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(pre-miRNAs). Pre-miRNAs are cropped by Dicer-like (DCL1) endoribonuclease activity yielding
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miRNA/miRNA* duplexes and the guide strand associated with Argonaute 1 (AGO1) to constitute
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a RISC, while the passenger strand miRNA*s are typically degraded (Kurihara and Watanabe, 2004;
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Voinnet, 2009). Fundamental roles are played by miRNAs in regulation of plant growth and
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development (Jones-Rhoades et al., 2006), as well as in adaptation to biotic and abiotic stresses
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(Sunkar et al., 2007; Ruiz-Ferrer and Voinnet, 2009; Sunkar et al., 2012).
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RNA silencing directed by siRNAs of 22 and 21nt generated, respectively, by dicing of
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virus-derived dsRNA by DCL2 and DCL4 is a potent antiviral mechanism (Deleris et al., 2006).
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Many plant viruses counteract this antiviral defense by expressing viral suppressors of RNA
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silencing (VSR) that inhibit or inactivate various components of the RNA silencing pathways
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(Voinnet et al., 1999; Roth et al., 2004; Li and Ding, 2006). Additionally, many VSRs disrupt
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miRNA pathways, resulting in the appearance of developmental defects in VSR-transgenic and
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virus-infected plants (Mallory et al., 2002; Chapman et al., 2004; Dunoyer et al., 2004; Zhang et al.,
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2006; Lewsey et al., 2007; Yang et al., 2008).
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It has been over a decade since the first plant miRNAs were reported (Llave et al., 2002; Park
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et al., 2002; Rhoades et al., 2002) but traditional genetic approaches are not well suited to functional
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studies of plant miRNAs because many miRNA families comprise multiple members having
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identical target sequences.
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regulation mechanism in Arabidopsis, termed target mimicry (TM), by which miR399 is inactivated
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by complementary pairing with the non-coding RNA transcribed from the gene, Induced by
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Phosphate Starvation 1 (IPS1). The complementary base-pairing raises a bulge at the expected
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miRNA cleavage site, preventing miR399-directed endonucleolytic cleavage of IPS1 by AGO1,
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which leads to increased accumulation of the miR399 target mRNA PHO2.
Franco-Zorrilla and colleagues (2007) discovered a novel miRNA
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Subsequently, the
Du et al. miR159 and CMV symptoms
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natural phenomenon of TM was developed into a technique to dissect miRNA function by stable
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expression of artificial TM sequences in transgenic Arabidopsis plants (Wu et al., 2009; Todesco et
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al., 2010; Debernardi et al., 2012). This approach was further refined by transgenic expression of
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transcripts comprising two TMs separated by a short spacer sequence, called a short tandem target
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mimic (STTM) sequence (Yan et al., 2012). Constitutive expression of STTM sequences was
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shown to be more effective at interfering with miRNA activity in transgenic Arabidopsis plants than
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the IPS1-based TM. We wondered if it would be possible to further enhance the efficiency and
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speed of TM technology for probing miRNA function by expressing TM sequences from a viral
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vector.
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Cucumber mosaic virus (CMV) is the type species of the genus Cucumovirus. CMV is an
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economically important pathogen with a host range exceeding 1200 plant species and is a
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well-characterized model for plant-virus interaction studies (Palukaitis and Garcia-Arenal, 2003;
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Jacquemond, 2012). The CMV genome comprises three positive-sense single-stranded RNAs and
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encodes five proteins (Palukaitis and Garcia-Arenal, 2003; Jacquemond, 2012).
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methyltransferase/helicase replicase component is translated from RNA1.
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another replicase component, the 2a RNA-dependent RNA polymerase, and also encodes the 2b
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VSR, which is translated from the RNA2-derived subgenomic mRNA, RNA4A.
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movement protein (MP) is translated directly from RNA3, whilst a second protein encoded by this
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genomic segment, the coat protein (CP), is translated from the RNA3-derived sub-genomic mRNA,
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RNA4. CMV 2b protein, which was one of the first VSRs identified, disrupts RNA silencing
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predominantly by sequestration of small double-stranded RNA (Brigneti et al., 1998; Goto et al.,
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2007; Gonzalez et al., 2010; Duan et al., 2012; Gonzalez et al., 2012).
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establishment of antiviral silencing, the 2b protein also interferes with salicylic acid-induced CMV
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resistance (Brigneti et al., 1998; Ji and Ding, 2001; Lewsey and Carr, 2009), it disrupts signaling
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mediated by jasmonic acid and abscisic acid (Lewsey et al., 2010; Westwood et al., 2013a), and it
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modifies plant-aphid interactions (Ziebell et al., 2011; Westwood et al., 2013b). The 2b VSR also
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affects dynamics of local and systemic CMV movement and functions as a virulence determinant
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(Ding et al., 1995; Soards et al., 2002; Wang et al., 2004).
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The 1a
RNA2 is the mRNA for
The 3a
In addition to inhibiting the
Numerous CMV strains and isolates have been characterized and can be divided into three
subgroups IA, IB and II (Roossinck et al., 1999).
Generally, subgroup IA and IB strains are more
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Du et al. miR159 and CMV symptoms
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virulent than subgroup II strains (Wahyuni et al., 1992; Zhang et al., 1994; Cillo et al., 2009).
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Experiments with transgenic Arabidopsis plants showed that constitutive expression of the 2b gene
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from subgroup IA strain Fny, but not its orthologs from subgroup II strains Q or LS, impaired
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miRNA regulation on gene expression, leading to developmental defects, concomitant with
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increased steady-state accumulation of mature miRNA and miRNA* (Chapman et al., 2004; Zhang
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et al., 2006; Lewsey et al., 2007). This effect was proposed as an explanation of differences in
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viral symptoms between subgroup IA and subgroup II strains (Zhang et al., 2006; Lewsey et al.,
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2007).
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symptoms by subgroup IA strains, including Fny-CMV, we explored the problem using a viral
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vector carrying a TM sequence.
Since it still remains unknown which miRNA(s) is associated with the induction of severe
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RESULTS
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Constitutive expression of CMV 2b protein or CMV infection disrupted miR159 functions.
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In line with previous findings, infection of Arabidopsis plants of ecotype Col-0 with Fny-CMV
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caused severe distortion of systemically infected leaves, while infection with LS-CMV caused only
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mild stunting (Fig. 1A) (Lewsey et al., 2007). We noted that Fny2b-transgenic lines exhibited
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developmental defects that phenocopied those previously observed by Allen and colleagues (2007)
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for mir159ab double mutant plants, which included severe stunting and up-curled rosette leaves
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(Fig. 1B).
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miR159c, which are encoded by distinct MIR genes at separate loci (Park et al., 2002; Rhoades et
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al., 2002). The sequences of miR159a and miR159b differ by one nucleotide but interact with the
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transcripts of the GAMYB-like genes MYB33 and MYB65 in a functionally redundant manner (Allen
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et al., 2007).
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nucleotides 2 and 1, respectively, it does not regulate MYB33 and MYB65 and mutation of its gene
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has no effect on Arabidopsis development (Allen et al., 2010).
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The Arabidopsis miR159 family contains three members, miR159a, miR159b and
Although miR159c differs in sequence from miR159a and miR159b only at
There is no documented investigation of miR159 accumulation or activity in 2b-transgenic or
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CMV-infected plants.
Therefore, we examined steady-state levels of miR159 at 15, 30 and 50
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days post-germination in Fny2b-transgenic as well as in Fny unt2b-transgenic plants, which express
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a non-translatable Fny2b transcript and retain a normal phenotype (Lewsey et al., 2007). RNA gel
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Du et al. miR159 and CMV symptoms
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blotting showed that, compared with Fny unt2b-transgenic plants, Fny 2b-transgenic plants had a
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higher level of mature miR159 and miR159* at 15 and 50 days post-inoculation (dpi) (Fig. 1C).
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This increased accumulation of miR159* and miR159 is likely to be the consequence of
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miR159/miR159* duplex stabilization by binding with Fny2b (Goto et al., 2007; Gonzalez et al.,
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2010; Duan et al., 2012; Gonzalez et al., 2012; Hamera et al., 2012).
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prevents recruitment of miR159 strands into RISC; an idea supported by increased steady-state
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levels of the miR159 target transcripts MYB33 and MYB65 in Fny2b-transgenic plants (Fig. 1D).
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We next analyzed the effects of CMV infection on the steady-state levels of miR159 and its target
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transcripts.
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which was not seen in plants infected by LS-CMV (Fig. 1E).
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coupled quantitative PCR (RT-qPCR) showed that MBY65 was most markedly elevated by
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Fny-CMV infection (Fig. 1F), consistent with the increased miR159* accumulation. Thus,
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constitutive expression of Fny2b and Fny-CMV infection disrupted miR159 accumulation and
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activity.
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Fny-CMV.
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Development of a mild strain LS-CMV-based target mimic system
Duplex stabilization likely
The clearest effect of Fny-CMV infection was elevated accumulation of miR159*,
Analysis by reverse transcription
This led us to investigate if miR159 was involved in induction of severe symptoms by
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We speculated that if the difference in viral symptoms between Fny-CMV and LS-CMV results
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specifically from differential effects of their respective 2b proteins on miR159ab, then engineering
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LS-CMV to carry a corresponding TM sequence would produce an agent that would induce disease
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symptoms resembling those caused by Fny-CMV.
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engineered LS-CMV to carry a TM sequence to deplete a specific miRNA that has a known and
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well-understood activity in Arabidopsis.
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(amiR-triOX-transgenic) line, plants of which have an easily observable phenotype (enhanced
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trichome clustering on its leaves) that results from transgenic expression of an artificial miRNA
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(amiRNA), amiR-trichome (Wang et al., 2011). We designed an amiR-trichome TM sequence
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(MIM-trichome) according to the design rule described previously (Todesco et al., 2010), and in
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parallel designed an arbitrary sequence (MIM-CK) unrelated to any known Arabidopsis miRNA
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sequence to use as a control (Fig. 2A).
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levels of RNA3 and its subgenomic RNA (RNA4) that occur in LS-CMV-infected plants, we
As a proof of concept for a viral TM vector we
For this, we selected the Arabidopsis ema1-1 mutant
To take advantage of the relatively high accumulation
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Du et al. miR159 and CMV symptoms
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introduced MIM-trichome or MIM-CK between the CP gene and 3’ un-translated region (UTR) of
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RNA3 as shown in Fig. 2A, to generate the LS-CMV-derived variants LS-MIM-trichome and
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LS-MIM-CK, respectively.
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Infection with LS-MIM-CK or wild-type LS-CMV had no discernable effect on the extreme
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trichome clustering phenotype on leaves of ema1-1 plants (Fig. 2B).
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LS-MIM-trichome infection strongly inhibited trichome clustering, demonstrating that expression
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of the TM sequence interfered with the activity of the amiRNA expressed in ema1-1 plants (Fig.
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2B). RNA blot analysis of viral RNAs showed that the viruses carrying either MIM-trichome or
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MIM-CK sequences on RNA3 replicated as efficiently as wild-type LS-CMV (Fig. 2C). Recent
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studies demonstrate that TM expression reduced stability of targeted miRNAs in transgenic
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Arabidopsis plants (Wu et al., 2009; Todesco et al., 2010; Debernardi et al., 2012). Therefore, we
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used RNA gel blotting to analyze accumulation of amiR-trichome in the infected plants.
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with LS-CMV or LS-MIM-CK had no obvious effect on accumulation of either amiR-trichome or
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its corresponding star strand amiR-trichome*.
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modest decrease in mature amiR-trichome, with the 24nt form affected more than the 21nt molecule;
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however, it did not decrease amiR-trichome* accumulation (Fig. 2D).
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In contrast,
Infection
Infection with LS-MIM-trichome caused only a
The target transcripts of amiR-trichome are CAPRICE (CPC), TRIPTYCHON (TRY), and
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ENHANCER of TRIPTYCHON AND CAPRICE2 (ETC2) (Wang et al., 2011).
Analysis by
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RT-qPCR of steady-state levels of these mRNAs in ema1-1 plants showed that infection with
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LS-CMV or LS-MIM-CK markedly increased accumulation of TRY, and had a limited effect on
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ETC2 accumulation.
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(Fig. 2E), demonstrating that MIM159 had indeed reversed the inhibition of CPC and TRY
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expression by amiR-trichome.
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(Fig. 2E).
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LS-MIM-CK-infected ema1-1 plants resulted from disruption of amiR-trichome regulation by these
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viruses, we tested the responses of amiR-trichome targets to LS-CMV infection in wild-type and
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ema1-1 mutant plants. RT-qPCR results showed that infection with LS-CMV had no significant
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effect on accumulation of these three transcripts in wild-type plants (Fig. 2F).
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data shown above, accumulation of TRY, but not CPC or ETC2, was altered significantly in the
Plants infected with LS-MIM-trichome had higher levels of CPC and TRY
In contrast, infection with LS-MIM159 reduced ETC2 expression
To determine whether changes in accumulation of ETC2 and TRY in LS-CMV- or
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Consistent with the
Du et al. miR159 and CMV symptoms
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ema1-1 plants when infected with LS-CMV (Fig. 2F).
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LS-CMV or LS-MIM-CK disrupted repression by amiR-trichome of its targets. To some extent
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this may have been due to the silencing suppression activity of its 2b protein. Additionally, we
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noticed that, in contrast to CPC and TRY, ECT2 was up-regulated in mock-inoculated ema1-1 plants,
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compared with levels seen in mock-inoculated wild-type plants (Fig. 2F).
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in ema1-1 plants, CPC and TRY but not ETC2, were negatively-regulated by the artificial miRNA
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amiR-trichome, and suggested that there could be a CPC-TRY-ETC2 triple interaction at the
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transcriptional
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LS-MIM159-infected ema1-1 plants, where CPC and TRY were up-regulated (Fig. 2E).
level.
This
may
explain
the
This demonstrated that infection with
decreased
This demonstrated that
accumulation
of
ETC2
in
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Although expression of the MIM-trichome sequence from LS-CMV had limited effects on the
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level of 21nt amiR-trichome, which is responsible for target cleavage, it clearly altered expression
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levels of the amiR-trichome targets and completely inhibited trichome clustering.
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that MIM-trichome functions mainly by inactivating amiR-trichome, rather than by reducing its
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stability.
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(Franco-Zorrilla et al., 2007).
This implies
Such an effect has been observed in the case of the endogenous IPS1-miR399 interaction
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To further demonstrate the efficacy of LS-CMV-based TM expression we selected the endogenous
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Arabidopsis miR165/166 as a model because inhibition of miR165/166 functions by a short tandem
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target mimic leading to alteration of developmental (morphological) phenotypes (Yan et al., 2012).
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We adopted a previously described target mimic sequence for miR165/166 (MIM165/166) (Todesco
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et al., 2010) for introduction into LS RNA3 at the same position between the CP gene and 3’ UTR
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used to generate LS-MIM-CK and LS-MIM-trichome (Fig. 2A), to generate LS-MIM165/166. We
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compared the effects of this construct with those of LS-CMV and LS-MIM-CK in wild-type
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Arabidopsis plants. Infection with either LS-CMV or LS-MIM-CK caused mild distortion of
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rosette leaves and mild stunting, which had become apparent by 28 dpi (Fig. 3A).
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upper, young leaves of plants infected with LS-MIM165/166 grew more vertically than equivalent
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leaves on wild-type plants, which were apparent by 14 dpi, and leaves were shaped like trumpets by
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28 dpi. The majority of trumpet-like leaves had outgrowths (enations) from their abaxial sides.
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These disease symptoms are typical phenotypes of the phb-1d mutant, which has a gain of function
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mutation disrupting miR165/166 binding sites in the PHABULOSA (PHB) gene (McConnell and
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Strikingly,
Du et al. miR159 and CMV symptoms
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Barton, 1998).
Expression of the MT sequence MIM165/166 has been shown to modestly reduce
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miR165/166 levels in transgenic Arabidopsis (Todesco et al., 2010; Yan et al., 2012). Using RNA
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gel blotting we found that while all three viruses accumulated to similar levels (Fig. 3B),
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miR165/166 was substantially reduced only in the plants infected with LS-MIM165/166 but not in
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mock-inoculated plants or plants infected with LS-CMV or the control construct MIM-trichome
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(Fig. 3C).
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expression was specific (Fig. 3C).
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PHB and PHAVOLUTA (PHV) were strongly up-regulated in the plants infected with
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LS-MIM165/166 but not in plants infected with either the control virus LS-MIM-CK or the parental
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virus LS-CMV (Fig.3D).
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MIM165/166 in LS-CMV effectively reduced miR165/166 levels and de-repressed expression of its
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target transcripts, leading to appearance of disease symptoms resembling phb-1d mutant plant
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phenotypes.
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Infection with an LS-CMV variant expressing miR159 target mimic sequence induced
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symptoms resembling those induced by the severe strain Fny-CMV
LS-MIM165/166 had no effect on miR167 levels, showing that the effect of TM
RT-qPCR experiments showed that the miR165/166 targets
All these data strongly supported the conclusion that expression of
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Having validated the LS-CMV-based TM expression system, we investigated the effect of
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disrupting miR159 functions on viral symptoms by expressing a miR159 TM sequence from the
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virus.
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LS-MIM159.
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LS-MIM-CK, LS-MIM159, and Fny-CMV.
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that were similar to those induced by Fny-CMV, while those induced by LS-CMV or LS-MIM-CK
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were very mild, as observed in the previous experiments (Fig. 4A).
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by RNA blot analysis as shown in Fig. 4B. Next, we conducted RNA gel blotting to analyze
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miRNA accumulation in the plants at 10 dpi. Mock-inoculated plants and plants infected with
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LS-CMV or LS-MIM-CK possessed similar levels of miR159, which were markedly higher than in
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plants infected with LS-MIM159 (Fig. 4C).
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comparable in all these plants, showing the effect of the MIM159 sequence on miR159
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accumulation was specific (Fig. 4C).
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targets MYB33, MYB65 and MYB101 using RT-qPCR.
The MIM159 sequence was introduced into LS-CMV RNA3 as before, generating
We compared viral symptoms on Arabidopsis plants infected with LS-CMV,
Infection with LS-MIM159 caused disease symptoms
Viral infection was confirmed
In contrast, comparable levels of miR167 were
We also analyzed relative accumulation of the miR159
As expected, infection with LS-CMV or
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Du et al. miR159 and CMV symptoms
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LS-MIM-CK had no obvious effect on steady-state levels of these target transcripts but infection
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with LS-MIM159 markedly increased accumulation of MYB33 and MYB65, but had no effect on
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MYB101 (Fig. 4D), which was consistent with the decreased level of miR159.
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Previous studies demonstrated that miR159a and miR159b only target MYB33 and MYB65, and
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deregulation of MYB33 and MYB65 is responsible for the phenotypes exhibited by mir159ab mutant
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plants (Allen et al., 2007).
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were not identical to the phenotypes of mir159ab mutant plants (compare Fig. 4A with Fig. 1B).
264
To further ascertain that the disease symptoms were specifically a consequence of disrupting
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miR159 functions by the MIM159 sequence, we compared viral symptoms on Arabidopsis
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wild-type and myb33/myb65 double mutant plants infected with LS-CMV, LS-MIM-CK and
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LS-MIM159.
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LS-MIM159 were largely ameliorated in the myb33/myb65 mutant plants infected with the same
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virus.
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comparable to that elicited by LS-CMV or LS-MIM-CK (Fig. 5).
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that the severe disease symptoms were the outcome of deregulating miR159 targets MYB33 and
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MYB65.
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The myb33/myb65 alleles moderate disease symptoms caused by infection with the severe
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strain Fny-CMV
Our results showed that disease symptoms induced by LS-MIM159
As expected, severe disease symptoms seen in wild-type plants infected with
LS-MIM159 infection caused mild leaf distortion in the mutant plants, which was
This was convincing evidence
275
As shown above, constitutive expression of Fny2b or infection with Fny-CMV negatively
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regulated miR159 activity, leading to increase of miR159 targets MYB33 and MYB65 (Fig. 1). To
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determine whether de-repression of MYB33 and MYB65 by impairing miR159 activity is
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responsible for induction of severe symptoms by Fny-CMV we inoculated Arabidopsis wild-type
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and myb33/myb65 mutant plants with Fny-CMV.
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majority (80%) of myb33/myb65 plants infected with Fny-CMV showed less deformation of upper,
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young systemically infected leaves than wild-type plants with the virus infection at 10 dpi (Fig. 6A).
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RNA gel blotting showed that there was little difference in accumulation of Fny-CMV RNA
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between the wild-type and mutant plants (Fig. 6B).
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infection, MYB33 and MYB65 expression is deregulated by the Fny 2b protein through inhibition of
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miR159 activity but that while this contributes to viral symptom induction, it does not enhance
Observation of viral symptoms showed that a
This strongly suggests that during Fny-CMV
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Du et al. miR159 and CMV symptoms
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virus titer.
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DISCUSSION
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This work demonstrated the biological significance of miR159 in the induction of viral symptoms
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by a severe subgroup IA strain of CMV by developing a mild strain LS-CMV-based TM system and
291
subsequently using the system to express a miR159 target mimic sequence.
292
demonstrated the potential of a virus-based TM system to investigate miRNA function, not only in
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plant responses to stress (in this case, infection by a highly virulent virus strain), but also in control
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of plant development.
The work also
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Previous studies showed that CMV 2b is not the only factor mediating CMV virulence. For
296
example, viral mutants that are unable to express the 2b protein (CMVΔ2b) are symptomless in
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wild-type Arabidopsis but induce disease in Arabidopsis dcl2/dcl4 or dcl2/dcl3/dcl4 mutant plants,
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co-incident with a marked increase in the titer of these CMV mutants (Diaz-Pendon et al., 2007;
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Ziebell and Carr, 2009). Our present data provides strong evidence that 2b proteins from severe
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CMV strains contribute directly to pathogenicity in wild-type plants by perturbing miR159 activity.
301
This is consistent with findings for other VSRs, including the HC-Pro of turnip mosaic virus, which
302
contributes to viral symptom induction by inhibiting miR167-mediated effects on the transcript
303
AUXIN RESPONSIVE FACTOR 8 (Jay et al., 2011). There is a high sequence similarity between
304
the miR159 and miR319 families and expression of MIM159 by LS-CMV may have disrupted
305
regulation by miR319 of its target transcripts encoding TCP transcription factors (Palatnik et al.,
306
2007).
307
were associated with de-repression of miR159ab targets MYB33 and MYB65.
308
whatever effect LS-MIM159 has on miR319 functions, it can be concluded that MIM159
309
expression by the vector derived from LS-CMV substantially reduced miR159 levels, and elevated
310
accumulation of MYB33 and MYB65, leading to production of severe viral symptoms, resembling
311
the disease symptoms caused by the severe strain Fny-CMV.
Our data demonstrated that the exacerbated viral symptoms caused by MIM159 expression
Thus, despite
312
The use of LS-CMV-based TM is an effective technology to assess miRNA functions in planta.
313
It is worth noting that 21nt and 24nt amiR-trichome molecules produced from the primary transcript
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Du et al. miR159 and CMV symptoms
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of amiR-trichome had different stabilities when exposed to the TM MIM-trichome (Fig. 2D).
315
MIM-trichome was originally designed to affect the 21nt miRNA, but worked better in reducing
316
stability of 24nt molecules.
317
miRNA molecule.
318
NUCLEASES (SDN), one of which (SDN1) was shown to specifically degrade single stranded
319
miRNAs using its 3’-5’ exonuclease activity (Ramachandran and Chen, 2008).
320
base pairing of 3’ terminal nucleotides of miRNA with its TM largely determines miRNA fate
321
(survival or degradation) and this could be one of the reasons that various miRNA-TM interactions
322
exhibit different effects on miRNA stability.
323
corresponding to miR165 and miR166, Yan and colleagues (2012) found that STTM165/166 with a
324
48nt spacer is most effective in destabilization of miR165/166.
325
suggested to be due to STTM transcripts being more stable than IPS1-based TM transcripts in vivo.
326
However, when IPS-based TM was expressed from LS-CMV, its efficacy was apparently greater
327
than that of STTM, as exemplified by its effects on miR165/166. The increased efficacy of TM
328
when expressed from a CMV vector could be attributed to be either increased stability of TM
329
sequences when present in CMV RNAs or the higher expression levels achieved by expression from
330
a virus, or both. This demonstrates that CMV-based TM is more effective than transgenic TM in
331
impairing miRNA functions. The fact that LS-CMV 2b has no or little effect on miRNAs in planta
332
and that CMV has an extremely broad host range would make LS-CMV-based TM a technology
333
potentially useful for analysis of miRNA functions in a variety plant species; not just in Arabidopsis.
334
Additionally, virus-based TM has other advantages since it is time-saving (compared to generation
335
of stably-transformed plants) and has the potential for high-throughput investigation of miRNA
336
function. Therefore, we suggest that virus-based TM technology could be used as a high-throughput
337
method to initially identify the effects of specific miRNAs and would be followed up by focussed
338
studies using the transgenic plant miRNA decoy approach in combination with specific knockouts
339
(or VIGS for silencing specific mRNAs) to identify the mRNA target(s) responsible for a given
340
phenotype.
The 24nt miRNA has three more nucleotides at its termini than a 21nt
In Arabidopsis, mature miRNA is degraded by SMALL RNA DEGRADATION
This implies that
Recently, by optimizing mimic sequences
The greater effectiveness was
341
342
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Du et al. miR159 and CMV symptoms
343
MATERIALS AND METHODS
344
Plant materials and plant growth conditions
15
345
Plants of Arabidopsis thaliana Heyn. ecotype Col-0, mutant plants and transgenic plants were
346
grown under an 8h photoperiod with a light intensity of 150-200 μE.m-2.s-1 at 22°C. Arabidopsis
347
mutants ema1-1 and myb33/myb65 have been described previously (Allen et al., 2007; Wang et al.,
348
2011). Nicotiana glutinosa and N. tabacum plants were grown under a 16h photoperiod with a
349
light intensity of 150-200 μE.m-2.s-1 at 25°C.
350
DNA constructs
351
Infectious clones pLS109, pLS209 and pLS309 corresponding to RNA1, RNA2 and RNA3 of
352
LS-CMV were described previously (Zhang et al., 1994).
To generate pLS309-derived infectious
353
RNAs expressing miRNA target mimic (TM) sequences or an arbitrary sequence as a control (CK)
354
(Fig. 2A) we introduced the desired DNA sequence immediately downstream of the CP sequence in
355
pLS309 by overlapping PCR using partially complementary oligonucleotides.
356
fragment I was amplified using pLS309 as template with forward primer LSR3F1393 and reverse
357
primer complementary to the 3’ end sequence of CP and target mimic.
358
amplified using pLS309 as template with reverse primer LSR3R2198 and forward primer
359
corresponding to target mimic and 5’ end of 3’ UTR. Fragments I and II were mixed together as
360
overlapping PCR template to generate a DNA fragment that was subsequently digested using
361
HindIII and PstI.
362
infectious
363
pLS309-MIM319, and pLS309-MIM-CK (control).
364
shown in Table S1.
365
Viruses and viral inoculation
Briefly, DNA
DNA fragment II was
The restriction product was ligated into pre-digested pLS309 to generate
clones,
pLS309-MIM-trichome,
pLS309-MIM165/166,
pLS309-MIM159,
All primers used for making constructs are
366
LS-CMV variants were constituted by co-inoculating N. glutinosa plants with in
367
vitro-synthesized transcripts of pLS109 and pLS209 together with transcripts of pLS309-derived
368
constructs carrying TM sequences or the control sequence (MIM-CK). Genetic stability of these
369
mutants was confirmed by DNA sequencing of RT-PCR products of viral RNA3 extracted from
370
systemically infected leaves of the inoculated plants.
371
infected N. glutinosa to N. tabacum plants for virus propagation and virus purification.
372
purification was carried out as described by Ng and Perry (2004).
These viruses were passaged from the
Virion
Purified virions at a
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Du et al. miR159 and CMV symptoms
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373
concentration of 100 ng/μl were mechanically inoculated onto Arabidopsis seedlings at the 5-6
374
true-leaf stage using Carborundum as an abrasive.
375
RNA gel blotting
376
Total RNA was extracted from a pool of leaf tissues harvested from five plant individuals using
377
TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA gel blotting for
378
analysis of CMV genomic RNAs was performed as described previously (Xu et al., 2013). For
379
analysis of low-molecular weight RNAs, including miRNAs and U6, 15 μg total RNA was used for
380
RNA blot analysis according to the protocol described in the instructions of the miRVana miRNA
381
isolation Kit (Ambion).
382
miRNA*, or U6 were labeled with digoxigenin at their 3’ ends using the Dig oligonucleotide tailing
383
kit generation II (Roche) following the manufacturer’s instructions, and purified using a G25
384
Sephadex column (GE) according to the manufacturer’s instructions.
385
for detection of corresponding miRNA or miRNA* using the Dig luminescence detection kit
386
(Roche) according to the manufacturer’s instructions. The sequence of the U6 probe has been
387
described previously (Wu et al., 2006).
388
Reverse transcription - quantitative PCR
389
DNA oligonucleotides completely complementary to mature miRNA,
The purified probe was used
For analysis of relative accumulation of miRNA target transcripts, RT-qPCR was conducted
390
using the protocol previously described (Westwood et al., 2013a,b).
Prior to reverse transcription,
391
total RNA preparations were incubated with Turbo DNase (Ambion). The primers and PCR
392
conditions for detection of amiR-trichome targets CPC, ETC2, TRY, miR165/166 targets PHB, PHV,
393
and miR159 targets MYB33, MYB65, MYB101 were described previously (Allen et al., 2007; Wang
394
et al., 2011; Yan et al., 2012).
395
396
SUPPLEMENTAL MATERIALS
397
Table S1 Primers used for generation of DNA constructs.
398
399
ACKNOWLEGEMENTS
400
We thank Dr. Anthony Millar for providing seeds for myb33/myb65 mutant, and Dr. Yijun Qi for
401
providing seeds for ema1-1 mutant. We also thank Drs. Alex. M. Murphy, Simon C. ‘Niels’ Groen,
402
and Qiansheng Liao for useful discussions.
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Du et al. miR159 and CMV symptoms
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Du et al. miR159 and CMV symptoms
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FIGURE LEGENDS
553
Figure 1. The effects of CMV 2b protein or CMV infection on miR159 function in Arabidopsis.
554
A, Viral symptoms on Arabidopsis plants inoculated with Fny-CMV, LS-CMV or sterile water
555
(Mock) at 10 dpi.
556
non-translatable Fny2b transcript), Fny 2b-transgenic and mir159ab mutant plants. The plants
557
were photographed when approximately 35 days old. C, RNA gel blot analysis of accumulation of
558
miR159 and its complementary strand miR159* in Fny unt2b-transgenic and Fny 2b-transgenic
559
Arabidopsis plants at 15, 30 and 50 days old.
560
blot analysis of accumulation of miR159 and miR159* in Arabidopsis plants inoculated with
561
Fny-CMV, LS-CMV or sterile water (Mock) at 10 dpi. E, RT-qPCR analyses of accumulation of the
562
miR159 target transcripts MYB33, MYB65 and MYB101 in Fny unt2b-transgenic (black bars) and
563
Fny 2b-transgenic (white bars) in Arabidopsis plants at 15, 30 and 50 days old. F, RT-qPCR
564
analyses of accumulation of MYB33, MYB65 and MYB101 in Arabidopsis plants inoculated with
565
Fny-CMV (gray bars), LS-CMV (white bars) or sterile water (Mock, black bars) at 10 dpi.
566
panels D & F, the Duncan new multiple range test was used to analyze statistical differences.
567
Asterisk (*) indicates statistically different values (P<0.05) and error bars represent standard error
568
about the mean.
B, Phenotypes of non-transgenic, Fny unt2b-transgenic (expressing a
U6 RNA served as a loading control.
D, RNA gel
In the
569
570
Figure 2.
Expression of the target mimic sequence MIM-trichome using LS-CMV inactivated the
571
artificial miRNA causing trichome clustering in Arabidopsis. A, Diagram of LS-CMV RNA3
572
harboring amiR-trichome target mimic (MIM-trichome) or mimic control (MIM-CK) sequences
573
inserted between the coat protein (CP) gene and the 3’ untranslated region (UTR).
574
red MIM-trichome indicate the introduced nucleotides for raising a bulge to prevent cleavage.
575
Asterisk indicates complementary nucleotide between amiR-trichome and MIM-triOX or MIM-CK.
576
MP indicates the open reading frame for the CMV movement protein. B, Alteration of trichome
577
clustering on Arabidopsis ema1-1 mutant plants inoculated with LS-CMV, LS-MIM-CK,
578
LS-MIM-trichome, or sterile water (Mock).
579
LS-MIM-trichome harbor MIM-CK and MIM-trichome, respectively.
580
14 dpi. Bar represents 1 cm. C, RNA gel blot analysis of accumulation of viral progeny RNAs
581
in the infected plants.
Nucleotides in
RNA3 segments of LS-MIM-CK and
Photographs were taken at
A biotin-labeled DNA oligonucleotide complementary to the conserved
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Du et al. miR159 and CMV symptoms
23
582
sequence common to the 3’ UTRs of the CMV genomic and subgenomic RNAs was used to detect
583
viral RNA.
584
analysis of accumulation of mature amiR-trichome and its opposite strand amiR-trichome*.
585
RNA served as a loading control. nt indicates nucleotide. E, RT-qPCR analyses of accumulation
586
of amiR-trichome target transcripts CAPRICE (CPC),
587
CAPRICE2
588
accumulation of CPC,
589
LS-CMV (white bars) or sterile water (Mock, black bars). The ANOVA Duncan new multiple
590
range test was used to analyze statistical differences.
591
different values (P<0.05), and error bars represent standard error about the mean. Total RNA
592
samples used in the experiments shown in C-F were prepared from pooled upper, non-inoculated
593
leaves harvested from five individual plants at 14 dpi.
Equal loading was confirmed by ethidium bromide staining.
D, RNA gel blot
U6
ENHANCER OF TRYPTYCHON AND
(ETC2) and TRYPTYCHON (TRY) in ema1-1 plants. F, RT-qPCR analysis of
ETC2 and TRY in wild-type (WT) and ema1-1 plants inoculated with
Different letters indicate statistically
594
595
Figure 3.
Expression of a miRNA165/166 target mimic sequence using LS-CMV substantially
596
reduced miR165/166 accumulation, leading to dramatic alteration to viral symptoms. A,
597
Arabidopsis plants showing viral symptoms induced by the infection with LS-CMV, LS-MIM-CK
598
and LS-MIM165/166.
599
were taken at 14 dpi and 28 dpi as indicated to the left of the images. A close-up view of
600
LS-MIM165/166-infected plants showed outgrowths (enations) from leaf abaxial sides indicated by
601
arrowheads in red. Scale bars represent 1 cm. B, RNA blot analysis of accumulation of viral
602
progeny RNAs in the infected plants.
603
conserved sequence common to the 3’ UTRs of the CMV genomic and subgenomic RNAs was used
604
to detect viral RNA.
605
analysis of accumulation of miR165/166 and miR167 in the infected plants and mock-inoculated
606
plants at 14 dpi.
607
accumulation of miR165/166 target transcripts PHABULOSA (PHB) and PHAVOLUTA (PHV) at 14
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dpi. Total RNA samples used in the experiments of B, C and D were prepared from pooled upper,
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non-inoculated leaves harvested from five individual plants at 14 dpi.
‘Mock’ plants were mock-inoculated with sterile water. Photographs
A biotin-labeled DNA oligonucleotide complementary to the
Equal loading was confirmed by ethidium bromide staining. C, RNA blot
U6 RNA was used as a loading control. D, RT-qPCR analysis of relative
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Figure 4. Infection with an LS-CMV variant expressing a miR159 target mimic sequence caused
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
Du et al. miR159 and CMV symptoms
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viral symptoms resembling those induced by the severe strain, Fny-CMV.
A, Viral symptoms in
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Arabidopsis plants inoculated with LS-CMV, LS-MIM-CK, LS-MIM159, or Fny-CMV.
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plants were inoculated with sterile water.
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B, RNA gel blot analysis of viral progeny RNAs in the infected plants. A biotin-labeled DNA
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oligonucleotide complementary to the conserved sequence common to the 3’ UTRs of the CMV
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genomic and subgenomic RNAs was used to detect viral RNA.
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ethidium bromide staining. C, RNA gel blot analysis of accumulation of miR159 and miR167 in the
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infected plants as well as mock plants. U6 RNA was used as a loading control. D, RT-qPCR
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analysis of relative accumulation of selected miR159 target transcripts MYB33, MYB65, and
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MYB101 in the infected plants as well as mock plants. Total RNA samples used in the experiments
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of B, C, D were prepared from pooled upper, non-inoculated leaves harvested from five individual
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plants at 10 dpi.
Photographs were taken at 10 dpi.
Mock
Bar represents 1 cm.
Equal loading was confirmed by
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Figure 5.
Mutant alleles for the miR159ab targets MYB33 and MYB65 diminished disease
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symptoms caused by LS-MIM159. Arabidopsis wild-type and myb33/myb65 mutant plants were
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inoculated with LS-CMV, LS-MIM-CK, LS-MIM159, or sterile water (Mock-inoculated), and
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photographed at 14 dpi.
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Figure 6. myb33/myb65 mutant alleles moderate disease symptoms induced by the severe strain
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Fny-CMV.
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with Fny-CMV or sterile water (Mock).
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1 cm. B, RNA gel blot analysis of viral accumulation in Arabidopsis wild-type and myb33/myb65
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mutant plants. Three virus-infected individual Arabidopsis wild-type or myb33/myb65 mutant
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plants were tested. Total RNA samples were prepared from upper, non-inoculated leaves at 10 dpi.
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A biotin-labeled DNA oligonucleotide complementary to the conserved sequence common to the 3’
637
UTRs of the CMV genomic and subgenomic RNAs was used to detect viral RNA. Equal RNA
638
loading was confirmed by ethidium bromide staining.
A, Arabidopsis wild-type and myb33/myb65 double mutant plants were inoculated
The plants were photographed at 10 dpi. Bars represent
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