Plant Physiology Preview. Published on October 9, 2014, as DOI:10.1104/pp.114.246694
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Running head: GbWRKY1 prioritizes development over defense
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Corresponding author: Longfu Zhu, National Key Laboratory of Crop Genetic
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Improvement, Huazhong Agricultural University, Wuhan, Hubei 430070, China
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Telephone number: +86-27-8728-3955
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Fax number: +86-27-8728-0196
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E-mail: [email protected]
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Research area: Ecophysiology and Sustainability
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Copyright 2014 by the American Society of Plant Biologists
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GbWRKY1 mediates plant defense-to-development transition during infection of
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cotton by Verticillium dahliae by activating JAZ1 expression
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Chao Li, Xin He, Xiangyin Luo, Li Xu, Linlin Liu, Ling Min, Li Jin, Longfu Zhu*,
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Xianlong Zhang
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Address: National Key Laboratory of Crop Genetic Improvement, Huazhong
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Agricultural University, Wuhan, Hubei 430070, China
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One sentence summary: GbWRKY1 is involved in a plant defense-to-development
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transition during Verticillium dahliae infection by regulating JAZ1 expression.
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Footnotes:
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Financial sources: This work was supported by funding from the National High-tech
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Program
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(2014ZX0800503B) and Program of Introducing Talents of Discipline to Universities
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in China (B14032).
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Corresponding author: Longfu Zhu; [email protected]
(863,
2013AA102601-4),
Ministry
of
Agriculture
of
China
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Keywords
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Gossypium barbadense, Verticillium dahliae, GbWRKY1, GhJAZ1, defense and
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development, gibberellic acid (GA) and jasmonic acid (JA)
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
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Abstract
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Plants have evolved an elaborate signaling network to ensure an appropriate level of
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immune response to meet the differing demands of developmental processes. Previous
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research has demonstrated that DELLA proteins physically interact with JAZ1 and
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dynamically regulate the interaction of the gibberellic acid (GA) and jasmonic acid
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(JA) signaling pathways. However, whether and how the JAZ1-DELLA regulatory
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node is regulated at the transcriptional level in plants under normal growth conditions
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or during pathogen infection is not known. Here, we demonstrate multiple functions
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of Gossypium barbadense GbWRKY1 in the plant defense response and during
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development. Although GbWRKY1 expression is induced rapidly by MeJA and
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infection by Verticillium dahliae, our results show that GbWRKY1 is a negative
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regulator of the JA-mediated defense response and plant resistance to the pathogens
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Botrytis
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GbWRKY1-overexpressing lines displayed GA-associated phenotypes, including
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organ elongation and early flowering, coupled with the downregulation of the putative
46
targets
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GbWRKY1-overexpressing plants depend on the constitutive expression of Gossypium
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hirsutum GhJAZ1. We also show that GhJAZ1 can be trans-activated by GbWRKY1
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through TGAC core sequences, and the adjacent sequences of this binding site are
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essential for binding specificity and affinity to GbWRKY1 as revealed by
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dual-luciferase reporter assays and electrophoretic mobility shift assays. In summary,
52
our data suggest that GbWRKY1 is a critical regulator mediating the plant
53
defense-to-development transition during V. dahliae infection by activating JAZ1
54
expression.
cinerea
of
and
DELLA.
V.
We
dahliae.
show
Under
that
the
normal
growth
GA-related
conditions,
phenotypes
of
55
56
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Introduction
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To survive and thrive, plants have evolved sophisticated mechanisms to allocate
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limited resources to rapidly activate both local and systemic immune responses
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(Robert-Seilaniantz et al., 2011). It has been stated that the activation of defense
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pathways occurs at the expense of plant growth (Kazan and Manners, 2009, 2012).
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However, vegetative growth and reproductive success in plants are directly related to
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population development and so plants require optimal immune activation to maintain
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growth and development (Walters and Heil, 2007; Pieterse et al., 2009).
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Jasmonic acid (JA) is a fatty acid-derived plant hormone that is perceived by an
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F-box protein, CORONATINE INSENSITIVE1 (COI1), a component of the SCF E3
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ubiquitin ligase complex (Katsir et al., 2008; Yan et al., 2009; Sheard et al., 2010).
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JASMONATE ZIM-Domain (JAZ) family proteins are transcriptional repressors that
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negatively regulate JA signaling via direct interaction with several transcription
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factors, such as bHLH subgroup IIIe transcription factors (MYC2, MYC3 and MYC4)
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and R2R3-MYB transcription factors (MYB21, MYB24 and MYB57) (Cheng et al.,
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2009; Cheng et al., 2011; Fernandez-Calvo et al., 2011; Niu et al., 2011; Qi et al.,
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2011; Song et al., 2011; Song et al., 2013). In the presence of JA, JAZ proteins are
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targeted by the SCFCOI1 complex for ubiquitination and degradation, which
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consequently relieves the repression and rapid activation of JA responses (Chini et al.,
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2007; Thines et al., 2007; Howe, 2010). Moreover, a recent study showed that JAZ1
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physically interacts with DELLA proteins, repressors of gibberellic acid (GA)
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signaling, to dynamically coordinate the balance between GA signaling and JA
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signaling (Hou et al., 2010; Robert-Seilaniantz et al., 2011). The attenuation of JA
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signaling via the overexpression of several JAZ proteins can activate the GA signaling
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pathway, which is consistent with the common phenomenon of the so-called
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growth-defense conflict (Kazan and Manners, 2009, 2012; Yang et al., 2012).
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However, one of the open questions is, which regulators are involved in the
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orchestration of development and disease resistance?
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WRKY transcription factors have been implicated in various transcriptional
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programs, including biotic and abiotic stress responses, growth and development
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(Pandey and Somssich, 2009; Rushton et al., 2010; Rushton et al., 2012; Van Aken et
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al., 2013). WRKY family members are known for their conserved DNA-binding
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region, which is called the WRKY domain, usually found at the N-terminus (Rushton
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et al., 1995; Rushton et al., 2010). Based on the number of WRKY domains and the
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structural features of the zinc-finger motifs at the C-terminus, WRKY transcription
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factors are classified into three groups (Eulgem et al., 2000). Intensive studies have
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demonstrated that most WRKY transcription factors specifically recognize the DNA
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binding site termed the W box (TTGACC/T) (Rushton et al., 1995; Rushton et al.,
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2010). However, a recent study demonstrated that the TGAC core sequence is
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sufficient to be targeted for binding by the AtWRKY28 protein (van Verk et al., 2011).
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Some other WRKY transcription factors, such as OsWRKY13 and HvWRKY46, can
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bind to non-W box sequences (Cai et al., 2008; Mangelsen et al., 2008). Gel shift
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experiments also suggest that different groups of WRKY transcription factors show
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different binding site preferences to recognize TTGACC and TTGACT (Ciolkowski
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et al., 2008). Despite the W-box being required for the binding of the WRKY protein,
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the sequences that are adjacent to the W-box also play an important role in binding
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selectivity (Ciolkowski et al., 2008).
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Accumulating evidence indicates that some WRKY transcription factors exhibit
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multiple regulatory roles, and are involved in several seemingly disparate processes.
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HvWRKY1/38 is a key regulator of cold and drought responses, immune response and
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seed germination (Marè et al., 2004; Shen et al., 2007; Zou et al., 2008). OsWRKY13
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is involved in plant resistance to bacterial infection and responds to drought stress by
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selectively binding to different target genes (Deng et al., 2012b; Xiao et al., 2013).
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AtWRKY75 was first identified as a critical modulator of phosphate (Pi) uptake and Pi
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stress responses (Devaiah et al., 2007). Subsequently, AtWRKY75 has been implicated
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in plant defense to Pseudomonas syringae (Pst), and wrky75 mutant plants display
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increased susceptibility to different Pst strains (Caballero et al., 2009). Moreover, the
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overexpression of AtWRKY75 and its rice orthologue OsWRKY72 causes early
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flowering in Arabidopsis (Arabidopsis thaliana) (Yu et al., 2010). In addition to
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AtWRKY75, other WRKY transcription factors, such as WRKY6, WRKY7, WRKY18
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and WRKY70, also exhibit multiple regulatory roles in immune response and
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flowering time control (Chen and Chen, 2002; Robatzek and Somssich, 2002; Li et al.,
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2004; Kim et al., 2006; Kim et al., 2008), although the molecular mechanism of such
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a transition remains unclear.
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With the limitation of energy resources, plants have evolved an elaborate signaling
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network to prioritize biotic and abiotic stress responses and growth development for
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survival (Kazan and Manners, 2009; Pauwels et al., 2009; Alcázar et al., 2011; Kazan
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and Manners, 2012; Claeys and Inze, 2013). Therefore, new information on how the
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multiple regulatory functions of WRKY transcription factors are integrated into a
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dynamic web is fundamentally important for our understanding of how plants
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coordinate growth and development in response to environmental cues.
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To identify genes that are differentially expressed in cotton (Gossypium barbadense)
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resistance to Verticillium dahliae, we constructed a suppression subtractive
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hybridization (SSH) cDNA library of cotton after inoculation with V. dahliae, from
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which GbWRKY1 was isolated and characterized as having similar regulatory
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functions in phosphate stress responses to its Arabidopsis homolog AtWRKY75 (Xu et
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al., 2012b). In the current study, we report the characterization of multiple roles for
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GbWRKY1 in JA-mediated disease resistance and GA-mediated development. Our
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results indicate that the dual regulatory roles of GbWRKY1 depend on the expression
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of Gossypium hirsutum GhJAZ1, which we propose is trans-activated by GbWRKY1
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in vivo. We further demonstrate that the TGAC core sequence of the GhJAZ1
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promoter and its adjacent DNA sequences are essential for the binding specificity and
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affinity for GbWRKY1.
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Results
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Down-regulation of Gossypium barbadense GbWRKY1 enhances plant resistance
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to Verticillium dahliae and Botrytis cinerea
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We previously identified a subset of genes that are involved in the cotton immune
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response to V. dahliae by screening a cDNA library from the cotton cultivar
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Gossypium barbadense cv. ‘7124’ (Xu et al., 2011a). Among these genes, GbWRKY1
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was induced by V. dahliae strain ‘V991’ at the early stage of infection (Supplemental
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Figure 1). To elucidate the putative role of GbWRKY1 during cotton defense to V.
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dahliae, Tobacco Rattle Virus (TRV)-based virus-induced gene silencing (VIGS) was
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employed to knock down the transcript of GbWRKY1 in cotton. Two weeks after
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inoculation with the recombinant viruses, the expression of GbWRKY1 was efficiently
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suppressed compared with the TRV:00 control through RT-PCR analysis (Figure 1A).
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Subsequently, the plants were either inoculated with the V. dahliae strain ‘V991’ or
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received a mock treatment. Typical disease symptoms caused by V. dahliae, including
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extensive chlorosis, necrosis of leaves and dark brown streaks in the stem, were more
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severe in TRV:00 plants compared to those in TRV:GbWRKY1 plants (Figure 1A and
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Supplemental Figure 2A). The lower disease index also indicated that the
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down-regulation of GbWRKY1 through VIGS could increase the resistance of cotton
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to V. dahliae (Supplemental Figure 2C). We also infected with the typical necrotroph
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Botrytis cinerea to examine the role of GbWRKY1 in plant immunity. Cotton leaves
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from the same stem nodes were detached and infected with 8 L of B. cinerea spore
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suspension (2×105 spores/mL). As shown in Supplemental Figure 2B and 2C, the
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disease lesions in the TRV:00 leaves extended more rapidly compared to those in the
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TRV:GbWRKY1 leaves.
μ
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We further generated stable transgenic cotton lines to upregulate or downregulate
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the expression of GbWRKY1 via overexpression or RNA interference (RNAi)
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strategies respectively via Agrobacterium tumefaciens-mediated transformation. After
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two generations of selfing, we identified two GbWRKY1-silencing lines (Ci2 and Ci3)
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and two GbWRKY1-overexpressing lines (COV4 and COV7), which were from
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independent transgenic events, for further experiments (Figure 1B). Similar to the
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results through VIGS, silencing of GbWRKY1 improved cotton resistance to B.
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cinerea and V. dahliae, while the overexpression of GbWRKY1 compromised this
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resistance (Figure 1C and 1D). The corresponding disease index and average lesion
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diameter data also support these results (Supplemental Figure 2D).
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To better understand the molecular mechanisms of GbWRKY1 in the immune
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response, GbWRKY1-overexpressing Arabidopsis plants were also generated.
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Northern blotting indicated that the GbWRKY1 transcript was significantly
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up-regulated in the two independent lines AOV4 and AOV9 (Figure 1E). Similar to
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the cotton disease assay, the overexpression of GbWRKY1 in Arabidopsis also
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compromised resistance to B. cinerea and V. dahliae (Figure 1F, Supplemental Figure
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2E and Figure 1G). Meanwhile, GbWRKY1-overexpressing plants showed an early
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flowering phenotype, whether inoculated with V. dahliae or not (Figure 1G). This
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indicates that GbWRKY1 is involved not only in the regulation in plant immunity but
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also in plant development.
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GbWRKY1 negatively regulates the JA signaling pathway
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The central role of JA in plant responses to necrotrophic pathogens has been
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demonstrated in a number of plant species (Veronese et al., 2006; Mengiste, 2012).
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Moreover, Ve1-mediated resistance is compromised in jar1–1 mutants in Arabidopsis
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(Fradin et al., 2011), and the JA signaling pathway can be activated in cotton by
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inoculation with V. dahliae (Gao et al., 2013), suggesting that JA plays an important
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role in cotton resistance to V. dahliae.
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To further evaluate the role of JA in cotton’s defense against V. dahlia, cotton
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seedlings were treated with MeJA (which can be converted to JA) two days before
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inoculation with V. dahlia. Typical chlorosis and necrosis of infected leaves were
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delayed in MeJA-treated plants compared to control plants, suggesting that the JA
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signaling pathway positively contributes to cotton resistance to V. dahliae (Figure 2A
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and 2B). To test the potential involvement of GbWRKY1 in JA signaling, we analyzed
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the expression of GbWRKY1 in response to MeJA and measured the content of JA in
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GbWRKY1-silencing and -overexpressing cotton plants. Interestingly, the GbWRKY1
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transcript was significantly and rapidly induced by MeJA (Supplemental Figure 3),
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while no significant differences in the endogenous level of JA were found between the
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wild type (WT) and GbWRKY1 transgenic cotton lines (Supplemental Figure 4).
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To determine whether GbWRKY1 could negatively affect the JA-mediated defense
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response, we checked the expression of GbPR4, which is a molecular marker of the
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JA signaling pathway in cotton (Xu et al., 2011b; Gao et al., 2013). qRT-PCR analysis
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demonstrated that the expression of GbPR4 was highly induced by MeJA in
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GbWRKY1-silencing cotton line Ci2, while only a slight upregulation of GbPR4
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transcript could be found in the GbWRKY1-overexpressing cotton line COV4 after
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MeJA treatment (Figure 2C), indicating that overexpression of GbWRKY1 suppressed
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the MeJA induction of GbPR4. Meanwhile, the overexpression of GbWRKY1
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appeared to repress the expression of PDF1.2 in Arabidopsis plants (Figure 2D).
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To further verify the role of GbWRKY1 in the JA signaling pathway, JA-induced
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anthocyanin accumulation was compared between WT and GbWRKY1-overexpressing
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Arabidopsis lines (Qi et al., 2011). Anthocyanin accumulated nearly 19-fold in WT
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plants and only 8-fold in GbWRKY1-overexpressing plants in the presence of MeJA
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compared to mock treatments (Figure 2E and 2F). JA-regulated anthocyanin
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accumulation was mediated by JAZ proteins marked by the downregulation of three
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anthocyanin biosynthetic genes (UF3GT, LDOX and DFR) in the dominant-negative
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transgenic plant JAZ1△3A (Qi et al., 2011). Consistent with this result, the
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expressions of UF3GT, LDOX and DFR were also downregulated by GbWRKY1 after
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MeJA treatment (Figure 2G). These results demonstrate that GbWRKY1 might be an
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important negative regulator in the JA signaling pathway.
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GbWRKY1-overexpressing plants display GA-related phenotypes coupled with
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the down-regulation of putative DELLA target genes
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Although the transcript of GbWRKY1 barely responded to GA treatment (data not
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shown), the overexpression cotton lines exhibited morphological phenotypes similar
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to those of GA-overdose cotton plants under normal growth conditions as described
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by Xiao et al. (2010), including petiole elongation and pale green leaves (Figure 3A,
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3C and 3E). GA-mediated elongation and the loss of green color rely on the alteration
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of cell length and chlorophyll accumulation, respectively (Cheminant et al., 2011; de
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Saint
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GbWRKY1-overexpressing cotton plants display longer petiole epidermal cells than
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those of the WT as observed under scanning electron microscope. Moreover, the
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chlorophyll level was reduced in GbWRKY1-overexpressing plants (Figure 3F),
Germain
et
al.,
2013).
As
shown
in
Figure
3B
and
3D,
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237
indicating that the effects of GbWRKY1 on the petiole length and leaf green color are
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likely to be due to the changes in cell length and chlorophyll accumulation.
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To verify this result, the sensitivity to the GA biosynthesis inhibitor paclobutrazol
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(PAC) was evaluated by comparing the hypocotyl length in the dark as previously
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described (Zhang et al., 2011). The results demonstrate that the upregulation of
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GbWRKY1 dramatically promotes cotton hypocotyl elongation not only under mock
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conditions but also under PAC treatment conditions compared to WT and
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GbWRKY1-RNAi plants (Figure 3G). The WT hypocotyl length was significantly
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inhibited by PAC treatment, with an inhibition ratio of up to 75%, compared with a
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ratio of 58% in the GbWRKY1-overexpressing plants (Figure 3H). This suggests that
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overexpression of GbWRKY1 leads to a reduced sensitivity to PAC in cotton.
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Interestingly, no observable differences in terms of plant morphology and sensitivity
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to PAC could be found between WT and GbWRKY1-RNAi plants (Supplemental
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Figure 5, Figure 3G and 3H), possibly due to the indirect involvement of GbWRKY1
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in the GA pathway. Similarly, the positive role of GbWRKY1 in the GA pathway was
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further confirmed from the phenotypic investigation of transgenic Arabidopsis plants.
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The GbWRKY1-overexpressing Arabidopsis lines AOV4 and AOV9 also displayed
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GA-related phenotypes, such as early flowering and longer hypocotyls (Figure 4A,
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Supplemental Figure 6A, 6B and 6C).
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Given the consistent GA-associated effects in GbWRKY1-overexpressing
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Arabidopsis plants and cotton plants, the transcripts of genes that are involved in GA
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biosynthesis and catabolism were analyzed in Arabidopsis seedlings to determine
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whether GbWRKY1 contributes to GA metabolism. The results indicate that the GA
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catabolic
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(Supplemental Figure 7A).
gene
AtGA2ox2
was
up-regulated
in
35S:GbWRKY1
seedlings
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GA is known to promote GA signaling transduction by destabilizing a number of
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GA-repressors, the DELLA proteins (GAI, RGA, RGL1, RGL2, and RGL3) in
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Arabidopsis (Harberd et al., 2009). Recently, chromatin immunoprecipitation (ChIP)
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and microarray analyses have identified a group of putative RGA targets (GID1a,
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GID1b, XERICO, SCL3, bHLH137, LBD40, MYB) (Zentella et al., 2007), which are
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down-regulated in quadruple-DELLA mutant plants (Josse et al., 2011). To determine
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whether the GA signaling pathway is influenced by GbWRKY1, the transcripts of
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these putative DELLA targets (GID1a, GID1b, XERICO, SCL3, bHLH137, LBD40,
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MYB) were detected in Arabidopsis through qPCR. Strikingly, most of their transcript
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levels were repressed by GbWRKY1, and the expression of AtGID1a and AtGID1b
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was repressed approximately 2- to 4.5-fold in GbWRKY1-overexpressing plants
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(Figure 4B and Supplemental Figure 7B). The homologs of AtGID1a and AtGID1b in
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cotton, GhGID1-a and GhGID1-b, have been isolated and are strongly inhibited by
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GA treatment in cotton ovules (Aleman et al., 2008). Consistent with a previous study,
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our qRT-PCR analysis demonstrated that the expression of GhGID1-a/GhGID1-b was
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strongly suppressed in cotton leaves after GA treatment (Supplemental Figure 7C),
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similar to AtGID1a and AtGID1b in Arabidopsis (Zentella et al., 2007). In addition,
279
the transcript levels of GhGID1-a and GhGID1-b were also significantly inhibited via
280
the overexpression of GbWRKY1 (Figure 4C). This implies that the positive
281
regulatory role of GbWRKY1 in the GA pathway is probably via an activation of the
282
GA-responsive pathway downstream of DELLA.
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A recent study demonstrated that DELLA proteins modulate flowering time by a
284
direct binding of RGA to SQUAMOSA PROMOTER BINDING–LIKE (SPL) to
285
attenuate the SPL transcriptional activities of SUPPRESSOR OF OVEREXPRESSION
286
OF CO1 (SOC1) (Yu et al., 2012). Our results demonstrate that the expression of
287
SOC1 is consistently up-regulated by the overexpression of GbWRKY1 during
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seedling development (Figure 4D). To examine the genetic relationship between
289
GbWRKY1 and SOC1 we employed an F2 population generated by crossing AOV9
290
with a soc1 mutant. The results demonstrate that the early flowering phenotype of
291
AOV9 in the soc1 background is similar to that in the soc1 mutant (Figure 4E),
292
indicating that the early flowering phenotype from the overexpression of GbWRKY1
293
depends on the activation of SOC1.
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GbWRKY1-orchestrated balance between the JA and GA signaling pathways
296
depends on the constitutive activation of Gossypium hirsutum GhJAZ1
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In accordance with the reports that an antagonistic interaction of JA/GA exists in
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defense and development (Navarro et al., 2008; Kazan and Manners, 2012), our
299
results demonstrate that GbWRKY1 contributes opposite functions to JA-mediated
300
immune responses and GA-mediated development. To examine how GbWRKY1
301
coordinates the crosstalk between the JA/GA signaling pathways, flowering time was
302
investigated after treatment with MeJA. The early flowering phenotype by GbWRKY1,
303
which is mediated possibly through the GA pathway, could be partially suppressed by
304
MeJA (Figure 5A, Supplemental Figure 8), suggesting that the GA-related phenotypes
305
of GbWRKY1-overexpressing plants might be indirectly regulated by JA.
306
To validate the participation of GbWRKY1 in the crosstalk between the JA and GA
307
signaling pathways, the expression of GhGID1-a/GhGID1-b was examined in
308
transgenic cotton plants under MeJA treatment. In WT plants, the expression level of
309
GhGID1-a and GhGID1-b increased by 2.7- and 2.4-fold after JA-treatment,
310
respectively (Figure 5B). Meanwhile, the transcript levels of these two genes was
311
up-regulated by 4.5- and 7.6-fold in GbWRKY1 knock-down cotton plants (Figure 5B),
312
respectively. However, the overexpression of GbWRKY1 appears to attenuate the
313
induction of GhGID1-a and GhGID1-b in response to MeJA (Figure 5B).
314
Reciprocally, the expression of the JA responsive gene GbPR4 was also examined in
315
GbWRKY1 transgenic plants after GA treatment (Supplemental Figure 9), and showed
316
that the GA-mediated change in expression of GbPR4 in WT and GbWRKY1
317
transgenic plants was similar to both mock treatment and the effect of JA (Figure 2C),
318
although the expression of GbPR4 showed opposite regulatory pattern after GA
319
treatment and JA treatment. These expression analyses indicate that GbWRKY1 most
320
likely functions in the JA pathway to indirectly regulate GA pathway, and not the
321
other way round.
322
Recent studies have demonstrated that the physical interaction between
323
JAZ1-DELLA and their dynamic balance contributes to the antagonism between JA
324
and GA (Hou et al., 2010). The up-regulation of a selected group of JAZ genes
325
(including AtJAZ1, 3, 4, 9, 10, and 11) in Arabidopsis can promote GA-mediated
326
development, such as hypocotyl elongation and flowering time (Yang et al., 2012). To
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327
examine whether there is a similar mechanism in regulating the GbWRKY1-mediated
328
orchestration between defense and development, the expression of a series of JAZ
329
genes (AtJAZ1, 3, 4, 9, 10, and 11) was characterized. Interestingly, only AtJAZ1 was
330
up-regulated via the overexpression of GbWRKY1 in Arabidopsis, and similar results
331
were also found in cotton, in which GhJAZ1 showed approximately 3.7- and 1.7-fold
332
activation in two independent GbWRKY1-overexpressing cotton lines (Figure 5C and
333
Supplemental Figure 10). We then used GhJAZ1-overexpressing lines (J92 and J131)
334
and GhJAZ1-RNAi transgenic cotton lines (JR1 and JR3) to assess the interaction
335
between GbWRKY1 and GhJAZ1 in cotton (Supplemental Figure 11). The results
336
demonstrate that the transcript level of GbWRKY1 was not significantly influenced in
337
the GhJAZ1 overexpression lines and was slightly up-regulated in the GhJAZ1-RNAi
338
lines (Figure 5D and 5E). This suggests that GbWRKY1 promotes GA-related growth,
339
potentially via activation of JAZ1.
340
To further elucidate the genetic interaction between GbWRKY1 and GhJAZ1, a
341
GbWRKY1-overexpressing line (COV4) was crossed with WT cotton and a
342
GhJAZ1-RNAi line (JR3), respectively, and the cotyledon petiole length was
343
compared in the F1 hybrid seedlings. As shown in Figure 5F, GbWRKY1-mediated
344
petiole elongation was rescued in the GhJAZ1 knock-down background. These data
345
indicate that GhJAZ1 is genetically epistatic to GbWRKY1 in cotton. This interaction
346
was
347
amiRNATIFY10A (an AtJAZ1 knock down line; Grunewald et al., 2009) via
348
cross-pollination, and the flowering time was investigated in AOV9 and
349
amiRNAJAZ1 hybrid progeny. The flowering time of AOV9 was rescued following
350
downregulation of the expression of AtJAZ1 (Figure 5G). Our genetic analyses
351
indicate that the GbWRKY1-orchestrated trade-off between the JA and GA signaling
352
pathways depends on the constitutive activation of JAZ1 both in cotton and in
353
Arabidopsis.
further
confirmed
in
Arabidopsis
by
introducing
GbWRKY1
into
354
355
GbWRKY1 transactivates expression of GhJAZ1 and AtJAZ1 in vivo
356
WRKY proteins specifically bind to the W-box (TTGACC/T) in most instances
14
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
357
(Rushton et al., 2010). To examine the possibility that GbWRKY1 can bind to the
358
promoter of GhJAZ1, the promoter region of GhJAZ1 was analyzed. Unexpectedly,
359
only one typical W-box (TTGACC) was found within 2-kb upstream of the GhJAZ1
360
start codon. However, four TTGACT sequences were found within the 2-kb promoter
361
sequence of AtJAZ1 according to the Arabidopsis cis-regulatory element database
362
(AtcisDB).
363
To test the interaction between GbWRKY1 and the promoters of JAZ1 genes, the
364
GhGS1 (Gossypium hirsutum sequence for gel shift assay, from -663 to -698)
365
sequence from the GhJAZ1 promoter and the AtGS1(Arabidopsis sequence for gel
366
shift assay, from -665 to -721) sequence from the AtJAZ1 promoter were synthesized
367
for an electrophoretic mobility shift assay (EMSA) (Figure 6A). Surprisingly,
368
GbWRKY1 binding to the AtJAZ1 promoter was detected (Figure 6C), whereas no
369
detectable binding was found in the case of the GhJAZ1 promoter (Figure 6B).
370
A recent study demonstrated that the TGAC core sequence is sufficient to bind to
371
the AtWRKY28 protein (van Verk et al., 2011); we found nine core sequences within
372
the GhJAZ1 promoter in the -711 to -1649 region. The fragment containing two core
373
sequences (GhGS2, from -1599 to -1654) was synthesized as a target probe to assay
374
the interaction between GbWRKY1 and the GhJAZ1 promoter (Figure 6A), and a
375
shifted band for GbWRKY1 binding to the GhJAZ1 promoter was detected (Figure
376
6D). A competitive experiment and a TGAC-mutated EMSA experiment further
377
demonstrated that GbWRKY1 could specifically bind to the TGAC core sequences of
378
GhJAZ1 (Figure 6D).
379
However, the binding selectivity of GbWRKY1 with regard to GhGS1 and AtGS1
380
led us to speculate that the sequences that are adjacent to the binding site may
381
determine whether the site can be bound. To test this hypothesis, we synthesized a
382
modified version of GhGS1 (designated as GhGS3), for which the W-box adjacent
383
sequences were replaced by the partial sequences adjacent to the GhGS2 TGAC core
384
(Figure 6A). After replacement, however, the GhGS3 probe was bound by
385
GbWRKY1, and this binding could be competed by non-labeled probes (Figure 6E).
386
Next, we synthesized several variants of GhGS3 (Figure 6A). As expected, a mutation
15
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
387
within the TTGACT sequence (designated as GhGS3-M1) could completely block
388
binding of GbWRKY1 (Figure 6E). However, the binding affinity of GhGS3
389
significantly decreased after the mutation of GhGS3 oligonucleotide at positions
390
-1/-2/-3/-4 from the TTGACT sequence (designated as GhGS3-M2). The mutation at
391
positions +1/+2/+3/+4 from the TTGACT sequence (designated as GhGS3-M3) did
392
not affect the binding capability of GhGS3. Because GhGS1 and GhGS3 had the same
393
nucleotides at positions -1/-2 from the binding site (Figure 6A), we next determined
394
the effect of positions -3/-4 on binding to GbWRKY1, and a similar binding
395
abundance between GhGS3-M2 and GhGS3-M4 suggested that positions -3/-4 were
396
responsible for the decreased binding of GhGS3-M2 (Figure 6E). Our results
397
therefore demonstrate that GbWRKY1 not only targets the W-box but also TGAC
398
core sequences and that the adjacent DNA sequences of the binding site are also
399
important for the binding selectivity of GbWRKY1.
400
To validate the possibility that the TGAC core sequences are required for binding
401
with GbWRKY1, the 419-bp promoter sequence (see Methods) of GhJAZ1 containing
402
four TGAC sequences was cloned into the pHisi-1 yeast one-hybrid bait vector, and
403
the full-length cDNA of GbWRKY1 was cloned into the pDEST22 prey vector. As
404
shown in Figure 6F, GbWRKY1 was also able to bind to the wild-type GhJAZ1
405
promoter in yeast, whereas mutations within the four TGAC core sequences abolished
406
GbWRKY1 binding to the GhJAZ1 promoter in the presence of 15 mM
407
3-amino-1,2,4-triazole (3-AT), demonstrating that the TGAC sequence is required for
408
GbWRKY1 binding.
409
We also employed the dual-luciferase reporter (DLR) system in tobacco to examine
410
the transcriptional activity of GbWRKY1 in vivo. The wild-type or mutated GhJAZ1
411
promoters (see Methods) were cloned and fused with the firefly luciferase (LUC)
412
reporter gene, and the relative LUC activity was measured after transfection. Figure
413
6G shows that the overexpression of GbWRKY1 trans-activated the activity of the
414
LUC reporter under the GhJAZ1 promoters, whereas this activity was abolished when
415
the TGAC cores in the GhJAZ1 promoter were disrupted.
416
To further assess the role of W-boxes (TTGACT) and the TGAC motif in
16
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
417
GbWRKY1 transactivation, the 1229-kb wild-type AtJAZ1 promoter (containing four
418
W-boxes and five TGAC core sequences) and a modified AtJAZ1 promoter (the four
419
W-boxes were mutated but not the TGAC core sequences) were fused with the LUC
420
reporter gene. These were used to examine the effect of GbWRKY1 on AtJAZ1
421
transcription. The data show that wild-type AtJAZ1 promoter was trans-activated by
422
approximately 3.2-fold after cotransfection with the effector (35S:GbWRKY1; Figure
423
6G). With the W-box-mutated version promoter, overexpression of GbWRKY1 only
424
caused a 1.7-fold increase in the activity of LUC reporter (Figure 6G). These results
425
show that GbWRKY1 is capable of transactivating GhJAZ1 via binding to the TGAC
426
core sequences but AtJAZ1 binds via the W-box (TTGACT) and the TGAC core
427
sequence.
428
429
Discussion
430
Due to the sessile lifestyle of plants and the limitation of available energy, plants have
431
evolved an elaborate regulatory network to effect a trade-off between growth and
432
immune responses, to ensure plant survival under fluctuating environments (Kazan
433
and Manners, 2009; Fan et al., 2014). Verticillium dahliae is notorious for its infection
434
strategies and great genetic plasticity, and causes significant economic losses for a
435
broad range of crops, such as cotton, strawberry, oilseed rape, tomato and potato
436
(Zhou et al., 2006; Klosterman et al., 2009). Typical symptoms caused by V. dahliae
437
include leaf necrosis, premature defoliation, altered flowering time and severe growth
438
stunting (Veronese et al., 2003; Fradin et al., 2009; Xu et al., 2011b). Recent studies
439
have identified signaling pathways that play essential roles in cotton resistance to V.
440
dahliae (Xu et al., 2011b; Gao et al., 2013; Zhang et al., 2013), but how plants
441
orchestrate development under V. dahliae infection remains elusive. By mining genes
442
that are expressed differentially in response to V. dahliae from the cotton cultivar G.
443
barbadense cv ‘7124’, the WRKY transcription factor gene GbWRKY1 was identified
444
as putatively functioning in the Pi starvation and resistance response (Xu et al., 2011a;
445
Xu et al., 2012b). In this study, we provide molecular and genetic evidence to
446
demonstrate that GbWRKY1 might be a key regulator in the orchestration of plant
17
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
447
defense and development.
448
To determine the precise role of GbWRKY1 in the resistance response in cotton, we
449
generated GbWRKY1-overexpressing transgenic cotton and Arabidopsis plants and
450
GbWRKY1 knock-down cotton plants through VIGS and RNAi respectively. All the
451
inoculation assays consistently demonstrated that overexpressing GbWRKY1 in cotton
452
and Arabidopsis attenuated resistance to B. cinerea and V. dahliae (Figure 1 and
453
Supplemental Figure 2). However, when GbWRKY1 was knocked down through
454
VIGS or RNAi, the plants showed more resistance to B. cinerea and V. dahliae
455
(Figure 1 and Supplemental Figure 2). Generally, the JA signaling pathway plays a
456
central role in plant resistance to necrotrophic pathogens. Several lines of evidence
457
have suggested that JA signaling is also required for plant resistance to V. dahliae, as
458
revealed by the exogenous application of MeJA (Figure 2A and 2B) and by genetic
459
and molecular biology analyses in Arabidopsis and cotton (Fradin et al., 2011; Gao et
460
al., 2013). Furthermore, GbWRKY1 showed clear negative regulation in JA-mediated
461
defense gene expression and anthocyanin accumulation (Figure 2). These results
462
suggest that GbWRKY1 acts as a negative regulator in the plant disease resistance
463
response, possibly by attenuating the JA signaling pathway.
464
Previous studies have demonstrated that the transition from vegetative to
465
reproductive growth is a critical phase for Verticillium invasion in oilseed rape
466
(Brassica napus; Zhou et al., 2006). This is in agreement with the common
467
observation that the prevalence of cotton Verticillium wilt is most severe after
468
flowering in the field (Ma et al., 2000; Wang et al., 2012). Meanwhile, the regulation
469
of flowering time seems to be a crucial strategy that plants select for survival under
470
pathogen infection. Susceptible ecotypes exhibit a pathogen-induced early flowering
471
phenotype, whereas more tolerant ecotypes exhibit delayed flowering after
472
Verticillium inoculation (Veronese et al., 2003; Haffner et al., 2010). It is reported that
473
VET1 (Verticillium dahliae-tolerance) plays a negative role in flowering time and a
474
positive role in pathogen resistance (Veronese et al., 2003). By contrast, GbWRKY1
475
appears to possess functions antagonistic to VET1 (Verticillium dahliae-tolerance), as
476
its overexpression in Arabidopsis resulted in early flowering and attenuation of
18
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
477
resistance to V. dahliae. Moreover, the overexpression of other defense-related
478
WRKY transcription factors, such as WRKY 6, WRKY7 and WRKY18, also can alter
479
flowering time (Chen and Chen, 2002; Robatzek and Somssich, 2002; Kim et al.,
480
2006; Kim et al., 2008). Therefore these WRKY transcription factors may have
481
multifaceted functions in the coordination of plant development and immune
482
response.
483
Interestingly, GbWRKY1-overexpressing cotton plants did not exhibit observable
484
differences in flowering time (Supplemental Figure 6D), although the overexpression
485
of GbWRKY1 appears to promote GA-related development in cotton seedlings, and
486
GA enhances flowering time in Arabidopsis. Additionally, the flowering time of
487
cotton plants did not display any alteration after exogenous application of GA but
488
showed severely delayed flowering in the presence of its biosynthesis inhibitor
489
paclobutrazol (PAC; Supplemental Figure 6E), which is consistent with previous
490
observations that flowering time was delayed by down-regulation of the cotton GA
491
biosynthesis gene GhGA20ox1, but shows little change when GhGA20ox1 was
492
overexpressed (Xiao, 2004). Thus, GA may exert a different regulatory role in
493
controlling the development of different plant species.
494
In agreement with the premise that the activation of plant immune responses is at
495
the expense of growth (Kazan and Manners, 2012), JA-incubated cotton plants were
496
slightly smaller than were the mock plants after inoculation with V. dahliae (Figure
497
2A). Meanwhile, JA-treated Arabidopsis plants showed retarded vegetative growth
498
and a late-flowering phenotype (Supplemental Figure 8 and Figure 5A). A previous
499
study demonstrated that the JA-mediated inhibitory effect on Arabidopsis hypocotyl
500
elongation was partly mediated by DELLA proteins (Hou et al., 2010). We found that
501
two GA-repressed genes, GhGID1-a/GhGID1-b, that are homologs of putative
502
DELLA targets in Arabidopsis were dramatically up-regulated by JA (Figure 5B).
503
These results indicate that JA exerts its suppressive role on plant growth partly by
504
suppressing GA signaling (Figure 7). We suggest that GbWRKY1 may be a pivotal
505
molecular switch in the orchestration of cotton development and defense during V.
506
dahliae infection.
19
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507
Studies in Arabidopsis have demonstrated that changes in GA signaling
508
transduction can affect the expression of GA biosynthetic genes and GA catabolic
509
genes by feedback mechanisms, and AtGA2ox is induced in GA-overproducing plants
510
(Zhang et al., 2011). Similarly, the induction of AtGA2ox-2 was accompanied by the
511
activation of the GA signaling pathway downstream of DELLA (Supplemental Figure
512
7). Further genetic analysis suggests that the early flowering time phenotype of
513
GbWRKY1-overexpressing Arabidopsis depends on the expression of SOC1 (Figure
514
4E), which participates in DELLA-mediated flowering time control (Yu et al., 2012).
515
Interestingly, WT and GbWRKY1-RNAi plants did not show observable differences in
516
terms of plant morphology and sensitivity to PAC (Supplemental Figure 5, Figure 3G
517
and 3H), but the expression of GhGID1-a and GhGID1-b was induced to higher levels
518
by MeJA treatment in RNAi plants (Figure 5B). In addition, there was no obvious
519
effect on the expression of GbWRKY1 after GA treatment (data not shown). We can
520
infer that there is cross-regulation between defense and growth, and the alteration of
521
defense-related processes indirectly affects developmental processes (Alcázar et al.,
522
2011).
523
A recent study demonstrated that the overexpression of a selected group of
524
JAZ-encoding genes by the 35S promoter is able to activate the GA signaling pathway
525
through the physical interaction between JAZ and DELLA proteins (Yang et al., 2012).
526
Molecular and genetic analyses indicated that GbWRKY1 is likely to act upstream of
527
GhJAZ1 and trans-activate the activity of GhJAZ1 (Figure 5F and Figure 6G),
528
suggesting that a precise interaction occurs between GbWRKY1 and GhJAZ1.
529
The Arabidopsis transcription factor WRKY33 negatively regulates the JAZ1 and
530
JAZ5 transcripts through B. cinerea-infection–induced binding to their promoter
531
regions (Birkenbihl et al., 2012), although 35S:WRKY33 plants exhibit enhanced
532
resistance to B. cinerea and a slightly earlier flowering time compared to WT (Zheng
533
et al., 2006a).
534
In addition, an expression profile analysis demonstrated that the homolog of
535
GbWRKY1 in Arabidopsis, AtWRKY75, and three JAZ genes (AtJAZ1, AtJAZ5 and
536
AtJAZ10) share the same expression pattern in WT and wrky33 mutant plants during B.
20
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
537
cinerea infection (Birkenbihl et al., 2012). Interestingly, qRT-PCR analysis showed
538
that only AtJAZ1 was up-regulated in 35S:GbWRKY1 Arabidopsis plants, and similar
539
results were obtained in the transcriptional profiling of grapevine, showing that only
540
VvJAZ1.1 and VvJAZ1.2 were up-regulated by VvWRKY1, the grapevine homologue
541
gene of AtWRKY75 (Marchive et al., 2013). 35S::VvWRKY1 transgenic plants also
542
displayed pale green leaves, similar to 35S:GbWRKY1 plants, indicating that these
543
homologs of AtWRKY75 may share a similar regulatory mechanism to selectively
544
recognize their target promoters.
545
A previous study suggested that the sequences that are adjacent to W-box play
546
important roles in binding selectivity (Rushton et al., 2010). However, our EMSA
547
results demonstrated that the sequences that are adjacent to the binding site are
548
required not only to determine whether the binding site can be bound by GbWRKY1
549
but also to ensure its high-affinity binding. In addition to these cis-acting elements,
550
we cannot exclude the possibility that other interacting partners of GbWRKY1 were
551
also involved in determining the specific recognition, which remains a challenging
552
area for further investigation in cotton and in the model plant Arabidopsis.
553
JA is known to activate its signaling transduction through promoting JAZ protein
554
ubiquitination and degradation (Chini et al., 2007; Thines et al., 2007). However, once
555
JA signaling is activated, the expression of many JAZ genes is rapidly increased
556
(Zheng et al., 2006b). This suggests that a negative feedback inhibition mechanism
557
may exist to avoid the hyperactivation of JA signaling (Figueroa and Browse, 2012),
558
although how expression of JAZ genes is activated via the negative feedback loop is
559
still unknown. In our study, GbWRKY1 expression was rapidly induced by JA
560
treatment and was also slightly increased in the GhJAZ1-RNAi lines (Supplemental
561
Figure 3, Figure 5E and Figure 7). Through the direct binding of GbWRKY1 to the
562
JAZ1 promoter and transactivating expression of JAZ1, a feedback regulatory loop
563
might be formed to maintain the JA homeostasis in the plant (Figure 7). GbWRKY1
564
shows early expression after V. dahliae infection and prioritizes development over
565
pathogen defense through a known antagonistic interaction of the JA/GA pathway
566
(Figure 7), shedding new light on the interplay between cotton and V. dahliae.
21
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
567
568
Materials and Methods
569
Plant materials, VIGS experiments, and disease assays
570
The cotton plants Gossypium barbadense cv ‘7124’ and Gossypium hirsutum cv
571
‘YZ1’ were used in this study. For the VIGS experiments and disease assays, cotton
572
plants were grown in a growth room with a 16-h day and 8-h night cycle at 25°C.
573
cDNA sequence of GbWRKY1 was cloned into the TRV plasmid using BamHI and
574
KpnI to construct the TRV:GbWRKY1 VIGS silencing vector and was then
575
transformed into Agrobacterium tumefaciens GV3101 via electroporation as
576
previously described (Fradin et al., 2009). The TRV:00 (control) and TRV:GbWRKY1
577
vectors were agroinfiltrated into the cotyledons of 10-d-old YZ1 plants using a
578
needleless syringe as previously described (Gao et al., 2013). Almost 2 weeks after
579
infiltration, RNA was extracted from cotton roots to measure the expression of
580
GbWRKY1.
581
The Verticillium dahliae strain ‘V991’ and Botrytis cinerea were cultured on potato
582
dextrose agar (PDA) at 25°C for 4 d and then further incubated on new PDA medium
583
for another 7 d. The conidia of V. dahliae and B. cinerea were collected and
584
resuspended in distilled water and 1% Sabouraud maltose broth buffer, respectively.
585
The V. dahliae infection assays were performed by root dipping with spore suspension
586
(2×105 spores/mL) as previously described (Xu et al., 2011b; Gao et al., 2013). The
587
disease index (%) was measured to examine the susceptibility to V. dahliae as
588
previously described (Xu et al., 2012a). B. cinerea infection assays were performed
589
using either a spore suspension (2×105 spores/mL) or colonized agar plugs as
590
indicated in the text. The average lesion size (diameter) and plant decay were
591
measured as previously described (Coego et al., 2005; Veronese et al., 2006).
592
593
Gene cloning, vector construction, and plant transformation
594
The expressed sequence tag (EST) of GbWRKY1 was isolated from the cotton
595
cultivars G. barbadense cv ‘7124’ using suppression subtractive hybridization (SSH)
596
(Xu et al., 2011a). The full-length sequence was obtained through Rapid
22
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
597
Amplification of cDNA Ends (RACE)-PCR according to the SMART RACE cDNA
598
amplification kit user manual (Clontech, Terra Bella Ave. Mountain View, CA, USA)
599
and using V. dahliae-infected cotton roots cDNA as the template. The full-length
600
coding sequence of GbWRKY1 was inserted into the modified binary plant vector
601
pCAMBIA2300 (Cambia, Canberra, Australia) using XbaI and SacI to construct the
602
vector 35S:GbWRKY1 for overexpression (Xu et al., 2012b). The RNA interference
603
(RNAi) region containing a partially conserved domain and the specific 3 region of
604
GbWRKY1 was cloned into the RNAi vector pHellsgate 4 through recombination
605
reaction (Tan et al., 2013). The overexpression vector and RNAi vectors were
606
introduced into cotton (YZ1) plants by Agrobacterium tumefaciens (strain
607
EHA105)–mediated transformation as previously described (Jin et al., 2006). The
608
overexpression vector was transferred into Agrobacterium tumefaciens strain
609
(GV3101) to transform the Arabidopsis thaliana ecotype Col-0 using the floral dip
610
method (Hao et al., 2012).
′
611
612
Nucleic acid extraction and expression analysis
613
Total RNA was isolated as previously described (Tan et al., 2013). For northern
614
blotting, twenty micrograms of total RNA per lane was separated on 1.2%
615
agarose-formaldehyde gels and transferred onto a nylon membrane. Blots were
616
hybridized with 32P-labeled GbWRKY1-specific probes. Hybridization was performed
617
in PerfectHyb hybridization solution (TOYOBO, Osaka, Japan). The procedures for
618
detecting the signal were performed as previously described (Tu et al., 2007). For
619
RT-PCR and qRT-PCR analyses, RNA was reverse-transcribed to cDNA using
620
SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). qRT-PCR was
621
performed using the ABI Prism 7000 system (Applied Biosystems, Foster City, CA,
622
USA). The values are given relative to the housekeeping genes ACTIN in Arabidopsis
623
or UB7 in cotton. The primers that were used in the northern blotting, RT-PCR, and
624
qRT PCR are listed in Supplemental Table 1.
625
626
Scanning electron microscopy and chlorophyll measurement
23
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
627
Seeds of the WT and GbWRKY1 transgenic lines were sown into individual pots (8
628
cm in diameter). At least 5 cm was spaced between the plants to avoid mutual shading.
629
After the second true leaves were fully expanded, cotyledon petioles were collected
630
and fixed in 2.5% (v/v) glutaraldehyde. The samples were pretreated as previously
631
described (Deng et al., 2012a) and photographed with a JSM-6390/LV scanning
632
electron microscope (JEOL). At the same time, the second true leaves were harvested
633
for chlorophyll measurement. Briefly, the leaves were weighed and ground into
634
powder in liquid nitrogen. The total chlorophyll was then extracted with 80% (v/v)
635
acetone and measured using a Beckman Coulter DU800 spectrophotometer. Total
636
chlorophyll content was calculated as previously described (Kim and Kim, 2013).
637
638
Protoplast transient transfection for dual-luciferase reporter assays
639
The transient dual-luciferase reporter assays were performed as previously described
640
(Hellens et al., 2005). The wild-type promoter sequence (from -1307 to -1726) of
641
GhJAZ1 containing five TGAC core sequences was amplified by PCR using YZ1
642
genomic DNA, and the relevant TGAC mutated version (mutation at positions -1333,
643
-1375,
644
(http://www.genscript.com/). The 1229-kb wild-type AtJAZ1 promoter (from -6 to
645
-1235 containing four W-boxes (TTGACT) and five TGAC core sequences) was
646
amplified by PCR using Arabidopsis genomic DNA, and the TTGACT-mutated
647
version (mutation at positions -428, -672, -679, -938) was obtained using overlap
648
extension PCR. These fragments were cloned into pGreenII 0800-LUC at the PstI and
649
BamHI
650
AtJAZ1PRO:LUC, and AtJAZ1PRO-M-LUC. The coding region of GbWRKY1 was
651
amplified by PCR and cloned into the pGreenII 62-SK at the PstI and BamHI sites to
652
generate 35:GbWRKY1. Protoplasts were isolated from tobacco (Nicotiana tabacum
653
cv Petite Havana) leaves according to Yoo et al. (2007). After transformation,
654
protoplasts were cultured for 16 h and collected. Firefly luciferase and Renilla
655
luciferase activities were quantified using the dual luciferase assay reagents (Promega,
656
USA) using a Multimode Plate Reader (PerkinElmer, USA). The primers that were
-1521,
sites
-1612,
to
-1645)
generate
was
synthesized
GhJAZ1PRO:LUC,
by
GenScript
GhJAZ1PRO-M-LUC,
24
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
657
used in dual-luciferase reporter assays are listed in Supplemental Table 1.
658
659
Yeast one-hybrid assay
660
A Y1H assay was performed as described by Ou et al. (2011). To generate reporter
661
strains, the 419-bp wild-type promoter sequences (from -1307 to -1726) and the
662
relevant TGAC-mutated version were amplified by PCR using GhJAZ1PRO:LUC
663
and GhJAZ1PRO-M-LUC plasmids as the template, respectively. Then, the
664
PCR-amplified fragments were cloned into the pHisi-1 Y1H bait vector to generate
665
GhJAZ1-PRO and GhJAZ1W-box-m PRO. After linearization by XhoI, the bait
666
vectors were integrated into the yeast strain YM4271 and selected on synthetic
667
dropout (SD) medium without His. In addition, the full-length cDNA of GbWRKY1
668
was cloned into the pDEST22 prey vector to generate AD-GbWRKY1 and was then
669
transferred to yeast strain Y187 followed by selection on SD medium without Trp.
670
The positive transformants of the bait plasmid and prey plasmid were transferred to
671
yeast extract-peptone-adenine-dextrose (YPAD) medium and mated for 24 h at 30°C.
672
A total of 8 L 1/10 dilution was dripped onto SD-Leu-Trp-His plates that were
673
supplemented with 15 mM 3-amino-1,2,4-triazole (3-AT) and incubated for 4 to 6 d at
674
30°C to determine the interaction between GbWRKY1 and the GhJAZ1 promoter.
675
μ
The primers that were used in the Y1H assay are listed in Supplemental Table 1.
676
677
Electrophoretic mobility shift assays
678
GbWRKY1 was cloned into a pET-28a inducible expression vector and expressed in
679
the Escherichia coli BL21 strain. The GbWRKY1 recombinant fusion protein was
680
purified using the MagneHis™ Protein Purification System (Promega, USA).
681
Synthesized oligonucleotides (Figure 6A) were labeled using a Roche DIG gel shift
682
kit (Roche). DNA-protein binding reactions were performed by incubating 100 ng of
683
purified GbWRKY1 recombinant protein with digoxigenin-labeled GhJAZ1 or AtJAZ1
684
promoter fragment as described in the Roche protocol manual. The protein-DNA
685
mixture was incubated at room temperature for 30 min and separated on a 12%
686
polyacrylamide gel in Tris-Gly buffer (25 mM Tris, 2 mM EDTA, and 380 mM Gly)
25
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
687
as described by Deng et al. (2012a). The probes were detected using the C-DiGit™
688
Blot Scanner (LI-COR Biosciences, USA).
689
690
Sensitivity to plant hormones and anthocyanin measurement
691
Cotton seeds were surface-sterilized with 0.1% HgCl2 (w/v) for 8 min and washed
692
three times with sterile distilled water. Seeds were then sown in a sterile plant culture
693
box containing 0.5X agar MS medium (Murashige and Skoog, 1962). After
694
germination at 28°C in the dark, approximately 16 seeds from the WT and GbWRKY1
695
transgenic lines were transferred to new 0.5X agar MS medium with or without 0.6
696
μ
M paclobutrazol (PAC) and allowed to grow for 5 d before scoring.
697
The anthocyanin content of seedlings was measured as previously described with
698
minor modifications (Qi et al., 2011). Surface-sterilized Arabidopsis seeds were sown
699
onto 0.5X agar MS medium with or without 8 M MeJA for 10 d and collected
700
directly in liquid nitrogen. The anthocyanin content was calculated as previously
701
described (Xu et al., 2012b).
μ
702
703
Supplemental data
704
Supplemental Figure 1. Response of GbWRKY1 to V. dahliae infection in cotton.
705
Supplemental Figure 2. Responses of GbWRKY1 transgenic plants to V. dahliae and
706
the necrotrophic pathogen B. cinerea.
707
Supplemental Figure 3. Response of GbWRKY1 to MeJA.
708
Supplemental Figure 4. Levels of JA in WT and GbWRKY1 transgenic cotton plants.
709
Supplemental Figure 5. Morphological phenotypes of GbWRKY1-RNAi plants.
710
Supplemental Figure 6. Morphological phenotypes of GbWRKY1 transgenic lines.
711
Supplemental Figure 7. Gene expression analyses of GA-related genes.
712
Supplemental Figure 8. Flowering phenotype of WT, AOV4 and AOV9 after MeJA
713
treatment.
714
Supplemental Figure 9. Evaluation of the expression of JA responsive gene (GbPR4)
715
after GA treatment.
26
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
716
Supplemental Figure 10. qRT-PCR analysis of a set of JAZs in GbWRKY1
717
transgenic plants.
718
Supplemental Figure 11. Examination of GhJAZ1 expression in GhJAZ1 transgenic
719
cotton lines.
720
Table S1. Primers that were used in this study.
721
722
Acknowledgements
723
The authors would like to thank Wim Grunewald and Tao Huang for sharing
724
Arabidopsis research materials. We also thank prof. Keith Lindsey (School of
725
Biological and Biomedical Sciences, University of Durham) for revising the
726
manuscript.
727
728
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Arabidopsis. J Biosci 35: 459-471
996
Zentella R, Zhang ZL, Park M, Thomas SG, Endo A, Murase K, Fleet CM,
997
Jikumaru Y, Nambara E, Kamiya Y, Sun TP (2007) Global analysis of
998
DELLA direct targets in early gibberellin signaling in Arabidopsis. Plant Cell
999
19: 3037-3057
1000
Zhang Y, Wang XF, Ding ZG, Ma Q, Zhang GR, Zhang SL, Li ZK, Wu LQ,
1001
Zhang GY, Ma ZY (2013) Transcriptome profiling of Gossypium barbadense
1002
inoculated with Verticillium dahliae provides a resource for cotton
1003
improvement. BMC Genomics 14: 637
1004
Zhang ZL, Ogawa M, Fleet CM, Zentella R, Hu JH, Heo JO, Lim J, Kamiya Y,
1005
Yamaguchi S, Sun TP (2011) SCARECROW-LIKE 3 promotes gibberellin
1006
signaling by antagonizing master growth repressor DELLA in Arabidopsis.
1007
Proc Natl Acad Sci U S A 108: 2160-2165
1008
Zheng W, Zhai Q, Sun J, Li C-B, Zhang L, Li H, Zhang X, Li S, Xu Y, Jiang H,
1009
Wu X, Li C (2006b) Bestatin, an Inhibitor of Aminopeptidases, Provides a
1010
Chemical Genetics Approach to Dissect Jasmonate Signaling in Arabidopsis.
1011
Plant Physiol 141: 1400-1413
1012
Zheng ZY, Abu Qamar S, Chen ZX, Mengiste T (2006a) Arabidopsis WRKY33
1013
transcription factor is required for resistance to necrotrophic fungal pathogens.
1014
Plant J 48: 592-605
1015
Zhou L, Hu Q, Johansson A, Dixelius C (2006) Verticillium longisporum and V.
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
1016
dahliae: infection and disease in Brassica napus. Plant Pathol 55: 137-144
1017
Zou X, Neuman D, Shen QJ (2008) Interactions of two transcriptional repressors
1018
and two transcriptional activators in modulating gibberellin signaling in
1019
aleurone cells. Plant Physiol 148: 176-186
1020
1021
Figure Legends
1022
Figure 1. GbWRKY1 is a negative regulator of cotton resistance to V. dahliae and B.
1023
cinerea.
1024
(A), TRV:00 plants and TRV:GbWRKY1 plants 7 d after inoculation with V. dahliae.
1025
The GbWRKY1 knock-down plants exhibited enhanced resistance to V. dahliae. (B),
1026
Northern blotting analysis to examine the expression of GbWRKY1 in two
1027
independent RNAi lines (Ci2 and Ci3) and two independent overexpression lines
1028
(COV4 and COV7). (C), WT and transgenic plants 4 d after inoculation with B.
1029
cinerea. (D), WT and transgenic plants 9 d after inoculation with V. dahliae. (E),
1030
Northern blotting analysis to examine the expression of GbWRKY1 in two
1031
independent overexpression lines (AOV4 and AOV9) in Arabidopsis. (F) and (G), 5 d
1032
and 15 d after inoculating B. cinerea and V. dahliae spore suspension (2×105
1033
spores/mL) on 20 d-old soil-grown Arabidopsis plants respectively by spraying or
1034
dipping method. Disease assays were repeated at least three times.
1035
1036
Figure 2. Evaluation of the role of GbWRKY1 in JA signaling.
1037
(A), MeJA-induced resistance to V. dahliae. WT plants were grown for 15-20 days
1038
and then infected with V. dahliae. Two days before infection, the roots of each cotton
1039
plant were drenched each day with 3 mL of MeJA solution or 3 mL of mock solution.
1040
(B), The disease index of mock- and MeJA-treated cotton plants was calculated 9 d
1041
after V. dahliae-infection. (C), qRT-PCR analysis of JA-responsive gene (GbPR4)
1042
expression in cotton lines with or without (Mock) 10 M MeJA treatment for 5 h.
1043
Mean ± SD, n=3. (D), qRT-PCR analysis of AtPDF1.2 in GbWRKY1-overexpressing
1044
Arabidopsis seedlings (AOV4 and AOV9). Mean ± SD, n=3. (E), Arabidopsis
1045
seedlings of WT, COV4, and COV9 grown on 1/2 MS medium with or without (Mock)
μ
37
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
1046
8 M MeJA treatment. (F), Anthocyanin content of the Arabidopsis seedlings in (E).
1047
(G),
1048
GbWRKY1-overexpressing Arabidopsis plants with or without (Mock) 10 M MeJA
1049
treatment for 8 h. Mean ± SD, n=3. The values are given relative to the housekeeping
1050
genes ACTIN in Arabidopsis or UB7 in cotton. Statistical analyses were performed
1051
using Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. WT, wild type; Ci2,
1052
GbWRKY1-RNAi cotton line; COV4, GbWRKY1-overexpressing cotton line.
μ
qRT-PCR
analysis
of
UF3GT,
LDOX,
and
DFR
in
WT
and
μ
1053
1054
Figure 3. Morphological phenotypes of GbWRKY1-overexpressing cotton.
1055
(A) and (C), Image and quantification of the petiole length of WT and
1056
GbWRKY1-overexpressing plants, respectively. (B) and (D), Image and quantification
1057
of the petiole epidermal cell length in (A), respectively. Bars: 50 m. (E), Image of
1058
cotton with two true leaves. (F), Quantification of chlorophyll contents in (E). (G),
1059
PAC susceptibility of WT and GbWRKY1 transgenic seedlings. (H), The susceptibility
1060
of WT and GbWRKY1 transgenic cotton to PAC was scored as relative inhibition
1061
ratios (%). The assays were repeated at least three times. The data in (C), (D), (F) and
1062
(H) represent the mean ± SD from a minimum of 16 plants. Statistical analyses were
1063
performed using Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. PAC,
1064
Paclobutrazol; WT, wild type; Ci2, GbWRKY1-RNAi cotton line; COV4,
1065
GbWRKY1-overexpressing cotton line.
μ
1066
1067
Figure 4. Evaluation of the role of GbWRKY1 in GA signaling.
1068
(A), Quantification of the flowering time of GbWRKY1-overexpressing lines (AOV4
1069
and AOV9) in Arabidopsis. (B), qRT-PCR analysis of a set of putative RGA target
1070
genes in 5 d-old Arabidopsis seedlings. Mean ± SD, n=3. (C), qRT-PCR analysis of
1071
the expression of GhGID1-a and GhGID1-b in GbWRKY1-overexpressing cotton
1072
lines. Mean ± SD, n=3. (D), qRT-PCR analysis of the expression of SOC1 in
1073
GbWRKY1-overexpressing Arabidopsis seedlings. Mean ± SD, n=3. (E), Flowering
1074
phenotype of AOV9:soc1 F2 progeny. Phenotype was evaluated from 3 replicates,
1075
each replicate contains at least 160 AOV9:soc1 F2 plants. 2 individual plants from F2
38
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2014 American Society of Plant Biologists. All rights reserved.
1076
population were imaged as representatives. PCR amplification of the genomic DNA
1077
from WT, GbWRKY1-overexpressing Arabidopsis plants (AOV9), Arabidopsis soc1
1078
mutant and AOV9:soc1 F2 progeny. The primer sets that were used in the PCR tests
1079
are listed in Supplemental Table 1. The assays were repeated three times. The data in
1080
(A) represent the mean ± SD from a minimum of 24 plants. Statistical analyses were
1081
performed using Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. The values are
1082
given relative to the housekeeping genes Actin in Arabidopsis or UB7 in cotton. WT,
1083
wild type; COV4 and COV7, GbWRKY1-overexpressing cotton lines.
1084
1085
Figure 5. Evaluation of the role of GbWRKY1 in coordinating the interaction of GA
1086
and JA.
1087
(A), Flowering time of WT and GbWRKY1-overexpressing Arabidopsis plants (AOV4
1088
and AOV9) after MeJA treatment. (B), Analysis of GA-responsive gene expression in
1089
WT and GbWRKY1 transgenic cotton plants with or without (Mock) 10 M MeJA
1090
treatment for 5 h. Mean ± SD, n=3. (C), qRT-PCR analysis of AtJAZ1 and GhJAZ1 in
1091
GbWRKY1 transgenic Arabidopsis plants and cotton plants, respectively. Mean ± SD,
1092
n=3. (D) and (E), qRT-PCR analysis of GbWRKY1 in GhJAZ1-overexpression lines
1093
(J92 and J131) and RNAi lines (JR1 and JR3), respectively. Mean ± SD, n=3. (F),
1094
Petiole length of the WT plant, GhJAZ1 RNAi plant (JR3), GbWRKY1 overexpression
1095
(COV4) cotton plant, F1 hybrid plant of WT and COV4 (COV4 & WT), and F1
1096
hybrid plant of COV4 and JR3 (COV4 & JR3). (G), Flowering phenotype of AOV9
1097
and
1098
AOV9/amiRNAJAZ1 are three homozygous lines from AOV9 and amiRNAJAZ1
1099
hybrid population. Data were from 3 replicates, each replicate contains at least 24
1100
plants. RT-PCR analysis to examine the expression of GbWRKY1 and AtJAZ1 in
1101
HY-AOV9, HY-amiRNAJAZ1, AOV9/amiRNAJAZ1 plants. The assays were
1102
repeated three times. Statistical analyses were performed using Student’s t test, *p <
1103
0.05, **p < 0.01, ***p < 0.001. WT, wild type; Ci2, GbWRKY1-RNAi cotton plants;
1104
COV4 and COV7, GbWRKY1-overexpressing cotton lines.
μ
amiRNAJAZ1
hybrid
progeny.
HY-AOV9,
HY-amiRNAJAZ1,
1105
39
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Copyright © 2014 American Society of Plant Biologists. All rights reserved.
1106
Figure 6. Assessment of the transactivation activity of GbWRKY1.
1107
(A), Promoter sequences of GhJAZ1, AtJAZ1, and different mutated and replaced
1108
versions as indicated in the text. The W-box element, TGAC core and mutated
1109
positions are indicated by short underlines. (B) to (E), EMSA of the binding of
1110
GbWRKY1 protein to the DiG-labeled oligonucleotide as shown in (A). (F),
1111
GbWRKY1 bound to the GhJAZ1 promoter in the yeast one-hybrid assay. (G),
1112
GbWRKY1 transactivates the promoter of GhJAZ1 and AtJAZ1 in vivo. The assays
1113
were repeated three times. The data in (G) represent the mean ± SD from three
1114
replicates. Statistical analyses were performed using Student’s t test, *p < 0.05, **p <
1115
0.01.
1116
1117
Figure 7. Schematic model of how GbWRKY1 prioritizes development over defense
1118
during V. dahliae infection.
1119
When plants detect V. dahliae invasion, JA signaling is rapidly activated to allocate
1120
energy resources to defense (Gao et al., 2013). However, activation of JA signaling is
1121
at the expense of growth and development. To avoid the hyperactivation of JA
1122
signaling and maintain the growth- and development-related processes, GbWRKY1 is
1123
rapidly induced by V. dahliae infection and subsequently up-regulates the
1124
transcription of a JAZ1-like gene in cotton, which promote GA-related phenotypes
1125
(such as flowering). This relieves the repression of DELLA and at the same time
1126
limits the extent of JA signaling activation, thereby prioritizing development and
1127
reproduction (flowering) over defense during V. dahliae infection.
40
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