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. 2022 Jun 11;190(1):828–842. doi: 10.1093/plphys/kiac275

SlVQ15 interacts with jasmonate-ZIM domain proteins and SlWRKY31 to regulate defense response in tomato

Huang Huang 1,2,#,✉, Wenchao Zhao 3,4,#, Chonghua Li 5,#, Hui Qiao 6, Susheng Song 7, Rui Yang 8, Lulu Sun 9,10, Jilin Ma 11, Xuechun Ma 12, Shaohui Wang 13,14,✉
PMCID: PMC9434178  PMID: 35689622

Abstract

Botrytis cinerea is one of the most widely distributed and harmful pathogens worldwide. Both the phytohormone jasmonate (JA) and the VQ motif-containing proteins play crucial roles in plant resistance to B. cinerea. However, their crosstalk in resistance to B. cinerea is unclear, especially in tomato (Solanum lycopersicum). In this study, we found that the tomato VQ15 was highly induced upon B. cinerea infection and localized in the nucleus. Silencing SlVQ15 using virus-induced gene silencing reduced resistance to B. cinerea. Overexpression of SlVQ15 enhanced resistance to B. cinerea, while disruption of SlVQ15 using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) technology increased susceptibility to B. cinerea. Furthermore, SlVQ15 formed homodimers. Additionally, SlVQ15 interacted with JA-ZIM domain proteins, repressors of the JA signaling pathway, and SlWRKY31. SlJAZ11 interfered with the interaction between SlVQ15 and SlWRKY31 and repressed the SlVQ15-increased transcriptional activation activity of SlWRKY31. SlVQ15 and SlWRKY31 synergistically regulated tomato resistance to B. cinerea, as silencing SlVQ15 enhanced the sensitivity of slwrky31 to B. cinerea. Taken together, our findings showed that the SlJAZ-interacting protein SlVQ15 physically interacts with SlWRKY31 to cooperatively control JA-mediated plant defense against B. cinerea.

A signaling module controls tomato defense against Botrytis cinerea in the jasmonate pathway.

Introduction

The necrotrophic pathogen Botrytis cinerea infects more than 200 economically important crop species and causes severe losses globally (Williamson et al., 2007; Dean et al., 2012; AbuQamar et al., 2017). The pathogen secretes multiple enzymes, toxins, and oxalic acid, and induces a burst of reactive oxygen species, all of which disrupt the plant immune system and kill host plants (van Kan, 2006; Cheung et al., 2020).

The VQ motif-containing transcription factors are defined by the presence of a conserved FxxhVQxhTG motif (Cheng et al., 2012). They regulate resistance to B. cinerea in Arabidopsis (Arabidopsis thaliana; Jing and Lin, 2015; Yuan et al., 2021). For instance, SIGMA FACTOR-BINDING PROTEIN 1 (SIB1/AtVQ23) and SIB2/AtVQ16 function as activators of AtWRKY33, and positively control Arabidopsis defense against B. cinerea (Lai et al., 2011). AtVQ10 interacts with AtWRKY8, and stimulates the transcriptional activity of AtWRKY8 to positively modulate resistance to B. cinerea (Chen et al., 2018). AtVQ12 and AtVQ29 form dimers, and repress defense responses to B. cinerea (Wang et al., 2015). The tomato (Solanum lycopersicum) genome contains 26 VQ proteins (Ding et al., 2019). Nevertheless, their functions in the regulation of tomato defense responses are poorly understood.

The lipid-derived phytohormones jasmonates (JAs) play essential roles in plant growth and developmental processes, and defense against biotic and abiotic stresses (Howe et al., 2018; Jang et al., 2020). JA-Ile, an active JA form (Fonseca et al., 2009), is perceived by the CORONATINE INSENSITIVE 1 (COI1)–JA ZIM-DOMAIN (JAZ) coreceptor complex (Xie et al., 1998; Yan et al., 2009; Sheard et al., 2010). COI1 can interact with ARABIDOPSIS SKP-LIKE 1/2 (ASK1/2), Cullin1, and RING-BOX PROTEIN 1 (Rbx1) to form SCFCOI1 E3 ligase complex (Xu et al., 2002). JAZ coreceptors also act as repressors (Chini et al., 2007; Thines et al., 2007; Yan et al., 2007), via interacting with and inhibiting downstream transcription factors including MYC2/3/4/5 (Cheng et al., 2011; Fernandez-Calvo et al., 2011; Niu et al., 2011; Qi et al., 2015), the WD-repeat/bHLH/MYB complexes (Qi et al., 2011), MYB21/MYB24 (Song et al., 2011), bHLH03/13/14/17 (Nakata et al., 2013; Sasaki-Sekimoto et al., 2013; Song et al., 2013), ETHYLENE INSENSITIVE 3 (EIN3) and EIN3-LIKE 1 (EIL1; Zhu et al., 2011), INDUCER OF CBF EXPRESSION 1 (ICE1) and ICE2 (Hu et al., 2013b), and FILAMENTOUS FLOWER 3 (FIL3; Boter et al., 2015). Upon perception of JA-Ile, JAZ repressors are degraded through the SCFCOI1-26S proteasome pathway (Chini et al., 2007; Thines et al., 2007), therefore, releasing JAZs-interaction transcription factors to modulate JA responses (Wasternack and Song, 2017).

Multiple studies revealed that JAs defend against B. cinerea (Yan and Xie, 2015). For instance, the Arabidopsis JA biosynthetic mutant allene oxide synthase (aos) and signaling mutant coi1-1 are susceptible to B. cinerea (Feys et al., 1994; Xie et al., 1998; Park et al., 2002; Mene-Saffrane et al., 2009; Song et al., 2013). Silencing of tomato JA biosynthesis gene 12-OXOPHYTODIENOATE REDUCTASE 3 (OPR3) abolishes JA-Ile production, and results in increased susceptibility to B. cinerea (Scalschi et al., 2015). Tomato mutants defective in the Arabidopsis COI1 homolog JAI1 show extreme sensitivity to B. cinerea (Li et al., 2004; Du et al., 2017). However, the molecular basis underlying JA-regulated defense against B. cinerea is not fully understood.

The crosstalk between JAs and VQ proteins in Arabidopsis defense against insect and pathogens has been investigated. AtVQ22/JAV1 functions as a negative regulator to control JA-mediated Arabidopsis defense against B. cinerea (Hu et al., 2013a). AtJAV1 interacts with AtJAZ8 and AtWRKY51 to form a JAV1–JAZ8–WRKY51 (JJW) complex, which represses JAs biosynthesis under normal condition. Injury triggers the phosphorylation of AtJAV1 to disrupt the JJW complex, resulting in rapid JAs accumulation for defense against insects (Yan et al., 2018). However, the interaction between JAs and VQ proteins in tomato defense against B. cinerea is largely unknown.

Here, we found that SlVQ15 was highly induced in response to B. cinerea. Silencing SlVQ15 via virus-induced gene silencing (VIGS) attenuated resistance to B. cinerea. SlVQ15 overexpression enhanced resistance to B. cinerea. We performed CRISPR/Cas9 system editing to generate slvq15 mutants and found that loss of function of SlVQ15 was more susceptible to B. cinerea. Furthermore, SlVQ15 formed homodimers, and interacted with 5 of 11 SlJAZ proteins and SlWRKY31. SlJAZ11 inhibited the interaction between SlVQ15 and SlWRKY31. The transcriptional activation activity of SlWRKY31 was promoted by SlVQ15, whereas this effect was attenuated by SlJAZ11. The gene-editing mutant slwrky31 presented increased susceptibility to B. cinerea. Additionally, we silenced the SlVQ15 gene in the slwrky31 background, and discovered that these mutants exhibited more susceptibility to B. cinerea than slwrky31 mutants. Our data demonstrate that the SlJAZ-interacting protein SlVQ15 interacts with SlWRKY31 to synergistically and positively control JA-mediated tomato defense against B. cinerea.

Results

VIGS of SlVQ15 affects the defense response against B. cinerea

To explore whether tomato VQ proteins are involved in the response to B. cinerea, we first used reverse transcription–quantitative polymerase chain reaction (RT–qPCR) to analyze the expression levels of multiple SlVQ genes in response to B. cinerea infection (Supplemental Figure S1). The results suggested that the expression levels of SlVQ2, SlVQ3, SlVQ5, SlVQ6, SlVQ10, SlVQ15, SlVQ18, SlVQ19, SlVQ20, SlVQ21, and SlVQ23 were differentially enhanced in response to B. cinerea, while the expression levels of SlVQ1, SlVQ7, SlVQ8, SlVQ14, SlVQ16, SlVQ17, SlVQ22, SlVQ24, SlVQ25, and SlVQ26 were significantly decreased. The expression levels of SlVQ4 and SlVQ9 were first upregulated and then downregulated, whereas the expression levels of SlVQ12, and SlVQ13 were first downregulated and then upregulated in response to B. cinerea infection. Of these SlVQs, the most highly induced genes upon the infection of B. cinerea were SlVQ3, SlVQ15, and SlVQ23. Therefore, we focused on investigating the potential function of the three genes by using VIGS technology, and discovered that silencing SlVQ15 confers decreased resistance to B. cinerea, as indicated by larger lesion area (Figure 1), suggesting that SlVQ15 may play a vital role in regulating defense against B. cinerea.

Figure 1.

Figure 1

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VIGS of SlVQ15 decreases tomato resistance to B. cinerea. A and B, Symptoms (A) and lesion areas (B) on detached leaves of TRV-control, and TRV-SlVQ15 plants at 3 days after inoculation with B. cinerea. Scale bar = 1 cm. Error bars represent se (n=15). The experiments were repeated three times with similar results. Asterisks represent Student’s t test significance compared with control (**P< 0.01).

Subcellular localization and expression patterns of SlVQ15

We next explored the subcellular localization of SlVQ15. The results in Figure 2A showed that SlVQ15-fused GFP was localized to the nucleus. We subsequently performed RT–qPCR to analyze the expression pattern of SlVQ15, and found that SlVQ15 was expressed in various tissues, including roots, stems, leaves, flowers and fruits, with strong expression in the leaves and roots (Figure 2B). These results suggest that SlVQ15 localizes to the nucleus, and is expressed in various tissues.

Figure 2.

Figure 2

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Subcellular localization and expression pattern of SlVQ15. A, SlVQ15 was localized in nucleus. GFP fluorescence was detected 50 h after infiltration of the N. benthamiana leaves with the Agrobacterium strain carrying the indicated constructs. Scale bars = 50 μm. B, RT–qPCR analysis of SlVQ15 in the roots, stems, leaves, flowers, and fruits of CM (WT). SlACTIN2 was used as the internal control. Data are means (±se) of three independent experiments.

Overexpression of SlVQ15 results in enhanced defense against B. cinerea in tomato

To confirm the function of SlVQ15 in defense, we generated two independent lines overexpressing SlVQ15 in the tomato cultivar Castlemart (CM) wild-type (WT) background (SlVQ15OE-1 and SlVQ15OE-2). The overexpression levels of SlVQ15 in these two lines were about five-fold and six-fold of WT level, respectively (Supplemental Figure S2). We inoculated B. cinerea spore suspension onto detached leaves of SlVQ15-overexpressing lines and WT plants. As shown in Figure 3, A and B, the SlVQ15-overexpresing lines exhibited increased resistance to B. cinerea, as the necrotic lesions on SlVQ15-overexpresing plants were smaller than those on WT plants. In tomato, B. cinerea infection increased the expression levels of THREONINE DEAMINASE (TD) and PR-STH2 (Du et al., 2017), which are wounding-responsive marker gene and pathogen-responsive marker gene, respectively (Marineau et al., 1987; Matton and Brisson, 1989; Despres et al., 1995; Chen et al., 2005). Consistent with the increased defense response, the expression of SlTD and SlPR-STH2 was highly induced in response to B. cinerea in the SlVQ15-overexpressing plants compared with those in WT plants (Figure 3, C and D). These results reveal that overexpression of SlVQ15 promotes tomato resistance to the necrotrophic pathogen B. cinerea.

Figure 3.

Figure 3

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SlVQ15 positively regulates tomato defense against B. cinerea. A and B, Phenotypes (A) and lesion areas (B) of detached leaves of CM (WT), and SlVQ15 overexpression lines (SlVQ15OE-1 and SlVQ15OE-2) after inoculation with B. cinerea for 3 days. Scale bars = 1 cm. Error bars represent se (n = 15). The experiments were repeated three times with similar results. Lower case letters indicate significant differences by ANOVA followed by Duncan’s multiple range test (P <0.05). C and D, RT–qPCR analysis of SlTD and SlPR-STH2 in CM (WT), and SlVQ15 overexpression plants (SlVQ15OE-1 and SlVQ15OE-2) inoculation without or with B. cinerea for 24 h. SlACTIN2 was used as the internal control. Data are means (±se) of three independent experiments. Lower case letters indicate significant differences by ANOVA followed by Duncan’s multiple range test (P<0.05). E and F, Symptoms (E) and lesion areas (F) on detached leaves of CM (WT), slvq15 mutants (slvq15-c-1 and slvq15-c-2) after inoculation with B. cinerea for 3 days. Scale bars = 1 cm. Error bars represent se (n = 15). The experiments were repeated three times with similar results. Lower case letters indicate significant differences by ANOVA followed by Duncan’s multiple range test (P <0.05). G and H, RT–qPCR analysis of SlTD and SlPR-STH2 in the indicated plants at 24 h after infection without or with B. cinerea. SlACTIN2 was used as the internal control. Data are means (±se) of three independent experiments. Lower case letters indicate significant differences by ANOVA followed by Duncan’s multiple range test (P <0.05).

slvq15 mutants display reduced resistance to B. cinerea

To further verify the function of SlVQ15, we generated slvq15 mutants (slvq15-c-1 and slvq15-c-2) using CRISPR/Cas9 gene editing technology. The slvq15-c-1 mutants have 100-bp deletions, and slvq15-c-2 mutants have 4-bp deletions in the first target region and 3-bp deletions in the second target region (Supplemental Figure S3, A and B), whereas they result in altered and truncated SlVQ15 proteins, respectively (Supplemental Figure S3C). We inoculated WT, slvq15-c-1, and slvq15-c-2 with B. cinerea, and found that the necrotic lesions on the slvq15-c-1 and slvq15-c-2 mutants were larger than those on the WT (Figure 3, E and F), indicating that slvq15 mutants display reduced resistance to B. cinerea. Consistently, upon B. cinerea infection, the expression levels of SlTD and SlPR-STH2 was lower in the slvq15-c-1 and slvq15-c-2 plants than those in WT (Figure 3, G and H).

Taken together (Figures 1 and 3), these results confirm that SlVQ15 is a positive factor controlling tomato defense against B. cinerea.

The C terminus of SlVQ15 is involved in homomeric interactions

A previous study showed that AtVQ proteins form homo- or heterodimers (Wang et al., 2015). We reasoned that SlVQ15 may also function as homodimers in tomato. We performed yeast two-hybrid (Y2H) assay to test this hypothesis. The full-length of SlVQ15 was fused with LexA DNA-binding domain (BD) to produce BD-SlVQ15, and with B42 activation domain (AD) to generate AD-SlVQ15. As shown in Figure 4A, BD-SlVQ15 interacted with AD-SlVQ15 in yeast. We further carried out pull-down assays to verify these results. Maltose-binding protein (MBP) and MBP fused SlVQ15 (MBP-SlVQ15) were expressed in Escherichia coli and purified using amylose resin. SlVQ15 fused with three flag tags (flag-SlVQ15) was transiently expressed in Nicotiana benthamiana leaves. As shown in Figure 4B, MBP-SlVQ15 pulled-down flag-SlVQ15, but MBP did not. Moreover, the firefly luciferase (LUC) complementation imaging (LCI) assays were performed (Figure 4C), and the results showed that cLUC-SlVQ15 interacted with SlVQ15-nLUC in N.benthamiana leaves. These results (Figure 4, A–C) demonstrate that SlVQ15 exhibits homodimerization in vitro and in vivo.

Figure 4.

Figure 4

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The C-terminal parts of SlVQ15 mediate dimeric interactions. A, Y2H assay to detect the dimer formation of SlVQ15. SlVQ15 was fused with the LexA DNA BD in pLexA or B42 AD in pB42AD. The interactions were assessed on Gal/raffinose/SD/–Ura/–His/–Trp/–Leu/X-β-Gal medium. B, Pull-down assay for detecting the interaction between SlVQ15 and itself. MBP and MBP-SlVQ15 were expressed in E. coli, and purified using amylose resin. flag-SlVQ15 was transiently expressed in N. benthamiana leaves, and pulled down by MBP-SlVQ15. C, Firefly LUC LCI assays verify that SlVQ15 forms homodimers in planta. SlVQ15 was fused to the N-terminal fragment of LUC (nLUC) or the C-terminal fragment of LUC (cLUC). Data were collected 50 h after coinfiltration. D, Schematic diagrams show the domain constructs of SlVQ15. The numbers indicate positions of the first and the last amino acids of the domain constructs. E, Y2H assays show that SlVQ15 interacts with SlVQ15CT. SlVQ15 was fused with BD. SlVQ15NT and SlVQ15CT were fused with AD. Interactions were assessed on Gal/raffinose/SD/–Ura/–His/–Trp/–Leu/X-β-Gal medium. F, LCI assays confirm that the C-terminus of SlVQ15 mediates the formation of homodimers. SlVQ15 was fused with nLUC, and SlVQ15CT was fused with cLUC. Data were collected 50 h after coinfiltration.

To identify the domain responsible for the dimerization, we divided SlVQ15 into N-terminal fragments (SlVQ15NT) containing the VQ domain, and C-terminal fragments (SlVQ15CT; Figure 4D). As shown in Figure 4E, the C-terminal region (SlVQ15CT), but not the N-terminal region (SlVQ15NT) interacted with the full length of SlVQ15 in yeast. Furthermore, we performed LCI assays and verified that the C-terminus of SlVQ15 interacted with SlVQ15 in planta (Figure 4F), demonstrating that the C-terminus of SlVQ15 is responsible for its homomeric interaction.

Taken together, these results (Figure 4) confirm that SlVQ15 forms homodimers, and that its C-terminus is necessary and sufficient for the homomeric interaction.

SlVQ15 physically interacts with SlJAZs

Multiple lines of evidence indicate that VQs interact with various proteins in the regulation of plant developmental processes and plant defense responses (Lai et al., 2011; Cheng et al., 2012; Li et al., 2014; Jing and Lin, 2015; Uji et al., 2019; Yuan et al., 2021). To identify SlVQ15-interacting proteins, we employed the Y2H system with SlVQ15 as bait to screen a tomato cDNA library, and isolated SlJAZ11 as a SlVQ15-interacting protein (Figure 5A).

Figure 5.

Figure 5

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SlVQ15 physically interacts with SlJAZs. A, Y2H assays to test the interactions of SlVQ15 with SlJAZs. SlJAZs were fused with B42 AD in pB42AD, and SlVQ15 was fused with the LexA DNA BD in pLexA. Interactions were assessed on Gal/raffinose/SD/–Ura/–His/–Trp/–Leu/X-β-Gal medium. B, LCI assays show that SlVQ15 interacts with SlJAZ2, SlJAZ5, SlJAZ7, and SlJAZ11. SlJAZ2, SlJAZ5, SlJAZ7, and SlJAZ11 were fused with nLUC to generate SlJAZ2-nLUC, SlJAZ5-nLUC, SlJAZ7-nLUC, and SlJAZ11-nLUC, respectively. SlVQ15 was fused with cLUC to produce cLUC-SlVQ15. The leaves of N. benthamiana were infiltrated with Agrobacterium strains containing the indicated construct pairs. The data were recorded 50 h after infiltration. C, Pull-down assays show that SlVQ15 interacts with SlJAZ7 and SlJAZ11. MBP and MBP-SlVQ15 were respectively expressed in E. coli, and purified using amylose resin. myc-SlJAZ7 or myc-SlJAZ11 was, respectively, transiently expressed in N. benthamiana leaves, and pulled down by MBP-SlVQ15. D, Schematic diagrams display the domain constructs of SlJAZ7 and SlJAZ11. The numbers indicate positions of the first and the last amino acids of the domain constructs. E, Y2H assays show that SlVQ15 interacts with SlJAZ7NT and SlJAZ11NT. SlJAZ7NT, SlJAZ7CT, SlJAZ11NT, and SlJAZ11CT were fused with AD. SlVQ15 was fused with BD.

We further analyzed whether SlVQ15 interacted with other SlJAZs. As shown in Figure 5A, SlVQ15 interacted with five SlJAZs (SlJAZ2, SlJAZ5, SlJAZ6, SlJAZ7, and SlJAZ11), but not with other remaining SlJAZs (SlJAZ1, SlJAZ3, SlJAZ4, SlJAZ8, SlJAZ9, and SlJAZ10) in yeast. We next performed LCI assays to verify the interaction between SlVQ15 and SlJAZs in planta. SlJAZ2, SlJAZ5, SlJAZ7, and SlJAZ11 were used as representatives in the LCI assay (Figure 5B). We discovered that the coexpression of cLUC-SlVQ15 and SlJAZ2-nLUC in N.benthamiana leaves showed strong LUC activity. Similar results were observed for the coexpression of cLUC-SlVQ15 with SlJAZ5-nLUC, SlJAZ7-nLUC, and SlJAZ11-nLUC. We further conducted pull-down assays and discovered that transiently expressed myc-SlJAZ7 or myc-SlJAZ11 could be pulled down by MBP-SlVQ15, but not by MBP (Figure 5C).

To identify the SlJAZs region responsible for interaction with SlVQ15, we divided SlJAZ7 and SlJAZ11 into N-terminal fragments including the ZIM domain (SlJAZ7NT, SlJAZ11NT), and C-terminal fragments including the Jas domain (SlJAZ7CT, SlJAZ11CT; Figure 5D). The results showed that SlVQ15 interacted with the N-terminal region of SlJAZ7 and SlJAZ11 in yeast (Figure 5E), indicating that the N-terminus of SlJAZs is required for the interaction with SlVQ15.

Taken together (Figure 5), the Y2H, LCI, and pull-down assays demonstrate that SlVQ15 is a direct target of SlJAZ proteins in tomato.

SlVQ15 physically interacts with SlWRKY31, and SlJAZ11 interferes with this interaction

Our Y2H screening also identified SlWRKY31 as a SlVQ15-interaction protein (Figure 6A). We adopted pull-down assays to study the interaction of SlVQ15 with SlWRKY31 (Figure 6B). MBP-SlVQ15 could pull down the transiently expressed flag-SlWRKY31, whereas MBP could not. Additionally, LCI assays indicated that the coexpression of SlVQ15-nLUC and cLUC-SlWRKY31 in N. benthamiana leaves reconstituted strong LUC activity, whereas the negative controls did not (Figure 6C). These results demonstrate that SlVQ15 interacts with SlWRKY31.

Figure 6.

Figure 6

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SlVQ15 physically interacts with SlWRKY31, and SlJAZ11 inhibits this interaction. A, Y2H assays to detect the interactions of SlVQ15 with SlWRKY31. SlVQ15 and SlVQ15NT were fused with the LexA DNA BD in pLexA. SlWRKY31 and its related domains were fused with B42 AD in pB42AD. B, Pull-down assays suggest that SlVQ15 interacts with SlWRKY31 in vitro. flag-SlWRKY31 was transiently expressed in N. benthamiana leaves, and pulled down by MBP-SlVQ15. C, LCI assays show that SlVQ15 interacts with SlWRKY31 in vivo. SlVQ15 was fused with nLUC, and SlWRKY31 was fused with cLUC. Data were collected 50 h after coinfiltration. D, Schematic diagrams show the domain constructs of SlWRKY31. The numbers indicate the positions of the first and the last amino acids of the domain constructs. E and F, LCI assays display that SlVQ15 interacts with SlWRKY31CT (E), and that SlVQ15NT interacts with SlWRKY31 (F). SlVQ15 and SlVQ15NT were fused with nLUC. SlWRKY31 and SlWRKY31CT were fused with cLUC. Data were collected 50 h after coinfiltration. G, LCI assays show that SlJAZ11 interferes with the interaction of SlVQ15 and SlWRKY31. The indicated combinations (SlVQ15-nLUC/cLUC-SlWRKY31, SlVQ15-nLUC/cLUC-SlWRKY31 plus myc-SlJAZ11 or myc-GFP) were infiltrated into the same leaf of N. benthamiana. Identical amounts of Agrobacterium bacteria carrying SlVQ15-nLUC and cLUC-SlWRKY31 were used. The experiments were repeated three times with similar results. H, Quantitative analysis of luminescence intensity in (G). Ten independent luminescence values were assessed for luminescence intensity. Error bars represent se. Lower case letters indicate significant differences by ANOVA followed by Duncan’s multiple range test (P <0.05).

We further investigated which domains are required for the interaction of SlVQ15 with SlWRKY31. SlWRKY31 was divided into N-terminal part (SlWRKY31NT), and C-terminal part containing two WRKY domains (SlWRKY31CT; Figure 6D). As shown in Figure 6A, Y2H assays showed that AD-fused SlWRKY31CT interacted with BD-fused SlVQ15, whereas AD-fused SlWRKY31NT did not. LCI assays further displayed that the coexpression of cLUC-SlWRKY31CT and SlVQ15-nLUC resulted in strong LUC activity, whereas the negative controls did not (Figure 6E). These results demonstrate that the C-terminal region of SlWRKY31 is responsible for its interaction with SlVQ15. Moreover, Y2H assays exhibited that BD-SlVQ15NT interacted with both AD-SlWRKY31 and AD-SlWRKY31CT in yeast (Figure 6A). Our LCI assay showed that the coexpressing of SlVQ15NT-nLUC and cLUC-SlWRKY31 in the N. benthamiana leaves exhibited strong LUC activity (Figure 6F). These results reveal that the N-terminus of SlVQ15 is involved in the interaction with SlWRKY31.

We were further interested in whether SlWRKY31 could interact with SlJAZs. SlWRKY31 was fused with LexA DNA BD and B42 AD, respectively, and we found that BD-SlWRKY31 had strong auto-activation (Supplemental Figure S4A). AD-SlWRKY31 was used to detect interactions with BD-SlJAZs. As shown in Supplemental Figure S4B, AD-SlWRKY31 could not interact with SlJAZs in yeast.

Because SlJAZ repressors associate with SlVQ15 (Figure 5), we carried out LCI assays to explore the effect of SlJAZs (SlJAZ11 was used as the representative) on the interaction between SlVQ15 and SlWRKY31. As shown in Figure 6, G and H, the coexpression of SlJAZ11 with SlVQ15 and SlWRKY31 dramatically decreased the luminescence intensity of SlVQ15 and SlWRKY31 coexpression, whereas the coexpression of the control GFP with SlVQ15 and SlWRKY31 did not affect the luminescence intensity. These results show that SlJAZ11 interferes with the SlVQ15–SlWRKY31 interaction.

SlJAZ11 represses the ability of SlVQ15 on the promotion of the transcriptional function of SlWRKY31

Having demonstrated the SlVQ15–SlWRKY31 interaction (Figure 6), we wondered whether SlVQ15 could affects the transcriptional function of SlWRKY31 using the protoplast transient expression system based on the GAL4 DNA-binding domain (GAL4DB) and its binding sites [GAL4(4X)-D1-3(4X)]. We discovered that GAL4DB-fused SlWRKY31 significantly activated GUS reporter activity (Figure 7, A and B), and that the coexpression of SlVQ15 with GAL4DB–SlWRKY31 increased the GUS reporter activity compared with GAL4DB–SlWRKY31 expression control (Figure 7, A and B). These results suggest that SlWRKY31 acts as a transcriptional activator and that SlVQ15 promotes the transcriptional activation function of SlWRKY31.

Figure 7.

Figure 7

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SlJAZ11 attenuates SlVQ15-enhanced transcriptional activation activity of SlWRKY31. A, Schematic diagrams show the constructs used in the transient expression assays in (B and C). B, SlVQ15 promotes the transcriptional activation activity of SlWRKY31, and this effect is repressed by SlJAZ11. The GUS/LUC ratios were measured in N. benthamiana leaf protoplasts. Data are means (±se) of three independent experiments. Lower case letters indicate significant differences by ANOVA followed by Duncan’s multiple range test (P <0.05). C, The ability of SlVQ151–77aa on promoting the transcriptional activity of SlWRKY31 is significantly reduced compared with SlVQ15. The GUS/LUC ratios were measured in N. benthamiana leaf protoplasts. Data are means (±se) of three independent experiments. Lower case letters indicate significant differences by ANOVA followed by Duncan’s multiple range test (P <0.05).

Moreover, we discovered that SlJAZ11 did not affect the transcriptional activity of SlWRKY31 alone (Figure 7B), while the coexpression of SlJAZ11 with SlVQ15 and GAL4DB–SlWRKY31 significantly reduced the GUS reporter activity compared with the coexpression of SlVQ15 and GAL4DB–SlWRKY31 without SlJAZ11 (Figure 7B), demonstrating that SlJAZ11 cannot affect the transcriptional activation activity of SlWRKY31, but inhibits the SlVQ15-promoted transcriptional activation activity of SlWRKY31.

The slvq15-c-1 and slvq15-c-2 mutants contain the altered and truncated SlVQ15 protein (Supplemental Figure S3), which both contain the same N-terminal part (1–77 amino acid [aa]). Therefore, we tested whether SlVQ151–77aa and SlVQ1578–160aa can promote the transcriptional activity of SlWRKY31. The results in Figure 7C revealed that although SlVQ151–77aa could enhance the transcriptional function of SlWRKY31 at a certain level, its effect was largely reduced compared with the full-length of SlVQ15, and that SlVQ1578–160aa could not promote the transcriptional function of SlWRKY31. These results suggested that both SlVQ151–77aa and SlVQ1578–160aa are necessary for the function of SlVQ15 on promoting the transcriptional function of SlWRKY31.

SlVQ15–SlWRKY31 cooperatively regulate defense against B. cinerea

Additionally, we generated a slwrky31 mutant (slwrky31-c) using the CRISPR/Cas9 gene editing technology. The slwrky31-c mutant, which harbors a 1-bp insertion in the first target and has a 2-bp deletion in the second target of SlWRKY31, produces a truncated SlWRKY31 proteins (Supplemental Figure S5). Phenotypic analysis showed that the slwrky31-c mutant displayed increased susceptibility to B. cinerea (Figure 8, A and B), which is consistent with the findings of previous studies (Liu et al., 2014; Zhou et al., 2015). We further silenced SlVQ15 in slwrky31-c background to investigate the function of SlVQ15-SlWRKY31 in defense response, and discovered that silencing SlVQ15 in slwrky31-c mutant caused decreased resistance to B. cinerea compared with slwrky31-c mutant (Figure 8, C and D), suggesting that SlVQ15 coordinates with SlWRKY31 to positively regulate defense resistance against B. cinerea.

Figure 8.

Figure 8

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SlVQ15–SlWRKY31 coordinately regulates tomato defense against B. cinerea. A, B, Symptoms (A) and lesion areas (B) on detached leaves of CM (WT) and slwrky31-c mutants after infection with B. cinerea for 3 days. Scale bars = 1 cm. Error bars represent se (n=15). The experiments were repeated three times with similar results. Asterisks represent Student’s t test significance compared with WT (**P < 0.01). C and D, Symptoms (C) and lesion areas (D) on detached leaves of the indicated plants at day 3 after infection with B. cinerea. Scale bars = 1 cm. Error bars represent se (n = 15). The experiments were repeated three times with similar results. Asterisks represent Student’s t test significance compared with the control (**P < 0.01).

Discussion

Botrytis cinerea, one of the top 10 fungal pathogens in the world, causes great losses to global agricultural production every year (Williamson et al., 2007; Dean et al., 2012). In Arabidopsis, several members of the VQ motif-containing family have been reported to control resistance to B. cinerea (Wang et al., 2015; Chen et al., 2018). However, little is known about the biological function of VQ motif-containing proteins in tomato. In this study, we first analyzed the expression patterns of 26 tomato VQ genes, and discovered that most responded to B. cinerea (Supplemental Figure S1). Silencing of SlVQ15 via VIGS-based gene silencing technology suggested that SlVQ15 may positively regulate defense against B. cinerea (Figure 1). Moreover, we generated slvq15 mutants using the CRISPR/Cas9 system and SlVQ15-overexpression lines. Phenotypic analysis showed that SlVQ15-overexpression lines displayed significantly decreased disease symptoms compared with those of the WT (Figure 3, A and B), whereas slvq15 mutants exhibited enhanced susceptibility against B. cinerea, with a larger lesion area compared with the WT (Figure 3, E and F). Our results demonstrate that SlVQ15 acts as a positive regulator of tomato defense against B. cinerea.

A previous study revealed that AtVQ12 and AtVQ29 associate with other Arabidopsis VQ proteins or themselves to form heterodimers or homodimers via their C-terminal parts, but not via the VQ motif in N-terminus (Wang et al., 2015). In this study, our Y2H, pull down, and LCI assays revealed that SlVQ15 interacts with itself to form homodimers both in vitro and in vivo (Figure 4, A–C). Furthermore, we carried out Y2H and LCI assays to demonstrate that the C-terminal fragment (not containing the VQ motif) of SlVQ15 is responsible for the formation of homodimers (Figure 4, E and F). It would be interesting to investigate whether SlVQ15 interacts with the other 25 SlVQs to exert its function.

In addition to forming homodimers or heterodimers, VQ proteins interact with a variety of other proteins to exercise their function. For instance, AtVQ29 physically interacts with PHYTOCHROME-INTERACTING FACTOR 1 (PIF1) to coordinately activate the cell elongation-related gene XYLOGLUCAN ENDOTRANSGLYCOSYLASE 7 (XTR7), which controls the light-mediated inhibition of hypocotyl elongation (Li et al., 2014). AtVQ22/JAV1 interacts with AtJAZ8, and AtWRKY51 to form the JJW complex. The injury-activated calmodulin-dependent phosphorylation of the JJW complex controls JAs biosynthesis and resistance to herbivory (Yan et al., 2018). OsVQ14 and OsVQ32 physically associate with MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) in rice (Oryza sativa) and act as substrates of the OsMPKK6-OsMPK4 module to positively regulate defense against Xanthomonas oryzae pv. oryzae (Xoo; Li et al., 2021). In this study, we verified that SlVQ15 interacted with SlJAZs via the N-terminus of SlJAZs (Figure 5), indicating that SlVQ15 participates in the JA signaling pathway to modulate tomato resistance to B. cinerea. Moreover, we demonstrated that SlVQ15 interacted with SlWRKY31 through the N-terminal region (containing the VQ motif) of SlVQ15 and the C-terminal region (containing the WRKY motif) of SlWRKY31 (Figure 6, A–F), whereas SlJAZ11 attenuated the interaction of SlVQ15 and SlWRKY31 (Figure 6, G and H).

Several studies have shown that VQ proteins either promote or repress the transcriptional activity of WRKYs. For instance, AtVQ10 interacts with AtWRKY8 and enhances its transcriptional activity (Chen et al., 2018). AtVQ20 interacts with AtWRKY2 and AtWRKY34 to promote their transcriptional repression function (Lei et al., 2017). SIB1/AtVQ23 and SIB2/AtVQ16, act as interactors of AtWRKY75 to inhibit its transcriptional repression function (Zhang et al., 2022). Here, we discovered that the transcriptional activation activity of SlWRKY31 was enhanced by SlVQ15 (Figure 7B), while this process was repressed by SlJAZ11 (Figure 7B). Furthermore, the results in Figure 7C showed that the ability of SlVQ151–77aa on promoting the transcriptional activity of SlWRKY31 was largely abolished compared with SlVQ15, and that SlVQ1578–160aa had no effect on the transcriptional function of SlWRKY31. Although SlVQ151–77aa harbors the region responsible for interaction with SlWRKY31 (Figure 6, A and F), both SlVQ151–77aa and SlVQ1578–160aa are necessary for promoting the transcriptional activity of SlWRKY31 (Figure 7C). Consistently, the slvq15 mutants, retaining its N-terminal parts (1–77aa) but altering or losing C-terminus (78–160aa), decreased tomato resistance to B. cinerea (Figure 3, E and F).

Liu et al. (2014) discovered that SlDRW1/SlWRKY31 is localized in the nucleus and is highly induced by B. cinerea infection and JA treatment, and revealed that silencing SlDRW1 via TRV-based gene silencing technology represses the tomato defense response to B. cinerea. Additionally, Zhou et al. (2015) found that tomato infected with TRV-SlWRKY33A/SlWRKY31 is more susceptible to B. cinerea, and that atwrky33 transformed with SlWRKY33A/SlWRKY31 confers more resistance against B. cinerea. In this study, using the CRISPR/Cas9 system, we generated the slwrky31 mutant, which resulted in a truncated SlWRKY31 protein. Consistently, the slwrky31 mutant exhibited reduced resistance to B. cinerea, with larger lesion areas than the WT (Figure 8, A and B), suggesting that SlWRKY31 functions as a positive regulator of resistance to B. cinerea. Furthermore, we generated slwrky31 TRV-SlVQ15 plants by disrupting SlVQ15 in the slwrky31 mutant background, and discovered that slwrky31 TRV-SlVQ15 plants had significantly reduced resistance to B. cinerea compared with slwrky31 mutants (Figure 8, C and D), suggesting that SlVQ15 interacts with SlWRKY31 to cooperatively control the defense against B. cinerea. Elucidating which genes are targeted by SlVQ15–SlWRKY31 to control tomato defense will benefit us in clarifying the mechanism of SlVQ15–SlWRKY31 in the regulation of tomato defense.

In summary, we propose a model for the function of SlVQ15 in tomato response to B. cinerea infection (Figure 9). In this model, SlVQ15 promotes the transcriptional activation function of SlWRKY31 through its interaction via the N-terminus of SlVQ15 and the C-terminus of SlWRKY31, whereas SlJAZ proteins repress the interaction of SlVQ15 and SlWRKY31 by directly interacting with SlVQ15 via the N-terminus of SlJAZs, and also attenuate the SlVQ15-enhanced transcriptional activation activity of SlWRKY31. The SlCOI1-SlJAZ coreceptor perceives JA-Ile, and induces the degradation of SlJAZ repressors through the SCFCOI1 complex, which releases SlVQ15-SlWRKY31 to cooperatively and positively mount the defense against B. cinerea.

Figure 9.

Figure 9

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A simplified model for the functions of SlVQ15 in JA signaling in tomato. SlVQ15 associates with SlWRKY31 to coordinately regulate resistance to B. cinerea in tomato. SlJAZs (e.g. SlJAZ11) interact with SlVQ15, inhibit the interaction of SlVQ15 and SlWRKY31, and attenuate SlVQ15-increased transcriptional activation activity of SlWRKY31. The coreceptor SlCOI1–SlJAZ complex perceives JA-Ile, a bioactive form of JA, which induces the degradation of SlJAZ repressors to release SlVQ15–SlWRKY31 to coordinately defend against B. cinerea.

Materials and methods

Plant materials and growth conditions

Tomato (Solanum lycopersicum) cv CM was used as the WT. To generate TRV-SlVQ15 plants, 300-bp fragments of SlVQ15 were amplified and cloned into the pTRV2 vector to generate the TRV-SlVQ15 construct. This construct was subsequently transferred into Agrobacterium tumefaciens strain GV3101, which was then transformed into WT plants or slwrky31 mutants using the sprout vacuum-infiltration method according to a previously published protocol (Yan et al., 2012). The primer pairs used for vector construction are listed in Supplemental Table S1.

To generate SlVQ15-overexpressing lines, the full-length CDS of SlVQ15 was fused to a modified pCAMBIA1300 vector that contained a 35S promoter and three flag tags. The recombinant vector was transferred into Agrobacterium strain GV3101, which was then transformed into WT plants. Transformants were selected based on the resistance to hygromycin B and were genotyped. Homozygous SlVQ15-overexpressing plants were used in this study. The primer pairs used for vector construction are listed in Supplemental Table S1.

To generate slvq15 mutants, we selected two targets (220–239 bp and 334–353 bp) of SlVQ15 for genome editing. To generate slwrky31 mutants, we selected two targets in the first and second exons of SlWRKY31 for genome editing. The construct was subsequently transformed into CM plants by Agrobacterium-mediated transformation. Cas9-free slvq15 and slwrky31 mutants were identified by PCR and sequencing, and used in this study.

TRV-SlVQ15 and slwrky31 TRV-SlVQ15 plants were grown in a greenhouse under 16 h (22°C)/8 h (20°C) light/dark conditions. The SlVQ15-overexpressing plants, slvq15 and slwrky31 mutants were grown under 16-h (26°C)/8-h (18°C) light/dark conditions. Nicotiana benthamiana plants were grown in a greenhouse under 16 h (22°C)/8 h (18°C) light/dark conditions.

Y2H screening and Y2H assays

The Y2H screening method was performed according to the manufacturer’s instructions (Clontech). For Y2H assays, the CDSs of SlJAZ1, SlJAZ2, SlJAZ3, SlJAZ4, SlJAZ5, SlJAZ6, SlJAZ7, SlJAZ8, SlJAZ9, SlJAZ10, and SlJAZ11 were inserted into the pB42AD or pLexA vector. The full-length CDS of SlVQ15, and its related domains were cloned into pLexA or pB42AD vectors. The full-length CDS of SlWRKY31, and its related domains were cloned into pB42AD vectors. The indicated plasmid pairs were transformed into the yeast (Saccharomyces cerevisiae) strain EGY48 according to the manufacturer’s instructions (Clontech). Transformants were grown on the medium consisting of 2% (w/v) Gal/1% (w/v) raffinose/SD/−Ura/−His/−Trp/−Leu/X-β-Gal to test the interactions of the proteins. The primers used for vector construction are listed in Supplemental Table S1.

Pull-down assays

The CDS of SlVQ15 was amplified and fused into the pMAL-c5X vector to generate MBP-fused SlVQ15. The primer pairs used for vector construction are listed in Supplemental Table 1. This recombinant vector and pMAL-c5X vector were transformed into E.coli strain Transetta (DE3), induced with 0.3-mM isopropyl-β-D-thiogalactoside, and purified by amylose resin (NEB).

The CDSs of SlJAZ7 and SlJAZ11 were cloned into the pROK2 vector to produce myc-JAZ7 and myc-JAZ11, respectively. The CDSs of SlVQ15 and SlWRKY31 were cloned into the modified pCAMBIA1300 vector to produce flag-SlVQ15, and flag-SlWRKY31, respectively. These vectors were transformed into N.benthamiana leaves via Agrobacterium strains to transiently express the myc-SlJAZ7, myc-SlJAZ11, flag-SlVQ15, or flag-SlWRKY31 protein. These proteins were extracted from 5 g of N.benthamiana leaves with RB buffer that included 50-mM Tris–HCl, pH 7.8, 100-mM NaCl, 25-mM imidazole, 10% (v/v) glycerol, 0.1% (v/v) Tween-20, EDTA-free complete mini protease inhibitor cocktail, and 20-mM 2-mercaptoethanol.

For pull-down assays, MBP and MBP-fused SlVQ15 proteins were incubated in amylose resin for 2 h at 4°C. After washing five times with RB buffer, incubated with myc-SlJAZ7, myc-SlJAZ11, flag-SlVQ15, or flag-SlWRKY31 fused proteins for 2 h at 4°C. The mixtures were washed five times using RB buffer, resuspended in sodium dodecyl sulfate (SDS) loading buffer, boiled at 100°C for 5 min, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride membranes. The anti-myc antibody was used to detect the myc-SlJAZ7 or myc-SlJAZ11 protein, and the anti-flag antibody was used to detect the flag-SlVQ15, or flag-SlWRKY31 protein.

LCI assays

For LCI assays, the CDSs of SlJAZ2, SlJAZ5, SlJAZ7, SlJAZ11, SlVQ15, and SlVQ15NT were inserted into pCAMBIA-nLUC, the CDSs of SlVQ15, SlVQ15CT, SlWRKY31, and SlWRKY31CT were cloned into pCAMBIA-cLUC, and the CDSs of SlJAZ11 and GFP were inserted into the pROK2 vector. The Agrobacterium strains (GV3101) were incubated overnight and resuspended in infiltration buffer (10-mM MES, 0.2-mM acetosyringone, and 10-mM MgCl2) to a concentration of OD600=0.5. Equal volumes of the indicated combined Agrobacterium suspensions were mixed and infiltrated into N.benthamiana leaves. Fifty hours later, the leaves were sprayed with 0.1 mM luciferin solution and then kept in the dark for 5 min. The low-light cooled CCD imaging apparatus (Tanon 5200Multi) was used to capture the LUC images and to count luminescence intensity. The primer pairs used for vector construction are listed in Supplemental Table S1.

RT–qPCR

The HiPure Plant RNA Mini Kit (Magen, China) and TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, China) were used to extract the total RNA and synthesize cDNA, respectively, according to the manufacturer’s instructions. RT–qPCR experiments were carried out with SYBR Green Mix using a Bio-Rad CFX96 qPCR instrument. The PCR programs were 95°C for 2 min, followed by 39 cycles at 95°C for 10 s, 56°C for 10 s, and 72°C for 15 s. SlACTIN2 was used as an internal control. The primers used for RT–qPCR analysis are listed in Supplemental Table S2.

Subcellular localization

The coding region of SlVQ15 was fused to the pEGAD vector under the control of the 35S promoter. The recombinant vector and the control vector were introduced into Agrobacterium strain GV3101, which was then injected into N.benthamiana leaves. 50 h after infection, GFP fluorescence was observed using a laser confocal microscope (488 nm excitation, 10% laser power, 100 nm collection bandwidth, master gain = 500, and digital gain = 1). The primer pairs used for vector construction are listed in Supplemental Table S1.

Transcriptional activity assays

The CDS of SlWRKY31 was cloned into the GAL4DB vector. SlVQ15 and its related domains, and SlJAZ11 were cloned into the pGreenII 62-SK vector. The GUS (β-glucuronidase) gene controlled by the GAL4DB binding sites [GAL4(4x)-D1–3(4x)] was used as the reporter. The luciferase (LUC) gene controlled by the 35S promoter acted as the internal control. The protoplast in the transcriptional activity assays was isolated from N.benthamiana leaves. Protoplast isolation and transfection were performed as described previously (Yoo et al., 2007). The primer pairs used for vector construction are listed in Supplemental Table S1.

Inoculation with B. cinerea

Botrytis cinerea was grown on potato dextrose broth (PDB) agar medium at 20°C under weak light conditions. After 10 days, B. cinerea spores were collected and resuspended in PDB liquid medium until the spore concentration was 106 spores mL−1. The detached leaves were placed in Petri dishes that contained 1% agar, and inoculated with 5 μL of B. cinerea spores. Afterwards, the dishes were covered with lids and maintained in a growth chamber with the same conditions as those for plant growth. Three days after inoculation, the lesion areas of the infected leaves were measured using Digimizer software (MedCalc Software, Belgium).

Accession numbers

Sequence data from this article can be found in the Sol Genomics Network Initiative under the following accession numbers: SlVQ15 (Solyc07g043250), SlWRKY31 (Solyc06g066370), SlJAZ1 (Solyc07g042170), SlJAZ2 (Solyc12g009220), SlJAZ3 (Solyc03g122190), SlJAZ4 (Solyc12g049400), SlJAZ5 (Solyc03g118540), SlJAZ6 (Solyc01g005440), SlJAZ7 (Solyc11g011030), SlJAZ8 (Solyc06g068930), SlJAZ9 (Solyc08g036640), SlJAZ10 (Soly08g036620), SlJAZ11 (Solyc08g036660), SlTD (Solyc09g008670), SlPR-STH2 (Solyc05g054380), and SlACTIN2 (Solyc11g005330).

Supplemental data

The following materials are available in the online version of this article

Supplemental Figure S1. Expression analysis of SlVQs with B. cinerea infection.

Supplemental Figure S2. RT–qPCR analysis of SlVQ15 in SlVQ15-overexpressing plants.

Supplemental Figure S3. Generation of the slvq15 mutants using CRISPR/Cas9 technology.

Supplemental Figure S4. SlWRKY31 cannot interact with SlJAZs in yeast.

Supplemental Figure S5. Generation of the slwrky31 mutant using CRISPR/Cas9 technology.

Supplemental Table S1. Primers used for vector construction.

Supplemental Table S2. Primers used for RT–qPCR.

Supplementary Material

kiac275_Supplementary_Data
Click here for additional data file. (365.3KB, zip)

Acknowledgments

We thank Professor Ji Tian for providing the vectors used in VIGS assays and Jiaojiao Wang for technical assistance or help.

Funding

The work was supported by the National Natural Science Foundation of China (31902026 and 31672201), Beijing Natural Science Foundation (6194030), and Scientific Research Project of Beijing Municipal Commission of Education (KM201910020013).

Conflict of interest statement. The authors declare that they have no conflicts of interests.

Contributor Information

Huang Huang, Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China; Beijing Key Laboratory for Agricultural Application and New Technique, Beijing University of Agriculture, Beijing 102206, China.

Wenchao Zhao, Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China; Beijing Key Laboratory for Agricultural Application and New Technique, Beijing University of Agriculture, Beijing 102206, China.

Chonghua Li, Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China.

Hui Qiao, Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China.

Susheng Song, College of Life Sciences, Capital Normal University, Beijing 100048, China.

Rui Yang, Beijing Key Laboratory for Agricultural Application and New Technique, Beijing University of Agriculture, Beijing 102206, China.

Lulu Sun, Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China; Beijing Key Laboratory for Agricultural Application and New Technique, Beijing University of Agriculture, Beijing 102206, China.

Jilin Ma, Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China.

Xuechun Ma, Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China.

Shaohui Wang, Plant Science and Technology College, Beijing University of Agriculture, Beijing 102206, China; Beijing Key Laboratory for Agricultural Application and New Technique, Beijing University of Agriculture, Beijing 102206, China.

 

S.W. and H.H. conceived the study and designed the research. H.H., W.Z., and S.S. analyzed data. H.H. and W.Z. wrote the manuscript and S.S. modified the manuscript. H.H., W.Z., C.L., H.Q., R.Y., L.S., J.M., and X.M. performed the experiments.

The author responsible for the distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is: Shaohui Wang (wangshaohui@bua.edu.cn).

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