BIN2 negatively regulates plant defence against Verticillium dahliae in Arabidopsis and cotton

Abstract Verticillium wilt is caused by the soil‐borne vascular pathogen Verticillium dahliae, and affects a wide range of economically important crops, including upland cotton (Gossypium hirsutum). Previous studies showed that expression levels of BIN2 were significantly down‐regulated during infestation with V. dahliae. However, the underlying molecular mechanism of BIN2 in plant regulation against V. dahliae remains enigmatic. Here, we characterized a protein kinase GhBIN2 from Gossypium hirsutum, and identified GhBIN2 as a negative regulator of resistance to V. dahliae. The Verticillium wilt resistance of Arabidopsis and cotton were significantly enhanced when BIN2 was knocked down. Constitutive expression of BIN2 attenuated plant resistance to V. dahliae. We found that BIN2 regulated plant endogenous JA content and influenced the expression of JA‐responsive marker genes. Further analysis revealed that BIN2 interacted with and phosphorylated JAZ family proteins, key repressors of the JA signalling pathway in both Arabidopsis and cotton. Spectrometric analysis and site‐directed mutagenesis showed that BIN2 phosphorylated AtJAZ1 at T196, resulting in the degradation of JAZ proteins. Collectively, these results show that BIN2 interacts with JAZ proteins and plays a negative role in plant resistance to V. dahliae. Thus, BIN2 may be a potential target gene for genetic engineering against Verticillium wilt in crops.


Introduction
Verticillium wilt, a highly destructive vascular plant disease, is primarily caused by the soil-borne plant pathogenic fungus Verticillium dahliae (Deketelaere et al., 2017;Fradin and Thomma, 2006). This vascular pathogen can attack a wide spectrum of globally important crops including cotton (Gossypium spp.) and cause tremendous economic losses (Pegg et al., 2002;Yang et al., 2015). Because of the long-term viability V. dahliae in soil, the lack of resistance genes in upland cotton (G. hirsutum), Verticillium wilt is notoriously difficult to prevent and control, earning it the name 'cancer of cotton' (Aguado et al., 2008;Su et al., 2018;Zhang et al., 2012). To respond to fungal and other pathogens, plants have evolved a wide spectrum of defences, including physical and chemical barriers, and some inducible responses (L opez et al., 2008;Pieterse et al., 2009). Among these, phytohormones play a crucial role in protecting plants from fungal pathogen infestation.
Phytohormones brassinosteroids (BRs) function as master regulators of plant immunity (Anwar et al., 2018;Nawaz et al., 2017;Wang, 2012;Yu et al., 2018). BRs were perceived by the receptor-like kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) and activated a signal transduction cascade to regulate BRresponsive genes (Belkhadir and Jaillais, 2015;Kim and Wang, 2010). Exogenous application of low concentrations of BR significantly improves the resilience of cucumber (Cucumis sativus L.), tobacco (Nicotiana tabacum L.), tomato (Solanum lycopersicum), and cotton (Gossypium spp.) to viral and fungal pathogens (Bajguz and Hayat, 2009;Gao et al., 2013;Wang et al., 2015). Several studies reported that BRs modulated plant immunity by regulating the JA signalling pathway. For instance, the BR-mediated susceptibility to Rice Black-Streaked Dwarf Virus (RBSDV) infection can be suppressed by the JA-mediated defence in rice (Oryza sativa) (He et al., 2017). Nicotiana attenuated BAK1, a co-receptor of BR receptor BRI1, is involved in modulating herbivory-induced JA accumulation . The GSK3-like kinase BIN2 (Brassinosteroid insensitive 2) is a key negative regulator of the BR signalling pathway. BIN2 phosphorylates and destabilizes the two homologous transcription factors BRASSINAZOLE RESISTANT1 (BZR1) and BZR2/BES1 to block BR signalling pathway (Li and Nam, 2002). The GSK3like kinase BIN2 is involved in plant multiple developmental and physiological processes (Saidi et al., 2012;Tong and Chu, 2018;Youn and Kim, 2015). Previous study had implicated that BIN2 expression was inhibited by V. dahliae infestation (Gao et al., 2013). However, the molecular mechanism of GSK3-like kinase BIN2-mediated antifungal defence remains unclear in plants.
Recent genetic evidence indicates that jasmonic acid (JA) biosynthesis and signalling pathway are integral to plant resistance to V. dahliae (Du et al., 2017;Goossens et al., 2016;Howe et al., 2018;Wang et al., 2019;Wasternack and Song, 2016). The JA signals are sensed by COI1-JAZ (CORONATINE INSENSITIVE-JASMONATE ZIM DOMAIN) co-receptors. Within the signalling cascade, JAZ proteins are degraded through the 26S proteasome, and then activate JA-related transcription factors (such as MYC2, MYC3, and MYC4), which subsequently regulate the downstream signalling cascades and modulate the respective plant responses (Chini et al., 2007;Sheard et al., 2010;Thines et al., 2007;Xu et al., 2002;Yan et al., 2007). Previous studies in Arabidopsis showed that unobstructed JA signal transduction helps to improve plant resistance to V. dahliae (Fradin et al., 2011). Mutants impaired in the JA signalling pathways become susceptible to V. dahliae infection (Campos et al., 2014;De Geyter et al., 2012;Fradin et al., 2011;Jiang and Yu, 2016;Thaler et al., 2004). During V. dahliae attack, JA and JA-Ile are significantly accumulated in plants, followed by increased expression of JA signal transduction genes (Hu et al., 2018a). JA largely contributes to cotton resistance to V. dahliae (Gao et al., 2013). Reduced transcription levels of GhSSN (SILENCE-INDUCED STEM NECROSIS) induce the accumulation of JA and JA-Ile and thus enhance cotton immunity to Verticillium wilt (Sun et al., 2014). GhCPK33 negatively regulates cotton sensitivity to V. dahliae by phosphorylating GhOPR3, a pivotal node gene of JA biosynthesis (Hu et al., 2018b). In Arabidopsis, constitutive JAZ expression increases susceptibility to the fungal pathogen (Thatcher et al., 2016). Gossypium barbadense WRKY1 transcription factor attenuates cotton resistance to V. dahliae by promoting JAZ1 expression . GhJAZ2 protein interacts with GhbHLH171 and inhibits its transcriptional activity, eventually restraining the JA-mediated defence against V. dahliae .
In this study, we identified the protein kinase BIN2 as a negative regulator of defence against V. dahliae in Arabidopsis and cotton. We provide evidence that BIN2 interacted with and phosphorylated JAZ proteins, which are negative regulators of the JA signalling pathway. We demonstrate that BIN2 phosphorylation destabilized JAZ1. Our study provides novel insights into the phosphorylation network of the BIN2 protein and the interaction between the BR and JA signalling pathways. In conclusion, this study showed that BIN2 directly participates in the regulation of plant biotic stress response and could be a potential molecular target for engineering next-generation cotton crops with improved resistance against Verticillium wilt disease.

Results
Identification of the GhBIN2 gene and its expression pattern BIN2 is a negative regulator of the BR signalling pathway (Li and Nam, 2002;Li et al., 2001;Xiong et al., 2017). To dissect the function of cotton BIN2, we identified a BIN2 gene (Accession number in GenBank: KM453729) in the upland cotton (Gossypium hirsutum) genome. The full-length cDNA of GhBIN2 consists of 1143 nucleotides and encodes a protein of 381 amino acids. There were five homologs of GhBIN2 in the upland cotton genome (Table S1). We performed multiple sequence alignment with GhBIN2 and other BIN2s from various plant species (Figure 1a). GhBIN2 showed high identity scores with BIN2 proteins from other species. The phylogenetic analysis with the protein sequences indicated that GhBIN2 was evolutionarily conserved, and the closest ortholog of GhBIN2 was GrBIN2 (Gossypium raimondii) (Figure 1b). Furthermore, we analysed the expression of GhBIN2 in various tissues of the cotton plant by QRT-PCR. Expression of the GhBIN2 gene was ubiquitous in cotton plants and showed relatively higher levels in petals, stems, and roots compared with those in leaves, anthers, and ovules ( Figure 1c). To investigate the involvement of GhBIN2 in disease responses, we examined the expression pattern of GhBIN2 in cotton roots after inoculation with V. dahliae (Figure 1d). GhBIN2 transcript abundance was down-regulated in roots after inoculation with V. dahliae. These results suggested that GhBIN2 was evolutionarily conserved and may be involved in the cotton defence against V. dahliae infection.
Silencing GhBIN2 expression increased cotton resistance to V. dahliae colonization In the current study and previous research, BIN2 was downregulated after inoculation with V. dahliae (Figure 1d; Gao et al., 2013). To better understand the putative function of GhBIN2 during the immune response against V. dahliae, we employed the tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) strategy to knock down the expression of GhBIN2. When the leaves of cotton seedlings inoculated with pTRV:GhPDS showed a photobleaching phenotype (Figure 2a), the TRV:00 and TRV:GhBIN2 cotton seedlings were harvested to determine the transcript levels of GhBIN2 (Figure 2b). The QRT-PCR results showed that the transcript abundance of GhBIN2 was significantly reduced in TRV:GhBIN2 plants, suggesting that GhBIN2 was successfully silenced in TRV:GhBIN2 plants. Two weeks after Agrobacterium infiltration, these cotton seedlings were challenged with the V. dahliae strain Vd07038 and monitored for the development of disease symptoms. We found that knock-down of GhBIN2 enhanced plant resistance to V. dahliae. Defoliation and yellowing disease symptoms were more severe in the TRV:00 plants than in the TRV:GhBIN2 plants. (Figure 2c and d). The disease index in the TRV:GhBIN2 plants was much lower than that of the control seedlings ( Figure 2e). Reactive oxygen species (ROS) burst is one of the canonical defence events during plant immune response (Hancock et al., 2002). We checked the ROS levels in the infected cotton leaves by DAB-peroxidase staining. The TRV:GhBIN2 cotton seedlings accumulated more spots of brown precipitates than did the TRV:00 plants, indicating a stronger defence response through the ROS pathway ( Figure 2f). We performed a recovery assay from stem sections of the inoculated plants to examine the extent of V. dahliae colonization. Significantly fewer fungal colonies were present on the TRV: GhBIN2 overexpression attenuates cotton resistance to Verticillium dahliae Thirteen independent transgenic cotton lines constitutively overexpressing GhBIN2 were obtained. Three lines with different expression levels of GhBIN2 were selected for the following analysis ( Figure S1). These GhBIN2 overexpression (OE) cotton seedlings along with the corresponding WT plants were subjected to challenge with V. dahliae (Figure 3). Disease symptoms were examined at 20 days post-inoculation. GhBIN2-overexpressing cotton plants OE7 displayed more severe wilting and yellowing symptoms than did the control plants (Figures 3a and b). By DABperoxidase staining of WT and OE7 leaves, we observed larger and more dark spots accumulated in WT plants than in OE7 plants (Figure 3c  To investigate whether BIN2 performs a similar function across plant species, we evaluated the sensitivity of BIN2 mutants to V. dahliae infection in the model plant Arabidopsis (Figure 4a). The disease symptoms caused by V. dahliae were more severe in the gain-of-function mutant bin2-1 seedlings compared with the control plants Col-0. The loss-of-function mutant bin2-3 and bin2-3 bil1 bil2 showed enhanced resistance to V. dahliae compared with the control plants Ws (Jonak and Hirt, 2002). We counted the numbers of stunted and chlorotic leaves in AtBIN2 mutants and WT plants, and found a relatively lower level of stunting and chlorosis in AtBIN2 loss-of-function mutants and Col-0 plants ( Figure 4a). Additionally, disease symptoms induced by V. dahliae on the rosette leaves of these seedlings grown in the pots showed the same pattern with those grown in the MS medium ( Figure S2). These results indicated that BIN2 negatively regulated plant resistance against V. dahliae in the model plant Arabidopsis, which is consistent with the functions of GhBIN2, indicating conserved functions of BIN2 against Verticillium wilt from Arabidopsis to cotton.
JA plays a vital role in plant response to V. dahliae (Fradin et al., 2011;Goossens et al., 2016;Howe et al., 2018;Veronese et al., 2006;Wasternack and Song, 2016). Importantly, studies have also reported that BRs participate in modulating plant immunity through regulating the JA signalling pathway (He et al., 2017;Yang et al., 2011). We investigated whether the BR negative regulator BIN2-mediated resistance to V. dahliae is related to the JA signalling pathway. First, we examined the endogenous JA and MeJA contents in AtBIN2 mutants ( Figure 4b). We found that BIN2 gain-of-function plants caused significant JA and MeJA accumulation, and the production of JA and MeJA was suppressed in the BIN2 loss-of-function mutants. Bikinin is one of the most specific inhibitors of BIN2 kinase protein, and previous studies showed that the expression levels of BIN2 or other BRrelated genes were significantly altered by treatment with bikinin (De Rybel et al., 2009;Rozhon et al., 2019). In the present study, bikinin was used to treat WT seedlings ( Figure 4c). More transcripts of JA-responsive marker genes including AOS, PDF1.2, and VPS2 accumulated under BIN2 inhibition. Furthermore, we examined the response of AtBIN2 mutants to the MeJA treatment ( Figure 4d). As indicated by the decreases in root length and fresh weight, bin2-1 showed enhanced sensitivity to JA signalling, and the sensitivity of bin2-3 and bin2-3 bil1 bil2 to JA was reduced. These results suggest that BIN2 negatively regulates plant resistance to V. dahliae and affects the JA signalling pathway.

AtBIN2 physically interacts with AtJAZ proteins
To understand the molecular mechanism modulated by AtBIN2 in regulating the JA signalling pathway, we used two-hybrid screening to discover potential BIN2-interacting proteins (Figure S3A). The BIN2 coding sequences were constructed into the bait vector pGBKT7. AtOPR3 and AtJAR1 genes play important roles in JA biosynthesis, and AtCOI1, AtNINJA, AtMYC2, and AtJAZ1 are key genes of JA signal transduction (Wasternack and Hause, 2013;Wasternack and Song, 2016). These JA-related genes were constructed into the vector pGADT7. The JAZ1 protein was identified as an interacting protein of BIN2 in twohybrid assays ( Figure S3A). Next, we examined the interaction between BIN2 and all JAZ family members by two-hybrid assays. Except for JAZ7, 12 of the 13 JAZ proteins interacted with BIN2 ( Figure 5a). GhBIN2 also interacted with GhJAZ2 protein (Figure S3B), which was reported to be involved in cotton resistance to V. dahliae . Furthermore, we observed that BIN2 homologs BIL1 and BIL2 interacted with JAZ proteins, suggesting that BIN2, BIL1, and BIL2 function redundantly in interaction with JAZ proteins ( Figure S3C). To further identify the functional domain responsible for their interaction, we made truncated versions of JAZ1 protein in yeast ( Figure 5b). The Nterminal domain (NT), the zinc-finger expressed in inflorescence meristem domain (ZIM), and the jasmonate-associated domain (Jas) of JAZ1 protein were individually deleted (Pauwels and Goossens, 2011;Wager and Browse, 2012). Deletion of the NT and Jas domains of JAZ1 did not eliminate the interaction between BIN2 and JAZ1, whereas deletion of the ZIM domain compromised their interaction, suggesting that the ZIM domain of JAZ1 is responsible for that interaction (Figure 5b).
We performed bimolecular fluorescence complementation (BiFC) assays in Nicotiana benthamiana leaves to further confirm the interaction between BIN2 and JAZ1 in vivo (Figure 5c). BIN2 was fused to an N-terminal yellow fluorescent protein fragment (nYFP), and JAZ1 was fused to a C-terminal YFP fragment (cYFP). Neither BIN2-nYFP nor JAZ1-cYFP co-transformation with the control vectors showed any fluorescence signal. When BIN2-nYFP and JAZ1-cYFP were co-expressed in N. benthamiana leaves, a bright fluorescence signal was observed in the cell nucleus, indicating that the interaction between BIN2 and JAZ1 occurred there. Additionally, we performed a GST pull-down assay to confirm their interaction in vitro (Figure 5d). We used GST agarose to precipitate GST or BIN2-GST proteins, and then incubated these GST proteins with purified His-JAZ1 proteins. Recombinant His-JAZ1 protein bound to GST-BIN2, but not to GST. These results indicate that BIN2 physically interacts with JAZ proteins both in vivo and in vitro, and this interaction occurs at the cell nucleus.

BIN2 phosphorylates JAZ proteins
As a kinase, BIN2 can phosphorylate most of the proteins with which it interacts (Youn and Kim, 2015). We further investigated whether JAZ1 was a substrate for phosphorylation by BIN2. S/ TxxxS/T (S/T corresponds to Ser or Thr and x denotes any other residue) is the conserved phosphorylation motif targeted by BIN2 (Ryu et al., 2010;Ryu et al., 2007;Wang et al., 2002;Woodgett, 2001). Bioinformatic analysis indicated that several putative BIN2 phosphorylation sites were identified in the JAZ1 protein sequence, suggesting that JAZ1 may be a substrate of BIN2 (Figure 6a). To verify the phosphorylation of JAZ1 by BIN2, we conducted in vitro phosphorylation assays (Figure 6b). These revealed that JAZ1 was specifically phosphorylated by BIN2 in vitro. Conversely, a kinase-dead mutant BIN2 K69R failed to phosphorylate JAZ1 protein (Figure 6b; Li and Nam, 2002). To identify the BIN2-mediated potential phosphorylation sites of JAZ1, we performed mass spectrometry analysis and found three potential phosphorylation sites T26, S28, T196 in JAZ1 protein ( Figure S4). To determine whether these amino acids of JAZ1 are phosphorylated by BIN2, we mutated these residues into alanine to make single-mutated forms of JAZ1 and performed in vitro kinase assays. JAZ1 and all the mutated forms were phosphorylated by BIN2; however, the phosphorylation levels of JAZ1 T196A by BIN2 were significantly reduced ( Figure 6c). These results indicate that JAZ1 T196 is a major phosphorylated site targeted by BIN2, while the other two sites are not. To investigate whether JAZ1 is phosphorylated in vivo, we expressed MYC-JAZ1/HF-BIN2 or MYC-JAZ1 alone in N. benthamiana protoplasts and performed phos-tag mobility shift assays. As shown in Figure 6d, a slow-migrating form of JAZ1 was detected and eliminated after incubating the protein samples with calf intestinal alkaline phosphatase (CIAP), suggesting that the band corresponded to phosphorylated JAZ1. These results indicate that JAZ1 is phosphorylated by BIN2 in planta. We also examined the effect of BIN2 on the phosphorylation of the mutated form MYC-JAZ1 T196A . We found that although the mutated form JAZ1 T196A was still phosphorylated by BIN2, the phosphorylation level was considerably reduced as compared with wild-type JAZ1 protein (Figure 6d and e). These results demonstrate that JAZ1 T196A is a major phosphorylation site of BIN2.

BIN2 promotes the degradation of the JAZ1 protein
Previous studies have shown that BIN2 phosphorylates and regulates the stability of its substrates in plants (Cheng et al., 2014;Gudesblat et al., 2012;He et al., 2002;Vert et al., 2008;Ye et al., 2019;Yin et al., 2002). To investigate whether BIN2 regulates the stability of JAZ1, we conducted cell-free protein degradation assays (Figure 7a and b). His-JAZ1 recombinant proteins were incubated with an equal amount of total proteins extracted from Col-0 and BIN2-overexpressing transgenic plants (AtBIN2-MYC) supplemented with ATP. Interestingly, we found that the degradation rates of the JAZ1 protein in Col-0 were much slower than in BIN2-overexpressing transgenic plants. These results suggest that BIN2 destabilizes JAZ1 in vitro. Besides, we used the GUS (b-glucuronidase) reporter system to examine the stability of JAZ1 protein under BIN2 inhibition (Jefferson et al., 1987). 35S-JAZ1-GUS transgenic plants were generated and exposed to MeJA and bikinin (Figures 7c, d, and S5). MeJA promoted the degradation of JAZ1 protein, while bikinin treatment induced a significant increase in GUS activity in these transgenic lines. Besides, we examined the protein levels of JAZ1 in BIN2-related mutants. We found that the levels of JAZ1 were obviously lower in the bin2-1 mutants but more in the bin2-3 Figure 4 BIN2 is a negative regulator of resistance to V. dahliae in Arabidopsis. (a) Disease symptoms of WT and AtBIN2-related mutants inoculated with V. dahliae. The bin2-1 mutant is the gain-of-function mutant of BIN2. The bin2-3 and bin2-3 bil1 bil2 mutants are the BIN2 and its close homologs BIL1 and BIL2 loss-of-function mutants. Two-week-old Arabidopsis plants were inoculated with V. dahliae, and photographs were taken at 14 dpi. The extent of stunting and leaf chlorosis in these Arabidopsis plants at 14 dpi were investigated. (b) Measurement of JA and MeJA contents in two-week-old WT and AtBIN2-related mutants. Data shown are the means from at least 10 seedlings for each indicated plant line. (c) Expression patterns of JA-related marker genes under BIN2 inhibition. Relative expression levels of two JA responsive genes AtPDF1.2, AtVPS2, and one JA biosynthetic gene AtAOS were examined. The expression levels were assessed by QRT-PCR using AtActin2 as the internal control gene. Bikinin was dissolved in DMSO, so the expression levels of these genes in DMSO were set to 1. (d) Phenotypes of 10-day-old WT and AtBIN2-related mutant seedlings exposed to DMSO and 10 lM MeJA. The fresh weight and root length of these seedlings (at least 10 seedlings for each indicated plant line) were measured. Error bars represent AESD (n = 3). Asterisks (*P < 0.05, **P < 0.01) indicate significant differences from the control. bil1bil2 mutants than those in the corresponding wildtype plants (Figure 7e and f). These observations further confirm that BIN2 promotes the degradation of the JAZ1 protein in vivo.
Furthermore, cell-free protein degradation assays revealed that JAZ1 T196A was more stable than JAZ1 when incubated with total proteins extracted from Col-0 plants (Figures 7g and h). As  Figure 7i and j, the accumulation of JAZ1 protein was increased under the treatment of bikinin and can be partly eliminated by CIAP treatment. These results indicate that the phosphorylation mediated by BIN2 is required for JAZ1 degradation.

Phosphorylation of JAZ1 mediated by BIN2 influences JA transduction
Considering JAZ protein is a negative regulator of the JA signalling pathway, we investigated whether BIN2 phosphorylation affects JA signalling transduction. The coi1-2 mutants are leaky mutant alleles of JA signal receptor COI1, which exhibited reduced JA insensitivity and partial fertility (Xu et al., 2002). We found that the bikinin-mediated stabilization of AtJAZ1 was inhibited in coi1-2 mutants (Figure 8a and b), suggesting the involvement of AtCOI1 in AtBIN2-mediated AtJAZ1 stability.
PDF1.2 is a JA-responsive gene that contributes to plant response to pathogens and is negatively regulated by the JA signal transcription factor MYC2 (Dombrecht et al., 2007;Lorenzo et al., 2004). PDF1.2 was therefore used as a target to assess the roles of BIN2 by using a LUC assay in vivo (Figures 8c  and d). LUC activity was lower in plants transformed with MYC2 as compared with the empty vector control, suggesting that MYC2 indeed inactivates PDF1.2 expression. The inhibition effect of PDF1.2 imposed by MYC2 was significantly attenuated by the addition of JAZ1. When BIN2 was co-expressed with JAZ1 and MYC2, the fluorescence signal was lower than in the plants injected with JAZ1 and MYC2. Furthermore, plants injected with JAZ1 T196A , BIN2, and MYC2 induced LUC activity more strongly than plants injected with JAZ1, BIN2, and MYC2. These results suggest that the phosphorylation of JAZ1 by BIN2 influences JA signalling transduction. BIN2-related mutants. The AtJAZ1 protein was detected with anti-AtJAZ1 antibody and actin was used as a loading control. (g) and (h) The mutant protein JAZ1-T196A is more stable than JAZ1 in a cell-free degradation assay. Equal amounts of the recombinant proteins AtJAZ1-His and AtJAZ1-T196A-His were incubated with equal amounts of total proteins extracted from 14-day-old Col-0 wildtype plants in the in vitro cell-free degradation assays. Anti-His antibody was used to detect recombinant proteins, and actin was used as the internal control. Relative levels of AtJAZ1 and AtJAZ1-T196A at 0 h were defined as "1". (i) and (j) The degradation of AtJAZ1 is related with BIN2-mediated phosphorylation in vivo. 14-day-old Col-0 wild-type plants were treated with indicated combinations for different hours. Anti-AtJAZ1 antibody was used to detect JAZ1 proteins, and actin was used as the internal control. CIAP represents Calf Intestinal Alkaline Phosphatase.

Discussion
Cotton is the most important natural fibre crop in the world, widely cultivated in almost 150 countries and integral in economical production worldwide . However, cotton yield and quality are significantly reduced by cotton Verticillium wilt, which is also called "cancer of cotton". This destructive disease causes massive annual losses in cotton crop yields Wang et al., 2016). Many studies have suggested that the BR signalling pathway is involved in plant resistance to V. dahliae (Anwar et al., 2018;Gao et al., 2013;Nawaz et al., 2017;Wang, 2012;Yu et al., 2018). BIN2 is a negative regulator of the BR signalling pathway, and the expression level of BIN2 was significantly decreased in Gossypium barbadense after V. dahliae infestation (Gao et al., 2013). However, the function and molecular mechanism of BIN2 in plant resistance to V. dahliae were poorly understood.
In the current study, we revealed that transgenic cotton lines constitutively expressing GhBIN2 and AtBIN2 gain-of-function Arabidopsis mutants bin2-1 exhibited much more severe symptoms after V. dahliae infestation than did the control plants (Figures 3 and 4), while the GhBIN2-silenced plants and AtBIN2 loss-of-function mutants bin2-3 bil1 bil2 showed increased resistance capability against V. dahliae (Figures 2 and 4). These observations confirmed that BIN2 functioned as a negative regulator of plant defence against V. dahliae in both Arabidopsis and cotton. In previous study, overexpression of MsK1, a GSK from Medicago sativa, was demonstrated to increase susceptibility to the virulent bacterial pathogen Pseudomonas and to compromise MAP kinase activation with pathogen infection (Wrzaczek et al., 2007). Recent work revealed that GSK3 enhanced antiviral defence in rice (He et al., 2020). Therefore, we propose that BIN2 may feature prominently in the plant response to various pathogens. Further, BIN2 may be used as a candidate gene for generating disease-resistant crops by using the CRISPR/Cas9 genome editing system or RNAi approaches.
In Arabidopsis, BIN2 has two homologs, BIL1 and BIL2, which function redundantly (Jonak and Hirt, 2002). In cotton, analysis of the GSK gene family found six BIN2 homologous genes in G. hirsutum including GhSK21, GhSK22, GhSK23, GhSK24, GhSK25, and GhSK26 . GhSK24 corresponds to GhBIN2 in the current study (Table S1). GhBIN2 and the other five homologs existed high sequence similarities. This functional redundancy mediated by additional homologs may affect the effectiveness of genetic engineering, which should be taken into consideration when GhBIN2 is the target of RNA silencing or genetic editing in the crops. BIN2 is the first plant GSK3-like kinase characterized from genetic screening and negatively influences BR signalling to regulate plant growth and development (Li and Nam, 2002;Li et al., 2001). Editing BIN2 could Figure 8 AtBIN2-AtJAZ1 interaction affects JA pathway transduction. (a) and (b) Accumulation of AtJAZ1 induced by Bikinin was inhibited in coi1-2 mutants. 14-day-old Col-0 and coi1-2 mutants were treated with 50 lM Bikinin for 6 h. Total proteins were extracted at different times after treatments and were detected by anti-AtJAZ1 antibody. Actin was used as the internal control. (c) and (d) BIN2 attenuates JAZ1 inhibition on MYC2-mediated repression of PDF1.2 expression. The reporter construct pPDF1.2-LUC with the indicated effector constructs shown in (c) were co-transformed into N. benthamiana leaves for 72 h; leaves transformed with pPDF1.2-LUC and empty vector pGreenII 62-SK were used as a control. Relative LUC activity indicated the PDF1.2 expression level; the control value was set to 1.0. LUC activity indicates the accumulation level of PDF1.2 with JAZ1 or JAZ1-T196A in N. benthamiana leaves. Error bars represent AE SD (n = 3). Each independent experiment contains at least 10 plants per treatment. Asterisks (*P < 0.05, **P < 0.01) indicate statistically significant differences. generate undesired effects on growth and development in plants, but the available evidence shows that BIN2 silenced Arabidopsis plants show better growth performance than that of control plants , suggesting that BIN2 may play a role in balancing plant growth and defence.
In this study, two-hybrid screening, in vivo BiFC, and pulldown assays indicated that BIN2 interacted with twelve of the thirteen JAZ proteins and that the ZIM domain of the JAZ1 protein was responsible for their interactions ( Figure 5). Previous studies showed that the ZIM domain acted as a protein-protein interaction domain, which mediated homo-and heteromeric interactions between JAZ proteins as well as the interaction between JAZ and NINJA (Chini et al., 2009). The interaction between BIN2 and JAZ proteins may influence the function of JAZ dimerization and interaction with NINJA. GSK2 was recently reported to influence the JAZ4-NINJA complex and JAZ4-JAZ11 dimerization in the monocotyledon rice (He et al., 2020). Further experiments are required in dicotyledons, including cotton and Arabidopsis. Subsequent experiments showed that BIN2 could phosphorylate JAZ. Mass spectrometry, in vitro phosphorylation assays and phos-tag mobility shift assays showed that T196 was the phosphorylation site mediated by BIN2, and was required for this regulation ( Figures S4, 6 and 7). In apple (Malus domestica), MdSnRK1.1 was integral to sucrose-induced biosynthesis of anthocyanins due to its interaction with and phosphorylation of MdJAZ18 protein (Liu et al., 2017a). In our study, we showed that BIN2 could act as a new upstream protein kinase to phosphorylate JAZ proteins. This observation reveals that BIN2 facilitates the interaction between the BR and JA signalling pathways.
BIN2 phosphorylation plays dual roles in regulating the stability and activity of its substrates. BIN2 phosphorylates and destabilizes its substrates or inhibits their activities, including BES1, BZR1, SPCH (SPECHESS), EGL3 (ENHANCER OF GLABRA3), TTG1 (TRANSPARENT TESTA GLABRA1), ARF2 (AUXIN RESPONSE FACTOR2), ICE1 (INDUCER OF CBF EXPRESSION1) (Cheng et al., 2014;Gudesblat et al., 2012;He et al., 2002;Vert et al., 2008;Ye et al., 2019;Yin et al., 2002). BIN2 also positively regulates several proteins (Cai et al., 2014;Cho et al., 2014;Hu and Yu, 2014;Ye et al., 2012;Zhang et al., 2014). In this study, we revealed that BIN2 destabilized the JAZ protein by direct phosphorylation, and affected JA signalling transduction (Figures 7 and 8). Previous studies reported that SA played a crucial role in the activation of defence responses against biotrophic and hemibiotrophic pathogens (including V. dahliae) and that the SA and JA defence pathways were mutually antagonistic (Beckers and Spoel, 2006;Grant and Lamb, 2006;Mur et al., 2006). We propose that the JAZ protein phosphorylated by BIN2 activates the JA signalling pathway and thus inhibits the SA defence pathways, which enhances plant susceptibility to V. dahliae. In previous study, researchers have revealed that JA mutants like jar1, coi1-16 and cyp94B3 showed less severe disease symptom development than that of the WT control (Scholz et al., 2018). These observations were consistent with our results, showing that the activated JA signalling pathway enhanced plant susceptibility to V. dahliae. Taken together, our results demonstrate the negative role of BIN2 in plant defence against V. dahliae, and advance understanding of the interaction between BR and JA signalling. In the future study, more evidence is needed to clarify whether JA and SA antagonism is essential for BIN2-regulated the resistance to V. dahliae.

Plant materials and culture conditions
The Arabidopsis mutant lines bin2-1, bin2-3, bin2-3 bil1 bil2, and BIN2 overexpression transgenic lines were kindly provided by Dr. Jianming Li of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, and Dr. Shuhua Yang of College of Biological Sciences, China Agricultural University. The Arabidopsis seeds were sterilized according to established methods (Song et al., 2017). Arabidopsis thaliana plants were grown at 22°C under long-light conditions (16 h light/8 h dark) on 1/2 Murashige and Skoog (MS) medium plates containing 0.8% agar. WT Gossypium hirsutum (ZM24) was used in this study, and the cotton plants were grown in a growth chamber at 25°C under 16 h light/8 h dark conditions. The seeds of tobacco Nicotiana benthamiana were sown in soil and grown in a greenhouse under the conditions described in a previous study (Wu et al., 2012).

Quantitative real-time PCR (QRT-PCR)
Total RNA was extracted from Arabidopsis or cotton seedlings using the RNAprep Pure Plant kit (Tiangen, Beijing, China), followed by first-strand cDNA product generation using the PrimeScript RT reagent kit (Takara, Beijing, China). The relative transcript levels were measured using SYBR Premix Ex Taq TM (Tli RNase H Plus) (Takara) and ABI 7900 qRT-PCR System (Applied Biosystems, Foster City, CA). Expression levels of the target genes were normalized to AtACTIN2 in Arabidopsis or GhHISTONE3 in cotton. The 2 ÀDDCt method was applied to calculate the relative expression level of all target genes (Livak and Schmittgen, 2001). The specific primers used in this study were designed by Primer Premier version 5.0 (Table S2).

Virus-induced gene silencing (VIGS) experiments
The conserved cDNA fragments of GhBIN2 (GenBank No.: KM453729) were amplified from the cDNA of G. hirsutum (ZM24), and the corresponding primer pairs used were found in Table S2  . We constructed the pTRV2 vector that contains the coding sequence of GhBIN2 to suppress its expression. The pTRV2 vector (TRV:00) was used as a negative control, and the pTRV2 vector with the cDNA of GhPDS was used as a positive control to indicate the VIGS efficiency. The amplified fragments were cloned into the TRV2 plasmid at the EcoRI-BamHI sites using the In-Fusion HD Cloning Kit (Clontech) according to the manufacturers' protocol. The pTRV1, pTRV2, and pTRV2 derivatives harboring the cDNA fragments of target genes were transformed into Agrobacterium tumefaciens GV3101. Cotton VIGS experiments were performed following established procedures (Pang et al., 2013;Song et al., 2018). The vectors were agro-infiltrated into the cotyledons of 7-day-old cotton seedlings. Two weeks after infiltration, RNA was extracted from these cotton seedlings to measure the expression of the target genes.

Preparation and inoculation of fungal pathogens
The antagonistic defoliating Vd07038 isolate of V. dahliae was used in this study. The fungal strain was cultured on potato dextrose agar (PDA) medium at 25°C for 7 days. Next, the colonies were transformed into Czapek medium (3% (w/v) sucrose, 0.2% (w/v) NaNO3, 0.131% (w/v) K2HPO4, 0.05% (w/ v) KCl, 0.05% (w/v) MgSO4, and 0.002% (w/v) FeSO4), and inoculated on a shaker at 200 rpm at 25°C for 7 days. The conidial suspensions were adjusted with deionized water to a final spore concentration of 10 7 ML À1 for cotton. To inoculate the Arabidopsis, conidial suspensions were diluted with distilled water to 5 9 10 3 mL À1 , and drops (2 lL) were used to inoculate the roots of 2-week-old seedlings . The rate of diseased cotton and the disease index (DI) was calculated as previously described . For Arabidopsis, the DI, the extent of stunting and leaf chlorosis were calculated according to the formulas described previously (Veronese et al., 2003).

Reactive oxygen species (ROS) detection in cotton leaves
After the cotton seedlings were inoculated with V. dahliae for 24 h, cotton leaves were collected and washed using distilled water. The production and accumulation of ROS species were detected using the 3,3 0 -Diaminobenzidine (DAB) staining method as previously described (Reissig et al., 1955). An optical microscope (Nikon, Tokyo, Japan) was used to observe and photograph the stained ROS patches.

Verticillium dahliae recovery assay
To identify the effect of infection by V. dahliae, cotton stem fragments (4.5 cm) from the first stem node at 20 days postinoculation (dpi) were analysed. These stem segments were surface-sterilized with 75% ethanol for 1 min, followed by 5% NaClO for 45 s, and then rinsed with sterile water three times. These fragments were sliced into five parts, placed on potato dextrose agar plates, and incubated at 25°C. Each experiment was carried out using three biological replicates.

Two-hybrid screening
The full-length encoding sequences of AtBIN2, AtBIL1, AtBIL2, and GhBIN2 were cloned into the bait vector pGBKT7. The fulllength cDNA sequences of AtJAZs, AtOPR3, AtJAR1, AtCOI1, AtNINJA, AtMYC2, and GhJAZ2 were fused into the prey vector pGADT7. These constructs were transformed into the yeast strain Y2HGold (Clontech) and grown on SD-Leu-Trp (-LW) plates. The yeast transformants were screened on SD-Leu-Trp-His-Ade (-LWHA) selective medium. Primers used for constructing various clones in this study are listed in Table S2.

Bimolecular fluorescence complementation (BiFC) assays
The full-length cDNA sequences of AtBIN2 were amplified and cloned into pUC-SPYNE to generate an N-terminal in-frame fusion with nYFP. Full-length AtJAZ1 encoding sequences were inserted into pUC-SPYCE to generate a C-terminal in-frame fusion with cYFP. The corresponding primer pairs used are shown in Table S2. The resulting vectors were introduced into Agrobacterium tumefaciens strain GV3101 and transformed into N. benthamiana plants. Infected leaves were analysed 48 h after infiltration using a confocal microscope (Olympus FV1200).

Purification of recombinant proteins and pull-down assays
AtBIN2 full-length cDNA was cloned into the pGEX4T-1 vector with the GST-tag, and AtJAZ1 full-length cDNA was cloned into the pET32a vector with the His-tag. The resulting plasmids were transformed into E. coli strain BL21 (DE3). The recombinant proteins AtBIN2-GST and AtJAZ1-His were purified with Glutathione Sepharose TM 4 Fast Flow and Ni Sepharose TM 6 Fast Flow (GE Healthcare, Pittsburgh, PA), respectively, according to the manufacturers' instructions. The pull-down assays were performed as described in previous studies (Wang et al., 2011).
Proteins retained on the beads were analysed by immunoblotting with an anti-GST or anti-His antibody.

In vitro phosphorylation assay
The in vitro phosphorylation assay was performed according to standard methods (Ding et al., 2015). Purified recombinant protein combinations were incubated in protein kinase assay buffer with 20 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 100 mM NaCl, 25 mM ATP, 1 µCi [c-32 P] ATP, and 1 mM DTT at 30°C for 30 min. The reactions were halted by the addition of a 59 SDS loading buffer. The phosphorylated protein was visualized by autoradiography after separation in a 10% SDS-PAGE gel and detected by a Typhoon 9410 imager. Coomassie brilliant blue was used as a loading control.

Identification of phosphorylation site
To identify the putative phosphorylation sites of BIN2 in JAZ1, we performed LC-MS/MS experiments. Purified GST-BIN2 and His-JAZ1 proteins were incubated in 20 lL of protein kinase assay buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 100 mM NaCl, 25 mM ATP, and 1 mM DTT) at 30°C for 30 min. The reaction mixture was reduced by DTT, alkylated by iodoacetamide (IAM), and digested by trypsin overnight at 37°C. The Titansphere Phos-TiO Kit (GL Sciences) was used to enrich the phosphopeptides according to the manufacturer's protocol. The resulting phosphopeptides were analysed by LC-MS/MS, performed as previously described (Liu et al., 2017b).

Phos-tag mobility shift assay
The Phos-tag mobility shift assay was performed as described previously (Mao et al., 2011). Total proteins extracted from protoplasts were separated in a 12% SDS-PAGE gel containing 50 mM Phos-tag and 200 mM MnCl 2 . After electrophoresis, the gel was washed three times in transfer buffer (50 mM Tris, 40 mM Glycine) for 10 min each time. Then, the gel was transferred to a polyvinylidene fluoride membrane. The AtJAZ1-MYC was detected with the anti-MYC antibody. Anti-FLAG antibody was used to detect AtBIN2-HF. The CIAP treatments were conducted by adding CIAP with its reaction buffer to total proteins as described by the manufacturer before SDS-PAGE. The integrated optical density (IOD) values of the indicated signals were quantified using the software Image-Pro Plus (Media Cybernetics).

JA content measurements
Two-week-old Arabidopsis seedlings were harvested, and measurements of JA contents were performed as described previously (Song et al., 2017). Three biological experiments which consisted of 10 plants per experiment were measured.

GUS staining and GUS activity quantification
The GUS staining and activity quantification assay was performed as described previously (Menand et al., 2002). Images were captured using a stereomicroscope (OLYMPUS MVX10). We used a MarkerGene TM b-glucuronidase (GUS) reporter gene activity detection kit (Marker Gene Technologies) to quantify GUS activity. The Bradford assay was used for total protein quantification. Three biological experiments, each consisting of 20 plants per treatment, were measured.

Cell-free protein degradation assay
The cell-free protein degradation assay was performed as described previously (Liu et al., 2010). Total proteins were extracted from 14-day-old seedlings in a degradation buffer (50 mM Tris-MES pH 8.0, 0.5 M Sucrose, 1 mM MgCl 2 , 10 mM EDTA pH 8.0, and 5 mM DTT). Purified recombinant His-JAZ1 or His-JAZ1 T196A was added to an equal amount of the indicated proteins and incubated at 25°C for 0, 30, and 60 min. His-JAZ1 or His-JAZ1 T196A was separated on SDS-PAGE and detected with the anti-His antibody. The integrated optical density (IOD) values of the indicated signals were quantified using the software Image-Pro Plus (Media Cybernetics).

Dual-luciferase reporter assays
The transient dual-luciferase reporter assays were performed as described previously (Hellens et al., 2005). The promoter sequences of AtPDF1.2 were amplified and cloned into pGreenII 0800-LUC at the PstI and SpeI sites. The coding regions of AtMYC2, AtJAZ1, AtJAZ1 T196A , and AtBIN2 were amplified and cloned into pGreenII 62-SK at the SpeI and EcoRI sites. Vectors were co-transformed into 4-weeks-old N. benthamiana leaves and expressed for 72 h, and the images were captured by CCD. These leaves were collected and grounded in liquid nitrogen. Firefly luciferase and Renilla spp. luciferase activities were quantified using the Dual-Luciferase Reporter Assay System (Promega) with a multimode plate reader (PerkinElmer, Waltham, MA).

Antibody validation
Immunoblotting was performed to detect the polyclonal antibody anti-AtJAZ1 (Agrisesera, Sweden). The wildtype Col-0, AtJAZ1overexpression transgenic lines and jaz1 mutant were used for protein extraction, and one specific band for this antibody was detected within the total protein fraction of plants tested ( Figure S6).

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.

Figure S1
Disease index assessment of GhBIN2 over-expression transgenic lines. Figure S2 Phenotypes of A. thaliana BIN2 mutants in defense response to V. dahliae. Figure S3 BIN2, BIL1, and BIL2 function redundantly in interactions with JAZ proteins. Figure S4 Mass spectrometry analysis of BIN2 phosphorylation sites in JAZ1. Figure S5 BIN2 induces the degradation of AtJAZ1. Figure S6 Validation of AtJAZ1 antibody. Table S1 Characteristics of BIN2 genes in the cotton genome.