VaCRK2 Mediates Gray Mold Resistance in Vitis amurensis by Activating the Jasmonate Signaling Pathway

Cysteine-rich receptor-like kinases (CRKs) are ubiquitous plant receptor-like kinases, which play a significant role in plant disease resistance. Gray mold is an economically important disease of grapes caused by Botrytis cinerea. However, CRK genes and their function in gray mold disease resistance in grapes have not been elucidated. This study aimed to identify and characterize CRKs in grapes and determine their role in gray mold resistance. Four CRKs were identified in Vitis amurensis and named VaCRK1–VaCRK4 according to their genomic distribution. The four VaCRKs were ectopically expressed in Arabidopsis thaliana to study their function in defense response against B. cinerea. Heterologous expression of VaCRK2 in A. thaliana conferred resistance to B. cinerea. VaCRK2 expression in gray mold-resistant grape cultivar increased significantly after B. cinerea inoculation and methyl jasmonate treatment. Furthermore, the expression of jasmonic acid (JA) signaling pathway-related genes in VaCRK2 overexpression lines of A. thaliana was significantly increased after B. cinerea inoculation, leading to the upregulation of pathogenesis-related (PR) genes and reactive oxygen species (ROS) accumulation. Overall, these results suggest that VaCRK2 confers resistance to B. cinerea by activating PR gene expression and oxidative burst through the JA signaling pathway.


Introduction
Two layers of immune system response have evolved during the arms race between plants and phytopathogens, including bacteria, fungi and viruses [1]. One involves the recognition of specific molecular motifs conserved within a class of pathogens, also known as pathogen-associated molecular patterns (PAMP), through receptors located on the plant cell membrane, thereby activating the PAMP-triggered immunity (PTI) to resist pathogen invasion [2]. However, sometimes pathogens manage to bypass PTI by secreting effectors into the plant cell. In response, plants evolved another form of immune response involving resistance (R) proteins. These proteins perceive specific virulence effectors secreted by pathogens, thereby triggering the effector-triggered immunity (ETI), a more robust immune response against pathogen attack [3]. Recent studies have found that ETI can increase the expression of core PTI components, thereby amplifying the PTI and inducing a durable immune response [4]. For example, ETI enhances the expression of respiratory burst oxidase homolog D (RBOHD) protein, while PTI promotes the complete activation of RBOHD protein. This cooperation mechanism ensures that plants mount a rapid and precise response against pathogen infection while maintaining optimal growth and development [4].
PTI response is activated when receptors on the plant cell membrane interact with specific pathogen molecular patterns. Plants use a variety of plasma membrane-bound receptor proteins to sense exogenous immune signals [5]. These cell surface receptor proteins (PF01657) domain and Pkinase (PF00069) domain in V. amurensis genome with an E-value cutoff of e −5 [22]. SignalP-5.0 (http://www.grapeworld.cn/am/download/ (accessed on 15 December 2020)) and TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/ TMHMM/ (accessed on 15 December 2020)) were used to verify the presence of signal peptides and transmembrane domain, respectively [23,24]. Putative genes were further verified based on the SMART website and InterproScan database [25,26].

Analysis of Subcellular Localization of VaCRK2
The primers of VaCRK2 gene were designed according to the full-length VAG0104573.1 sequence. The full-length VaCRK2 gene was amplified by PCR using cDNA of V. amurensis cultivar (cv. Shuangyou) as template. Purified PCR products were then inserted into pBin-GFP4 vector, fused with GFP gene, under the control of cauliflower mosaic virus 35S promoter [6]. The vectors p35S:GFP and p35S:VaCRK2 were transiently expressed in tobacco leaf epidermal cells using the Agrobacterium tumefaciens infiltration method [12]. Laser scanning confocal microscope (LSMT-PMT) was used to examine the subcellular localization of the proteins at 488 and 510 nm excitation and emission wavelengths, respectively.

Grape Plant Materials and Treatments
Grape species V. amurensis cv. Shuangyou and V. vinifera cv. Red Globe were used for the experiments. Notably, cv. Shuangyou is resistant to B. cinerea, whereas cv. Red Globe is susceptible. B. cinerea strain BcSD3 (highly virulent strain) was cultured in potato dextrose agar (PDA) medium to produce conidia. Conidia were eluted with sterile deionized water and adjusted to a concentration of 2 × 10 6 conidia/mL. Grape leaves were inoculated with BcSD3 and harvested at 0, 2, 6, 12, 24, 48, 72 and 120 h time points to assess resistance to B. cinerea. Grape leaves were sprayed with 10 mM methyl jasmonate (MeJA), 10 mM salicylic acid (SA), 10 mM ethephon (ETH) and 100 mM abscisic acid (ABA) to explore related signaling pathways. Leaves of control plants were sprayed with sterile distilled water. The leaves were collected at 0, 2, 6, 12, 24, 48 and 72 h time points. Six leaves per replicate, repeated three times, were harvested at each time point, frozen in liquid nitrogen and stored at −80 • C for subsequent analysis.

Generation and Analysis of Transgenic A. thaliana
The ORF sequence of VaCRK2 gene was integrated into vector pBI121 under the control of cauliflower mosaic virus 35S promoter. The vector was then transformed into Agrobacterium tumefaciens strain GV3101 using freeze-thaw method [18]. VaCRK2 gene was introduced into A. thaliana wildtype Col-0 using A. tumefaciens mediated transformation [27]. Putatively transformed events were analyzed using PCR and RT-PCR to identify T 3 homozygous transgenic plants. Genomic DNA and cDNA from A. thaliana wildtype Col-0 were used as the control for PCR and RT-PCR detection [19].

Evaluation of Transgenic A. thaliana for Resistance against B. cinerea
A. thaliana wildtype Col-0 and VaCRK2 overexpression lines (OE1, OE2 and OE3) were used for the experiments. BcSD3 strain was cultured in PDA medium for five days. Conidia were eluted with sterile deionized water and adjusted to a concentration of 2 × 10 6 conidia/mL. Three-week-old A. thaliana plants were inoculated with B. cinerea conidia suspension. After inoculation, the plants were incubated at 22 • C under 16 h photoperiod with a humidity of 80-100%. A total of 24 leaves of six plants (four leaves per plant) were inoculated in one experiment, and the experiment was repeated thrice. The lesion diameter and fungal biomass were determined three days post-inoculation. Six leaves per replicate were harvested at each time point. The samples were frozen in liquid nitrogen and stored at −80 • C for subsequent analysis.

Oxidative Burst Assay in A. thaliana
A. thaliana wildtype Col-0 and VaCRK2 overexpression 1 line (OE1) were used for the experiments. Reactive oxygen species accumulation was determined by diaminobenzidine (DAB) staining. Three-week-old A. thaliana leaves were infiltrated with 10 µL conidia suspension of B. cinerea. The A. thaliana leaves were infiltrated with sterile water as a control. After 12 h, the leaves were immersed in DAB buffer and vacuum infiltrated at 25 • C in the dark. The reaction was terminated after 12 h, and the leaves were washed with sterile water, bleached using 75% ethanol, and then immersed in 30% glycerin. ImageJ software was used to determine the percentage of brown pixels in the leaves. Antioxidant enzymes, including peroxidase (POD) and catalase (CAT), were extracted from A. thaliana leaves and measured as described by Guo et al. [18].

Relative Gene Expression Analysis
PLANTeasy kit (YPH-Bio, Beijing, China) was used to extract total RNA. After extraction and quality verification, the RNA samples were reverse transcribed to cDNA using TIANSeq M-MLV (RNase H-) reverse transcriptase (Tiangen, Beijing, China) according to the manufacturer's instructions. Then, RT-qPCR analysis was performed using the SYBR Premix Ex Taq kit (TaKaRa, Kusatsu, Japan) on the QuantStudio 6 Flex qPCR System (Applied Biosystems, Foster City, CA, USA). The 2 −∆∆CT method was used to determine relative gene expression [28]. V. vinifera EF1γ (CB977561), A. thaliana UBQ5 (NM_116090.3) and APT1 (NM_102509.4) were used as the internal reference genes. The primer sequences are listed in Table S1.

VaCRK2 Enhances Resistance to B. cinerea in A. thaliana
Four cysteine-rich receptor-like kinase (CRK) genes were identified in V. amurensis genome and named VaCRK1-VaCRK4 according to their position in the genome. The protein encoded by VaCRK genes comprises 200 to 420 amino acids in length. The predicted molecular weight is 48 to 66 KDa, and the isoelectric point ranges from 3.7 to 5.2 (Table S2). The four VaCRK genes were transformed into A. thaliana wildtype Col-0, and at least three overexpression lines were obtained following PCR and RT-PCR analyses. The disease assay results showed that the lesion area and fungal biomass of VaCRK2 overexpression lines were 56% and 40% lower than those of the wildtype Col-0, respectively, indicating that the VaCRK2 overexpression lines were more resistant to B. cinerea than the wildtype Col-0 ( Figure 1). Notably, transgenic A. thaliana lines overexpressing the other three VaCRK genes VaCRK1, VaCRK3 and VaCRK4 showed no significant improvement in resistance to B. cinerea compared with the wildtype Col-0 ( Figure S1).

VaCRK2 Localizes to Plasma Membrane
Conserved domain analysis of VaCRK2 peptide sequence using SMART identified a typical CRK family protein with signal peptide and TM domain, which were confirmed by SignalP5.0 (1-25 aa) and TMHMM2.0 (266-288 aa), respectively ( Figure 2A). To further understand the function of VaCRK2, the subcellular localization was verified by transient expression of VaCRK2 and green fluorescent fusion protein (VaCRK2-GFP) in tobacco leaf epidermal cells. According to the results, the fluorescent signal of VaCRK2-GFP fusion protein was clearly localized to the cell membrane, in contrast to the fluorescence signal of GFP proteins, which was prevalent throughout the foliar cells in tobacco, indicating that VaCRK2 was localized on the cell membrane ( Figure 2B).

VaCRK2 Positively Responds to B. cinerea and MeJA Treatment
To determine the relationship between VaCRK2 expression and B. cinerea resistance, we compared the expression patterns of VaCRK2 between the resistant and susceptible grape cultivars following B. cinerea inoculation. VaCRK2 expression was significantly upregulated in resistant cv. Shuangyou from 2 to 24 h post-inoculation, after which the expression Agronomy 2021, 11, 1672 5 of 12 was reduced ( Figure 3A). In contrast, no significant change in VaCRK2 expression was observed in susceptible cv. Red Globe during the entire experimental period, indicating that VaCRK2 may mediate B. cinerea resistance in grapes. To explore VaCRK2-related signaling pathways, we analyzed the expression patterns of VaCRK2 after MeJA, SA, ETH and ABA treatments. VaCRK2 was significantly upregulated in resistant cv. Shuangyou after treatment with methyl MeJA within 120 h ( Figure 3B) and less affected by treatment with ET ( Figure 3D), but it was downregulated by treatment with SA or ABA at several points ( Figure 3C,E), which suggested that the VaCRK2 response of the resistant cultivar to B. cinerea is mediated by JA signaling pathway. Overall, these findings show that B. cinerea infection and exogenous MeJA induce VaCRK2 expression in gray mold-resistant cv. Shuangyou.

VaCRK2 Localizes to Plasma Membrane
Conserved domain analysis of VaCRK2 peptide sequence using SMART identified a typical CRK family protein with signal peptide and TM domain, which were confirmed by SignalP5.0 (1-25 aa) and TMHMM2.0 (266-288 aa), respectively (Figure 2A). To further understand the function of VaCRK2, the subcellular localization was verified by transient expression of VaCRK2 and green fluorescent fusion protein (VaCRK2-GFP) in tobacco leaf epidermal cells. According to the results, the fluorescent signal of VaCRK2-GFP fusion protein was clearly localized to the cell membrane, in contrast to the fluorescence signal of GFP proteins, which was prevalent throughout the foliar cells in tobacco, indicating that VaCRK2 was localized on the cell membrane ( Figure 2B).

VaCRK2 Mediates B. cinerea Resistance Via the JA Signaling Pathway
Jasmonic acid (JA) signaling pathway plays a vital role in plant disease resistance, especially against saprophytic fungal diseases [29]. Therefore, in order to verify whether VaCRK2 mediates resistance to B. cinerea through JA signaling pathway, we firstly detected the expression pattern of AtCRK2, a homolog of VaCRK2 in A. thaliana, and the results showed that the expression of AtCRK2 gene was not induced in A. thaliana wildtype Col-0 inoculated with B. cinerea ( Figure S2). Secondly, we overexpressed VaCRK2 gene in JA-deficient A. thaliana mutant jar1 (SALK_030821); compared with the mutant jar1, the overexpressed line could not improve the resistance to B. cinerea ( Figure S3). Thirdly, we Agronomy 2021, 11, 1672 6 of 12 examined the expression patterns of genes related to JA signaling pathway in A. thaliana wildtype Col-0 and VaCRK2 overexpression lines after inoculation with B. cinerea; the results showed that 11 JA signaling pathway-related genes, including AOC1, AOS, OPR3, LOX2, ACX, COI1, VSP2, ORA59, JAR1 and MYC2, were upregulated in both wildtype Col-0 and VaCRK2 overexpression lines but significantly more upregulated in VaCRK2 overexpression lines than in the wildtype Col-0 ( Figure 4, Figures S4 and S5). Altogether, these results indicate that VaCRK2 interacts with the JA signaling pathway to mediate B. cinerea resistance in A. thaliana.

VaCRK2 Positively Responds to B. cinerea and MeJA Treatment
To determine the relationship between VaCRK2 expression and B. cinerea resistance, we compared the expression patterns of VaCRK2 between the resistant and susceptible grape cultivars following B. cinerea inoculation. VaCRK2 expression was significantly upregulated in resistant cv. Shuangyou from 2 to 24 h post-inoculation, after which the expression was reduced ( Figure 3A). In contrast, no significant change in VaCRK2 expression was observed in susceptible cv. Red Globe during the entire experimental period, indicating that VaCRK2 may mediate B. cinerea resistance in grapes. To explore VaCRK2-related signaling pathways, we analyzed the expression patterns of VaCRK2 after MeJA, SA, ETH and ABA treatments. VaCRK2 was significantly upregulated in resistant cv. Shuangyou after treatment with methyl MeJA within 120 h ( Figure 3B) and less affected by treatment with ET ( Figure 3D), but it was downregulated by treatment with SA or ABA at several points ( Figure 3C,E), which suggested that the VaCRK2 response of the resistant cultivar to B. cinerea is mediated by JA signaling pathway. Overall, these findings show that B. cinerea infection and exogenous MeJA induce VaCRK2 expression in gray mold-resistant cv. Shuangyou.

VaCRK2 Overexpression Affects PR Gene Expression and ROS Accumulation
Pathogenesis-related (PR) gene expression and ROS accumulation in A. thaliana wildtype Col-0 and VaCRK2 overexpression lines were evaluated after B. cinerea inoculation to further verify that VaCRK2 facilities defense response against B. cinerea. The expression levels of JA signaling pathway defense-related genes (PDF1.2, PR3 and PR4) were significantly higher in the VaCRK2 overexpression lines than in the wildtype Col-0 after inoculation with B. cinerea ( Figure 5A, Figures S6 and S7). Furthermore, ROS accumulation in the leaves of VaCRK2 overexpression lines was significantly higher than in wildtype Col-0 relative to sterile water treatment ( Figure 5B,C). Peroxidase and catalase activity detection results showed that there was no significant difference in peroxidase and catalase activities between wildtype Col-0 and overexpression line of A. thaliana under control conditions. After inoculation with B. cinerea, the peroxidase and catalase activities in wildtype Col-0 and overexpression line of A. thaliana were increased, and the overexpression line showed significantly higher changes than wildtype Col-0 ( Figure S8). Altogether, these

VaCRK2 Mediates B. cinerea Resistance Via the JA Signaling Pathway
Jasmonic acid (JA) signaling pathway plays a vital role in plant disease resistance, especially against saprophytic fungal diseases [29]. Therefore, in order to verify whether VaCRK2 mediates resistance to B. cinerea through JA signaling pathway, we firstly detected the expression pattern of AtCRK2, a homolog of VaCRK2 in A. thaliana, and the results showed that the expression of AtCRK2 gene was not induced in A. thaliana wildtype Col-0 inoculated with B. cinerea ( Figure S2). Secondly, we overexpressed VaCRK2 gene in JA-deficient A. thaliana mutant jar1 (SALK_030821); compared with the mutant jar1, the overexpressed line could not improve the resistance to B. cinerea ( Figure S3). Thirdly, we examined the expression patterns of genes related to JA signaling pathway in A. thaliana wildtype Col-0 and VaCRK2 overexpression lines after inoculation with B. cinerea; the results showed that 11 JA signaling pathway-related genes, including AOC1, AOS, OPR3, LOX2, ACX, COI1, VSP2, ORA59, JAR1 and MYC2, were upregulated in both wildtype Col-0 and VaCRK2 overexpression lines but significantly more upregulated in VaCRK2 overexpression lines than in the wildtype Col-0 (Figures 4, S4 and S5). Altogether, these results indicate that VaCRK2 interacts with the JA signaling pathway to mediate B. cinerea resistance in A. thaliana.

VaCRK2 Overexpression Affects PR Gene Expression and ROS Accumulation
Pathogenesis-related (PR) gene expression and ROS accumulation in A. thaliana wildtype Col-0 and VaCRK2 overexpression lines were evaluated after B. cinerea inoculation to further verify that VaCRK2 facilities defense response against B. cinerea. The expression levels of JA signaling pathway defense-related genes (PDF1.2, PR3 and PR4) were significantly higher in the VaCRK2 overexpression lines than in the wildtype Col-0 after inoculation with B. cinerea ( Figures 5A, S6 and S7). Furthermore, ROS accumulation in the leaves of VaCRK2 overexpression lines was significantly higher than in wildtype Col-0 relative to sterile water treatment ( Figure 5B,C). Peroxidase and catalase activity detection results showed that there was no significant difference in peroxidase and catalase activities between wildtype Col-0 and overexpression line of A. thaliana under control conditions. After inoculation with B. cinerea, the peroxidase and catalase activities in wildtype Col-0 and overexpression line of A. thaliana were increased, and the overexpression line showed significantly higher changes than wildtype Col-0 ( Figure S8). Altogether, these results suggest that VaCRK2 mediates resistance to B. cinerea by upregulating PR genes and inducing oxidative burst in A. thaliana.

Discussion
Cysteine-rich receptor-like kinases (CRKs) have been shown to play an essential role in mediating disease resistance in several plants, including Arabidopsis [10,11,30], rice [13], barley [14], wheat [31] and Medicago truncatula [32]. However, CRK genes and their function in disease resistance have not been demonstrated in grapes. This study characterized CRK genes in V. amurensis (VaCRK) and evaluated their role in mediating B. cinerea resistance. The VaCRK genes identified in this study have a signal peptide and two DUF26 motifs at the N-terminus, a TM domain in the middle and a kinase domain at the C-terminus, which are typical domains of a CRK gene. Based on the CRK gene identification

Discussion
Cysteine-rich receptor-like kinases (CRKs) have been shown to play an essential role in mediating disease resistance in several plants, including Arabidopsis [10,11,30], rice [13], barley [14], wheat [31] and Medicago truncatula [32]. However, CRK genes and their function in disease resistance have not been demonstrated in grapes. This study characterized CRK genes in V. amurensis (VaCRK) and evaluated their role in mediating B. cinerea resistance. The VaCRK genes identified in this study have a signal peptide and two DUF26 motifs at the N-terminus, a TM domain in the middle and a kinase domain at the C-terminus, which are typical domains of a CRK gene. Based on the CRK gene identification criteria, five candidate genes were identified in V. amurensis. However, VAG0113336.1 and VAG0128137.1 genes were the same and were combined into one gene, VaCRK3 (Table S2). Among the four CRK genes identified in V. amurensis, VaCRK2 was the only one shown to confer resistance to B. cinerea effectively. VaCRK2 is located on the cell membrane and significantly increased the resistance of A. thaliana overexpression lines to B. cinerea (Figures 1 and 2). Studies have shown that ETI promotes PTI by inducing the expression of PTI signal components, while PTI ensures the normal resistance function of ETI through diverse signals [33]. Enhanced expression of immune system-related genes, especially PTI genes, is crucial in mediating plant disease resistance [34]. VaCRK2 expression was significantly increased in resistant cv. Shuangyou at several time points after B. cinerea inoculation, but not in susceptible cv. Red Globe ( Figure 3A), suggesting that VaCRK2 potentially mediates B. cinerea resistance and its expression is cultivar-dependent. At the same time, it is speculated that this result may be because the host increased the transcription level of VaCRK2 gene through immune regulation after sensing the signal of B. cinerea, especially in the early stage, to resist the infection of B. cinerea [16,17]. Perhaps because of this, different grape varieties have resistant and susceptible phenotypes to pathogens, which is consistent with the disease resistance mediated by CRKs in other plants [6].
JA is an essential signaling molecule in plants and facilitates various physiological reactions, including seed germination, damage response and biological stress response [29]. In this study, exogenous MeJA application induced the expression of VaCRK2 gene ( Figure 3B), indicating that B. cinerea resistance mediated by VaCRK2 may be related to the JA signaling pathway. VaCRK2 gene expression was upregulated at 12 and 24 h after ETH treatment and downregulated at 6, 12 and 72 h after SA treatment ( Figure 3C,D), which may be because ETH and JA act synergistically, and SA has negative feedback regulation on JA [29]. The JA signaling pathway activates diverse defense-related proteins and secondary metabolites in response to necrotrophic fungal invasion [29]. B. cinerea, the causative agent of gray mold disease, is a typical necrotrophic fungus infecting several plant species. Several genes related to JA signaling pathway have been cloned from different plants. These include JA self-synthesis genes, such as LOX2, AOS and OPR3, and defense-related genes, such as PDF1.2, PR3 and PR4 [35]. In this study, several genes related to JA signaling pathway were upregulated in A. thaliana VaCRK2 overexpression lines compared with A. thaliana wildtype Col-0 after B. cinerea inoculation (Figure 4, Figures S2 and S8). The JA signaling pathway has been associated with gray mold resistance in different plants, including A. thaliana, cucumber and tomato [36][37][38]. For example, A. thaliana phyB and SDG8 genes participate in the resistance defense against B. cinerea by regulating JA biosynthesis [39,40]. In addition, SlMYB75 plays an active role in the resistance of tomato to B. cinerea by regulating the JA signaling pathway [38]. Therefore, we speculated that the JA signaling pathway may be essential in VaCRK2-mediated resistance against B. cinerea.
PR gene expression is upregulated in many plants after inoculation with phytopathogens, including fungi, bacteria or viruses, to attack pathogens and inhibit pathogen infection [41]. Oxidative burst plays a vital role in plant disease resistance by directly neutralizing pathogens and amplifying signals of other immune events [4]. Notably, ROS accumulation can directly kill the invading pathogens and activate cell wall cross-linking to restrict further pathogen invasion [42]. Plant resistance proteins, including CRK proteins, recognize specific pathogenic effectors and initiate various defense responses, such as PR gene overexpression and ROS accumulation [43,44]. For instance, overexpression of CRK5, CRK6, CRK36 and CRK45 in A. thaliana significantly enhances the resistance to Pseudomonas syringae pv. tomato DC3000 by upregulating PR genes and enhancing ROS accumulation [9,30,45]. CRK6 and CRK7 genes play an essential function in extracellular ROS signal transduction in A. thaliana [46]. Various defense responses, including hormone induction, PR gene overexpression and ROS accumulation, are key to grape resistance against gray mold disease [16,47]. In this study, JA signaling pathway defense-related PR gene expression and ROS accumulation in A. thaliana VaCRK2 overexpression lines were significantly higher than in wildtype Col-0 after B. cinerea inoculation ( Figure 5, Figures S3 and S6), indicating that VaCRK2 mediates gray mold resistance through JA signaling pathway defense-related PR gene expression and ROS accumulation.

Conclusions
This study identified and characterized CRK genes in V. amurensis and validated their role in B. cinerea resistance. The overexpression of VaCRK2 significantly increased the resistance to B. cinerea in A. thaliana, suggesting that VaCRK2 may play a crucial role in gray mold resistance. Furthermore, VaCRK2 expression significantly induced the JA signaling pathway-related genes, PR gene expression and ROS accumulation after B. cinerea inoculation, indicating that VaCRK2 mediates B. cinerea resistance by interacting with the JA signaling pathway and activating oxidative burst. This study provides new insights into the role of CRKs in plant disease resistance and will inform future research on the molecular breeding of grapes for resistance against gray mold disease.