Strawberry WRKY Transcription Factor WRKY50 Is Required for Resistance to Necrotrophic Fungal Pathogen Botrytis cinerea

: WRKY protein is one of the largest plant-speciﬁc transcription factors that plays critical roles in plant stress responses, but few WRKY transcription factors have been functionally analyzed in strawberry. In this study, a Botrytis cinerea response WRKY gene, FvWRKY50 , was isolated from the woodland strawberry. Expression analysis indicated that the transcript of FvWRKY50 was gradually decreased with fruit ripening, but was signiﬁcantly induced by B. cinerea infection in mature strawberry fruit. Subcellular localization assay revealed that FvWRKY50 was localized in the nucleus. Several cis -elements related to pathogen responses were observed in the promoter region of FvWRKY50 . Pathogen infection assay indicated that overexpression of FvWRKY50 in strawberry fruit signiﬁcantly enhanced their resistance against B. cinerea , while the silencing of FvWRKY50 dramatically compromised their disease-resistant ability. The expression levels of several genes involved in jasmonic acid (JA) biosynthesis, signaling transduction, and antimicrobial protein biosynthesis were regulated to diverse extents in FvWRKY50 overexpressed and silenced fruit. Collectively, our study inferred that FvWRKY50 is a positive regulator that mediates resistance against B. cinerea through regulating some JA pathway and defense-related genes.


Introduction
Strawberry is an important fruit with high nutritional and economical value, which has been appreciated by consumers worldwide. Similar to other plants, the strawberries are frequently attacked by various pathogens such as viruses, bacteria, fungi, and nematodes during their growth processes [1]. Among these pathogens, fungi are the most harmful to strawberry. Several disastrous diseases are caused by fungi pathogen infection. For example, the Fusarium wilt disease caused by Fusarium oxysporum f. sp. Fragariae [2], anthracnose disease caused by Colletotrichum spp fungi [3], powdery mildew disease invaded by Podosphaera aphanis [4], and gray mold caused by Botrytis cinerea [1].
Among these diseases, B. cinerea, which causes gray mold, is considered to be one of the most serious diseases to the strawberry industry, for it can lead to the infected tissues rotting in a short period, which consequently results in large economical losses. Currently, the predominant method for controlling B. cinerea in strawberry production is applying fungicides [5], but the effects are usually inferior due to the rapid evolution of fungicide resistance of B. cinerea [6]. In addition, the frequent use of fungicides increases the risk of food safety [7]. Therefore, exploring the gray mold disease-resistant mechanisms and further breeding cultivars with high pathogen-resistant capability are effective and sustainable alternatives for the strawberry industry.
Plants have developed sophisticated defense mechanisms in response to pathogen infection. To date, a large set of genes involved in plant immunity have been identified. octoploid strawberry Fragaria x ananassa cultivar 'Benihoppe' were used in this study. Plants were grown in a greenhouse; tissues of diploid strawberry were used for tissue expression analysis and octoploid strawberry fruit were used for transient expression and B. cinerea infection assay. Strawberry fruit infection assay was performed as previously described [38]. Tissues used for gene expression analysis were harvested at the indicated time point, immediately frozen by liquid nitrogen and then stored at −80 • C until RNA extraction.

RNA Isolation and Gene Expression Analysis
Total RNA of woodland strawberry or cultivated strawberry fruit was extracted by Plant total RNA Isolation Kit (Foregene, Chengdu, China) following the manufacturer's protocol. Reagent for reverse transcription or quantitative reverse transcription polymerase chain reaction (RT-qPCR) were all produced by Takara (Dalian, China). M-MLV reverse transcriptase was used for cDNA synthesis, and SYBR Premix Ex Taq was used for RT-qPCR.
RT-qPCR was conducted on the real-time system (CFX96, Bio-Rad, Hercules, CA, USA). FvGAPDH2 was selected as the internal control for gene tissue expression assay, and FaActin was used as internal control for B. cinerea induction assay. The transcription level of detected genes was calculated using the 2 −∆∆CT method [39]. Three biological replicates and three technical replicates were conducted. All the primers were listed in the Supplementary Table S1.

Gene Cloning and Vector Construction
The full length of FvWRKY50 was cloned from woodland strawberry accession Havaii-4. After sequence verification, the gene was cloned into the binary vector pJX003 downstream of the CaMV 35S promoter in frame with the GFP tag (35S:FvWRKY50-GFP) for subcellular localization assay. For transient assay, FvWRKY50 was cloned into binary vector pJX001 under the control CaMV 35S promoter (35S:FvWRKY50) for overexpression. A specific fragment of FvWRKY50 was amplified and then cloned into pFGC5941 vector for gene silence assay. Primers were listed in the Supplementary Table S1.

Bioinformatics Analysis of FvWRKY50
For phylogenetic analysis, the protein sequences of FvWRKY50 and selected WRKY TFs from strawberry and Arabidopsis were aligned with the Clustal W algorithm and phylogenic tree was generated by MEGA software (version 7.0) through using neighbor-joining method with 1000 bootstrap replicates [40]. A 2000 bp fragment of the promoter region of FvWRKY50 was downloaded from JGI Phytozome 13 database (https://phytozome-next.jgi.doe.gov/, accessed on 16 September 2021). PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 13 July 2021) was used to analyze the cis-regulate elements of FvWRKY50 promoter.

Subcellular Localization Analysis of FvWRKY50
The binary vector containing 35S:FvWRKY50-GFP expression cassette was transiently expressed in tobacco leaves by Agrobacterium-mediated infiltration. The expressed fluorescence signal was detected using a laser scanning confocal microscope (FV3000, Olympus, Tokyo, Japan).

Strawberry Fruit Infiltration and Sample Treatment
Strawberry fruit transient expression assay was performed as described by Zhao et al. [41]. Strawberry fruit were divided into four treatments, i.e., fruit transiently expressed the FvWRKY50 overexpression vector (FvWRKY50-OE), the overexpression empty vector (EV-OE), FvWRKY50 gene silence vector (FvWRKY50-RNAi) and gene silence empty vector (EV-RNAi). Fruit that were infiltrated with empty vectors were defined as control. Three days after infiltration, 20% of the fruit of each treatment were randomly chosen to detect the expression level of FvWRKY50. After detection, the rest strawberry fruit were inoculated with B. cinerea for disease resistance assay. Photographs were taken at 48 h after Agrobacterium infiltration. The lesion area caused by B. cinerea infection on the strawberry fruit surface was calculated by the Image J software [42]. For each treatment, 30 fruit were analyzed, and three biological replicates were analyzed.

Analysis of the Expression of FvWRKY50 in Response to B. cinerea Infection
According to the transcriptome database developed by our previous study [36], we have observed that the expression level of FvWRKY50 is significantly up-regulated by B. cinerea infection at three detected time points (i.e., the 12th h, the 24th h, and the 48th h). Accordingly, we speculate that FvWRKY50 might be involved in strawberry defense response to B. cinerea. To verify our speculation, we first analyze the expression patterns of FvWRKY50 by RT-qPCR in this study. As shown in Figure 1, compared to that of time zero, FvWRKY50 is slightly depressed at the 12th h after B. cinerea infection, but significantly induced along with B. cinerea infection after 24 h and increased to 20 times higher at the 120th h after B. cinerea inoculation. These results reveal that FvWRKY50 is involved in response to B. cinerea infection in strawberry. Mature cultivated strawberry fruit were inoculated with spore suspension (5 µL of 1 × 10 6 spores) of B. cinerea. Tissues around the inoculated sites were collected for the detection of the expression level of FvWRKY50 by RT-qPCR analysis at 0, 12, 24, 48, 72, 96, and 120 h after inoculation. Error bars represent the standard deviations of three biological replicates. Asterisks above the columns represent a significant difference at ** p < 0.01 levels according to Student's t-test.

Analysis of Sequence and Phylogenesis of FvWRKY50
A cDNA fragment encoding FvWRKY50 was isolated from woodland strawberry Hawaii-4 with gene specific primers. The full length of FvWRKY50 gene contains 504 bp, which encodes a protein with 168 amino acids. Phylogenetic analysis indicates that FvWRKY50 belongs to subgroup II c ( Figure 2A). As the WRKY domain is critical for the function of WRKY domains, we analyze the WRKY domain sequence of the group II c FvWRKY TFs. The results show that all the group II c WRKYs, except FvWRKY7, FvWRKY46, and FvWRKY50, contain a conserved WRKYGQK hepta-peptide. In FvWRKY7, FvWRKY46, FvWRKY50, FaWRKY50, and AtWRKY50, this hepta-peptide has changed into WRKYGKK. In addition, the zinc-finger motif of all group II c WRKY TFs except FvWRKY50 are C-X4-X23-H-X-H type, but in FvWRKY50, a deca-peptide of RKIRAA-GAAD is inserted in the zinc-finger motif. Moreover, the first histidine in the zinc-finger motif of FvWRKY50 is substituted by arginine ( Figure 2B). The question of whether these two variations in the WRKY domain of FvWRKY50 might influence its function needs further investigation.

Analysis of Tissue Expression Pattern and Subcellular Localization of FvWRKY50
The expression profiles of genes generally reflect their function. Here, we employ RT-qPCR to analyze the expression pattern of FvWRKY50. The results indicate that this gene is expressed in all tissues, with high expression level in the leaves (2.8-fold) and low expression level in red fruit (0.15-fold) relative to those in the roots. Moreover, the expression level of FvWRKY50 is found decreasing gradually along with the ripening of fruit ( Figure 3A). Intriguingly, although the expression level of FvWRKY50 in the red fruit is the lowest, this gene is significantly up-regulated when the red fruit are inoculated with B. cinerea. As shown in Figure 1, it is gradually up-regulated to about 22-fold higher at the 5th day after inoculation. These results confirm our previous speculation that FvWRKY50 is in response to B. cinerea infection. The tobacco used for assay is a stable transgenic line that has been transformed with a specific nucleus-localized red fluorescent protein construct. The fluorescence was detected at 72 h after infiltration. The red fluorescence shows the localization of nucleus. Error bars represent the standard deviations of three biological replicates. Asterisks above the columns represent a significant difference at * p < 0.05 and ** p < 0.01 levels according to Student's t-test. Bar, 20 µm.
To identify the localization of FvWRKY50 in the cells, we fused FvWRKY50 to the N-terminal of green fluorescent protein under the control of CaMV35S promoter (35S:FvWRKY50-GFP). As shown in Figure 3B, the fluorescent signal of the fusion protein is specifically located in the nucleus. This result indicates that FvWRKY50 is a nuclear localization protein.

Analysis of Promoter of FvWRKY50
The spatial and temporal-specific expression of genes are generally determined by the cis-regulatory elements in their promoter and the later are specifically recognized and bound by transcriptional regulators of different classes. These regulators regulate gene expression in response to internal and/or external stimuli such as phytohormone, abiotic, and biotic stresses and others. To analyze the cis-regulatory elements in the promoter of FvWRKY50, a 2000 bp promoter sequence located in the upstream of the star codon of FvWRKY50 was predicted using PlantCARE online tool (http://bioinformatics.psb.ugent. be/webtools/plantcare/html/ (accessed on 30 September 2021)). The results indicates that beside a large number of the core promoter elements TATA-box (57 sites) and CAAT-box (35 sites), there are 22 other types of cis-regulatory elements in the promoter of FvWRKY50 (Table 1). Among these cis-regulatory elements, seven of them are light-response elements, i.e., the ACE, ATCT-motif, G-Box, GATA-motif, GT1-motif, TCT-motif and chs-CMA1a, and six of them are defense-related elements, i.e., ABRE, CGTCA-motif, TGACG-motif, WER3, WUN-motif, and W-box. In addition, other elements involved in abiotic stress response and development such as LTR, MBS, MYB, and STRE are also identified in the promoter of FvWRKY50. These data suggest that FvWRKY50 is probably involved in plant growth, development, and disease response.  ATCT-motif -561 (+) AATCTAATCC Part of a conserved DNA module involved in light responsiveness [46] CCGTCC motif -1027 (-) CCGTCC Cis-acting regulatory element related to meristem specific activation [47] CAT-box -986 (-) GCCACT Cis-acting regulatory element related to meristem expression [47] CGTCA-motif -1462 (

Effect of FvWRKY50 in Regulation of Defense to B. cinerea
To determine the function of FvWRKY50 in response to B. cinerea infection, we first transiently expressed it in strawberry fruit by Agrobacterium-mediated infiltration. As shown in Figure 4A, on the third day after agroinfiltration, compared with that in the fruit infiltrated with empty vector (EV-OE), the expression level of FvWRKY50 increases about four folds in the fruit infiltrated with the FvWRKY50 overexpression vector (FvWRKY50-OE). However, compared with the fruit infiltrated with RNAi empty vector (EV-RNAi), the transcription level of FvWRKY50 decreases about three folds in the fruit that transiently express FvWRKY50 gene silence vector (FvWRKY50-RNAi). After confirming the reliability of strawberry fruit transient expression system, we further inoculated these fruit with B. cinerea respectively to determine the disease resistance of FvWRKY50. As shown in Figure 4B, 48 h after B. cinerea inoculation, the lesion area on the fruit of FvWRKY50-OE is significantly smaller than those of EV-OE. Consistently, the lesion area on the fruit of FvWRKY50-RNAi is bigger than that of empty vector. These results demonstrate that overexpression of FvWRKY50 could increase the resistance of strawberry against B. cinerea ( Figure 4B). Values are means ± SD of three biological replicates. Asterisks above the columns represent a significant difference at ** p < 0.01 levels according to Student's t-test.

Effect of FvWRKY50 on the Transcript Levels of Disease Resistance Genes
Promoter analysis implying FvWRKY50 might be involved in JA signaling pathway in response to B. cinerea infection. To explore the regulation mechanism of FvWRKY50 in pathogen resistance, we detected the transcript levels of several defense-related genes in FvWRKY50 overexpression and silencing fruit, respectively. As shown in Figure 5, many genes have been affected by FvWRKY50. For example, the expression pattern of FaAOC, one key gene that involves in JA biosynthesis, is similar to FvWRKY50, suggesting significantly up-regulated in FvWRKY50-OE fruit and significantly suppressed in FvWRKY50-RNAi fruit, while the expression pattern of other genes such as FaAOS, FaLOX, FaOPR2 that involve in JA biosynthesis is not closely correlated with FvWRKY50 ( Figure 5). Interestingly, the expression pattern of FaJAZ5 and FaJAZ10, two genes involved in JA signaling pathway, are negatively relative to FvWRKY50. This suggests that they were suppressed in FvWRKY50-OE fruit but up-regulated in FvWRKY50-RNAi fruit. In addition, we observed that the transcript of FaJAZ1, FaJAZ4 and FaJAZ8 were strongly up-regulated in FvWRKY50-OE fruit but not influenced in FvWRKY50-RNAi fruit ( Figure 5). Accordingly, FvWRKY50 may have been involved in JA-mediated resistance against B. cinerea through regulating the expression of genes in biosynthesis and signaling pathway.
In addition, the expression of several transcription factor genes such as FaWRKY1, FaWRKY11, FaWRKY25, FaWRKY33, FaWRKY75, FaWHIRLY1, and FaWHIRLY2 were also detected. The results indicate that the expression pattern of FaWRKY1 is positively related to FvWRKY50, while the expression pattern of FaWRKY33 is negatively related to FvWRKY30. The expression pattern of other transcription factor genes is not closely related to FvWRKY50. Finally, several defense-related genes coding antimicrobial proteins (PR proteins) and fungi cell wall degradation hydrolases were also detected. The results show that FaCHI3-1, a chitinase coding gene, was significantly up-regulated in FvWRKY50-OE fruit and suppressed in FvWRKY50-RNAi fruit. In addition, FaPR1 gene was also significantly induced in FvWRKY50-OE fruit. Moreover, other defense-related genes including FaPR5-1, FaCHI2-2, FaBG2-2, FaBG2-3, and FaPGIP1 were down-regulated in FvWRKY50-RNAi fruit.

Discussion
WRKY gene family encodes one of the largest plant-specific transcription factors and plays a critical role in plant growth processes, especially in response to abiotic and biotic stresses. For example, AtWRKY8 was found to interact with AtVQ9 to regulate salt stresses [57]. Interestingly, AtWRKY8 was also found to positively regulate plant basal resistance against B. cinerea through interaction with AtVQ10, knockout of AtVQ10 decreased the resistance to B. cinerea, whereas plants that constitutively express AtVQ10 or AtWRKY8 enhanced resistance to B. cinerea [58]. In addition, Wei et al. reported that ectopic expression of FvWRKY42 in Arabidopsis could not only enhance the pathogenic resistance, but also improve salt and drought stress tolerance of the transgenic plants [35]. In addition, in Arabidopsis, AtWRKY33 was identified as a substrate of AtMPK3/MPK6. Upon B. cinerea infection, AtWRKY33 was phosphorylated by the two kinases, which subsequently activated the expression of camalexin biosynthesis genes [59]. In strawberry, transient expression of FaWRKY11 in strawberry fruit increased their resistance to B. cinerea by regulating several disease resistance transcription factors and metabolic genes [32].
Based on our previous study, we isolated and characterized a WRKY gene, FvWRKY50, from woodland strawberry accession Hawaii-4 in this study. Tissue expression pattern analysis indicates that FvWRKY50 is ubiquitously expressed in all detected tissues ( Figure 3A). Interestingly, despite the expression levels of FvWRKY50 being gradually decreased in strawberry fruit along with ripening, it was constantly up-regulated when the ripe fruit was infected by B. cinerea (Figure 1). This result implies that FvWRKY50 might be involved in regulating plant defense responses not only at the early stage, but also at the later stage. The induction pattern of FvWRKY50 was similar to that of FaWRKY11 which has been found positively regulating resistance to B. cinerea and whose expression has also been proved to be able to last for four to five days [34], implying the two strawberry WRKY genes might play similar roles in plant disease response. Further promoter sequence analysis also confirms that FvWRKY50 gene is involved in plant disease response, because several defense-related signal response cis-regulatory elements are identified in its promoter region (Table 1). Sequence analysis indicates that FvWRKY50 is classified into subgroup II c (Figure 2A). Interestingly, different from other subgroup II c strawberry WRKYs, two variations were observed in WRKY domain of FvWRKY50. The first variation means that the most conserved WRKYGQK hepta-peptide in FvWRKY50 is changed into WRKYGKK, which is located at the N-terminal of the WRKY domain. The second variation refers to the change of the conserved C-X 4 -C-X 23 -H-X-H type in the group II c WRKY TFs into C-X 4 -C-X 33 -R-X-H type in FvWRKY50, which is located at the zinc-finger motif region. Variation in the amino acid residues number of the zinc-finger has only been found in rice, moso bamboo and wheat group III WRKY TFs, but not been reported in other group WRKY TFs [60]. Numerous studies have demonstrated both the WRKYGQK region and the zinc-finger motif are indispensable for the DNA-binding function of WRKY proteins [60]. Therefore, it is worthwhile to further analyze whether these amino acid variations could influence the function of FvWRKY50. Interestingly, these variations did not affect its subcellular localization, as shown in Figure 3B which demonstrates that the fluorescent signal of FvWRKY5-GFP fusion protein is exclusively localized in the nucleus.
To explore the function of FvWRKY50 in response to B. cinerea infection, we transiently expressed this gene in strawberry fruit by Agrobacterium-mediated infiltration. Through analyzing the relationship between the expression level of FvWRKY50 and lesion area that is caused by B. cinerea infection, we conclude that FvWRKY50 may be able to positively regulate plant defense to B. cinerea. The reasons for this are that the lesion area on the fruit that overexpresses this gene is significantly smaller than that of the control, and that the opposite symptom was observed on the FvWRKY50 silenced strawberry fruit ( Figure 4B).
Numerous studies have revealed that a large portion of WRKY TFs participated in disease response through jasmonic acid (JA) signaling pathway [11]. To test whether FvWRKY50 is also involved in JA pathway to regulate disease response, we analyzed the expression levels of several JA biosynthesis and signaling pathway genes in FvWRKY50 overexpressed and silenced strawberry fruit. The results showed that when FvWRKY50 was overexpressed or silenced in fruit, the transcript pattern of FaAOC, one of the key gene involves in JA biosynthesis, was highly similar to FvWRKY50. Additionally, two JA signaling pathway genes, FaJAZ5 and FaJAZ10, exhibit opposite expression pattern to FvWRKY50 in FvWRKY50 overexpression and silencing fruit. Based on these results, we propose that FvWRKY50 might be involved in JA biosynthesis and signaling transduction to regulate plant defense. In addition, the expression levels of four FaWRKY genes in FvWRKY50 overexpressed and silenced fruit were varied ( Figure 5). For example, we observed when FvWRKY50 was overexpressed, FaWRKY1 was significantly up-regulated, while other two genes, FaWRKY25 and FaWRKY33, were down-regulated. As studies in Arabidopsis have revealed that AtWRKY57 and AtWRKY33 competitively binding to the W-box in the promoter of JAZ1 and JAZ5 to regulate response to B. cinerea infection via JA signaling pathway [61], further analyses are required to explore the roles of FvWRKY50 in JA signaling pathway. Fungi cell wall degradation enzymes and antimicrobial proteins play important roles in defense against plant pathogen [62]. Therefore, the transcript levels of FaPR1, FaPR5-1, FaPR10, FaCHI2-2, FaCHI3-1, FaBG2-1, FaBG2-2, FaBG2-3, FaPGIP1, and FaPGIP2 were detected. The results indicated that FaCHI3-1 and FaPR1 were significantly induced in FvWRKY50-OE fruit. Taken together, these data imply FvWRKY50 might increase the resistance ability of strawberry against B. cinerea through regulating JA biosynthesis and signaling transduction, fungi cell degradation enzymes, and antimicrobial proteins biosynthesis.
In summary, the present study has functionally characterized the FvWRKY50 gene in strawberry fruit by transient expression system. With this study, we have provided primary evidence for the relationship between FvWRKY50 and the resistance of strawberry against B. cinerea. Our results indicate that FvWRKY50 positively regulates strawberry resistance to B. cinerea. In addition, because several defense-related WRKY transcription factors are also found to play roles in against abiotic stresses, further studies to generate stable transgenic strawberry plants for overexpression and/or knockout of FvWRKY50 to confirm its functions are required in the future.