Genome Wide Transcriptomic Analysis of WRKY Gene Family Response to Biotic Stresses in Malus × domestica

Apple (Malus × domestica Borkh.) is a perennial woody plant that often suffers from various biological stresses. Many harmful pathogens can infect apple trees and lead to reduced production. We comprehensively identified the WRKY genes in the apple genome and analyzed their expression in response to several biological stressors, including Alternaria alternata, Pythium ultimum, Botryosphaeria dothidea, Erwinia amylovora, Penicillium expansum, Gymnosporangium yamadae, and Apple replant disease. There were 113 MdWRKYs identified in the apple genome. Twenty-two MdWRKYs were differentially expressed in response to at least five pathogens. Promoter sequence analysis showed that these genes carried many defenseand stress-responsive elements, such as MeJA-response elements, salicylic acid-response elements, and W-box elements, in their promoters. Transient expression assays showed that MdWRKY40a and MdWRKY54h played negative roles in defense against B. dothidea infection. WRKY40 and WRKY60 and the MdWKRY33s might play important roles in responding to pathogens and are conserved in some plants. These differentially expressed MdWRKYs might play key roles in the apple response to multiple pathogens.


Introduction
Apple (Malus × domestica Borkh.) is an important fruit crop cultivated on a great deal of land around the world. Many harmful pathogens infect apple trees and lead to a reduction in yields [1] [2]. Fungicides are commonly used in orchards to control and prevent fungal diseases. But fungicides have adverse effects on the environment and can result in pathogen resistance. Screening disease-resistant genetic resources and breeding disease-resistant cultivars combine to form one of the effective strategies to resist pathogens. Therefore, it is important to understand the molecular mechanism of pathogen infection in apple and to identify disease resistance genes.
WRKY transcription factors are known to participate in the defense responses of higher plants [3]. A growing number of WRKY transcription factors have been proved to play roles in host-pathogen interactions between different plants and pathogens. The WRKY transcription factors are characterized by the conserved 7-amino acid sequence WRKYGQK at the N-terminal and the zinc finger motif at the C-terminal. The WRKY family was divided into 3 groups based on the number of WRKY sequences and the zinc finger sequence. Group I WKRY proteins contain two WRKY domains and a C2H2 zinc-finger motif, while the group II and group III have only one WRKY domain and either a C2H2 or C2HC zinc-finger motif, respectively. The WRKY domain can bind to a W-box (TTGACC/T) ciselement in a promoter to stimulate or repress target gene expression. The W-box appears in the promoters of many plant genes that are associated with defense [4]. In Arabidopsis, several WRKY genes have been proved to associate with responses to pathogen infections. The Group IIa members AtWRKY18, AtWRKY40 and AtWRKY60 interact with each other to regulate defense pathways [5].
WRKY18, WRKY40, and WRKY33 each bind to the promoters of more than 1000 genes involved in signal perception and transduction not only during microbial-associated molecular pattern-triggered immunity (MTI) but also upon damage-associated molecular pattern-triggered immunity [6]. WRKY22 and WRKY29 are induced by the MAPK pathway involved in plant responses to both bacterial and fungal pathogens, and transient expression of WRKY29 in leaves leads to reduced disease symptoms [7]. WRKY53 and WRKY70 both positively modulate systemic acquired resistance (SAR) [8]. The Group I members WRKY3 and WRKY4 play positive roles in plant resistance to necrotrophic pathogens. WRKY4 has a negative effect on plant resistance to biotrophic pathogens [9]. The group IId members WRKY11 and WRKY17 are negative regulators of basal resistance in Arabidopsis [10]. Some of the apple WRKY genes have been demonstrated to be involved in plant defense. MdWRKYN1 and MdWRKY26 are targeted by miRNAs and are involved in apple resistance to leaf spot disease caused by Colletotrichum spp. [11]. MdWRKY100 positively regulates apple resistance to Colletotrichum gloeosporioides infection [12]. Ectopic expression of MdWRKY1 (homolog of AtWRKY15) in tobacco plants enhances resistance to Phytophthora parasitica tance to Botryosphaeria dothidea via the salicylic acid-mediated pathway by directly binding the MdICS1 promoter [14] (Zhao et al., 2020). MdWRKY46 enhances apple resistance to B. dothidea by activating the expression of MdPBS3.1 in the salicylic acid signaling pathway [15]. MdWRKY31 regulates plant resistance to B. dothidea through the SA signaling pathway by interacting with MdHIR4 [16].
Apple is a commercially cultivated fruit that is important economically and is favored by consumers, and thus is extensively studied. There are large amounts of publicly available data on apples, including genomic sequences, transciptomic and metabolic datasets. Although the WRKY gene family has been analysed genome-wide in several species, including Arabidopsis, wheat, grapes, poplar, and strawberry [4]

Phylogenetic Analysis and Classification of Apple MdWRKY Genes
The Arabidopsis and apple WRKY amino sequences were used for phylogenetic tree construction. The phylogenetic tree was constructed using the MEGA 7

Expression Analysis of the MdWRKY Genes in Apple
The expression of the MdWRKYs members in different tissues was determined by published transcriptomics data (Supplemental Table S1). qRT-PCR was also used to measure the expression of several MdWRKYs in the leaf, shoot, root, flower, and fruit from the 4 years old apple rootstock M9-T337. Primers for qRT-PCR were designed to amplify 100 -200 bp target fragments using NCBI Primer Blast (Supplemental Table S3 The expression responses of the MdWRKYs to apple replant disease (ARD), Alternaria alternata, Pythium ultimum, Botryosphaeria dothidea, Erwinia amylovora, Penicillium expansum, and Gymnosporangium yamadae were determined by the transcriptome data downloaded from the NCBI SRA (Supplemental Table S2).
After filtering low quality reads and contaminant sequences, the clean reads were aligned to the Malus × domestica genome GDDH13_1-1 using the HISAT2 software. The Stringtie software was used to assemble the transcripts [28]. Gene expression was calculated using the Fragments Per Kilobase of transcript per Million fragments mapped reads method (FPKM). DESeq2 software was used to estimate differentially expressed genes [29]. Genes with an FDR < 0.1 and |log 2 (fold change)| ≥ 1 between two samples were identified as differentially expressed genes.

Promoter Analysis for Cis-Acting Regulatory Elements
For each MdWRKY gene, a 2000-bp sequence upstream of the start codon was retrieved from the GDDH13_1-1 genome and was submitted to the PlantCARE website to search the cis-acting regulatory elements [30].
the overexpression vector SAK-227 to generate the vectors MdWRKY40a-OE and MdWRKY54h-OE. About 300-bp fragments specific to either MdWRKY40a or MdWRKY54h were ligated into the virus induced gene silence (VIGS) vector TRV2 to generate TRV-MdWRKY40a or TRV-MdWRKY54h. Agrobacterium tumefaciens transformed with the VIGS or OE recombinant vectors was injected into mature 'Pink Lady' apple fruits as described previously [31]. The empty vectors were the controls. After A. tumefaciens infiltration, the injection holes were inoculated with freshly grown B. dothidea mycelia. The apples inoculated with Agrobacterium tumefaciens and B. dothidea were stored in darkness at 28˚C, and the symptoms were recorded on 4 days post inoculation (dpi). Fifteen apples were inoculated with each treatment combination. Each apple was inoculated with two holes, one as control, and the other as silence or overexpression treatment, on the opposite side of the apple fruit peels. The area of each spot was measured and compared to control.

Identification and Classification of Apple MdWRKY Genes
A total of 113 members homologous to the WRKY transcription factor family were identified from the apple genome. All members were systematically numbered, as shown in Table 1, based on their similarity to genes in Arabidopsis thaliana. Three genes were mapped to the unassembled scaffolds.

Gene Structure and Motif Analysis of MdWRKYs
The predicted structures of the MdWRKY genes are shown in Figure 3

Expression Analysis of MdWRKYs in Different Tissues
The expression levels of the 113 MdWRKYs in different tissues were extracted from published transcriptome data ( Figure 4). MdWRKY11a, MdWRKY69b, MdWRKY44c, MdWRKY58a, MdWRKY1a, MdWRKY32a, MdWRKY15a, We further examined expression of eight MdWRKY genes in different tissues by qRT-PCR ( Figure 5). The results showed that MdWRKY33a, MdWRKY40a, MdWRKY51, and MdWRKY75b were highly expressed in the root. MdWRKY42a was detected in all examined tissues, with higher expression in the root and flower. MdWRKY54h showed higher expression in the leaf and shoot compared to other tissues. MdWRKY60c showed higher expression in the leaf and fruit. MdWRKY71b showed higher expression in the leaf and root. Error bars indicate SEs (standard errors) from 3 biological repetitions.

Expression Analysis of MdWRKYs in Response to Pathogens
The expression of the MdWRKYs in response to pathogens was determined ( Figure 6). Alternaria alternata can cause apple Alternaria blotch disease, which often results in defoliation of the tree. Transcriptome analysis was used to determine the response in apple leaves to A. alternata infection at 0, 12, 18, 36, and 72 hours post inoculation (hpi) [32]. There were 59 differentially expressed MdWRKYs after Alternaria infection (Figure 6(a)). MdWRKY61c, MdWRKY32b, Replanting apple trees in land previously used as apple orchards or nurseries often results in apple replant disease (ARD). ARD weakens apple trees and affects fruit yield and quality [33] [34]. Cultivating the ARD-susceptible apple rootstock M26 on ARD-affected soil significantly upregulated MdWRKY75b and MdWRKY51 expression in leaves (Figure 6(b)).
Pythium ultimum is a primary component of the ARD pathogen complex identified in orchard soil [35].  Penicillium expansum can infect apple fruit through wounds, causing blue mold disease that results in fruit rot. Transcriptomics was used to analyze the mature apple fruit of the susceptible 'Royal Gala' and resistant Malus sieversii-PI613981 in response to Pe. expansum inoculation at 6 hpi, 24 hpi, and 48 hpi [38]. In the Malus sieversii, most of the differentially expressed MdWRKYs were significantly downregulated at 48 hpi ( Figure 6(f)    B. dothidea, the TRV-MdWRKY40a and TRV-MdWRKY54h constructs significantly decreased the lesion size compared with the control (Figure 9(a) and Figure 9(b)). On the contrary, overexpression of MdWRKY40a-OE and MdWRKY54h-OE reduced resistance to B. dothidea (Figure 9(c) and Figure  9(d)). The disease spot size of apple fruits transiently expressing MdWRKY40a-OE and MdWRKY54h-OE were significantly larger than the control. These results indicated that MdWRKY40a and MdWRKY54h promote growth of B. dothidea or decrease plant resistance.

Discussion
In this paper, we systematically identified 113 MdWRKYs in the apple genome and analyzed their response to seven pathogens. Among these MdWRKYs, 22 MdWKRYs showed differential expression in response to at least 5 pathogens.  Fifteen apples were inoculated with each treatment combination.
plays a negative role in resistance to hemibiotrophic fungi in poplar but functions as a positive regulator of resistance toward the necrotrophic fungi in Arabidopsis [43]. GmWRKY40, from Glycine max L., enhances the resistance to Phytophthora sojae [44]. In Malus hupehensis, MhWRKY40b were induced by the powdery mildew (Podosphaera leucotricha) [37]. In Malus × domestica, 4 MdWRKY33s were induced by A. alternata, Pe. expansum, Py. ultimum, G. yamadae, and E. amylovora. MdWRKY33a and MdWRKY33d were also induced by ARD and B. dothidea, respectively. Arabidopsis WRKY33 is a key positive resistance regulator against the necrotrophic fungi Alternaria brassicicola and Botrytis cinerea [45] [46]. Hence, the group IIa members WRKY40 and WRKY60 and group I member WKRY33 may play important roles in responding to pathogens and are conserved in plants.
MdWRKY42a and MdWRKY42b showed differential expression in response to 5 pathogen infection. MdWRKY42a (named MdWRKY31 in [16]) regulates plant resistance to B. dothidea through the SA signaling pathway by interacting with MdHIR4. In rice, WRKY42 negatively regulates the rice response to Magnaporthe oryzae by suppressing JA signaling-related genes [50]. MdWRKY54h and MdWRKY40a showed differential expression after infection with B. dothidea, A. alternata, E. amylovora, G. yamadae, and Py. ultimum. In the transcriptome of apple fruit inoculated B. dothidea, MdWRKY54h was upregulated in the sensitive genotype. Transient expression assays showed that In Arabidopsis, apple and other plants, many WRKY genes are responsive to pathogen infection. About 75 MdWRKYs were differentially expressed in response to at least 2 pathogens. About one-quarter of the MdWRKYs contain a W-box element. The WRKY-WRKY regulation network complex has been characterized based on the auto-and cross-regulation patterns through the WKRY domain/W-box and physical interaction between WRKY members [5] [54]. Many WRKY promoters contain MeJA-and SA-responsive elements. Some WRKYs also enhance disease resistance by involvement in MeJA and SA synthesis or signal transduction [14] [44] [53]. Therefore, pathogens, WKRY proteins, and hormones come together in a regulatory network that may be the cause of the many different expression patterns seen for the WRKY gene family after inoculation with pathogens.

Conclusion
In short, we identified 113 MdWRKY members in the apple genome and analyzed their expression patterns in response to various biological stressors. Twenty-two MdWRKYs showed differential expression in response to at least five pathogens. MdWRKY40a and MdWRKY54h played negative roles in resistance to Botryosphaeria dothidea. Autoregulation, cross-regulation, and physical interaction between WRKY members and cross-regulation between pathogens, WRKY proteins, and hormones may work together to create the many MdWRKY expression patterns after inoculation with pathogens.   Figure S1. Motif consensus sequences for Figure 3.