Role of PsnWRKY70 in Regulatory Network Response to Infection with Alternaria alternata (Fr.) Keissl in Populus

WRKY is an important complex family of transcription factors involved in plant immune responses. Among them, WRKY70 plays an important role in the process of the plant defense response to the invasion of pathogens. However, the defense mechanism of PsnWRKY70 is not clear in Populus nigra. In this study, we showed that PsnWRKY70-overexpression lines (OE) had fewer leaf blight symptoms than PsnWRKY70-repressing lines (RE). PsnWRKY70 activated MAP kinase cascade genes (PsnM2K4, PsnMPK3, PsnM3K18), calcium channel proteins-related genes (PsnCNG3, PsnCNGC1, PsnCNG4), and calcium-dependent protein kinases genes (PsnCDPKL, PsnCDPKW, PsnCDPKS, PsnCDPKQ). Furthermore, 129 genes of PsnWRKY70 putative genome-wide direct targets (DTGs) were identified by using transcriptome (RNA-seq) and DNA affinity purification sequencing (DAP-seq). PsnWRKY70 directly binds to the promoters of homologous genes and LRR domain proteins to promote the expression of WRKY6, WRKY18, WRKY22, and WRKY22–1, LRR domain proteins LRR8, LRR-RLK, ADR1-like 2, NB-ARC, etc. Our study suggests that PsnWRKY70 enhances the resistance of A. alternata in poplar by activating genes in both pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).


Introduction
In the natural environment, plants are constantly confronted with diverse biotic and abiotic stresses that adversely affect their growth and development [1]. Pathogen attack is one of the most limiting factors affecting plant growth and thus poses a serious threat to the agroforestry industry worldwide [1][2][3]. Alternaria alternata is a necrotrophic fungal pathogen. Its multiple pathological races can infect many plants, such as chrysanthemum (Chrysanthemum morifolium), tobacco (Nicotiana attenuata), and poplar (Populus. tomentosa), causing huge national economic losses [3][4][5].
Plants have developed multitiered defense mechanisms for protection against pathogens. Plants have evolved pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) systems [6,7]. Usually, a reactive oxygen species (ROS) burst occurs by binding to cell membrane surface co-receptors and by phosphorylating RLCKs, MAPKs, and calcineurin kinases [8][9][10][11]. At the same time, intracellular nucleotide-binding LRR (NLR) receptors cooperate with lipase-like proteins such as EDS1, SAG101, and PAD4 to induce ETI through NLRs of the ADR1 family and the NRG1 family [12]. In N. benthamiana, the MAPK cascade activates WRKY7, WRKY8, WRKY9, and WRKY11. These WRKYs can bind to the W-box element in the RBOHB promoter to upregulate the RBOHB gene and cause a ROS burst [13]. Recent studies have shown that, in Arabidopsis, activation of the RPS2 gene leads to the accumulation of BIK1 and RBOHD proteins and increased transcription of WRKY29 and AZI [14]. Pathogen defense response is achieved through large-scale transcriptional activation of defense-related genes [15,16]. WRKY transcription factors play a crucial role in the regulatory network for defense against pathogenic infection [17]. WRKYs are mainly characterized by a highly conserved WRKYGQK motif and a zinc finger motif [18]. WRKYs exert their regulatory functions by binding to the DNA sequence of the W-box (TTGACT/C) [19,20]. PtrWRKY89 accelerates pathogenesis-related protein (PR) gene expressions and enhances pathogen resistance in transgenic poplar (Populus trichocarpa) and is a regulator of salicylic acid (SA)-dependent defense signaling in poplar [21]. Liu et al. identified BnWRKY33 as a S. sclerotiorum responsive gene by enhancing the expression of genes involved in camalexin synthesis and regulating SA and jasmonic acid (JA) to defend against the pathogen [22].
We previously showed that PsnWRKY70 overexpression improved leaf blight resistance in Populus. Fifteen days after A. alternata infection, the leaf disease index and MDA content of OE lines were significantly lower than those of WT lines, and the plant height was significantly higher than that of WT lines [23]. However, little is known about the defense mechanism of how poplar trees respond to the infection of A. alternata. The objective of the study was to investigate the defense mechanism of PsnWRKY70 and understand its role in the regulatory network response to infection with A. alternata. We performed RNA-seq analysis to investigate differentially expressed genes regulated by PsnWRKY70 during disease resistance in poplar. We also identified direct genome-wide target genes of PsnWRKY70 using DAP-seq. The key direct target genes regulated by PsnWRKY70 were verified in vivo by CHIP-PCR and qRT-PCR. Our results provide new insight into the molecular mechanism of the PsnWRKY70 transcription factor in forest disease resistance and could be used as one technical guidance for molecular breeding of forest disease prevention.

PsnWRKY70 Reduces Infection with A. alternata
To ensure the accuracy of gene transformation, PCR molecular detection was performed on the PsnWRKY70-transformed poplars ( Figure 1A). At the same time, qRT-PCR results show that compared with the WT line, the gene expression of PsnWRKY70 was significantly upregulated, 2.94 times in OE compared to that of WT ( Figure 1B); on the contrary, the relative expression of PsnWRKY70 in the RE was significantly lower than that in the WT ( Figure 1B). All the tested lines were susceptible to A. alternata in different degrees after 15 days of inoculation ( Figure 1C,D). The leaves of the OE line had fewer disease spots and more green leaf color. OE had a 16% disease index in comparison with 46.3% of the WT line. The RE line had a 62.8% disease index and leaves showed chlorosis and yellowing with serious disease.

Transcriptime Analysis
A total of about 86 Gb of clean data was obtained with an average of about 9.5 Gb per sample. The mapping rate was between 81.72% and 84.68% (Table S2). The thresholds of DEGs between OE vs. WT and RE vs. WT were used with fold change ≥ 2 and p-value < 0.05. There were 691 and 309 exclusively upregulated DEGs in OE and RE lines. There were 70 overlap DEGs between OE and RE ( Figure 2A). There were 804 and 322 exclusively downregulated DEGs in OE and RE lines. There were 55 overlap DEGs between OE and RE ( Figure 2B). GO analysis showed that the 691 upregulated DEGs in OE were enriched in protein phosphorylation, cellular protein modification process, and response to stimulus ( Figure 2C). The 309 upregulated DEGs for RE were enriched in protein phosphorylation, defense response to other organism, and immune response ( Figure 2E). The 804 downregulated DEG for OE were enriched in cellular process, metabolic process, and organic substance metabolic process ( Figure 2D). The 322 downregulated DEGs for RE were enriched in response to chemical, biological regulation, and cellular process ( Figure 2F). KEGG enrichment analysis showed that 691 DEGs upregulated for OE were enriched in plant-pathogen interaction, MAPK signaling pathway-plant, and ABC transporters ( Figure 3A). The 309 upregulated DEGs for RE were enriched in propanoate metabolism, alpha-Linolenic acid metabolism, and phosphonate and phosphinate metabolism ( Figure 3C). The 804 downregulated DEGs for OE were enriched in alpha-Linolenic acid metabolism, starch and sucrose metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis ( Figure 3B). The 322 downregulated DEGs for RE lines were enriched in ABC transporters, ubiquitin-mediated proteolysis, and plant hormone signal transduction ( Figure 3D).  response to other organism response to abiotic stimulus protein metabolic process organonitrogen compound metabolic process cellular protein metabolic process cellular process regulation of cellular process response to chemical response to oxygen-containing compound phosphate-containing compound metabolic process phosphorus metabolic process regulation of biological process macromolecule modification biological regulation response to external stimulus phosphorylation response to stimulus cellular protein modification process protein modification process protein phosphorylation log10(p value) 0 5 1 0 1 5 protein autophosphorylation defense response to bacterium defense response biological process involved in interspecies interaction between organisms response to biotic stimulus response to external biotic stimulus response to other organism macromolecule modification response to bacterium response to external stimulus cellular process protein modification process cellular protein modification process immune system process immune response defense response to other organism phosphorus metabolic process phosphate-containing compound metabolic process phosphorylation protein phosphorylation log10(p value) 0 5 1 0 negative regulation of transcription by RNA polymerase II cellular carbohydrate metabolic process metabolic process cellular metabolic process regulation of metabolic process cellular carbohydrate biosynthetic process cellular protein metabolic process response to oxygen-containing compound response to abiotic stimulus macromolecule metabolic process response to organic substance cellular macromolecule metabolic process response to stimulus regulation of biological process protein modification process cellular protein modification process macromolecule modification cellular process biological regulation response to chemical   response to other organism response to abiotic stimulus protein metabolic process organonitrogen compound metabolic process cellular protein metabolic process cellular process regulation of cellular process response to chemical response to oxygen-containing compound phosphate-containing compound metabolic process phosphorus metabolic process regulation of biological process macromolecule modification biological regulation response to external stimulus phosphorylation response to stimulus cellular protein modification process protein modification process protein phosphorylation log10(p value) 0 5 1 0 1 5 protein autophosphorylation defense response to bacterium defense response biological process involved in interspecies interaction between organisms response to biotic stimulus response to external biotic stimulus response to other organism macromolecule modification response to bacterium response to external stimulus cellular process protein modification process cellular protein modification process immune system process immune response defense response to other organism phosphorus metabolic process phosphate-containing compound metabolic process phosphorylation protein phosphorylation log10(p value) 0 5 1 0 negative regulation of transcription by RNA polymerase II cellular carbohydrate metabolic process metabolic process cellular metabolic process regulation of metabolic process cellular carbohydrate biosynthetic process cellular protein metabolic process response to oxygen-containing compound response to abiotic stimulus macromolecule metabolic process response to organic substance cellular macromolecule metabolic process response to stimulus regulation of biological process protein modification process cellular protein modification process macromolecule modification cellular process biological regulation response to chemical

PsnWRKY70 Activates the Expression of MAPK and Ca2 + Signaling-Related Genes
Genes that play important roles in disease resistance were regulated by PsnWRKY70 ( Figure 4A,B). After OE lines were infected with A. alternata, genes in the MAPK cascades, such as M2K4, MPK3, and M3K18, were significantly upregulated. Similarly, Ca2 + channel proteins CNG3, CNGC1, and CNG4, and calcium-dependent protein kinases CDPKL, CDPKW, CDPKS, and CDPKQ were significantly upregulated as well. In addition, transmembrane receptor protein-related genes NIK2, CRK35, CRPK1, LRK10L-1.2, CRCK2, CES101, and CRK23 were significantly upregulated. As a result, PsnWRKY70 enhances disease resistance by upregulating the expression of a MAP kinase cascade, calcium signal-related genes, membrane receptors, and other related genes. SA-related biological processes were enriched with upregulated DEGs in OE ( Figure 5). They were PAD4, CBP60C, and CBP60D, but they were not significantly changed in RE.

PsnWRKY70 Activates the Expression of MAPK and Ca2 + Signaling-Related Genes.
Genes that play important roles in disease resistance were regulated by PsnWRKY70 ( Figure 4A, B). After OE lines were infected with A. alternata, genes in the MAPK cascades, such as M2K4, MPK3, and M3K18, were significantly upregulated. Similarly, Ca2 + channel proteins CNG3, CNGC1, and CNG4, and calcium-dependent protein kinases CDPKL, CDPKW, CDPKS, and CDPKQ were significantly upregulated as well. In addition, transmembrane receptor protein-related genes NIK2, CRK35, CRPK1, LRK10L-1.2, CRCK2, CES101, and CRK23 were significantly upregulated. As a result, PsnWRKY70 enhances disease resistance by upregulating the expression of a MAP kinase cascade, calcium signal-related genes, membrane receptors, and other related genes. SA-related biological processes were enriched with upregulated DEGs in OE ( Figure 5). They were PAD4, CBP60C, and CBP60D, but they were not significantly changed in RE.

Genome-Wide Binding Targets of PsnWRKY70
The target protein of PsnWRKY70 was expressed by the wheat germ protein expression system and the protein expression was detected by Western blot. The detection results are shown in Figure 6A

PsnWRKY70 Activates the Expression of MAPK and Ca2 + Signaling-Related Genes.
Genes that play important roles in disease resistance were regulated by PsnWRKY70 ( Figure 4A, B). After OE lines were infected with A. alternata, genes in the MAPK cascades, such as M2K4, MPK3, and M3K18, were significantly upregulated. Similarly, Ca2 + channel proteins CNG3, CNGC1, and CNG4, and calcium-dependent protein kinases CDPKL, CDPKW, CDPKS, and CDPKQ were significantly upregulated as well. In addition, transmembrane receptor protein-related genes NIK2, CRK35, CRPK1, LRK10L-1.2, CRCK2, CES101, and CRK23 were significantly upregulated. As a result, PsnWRKY70 enhances disease resistance by upregulating the expression of a MAP kinase cascade, calcium signal-related genes, membrane receptors, and other related genes. SA-related biological processes were enriched with upregulated DEGs in OE ( Figure 5). They were PAD4, CBP60C, and CBP60D, but they were not significantly changed in RE.

Genome-Wide Binding Targets of PsnWRKY70
The target protein of PsnWRKY70 was expressed by the wheat germ protein expression system and the protein expression was detected by Western blot. The detection results are shown in Figure 6A

Genome-Wide Binding Targets of PsnWRKY70
The target protein of PsnWRKY70 was expressed by the wheat germ protein expression system and the protein expression was detected by Western blot. The detection results are shown in Figure 6A: the fusion protein is about 70 kD. After filtering the original DAP-seq data, a total of about 2.95 Gb of data was obtained, including 5.6~6.5 million clean reads per sample (Q30 ≥ 89.82%) ( Table S3). The mapping rate of the unique locus of the gene was 40.38-42.24%. The total mapping rate (including the multi-locus alignment of the genome) was 71.05-73.92% (Table S4). The 1 kb flanking sequences around the peaks of all genes were applied to the motif discovery tool MEME-CHIP to explore the PsnWRKY70 transcription factor binding motif ( Figure 6D). The results showed that the main motifs bound by PsnWRKY70 were "NMAAGTCAACNNNDN" (E-value = 1.7 × 10 −270 ) and "RGTCAAY" (E-value = 4.9 × 10 −268 ). PsnWRKY70 peaks in DAP-seq were distributed over gene regions. The transcription starting site is 2 kb downstream of the transcription termination. PsnWRKY70 binding sites were identified in 2103 gene regions. Among them, 65.56% were in the promoter region (−2 kb to +500 bp of TSS), 0.41% in the intergenic region, 14.09% in the intron region, 14.73% in the exon region, and 5.18% in the terminator region ( Figure 6C). The identification promoter site for PsnWRKY70 was based on the distance between each peak and its closest gene TSS. It was determined that the PsnWRKY70 binding site was significantly concentrated at 300 bp upstream of TSS in the core promoter region ( Figure 6B). These distribution patterns of PsnWRKY70 binding sites in DAP-seq experiments are consistent with the fact that PsnWRKY70 acts as a transcription factor.

Target Genes Were Regulated by PsnWRKY70
To find the downstream target genes directly regulated by PsnWRKY70, the Venn map showed that PsnWRKY70 directly regulated 43 target genes among 691 upregulated genes in OE lines ( Figure 7A). These include the WRKY family homologous genes WRKY6, WRKY18, WRKY22, and WRKY22-1. These WRKY family homologous genes play an important role in the process of resistance to pathogenic bacteria. It also includes some R genes that play an important role in disease resistance, such as leucine-rich repeat (LRR8), leucine-rich repeat receptor-like protein kinase family protein (LRR-RLK), ADR1-like 2, similar to putative disease resistance protein, LRR protein, NB-ARC domain, and LRR-containing proteins (Table 1). Among the 322 downregulated genes in RE lines, PsnWRKY70 directly regulated 12 target genes ( Figure 7B). These include LOB domain-containing protein 41, LRR-1, photosystem II subunit R, glycosyl transferase family 2 protein, and some others. These genes positively regulated by PsnWRKY70 play an important role in disease resistance. Similarly, among the 804 downregulated genes in the OE lines, PsnWRKY70 directly targeted 47 of them ( Figure 7A). Among the 309 upregulated genes in RE lines, PsnWRKY70 directly regulated 27 of them as target genes ( Figure 7B). These PsnWRKY70 negatively regulated genes may also play a role in disease resistance. qPCR validation showed that WRKY6, WRKY18, WRKY22, ADR1-like2, CBP60C, and LEA2 genes were significantly upregulated in OE lines (p < 0.05) ( Figure 7D). In addition, CHIP-PCR showed that WRKY6, WRKY18, WRKY22, WRKY22-1, ADR1-like2, NB-ARC, and LRR8 amplify clear bands. This suggests that PsnWRKY70 directly regulates WRKY6, PsnWRKY18, WRKY22, WRKY22-1, ADR1-like2, NB-ARC, and LRR8, thereby enhancing the resistance to A. alternata ( Figure 7C).

PsnWRKY70 Induces Other WRKYs to Enhance Disease Resistance
WRKYs typically contain functional W-boxes in their promoters and can be regulated by their own gene products or other WRKYs [24][25][26][27]. In Parsley (Petroselinum crispum), PcWRKY1 binds to the W-box element on its own promoter for self-regulation, and can also bind to the W-box element on the promoter of PcWRKY3 [25]. In Arabidopsis, WRKY46 interacts with NPR1 to bind to the W-box of the WRKY6 promoter to induce its expression in response to SA signaling. Overexpression of WRKY6 is sufficient to accelerate leaf senescence [26]. In Rice (Oryza sativa), WRKY45-2, WRKY13, and WRKY42 form a transcriptional regulatory cascade in response to Magnaporthe oryzae [27]. WRKY transcription factor plays an important role in disease resistance. Previous studies have shown that OsWRKY6 directly binds to WLE1 and W-box in defense-related gene promoters and regulates pathogen defense responses. OsWRKY6 enhances SA accumulation by activating rice OsICS1 gene expression involved in SA biosynthesis [15]. AtWRKY18 enhances PR gene expression and resistance to Pseudomonas syringae. However, excessive expression of AtWRKY18 also leads to severe abnormalities in plant growth [28]. Our data suggest that the PsnWRKY70 directly binds to the promoters of the homologous genes WRKY6, WRKY18, WRKY22, and WRKY22-1 to induce their expression after infection with A. alternata, thereby enhancing the resistance of poplar to necrotrophic fungi invasion.

PsnWRKY70 Plays a Key Role in PTI and ETI Pathways
Plant immunity has evolved into a complex, multilayered, and sophisticated system to fight against the threat of pathogenic microorganisms [29]. It is well-known that the plant's innate immune system mainly relies on two major pathogen recognition mechanisms: PTI and ETI [30]. In Arabidopsis, the EDS1-PAD4-ADR1 node is a convergence point for defense signaling cascades activated by both surface-resident and intracellular LRR receptors in conferring pathogen immunity through PTI and ETI [31]. Additionally, (Ca2 + ) signaling plays an important role in plant PTI and ETI processes [32]. OsCNGC9 enhances rice resistance to Magnaporthe oryzae by regulating extracellular Ca2 + influx to promote ROS burst and activate the expression of PTI-related genes [33]. The formation of calcium channels by CNGC2 and CNGC4 is phosphorylated and activated by BIK1. BIK1 triggers an increase in cytosolic calcium concentration, which in turn activates immune signaling [34]. SA is a phytohormone essential for signaling in the pattern PTI and ETI [35]. SARD1 is a plant immune activator that promotes the production of the hormone SA and the activation of defense gene expression [36,37]. In the uninfected stage, AtWRKY70 represses the expression by binding to the GACTTTT motif on the SARD gene promoter and indirectly repressing CBP60g and SID2. In the infected stage, CBP60g and SARD1 are significantly induced. ES4326, CBP60g, and SARD1 are significantly induced by Pseudomonas syringae pv. Maculicola [38]. In Figure 8, we show that the PsnWRKY70 gene acts as a regulator to mediate the activation of PTI and ETI pathways of innate immunity. Our study found that M2K4, MPK3, and M3K18 in the MAPK cascade pathway were significantly upregulated in OE lines, along with calcium channel protein genes CNG3, CNGC1, and CNG4, and calcium-dependent protein kinase (CPKs) genes CDPKL, CDPKW, CDPKS, and CDPKQ. In addition, it was also found that SA biosynthesis-related genes PAD4, CBP60C, and CBP60D were also significantly upregulated in OE lines. The overexpression of the PsnWRKY70 gene in the OE line upregulated the expression of the MAP kinase cascade, calcium signal-related genes, SA synthesis genes, membrane receptors, and some other genes, thereby enhancing the resistance to A. alternata.

Evaluation of Disease Severity
Plants for OE, RE, and WT lines were inoculated by spraying with A. alternata spore suspension (5.0 × 10 7 spores mL −1 ). Plants sprayed with water were used as controls for OE, RE, and WT lines. Each treatment had three biological replications. Disease severity scores were recorded and photographed after inoculation for 15 days. The disease index was calculated using the method [23]. The leaves were scored on a scale from 0 to 4: 0 (healthy leaves), 1 (<20% of leaves with spots), 2 (about 50% of leaves with spots), 3 (>80% of leaves with spots), and 4 (leaf is dead). Leaf disease index = ∑ (i × Ni) (Nt × Gmax)/ (Nt ×Gmax) × 100%, where i is the grade level (i.e., 0-4), Ni is the number of grades of i leaves, Nt is the total number of leaves, and Gmax is the highest grade level (i.e., 4) [23].

Plant Material
Transgenic plants (Populus simonii × Populus nigra) of PsnWRKY70 used in this study were described previously [23]. They are the overexpression line (OE), the repressed expression line (RE), and the wildtype (WT  Table S1.

Evaluation of Disease Severity
Plants for OE, RE, and WT lines were inoculated by spraying with A. alternata spore suspension (5.0 × 10 7 spores mL −1 ). Plants sprayed with water were used as controls for OE, RE, and WT lines. Each treatment had three biological replications. Disease severity scores were recorded and photographed after inoculation for 15 days. The disease index was calculated using the method [23]. The leaves were scored on a scale from 0 to 4: 0 (healthy leaves), 1 (<20% of leaves with spots), 2 (about 50% of leaves with spots), 3 (>80% of leaves with spots), and 4 (leaf is dead). Leaf disease index = ∑ (i × Ni) (Nt × Gmax)/ (Nt ×Gmax) × 100%, where i is the grade level (i.e., 0-4), Ni is the number of grades of i leaves, Nt is the total number of leaves, and Gmax is the highest grade level (i.e., 4) [23].

Transcriptome Analysis
Total RNA was extracted from the whole leaves of WT, OE, and RE transgenic plants using a universal plant total RNA extraction kit (BioTeke Corporation, Beijing, China) after A. alternata infection for 15 days. RNA samples were submitted for 150 bp paired−end reads on Illumina × 10 platform. To ensure the quality of the analysis data, the original sequences were filtered to obtain high-quality clean reads. Clean reads were mapped to the Populus trichocarpa genome using hisat2 [39]. The mapped reads were counted using Stringtie software [40]. Significance analysis of differentially expressed genes (DEGs) was performed using DEseq2 (p < 0.05, fold change ≥ 2) [41]. Each treatment sample had three sequencing biological replicates. DEGs were used for gene ontology (GO) enrichment analysis using the GO enrichment tool (http://geneontology.org (accessed on 1 February 2022)), with a p-value less than 0.01 as the threshold for significant enrichment [42][43][44]. DEGs were used for KEGG enrichment analysis using the KEGG enrichment tool (http: //kobas.cbi.pku.edu.cn/genelist/ (accessed on 1 February 2022)), with a p-value less than 0.05 as the threshold for significant enrichment [45].

Quantitative Real-Time Polymerase Chain Reaction (qPCR)
Total RNA was extracted using a universal plant total RNA extraction kit (BioTeke Corporation, Beijing, China), and cDNA was separately synthesized using the Toyobo Reverse Transcription Kit (ReverTra Ace ® qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan)). qRT-PCR was performed on an ABI-7500 quantitative PCR instrument using Toyobo SYBR ® Green Real-time PCR Master Mix Plus (Toyobo, Osaka, Japan). The qPCR data were analyzed by the 2 −∆∆CT method, and 18S was used as the internal reference gene [46]. Primers of target genes are listed in Table S1.

DAP-Seq Analysis
The DAP experiments were performed following the published protocol [47]. Briefly, the genomic DNA of WT leaves was extracted by the CTAB method [48], and the genomic DNA was fragmented about 100-400 bp by Covaris M220, and then the genomic DNA library was prepared. In parallel, the coding sequence of PsnWRKY70 was cloned into the pFN19K HaloTag T7 SP6 Flexi expression vector. The Halo-PsnWRKY70 fusion protein was expressed using a TNT SP6-coupled wheat germ extraction system (Promega, USA) in 50 µL reactions and incubated at 37 • C for 2 h. The proteins were captured directly using Magne Halo Tag Beads (Promega, Madison, WI, USA). Finally, the purified protein was incubated with the genomic DNA library. The non-specifically bound DNA was eluted. The DNA fragments that could bind to the PsnWRKY70 protein were eluted. The library was amplified with primers containing sample-specific barcodes. A sample with two biological replicates for PsnWRKY70 DAP was used for 150 bp paired-end sequencing on the Hiseq X Ten platform (Illumina, San Diego, CA, USA). Reads were mapped to the Populus trichocarpa genome sequence using bowtie2 [49]. Peak calling was performed using Macs2 [50]. Motif discovery was performed using MEME [51]. Associations of DAP-seq peaks located upstream or downstream of the transcription start site (TSS) within 3.5 kb were analyzed using Homer [52].

CHIP-PCR
The CHIP procedure followed the method in [53]. The leaves of OE were cross-linked in a vacuum with a cross-linking buffer containing 1% formaldehyde on ice for 15 min. Chromatin was sonicated into fragments between 300 and 500 bp. The supernatant was incubated with an anti-GFP antibody (Abcam, ab290). Immunoprecipitated DNA fragments were purified using the Qiaquick PCR purification kit (Qiagen, Hilden, Germany). CHIP-PCR was conducted using the immunoprecipitated DNA fragments that were obtained above, and the sheared genome DNA was used as a positive control (primers are listed in Table S1) [54].

Conclusions
Here, we reported previously unknown regulatory pathways that mediate the response of Populus nigra to resistance to A. alternata necrotrophic fungi. RNA-seq analysis showed that the PsnWRKY70 gene enhanced the disease resistance by upregulating the expression of the MAP kinase cascade, calcium ion signal-related genes, membrane receptors, and other related genes. In addition, 129 downstream target genes of PsnWRKY70 were identified by RNA-seq and DAP-seq analysis, and CHIP-PCR found that the PsnWRKY70 gene can activate homologous WRKY6, WRKY18, WRKY22, WRKY22-1, and R genes to confer higher disease resistance in Populus. Other members of WRKYs may also play a role in disease resistance. This study further demonstrated the role of PsnWRKY70 in the regulatory network response to infection with Alternaria alternata (Fr.) Keissl in Populus.