Molecular Mechanism Of PsnWRKY70 Gene Mediated Inhibition Of Alternaria Alternata In Populus Simonii × P.Nigra

WRKY family is one of the largest and most widely studied transcription factor families in higher plants,which is an important part of the signal network regulating many biological processes in plants.WRKY family plays an important role in plant growth and development,senescence,abiotic and biotic stresses.Previous studies have shown that over-expression of PsnWRKY70 gene,a member of WRKY family,can enhance the resistance to leaf blight in Populus simonii ×Populus nigra.However,the defense mechanism and defense pathway of PsnWRKY70 gene involved in resistance to plant blight are still unclear.In this study,overexpression(OE)and repressed(RE)of PsnWRKY70 and wild type of Populus simonii × P.nigra were used.Transcriptome sequencing(RNA-seq),DNA affinity purification sequencing(DAP-seq)and chromatin immunoprecipitation-PCR(Ch IP-PCR)were used to screen and verify the target genes regulated by PsnWRKY70.The 16 s amplicon sequencing,phyllosphere bacterial isolation and culture,and plate confrontation were used to reveal the changes in phyllosphere bacterial community composition,and to explore the probiotic bacteria of OE lines that can resist pathogen infection.The results are as follows:(1)Transcriptome analysis of the transgenic lines and the control lines showed that there were 691 differentially expressed genes in the OE line compared with the WT line.KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in plant-pathogen interaction,MAPK signaling pathway and other signaling pathways.Go enrichment analysis showed that the differentially expressed genes were mainly enriched in protein phosphorylation,cellular protein modification process and other biological processes.PsnWRKY70 gene can promote the expression of some resistance-related genes,including MAPK signaling pathway(PsnM2K4,PsnMPK3,PsnM3K18),calcium channel protein(PsnCNG3,PsnCNGC1,PsnCNG4).Calcium-dependent protein kinases(PsnCDPKL,PsnCDPKW,PsnCDPKS,and PsnCDPKQ),and SA synthesis pathway-related genes(PsnPAD4,PsnCBP60 C,and PsnCBP60D).Therefore,the overexpression of PsnWRKY70 gene can enhance the resistance to Alternaria alternata by increasing the expression of the above genes.(2)The results of DAP-seq analysis showed that there were 2103 gene promoter regions with PsnWRKY70 binding sites in Populus simonii × P.nigra.The major motifs for PsnWRKY70 binding are "TTGACT/C" and "A/GGTCAA".Therefore,PsnWRKY70 can regulate the expression of downstream genes by binding to W-box elements.RNA-seq,DAPseq and Ch IP-PCR analysis showed that,PsnWRKY70 can directly bind to the promoters of homologous genes(PsnWRKY6,PsnWRKY18,PsnWRKY22,and PsnWRKY22-1)to promote gene expression.PsnWRKY70 can also directly regulate some R genes that play an important role in disease resistance,such as PsnLRR8,PsnLRR-RLK,PsnADR1-like 2,PsnNB-ARC and so on.Therefore,the transcription factor PsnWRKY70 can enhance the resistance of Populus simonii × P.nigra to Alternaria alternata by directly regulating the expression of the above genes.(3)The 16s amplicon sequencing analysis of the phyllosphere bacterial communities of the transgenic lines and the control lines showed that the Shannon diversity index of the epiphytic bacterial communities of OE lines was significantly higher than that of WT and RE lines under Alternaria alternata treatment.PCoA analysis showed that there were significant differences in the epiphytic bacterial community structure among the tested lines under Alternaria alternata stress.The results of plate confrontation test showed that Chryseobacterium,Methylobacterium,Paracoccus,Brevundimonas,Pseudomonas and Flavobacterium could significantly inhibit the growth of Alternaria alternata.The bacteriostatic efficiency was 13.89%,18.06%,27.78%,33.68%,37.15% and 56.60%respectively.Thus,overexpression of the PsnWRKY70 gene can recruit beneficial bacteria to enhance their disease resistance.(4)Metabolome analysis of leaves of transgenic and control lines showed that OE lines had 12 up-regulated differential metabolites compared with WT.Scopolin and Fraxin can promote the growth of beneficial bacteria such as Chryseobacterium,Pseudomonas,Methylobacterium,Achromobacter and Flavobacterium.Scopolin has a significant inhibitory effect on the growth of Alternaria alternata.Therefore,Scopolin and Fraxin not only participate in the shaping of the phyllosphere microbiome,but also directly inhibit the growth of the pathogen Alternaria alternata.In conclusion,PsnWRKY70 is an important participant in the regulation of resistance to Alternaria alternata in Populus simonii × P.nigra.PsnWRKY70 enhanced the resistance of P.simonii × P.nigra to Alternaria alternata by directly regulating the expression of homologous genes,R genes,indirect calcium signaling pathway genes and MAPK cascade related genes.Meanwhile,the PsnWRKY70 gene can enrich some probiotic bacteria to resist the infection of Alternaria alternata by regulating plant metabolites.The results revealed the important role of PsnWRKY70 gene in the resistance of Populus simonii × P.nigra to Alternaria alternata infection.It also laid a theoretical foundation for the mechanism of action of forest phyllosphere microbiome against pathogenic fungi.