JrWRKY21 interacts with JrPTI5L to activate the expression of JrPR5L for resistance to Colletotrichum gloeosporioides in walnut

SUMMARY Walnut (Juglans regia L.) anthracnose, induced by Colletotrichum gloeosporioides, is a catastrophic disease impacting the walnut industry in China. Although WRKY transcription factors play a key role in plant immunity, the function of the WRKY gene family in walnut resistance to C. gloeosporioides is not clear. Here, through transcriptome sequencing and quantitative real‐time polymerase chain reaction (qRT‐PCR), we identified a differentially expressed gene, JrWRKY21, that was significantly upregulated upon C. gloeosporioides infection in walnut. JrWRKY21 positively regulated walnut resistance to C. gloeosporioides, as demonstrated by virus‐induced gene silencing and transient gene overexpression. Additionally, JrWRKY21 directly interacted with the transcriptional activator of the pathogenesis‐related (PR) gene JrPTI5L in vitro and in vivo, and could bind to the W‐box in the JrPTI5L promoter for transcriptional activation. Moreover, JrPTI5L could induce the expression of the PR gene JrPR5L through binding to the GCCGAC motif in the promoter. Our data support that JrWRKY21 can indirectly activate the expression of the JrPR5L gene via the WRKY21–PTI5L protein complex to promote resistance against C. gloeosporioides in walnut. The results will enhance our understanding of the mechanism behind walnut disease resistance and facilitate the genetic improvement of walnut by molecular breeding for anthracnose‐resistant varieties. Significance Statement Integrative transcriptomics and molecular biology approaches have revealed that JrWRKY21 not only interacts with JrPTI5L but also binds to the W‐box on its promoter to trigger its activity. JrWRKY21–JrPTI5L activates the expression of JrPR5L through JrPTI5L binding to the GCCGAC motif to promote resistance to C. gloeosporioides in walnut.