Functional analysis of NtWRKY70 regulating tobacco defense against Rhizoctonia solani AG3-TB

Rhizoctonia solani AG3-TB is a destructive necrotrophic pathogen causing tobacco target spot disease. WRKY70 acts as a key regulator integrating salicylic acid (SA) and jasmonic acid (JA) signals in plant immunity. Our previous study found that NtWRKY70 in Nicotiana tabacum L. is significantly induced by R. solani AG3-TB infection; however, its functional mechanism remains elusive. In this study, bioinformatics analysis showed that NtWRKY70 is a nuclear localized protein with conserved domains and transcriptional autoactivation activity. Further investigations using virus-induced gene silencing (VIGS), CRISPR/Cas9-mediated knockout and overexpression transgenic plants confirmed that NtWRKY70 acts as a positive regulator against R. solani AG3-TB. Based on RNA-seq data and differential expression genes (DEGs) screening, the total of 11 defense-related genes were significantly upregulated. Exogenous application of SA and methyl jasmonate (MeJA) induced NtWRKY70 expression and decreased the lesion diameter, which indicated that NtWRKY70 is potentially linked to the activation of downstream SA and JA signaling pathways. Collectively, our findings suggest that NtWRKY70 participates in tobacco defense against R. solani AG3-TB, potentially by coordinating SA and JA signaling to activate defense-related genes, which provides a valuable candidate for tobacco disease-resistant breeding.