Small RNAs function to regulate plant defense responses to pathogens. We previously showed that miR825 and miR825* downregulate Bacillus cereus AR156 (AR156)-triggered systemic resistance to Pseudomonassyringae pv. tomato DC3000 in Arabidopsis thaliana (Arabidopsis). Here, Northern blotting revealed that miR825 and miR825* were more strongly downregulated in wild type Arabidopsis Col-0 (Col-0) plants pretreated with AR156 than in nontreated plants upon Botrytis cinerea (B. cinerea) B1301 infection. Furthermore, compared with Col-0, transgenic plants with attenuated miR825 and miR825* expression were more resistant to B. cinerea B1301, yet miR825- and miR825*-overexpressing (OE) plants were more susceptible to the pathogen. With AR156 pretreatment, the transcription of four defense-related genes (PR1, PR2, PR5, and PDF1.2) and cellular defense responses (hydrogen peroxide production and callose deposition) were faster and stronger in miR825 and miR825* knockdown lines but weaker in their OE plants than in Col-0 plants upon pathogen attack. Also, AR156 pretreatment caused stronger phosphorylation of MPK3 and MPK6 and expression of FRK1 and WRKY53 genes upon B. cinerea B1301 inoculation in miR825 and miR825* knockdown plants than in Col-0 plants. Additionally, the assay of agrobacterium-mediated transient co-expression in Nicotiana benthamiana confirmed that AT5G40910, AT5G38850, AT3G04220, and AT5G44940 are target genes of miR825 or miR825*. Compared with Col-0, the target mutant lines showed higher susceptibility to B. cinerea B1301, while still expressing AR156-triggered induced systemic resistance (ISR). The two-way analysis of variance (ANOVA) revealed a significant (P < 0.01) interactive effect of treatment and genotype on the defense responses. Hence, miR825 and miR825*act as negative regulators of AR156-mediated systemic resistance to B. cinerea B1301 in Arabidopsis.
Keywords: Bacillus cereus AR156; Botrytis cinerea B1301; induced systemic resistance; miR825 and miR825*; plant innate immunity.