A novel member of miR169 family negatively regulates maize resistance against Bipolaris maydis

MicroRNAs (miRNAs) have been confirmed to play important roles in plant defense response. However, the key maize miRNAs involved in the defense response against Bipolaris maydis are very limited. In this study, a novel member of the miR169 family in response to B. maydis, named zma-miR169s, was discovered and investigated. The expression levels of pre-miR169s and zma-miR169s were significantly repressed during B. maydis infection. The CRISPR/Cas9-induced zma-miR169s mutant exhibited more resistance against B. maydis, whereas overexpression of zma-miR169s enhanced susceptibility, supporting that zma-miR169s might play a negative role in maize resistance. Moreover, RNA-seq and Gene Ontology analysis showed that differentially expressed genes were highly enriched in the oxidation-reduction process and plant hormone pathway. Hence, reactive oxygen species (ROS) and plant hormone levels were further investigated. ROS detection confirmed that the zma-miR169s mutant accumulated more ROS, while less ROS was detected in transgenic maize OE-miR169s. Furthermore, more remarkable changes in PR-1 expression levels and salicylic acid (SA) contents were detected in the zma-miR169s mutant compared with wild-type and transgenic maize during B. maydis infection. Additionally, nuclear transcription factors (NF-YA1 and NF-YA13) were identified as targets regulated by zma-miR169s through the agrobacterium-mediated transient expression method. Overexpression of ZmNF-YA13 enhanced Arabidopsis resistance to Pseudomonas syringae pv. tomato DC3000. Taken together, our results suggest that zma-miR169s negatively regulates maize defense responses by influencing ROS accumulation and the SA-dependent signaling pathway.