Exogenous anti-MoPMK1 microRNA and precursor formats reduce Magnaporthe oryzae infection in rice

Exogenous RNA application is emerging as a useful strategy to investigate plant-pathogen interactions. Most studies against fungal pathogens have focused on long double-stranded RNAs, whereas the potential of artificial miRNA and miRNA-precursor formats remains less explored. Here, we targeted the Magnaporthe oryzae MAP kinase gene MoPMK1, a central regulator of appressorium-mediated penetration, invasive growth and host tissue colonization, using three exogenous RNA formats: chemically synthesized mature miRNA duplexes, in planta-produced anti-MoPMK1-containing small RNA fractions and in vitro-synthesized miRNA precursors. All three anti-MoPMK1 formats reduced blast symptoms in rice leaves and were accompanied by lower accumulation of fungal MoPMK1 transcripts. Under the mass-based doses and assay conditions tested, the in vitro-synthesized precursor was associated with a marked reduction in lesion development despite being applied at a lower molar concentration than the mature duplex. Leaf sheath infection assays further showed altered fungal development after anti-MoPMK1 precursor treatment, whereas appressorium formation was reduced most clearly at the highest precursor concentration tested. In addition, application of the precursor through the leaf base reduced disease symptoms at a distal infection site in detached leaves. Direct incubation of fungal spores with the precursor in liquid culture did not reveal a clear inhibitory effect, suggesting that the infection context in plant tissue contributes to precursor activity. Together, these results identify mature and precursor anti-MoPMK1 microRNA formats as sequence-defined RNA tools with biological activity in the rice-M. oryzae interaction, while highlighting RNA format and plant tissue context as important determinants of exogenous RNA activity.