Argonaute1‐Dependent LtmilR2 Negatively Regulated Infection of <i>Lasiodiplodia theobromae</i> by Targeting a Guanine Nucleotide Exchange Factor in RAS Signalling

Lasiodiplodia theobromae (L. theobromae) is the causative agent of grapevine canker disease, which is a serious threat to global grape production. Currently, no effective fungicides are available to manage this disease. In this study, LtmilR2 was identified as an Argonaute1 (AGO1)-dependent small RNA produced by L. theobromae. Functional analysis revealed that both LtAGO1 and LtmilR2 negatively regulated the pathogenicity of L. theobromae. Through degradome sequencing, we identified LtRASGEF-a guanine nucleotide exchange factor involved in RAS signalling-as the target gene of LtmilR2. LtmilR2 suppressed the expression of LtRASGEF. Pathogenicity assays demonstrated that LtRASGEF was essential for fungal virulence. Furthermore, following infection, the expression of LtmilR2 rapidly decreased, while LtRASGEF expression quickly increased and positively regulated infection. Finally, we observed that the growth of L. theobromae was inhibited when an RNA duplex targeting LtmilR2 was delivered via the star polycation (SPc) nanocarrier, suggesting that LtmilR2 may serve as an RNA-based fungicide target for grapevine canker disease management.