Valsa mali effector Vm_04797 interacts with adaptor protein MdAP-2ß to manipulate host autophagy.

Apple Valsa canker, caused by the ascomycete fungus Valsa mali, employs virulence effectors to disturb host immunity and poses a substantial threat to the apple industry. However, our understanding of how V. mali effectors regulate host defense responses remains limited. Here, we identified the V. mali effector Vm_04797, which was upregulated during the early infection stage. Vm_04797, a secreted protein, suppressed Inverted formin 1 (INF1)-triggered cell death in Nicotiana benthamiana and performed virulence functions inside plant cells. Vm_04797 deletion mutants showed substantially reduced virulence toward apple. The adaptor protein MdAP-2ß positively regulated apple Valsa canker resistance and was targeted and degraded by Vm_04797 via the ubiquitination pathway. The in vitro analysis suggested that Vm_04797 possesses E3 ubiquitin ligase activity. Further analysis revealed that MdAP-2ß is involved in autophagy by interacting with Malus domestica autophagy protein 16 MdATG16 and promoting its accumulation. By degrading MdAP-2ß, Vm_04797 inhibited autophagic flux, thereby disrupting the defense response mediated by autophagy. Our findings provide insights into the molecular mechanisms employed by the effectors of E3 ubiquitin ligase activity in ascomycete fungi to regulate host immunity.

The Valsa Mali effector Vm1G-1794 protects the aggregated MdEF-Tu from autophagic degradation to promote infection in apple.

Macroautophagy/autophagy is a conserved degradation pathway in eukaryotes that is required for recycling unwanted intracellular components, maintaining homeostasis, and coping with biotic and abiotic stresses. Pathogens have evolved to subvert autophagic machinery by secreting host cell-entering effector proteins. Here, we provided evidence that an apple autophagy-related gene MdATG8i, activated autophagy and contributed to resistance against Valsa canker caused by Valsa Mali (Vm) when being overexpressed in apple. MdATG8i interacted with a plastid elongation factor Tu (MdEF-Tu) which became insoluble and aggregated during Vm infection and was degraded through the autophagy pathway. Intriguingly, we identified a highly-induced effector secreted from Vm, Vm1G-1794, which competitively interacted with MdATG8i, suppressed autophagy, and depleted MdEF-Tu out of MdATG8i complexes. The formation of stable MdEF-Tu aggregates caused by Vm1G-1794 promoted the susceptibility of apple to Vm. Overall, our study demonstrated that MdATG8i contributed to Vm resistance by targeting and degrading MdEF-Tu, and Vm1G-1794 competed with MdEF-Tu to target MdATG8i and prevent MdEF-Tu degradation, thus favoring infection.Abbreviations: 35S: cauliflower mosaic virus 35S promoter; AIM: ATG8-interacting motif; ATG8-PE: ATG8 conjugated with phosphatidylethanolamine; BiFC: biomolecular fluorescence complementation; Con A: concanamycin A; Co-IP: co-immunoprecipitation; DEPs: differentially expressed proteins; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; hpt: hours post-treatment; LCI: luciferase complementation imaging; MdATG8i: autophagy-related protein 8i in Malus domestica; MDC: monodansylcadaverine; MdEF-Tu: elongation factor Tu in Malus domestica; MdNBR1: neighbor of BRCA1 in Malus domestica; N. benthamiana: Nicotiana benthamiana; OE: overexpression; PAMP: pathogen-associated molecular pattern; PTI: pattern-triggered immunity; qRT-PCR: quantitative reverse transcription PCR; RFP: red fluorescent protein; RNAi: RNA interference; ROS: reactive oxygen species; Ub: ubiquitin; V. Mali: Valsa Mali; WT: wild-type plant; YFP: yellow fluorescent protein.