The distinct roles of Argonaute protein 2 in the growth, stress responses and pathogenicity of the apple tree canker pathogen

Abstract

SummaryValsa mali (V. mali), the causal agent of apple tree Valsa canker, severely damages apple production, a major economic crop in China. To date, our understanding of the molecular mechanisms associated with the pathogenicity of V. mali is still limited. RNA interference participates in various biological processes in multicellular organisms. The Argonaute proteins (AGOs), which are a core component of the RNA interference system, play key roles in vegetable growth, environmental responses and fungal pathogenicity. Previously, transcriptome analysis revealed that the AGO2 gene (VMAGO2) was up‐regulated during V. mali infection, suggesting that VMAGO2 plays a potential role in pathogenicity. In this study, we investigated the potential roles of VMAGO2 in the growth, stress responses and pathogenicity of V. mali. VMAGO2 was isolated and found to be orthologous to AGO2 of Neurospora crassa and Fusarium graminearum. Real‐time quantitative PCR analysis showed that VMAGO2 expression was 3.4‐fold higher than that of the control (mycelium) at 24 hr post‐inoculation (hpi). Six positive VMAGO2 mutants were generated using double‐joint PCR and PEG‐mediated transformation. Deletion of VMAGO2 did not result in any obvious phenotypic change when compared with that of wild‐type strain 03‐8. Furthermore, the colonial morphology was not obviously affected when the mutants were subjected to osmotic and pH stress treatments. However, the knockout mutants did not grow on PDA with 0.05% H2O2. More importantly, infection assays showed that the average lesion diameter/length resulting from mutant infections was 34.8% and 19.8% smaller on apple leaves and twigs, respectively, than those resulting from wild‐type infections. All six positive mutants showed a consistent phenotype, and the defects of the mutants were fully complemented by re‐introducing the WTVMAGO2 allele. Our results demonstrated that VMAGO2 plays an important role in H2O2 tolerance and the pathogenicity of V. mali.