PlAtg8-mediated autophagy regulates vegetative growth, sporangial cleavage, and pathogenesis in Peronophythora litchii

Abstract

ABSTRACT Autophagy is a conserved cellular metabolic process in eukaryotes that transports damaged or redundant intracellular organelles and proteins to lysosomes or vacuoles for degradation and recycling. Atg8 is not only essential for autophagosome formation but also required for fungal development and pathogenicity, but its function is unknown in Peronophythora litchii. In this study, PlAtg8, a yeast Atg8 homolog, is deleted by the CRISPR/Cas9 system coupled with PEG-mediated protoplast transformation in P. litchii. Compared with the wild-type strain, the absence of PlAtg8 affects autophagosome formation. The colony growth rate, number of sporangia, and zoospore release rate are significantly decreased, and the oospores are undetected in ΔPlatg8. The pathogenicity test shows that the lesion areas on litchi leaves and fruits caused by ΔPlatg8 are significantly attenuated compared with the wild type, indicating that PlAtg8 is also required for the pathogenicity of P. litchii. Moreover, the cleavage rate of sporangia in ΔPlatg8 is significantly decreased compared to the wild type by FM4-64 staining, which is probably the main reason for the lower zoospores release rate in ΔPlatg8. These results suggest that PlAtg8 not only regulates autophagy but also participates in the growth and development of P. litchii. IMPORTANCE Peronophythora litchii is the pathogen of litchi downy blight, which is the most serious disease in litchi. Autophagy is an evolutionarily conserved catabolic process in eukaryotes. Atg8 is a core protein of the autophagic pathway, which modulates growth and pathogenicity in the oomycete P. litchii. In P. litchii, CRISPR/Cas9-mediated knockout of the PlATG8 impaired autophagosome formation. PlATG8 knockout mutants exhibited attenuated colony expansion, sporangia production, zoospore discharge, and virulence on litchi leaves and fruits. The reduction in zoospore release was likely underpinned by impaired sporangial cleavage. Thus, in addition to governing autophagic flux, PlAtg8 is indispensable for vegetative growth and infection of P. litchii.

Keywords

• PlAtg8
• sporangial cleavage
• autophagosome formation
• pathogenicity
• Peronophythora litchii