Abstract:

Objective: This article aims to reveal the biological function of cysteine protease CfAtg4 in Colletotrichum fructicola on Camellia oleifera, so as to provide a candidate target site for the development of new prevention and control agents for anthracnose in C. oleifera. Method: The CfATG4 gene knockout fragment was constructed by Overlap PCR, and the knockout mutants and complemented strains were obtained by homologous recombination and PEG-mediated protoplast transformation. The sensitivity of wild-type, mutant and complemented strains to rapamycin was measured. The degree of autophagy was evaluated by observing the number of autophagosomes before and after starvation induction under an inverted fluorescent microscopy, and validating the microscopic observation results using the Western blot assays. The growth rate, conidia number, pathogenicity, spore germination rate, appressoria formation rate and response to external stresses were measured in the wild type, mutant and complemented strains. Result: 1) The CfATG4 knockout mutant, ΔCfatg4, was more sensitive to rapamycin and showed significantly fewer autophagosomes than wild-type CFLH16. The ΔCfatg4 mutant was not able to degrade the autophagy marker protein, GFP-CfAtg8, before and after starvation induction, exhibiting autophagy abolishment. 2) The biological phenotypic analysis revealed that ΔCfatg4 mutant grew slowly, and the aerial hyphae decreased on different nutrient media. ΔCfatg4 mutant significantly reduced conidiation, and lost pathogenicity on tea oil leave. 3) The spore germination rate and appressoria formation rate of ΔCfatg4 mutant were significantly reduced. 4) ΔCfatg4 mutant showed stronger tolerance to salt stresses (NaCl, KCl), and a cell wall integrity stress agent, Congo red (CR), but it was more sensitive to sodium dodecyl sulfate (SDS), another cell wall integrity stress agent. Furthermore, ΔCfatg4 mutant was more sensitive to oxidizing environment (H₂O₂), and however, more tolerant to reducing environment (dithiothreitol, DTT). Conclusion: The cysteine protease CfAtg4 is involved in regulating the growth and development, conidiation, appressoria formation, external stress responses, and pathogenicity through mediated autophagy. In this study, we have first elucidated that the CfAtg4-mediated autophagy regulates the pathogenicity of C. fructicola, which provides potential target sites for the development of novel fungicides.

Key words: Camellia oleifera, Colletotrichum fructicola, cysteine protease Atg4, pathogenicity