Functional characterization of cutinase genes NsCut1-NsCut4 in Neostagonosporella sichuanensis and their effects on fishscale bamboo

Mengyao Lu Fang Liang Lijuan Liu Yanji Yin Dongxin Xu Huan Zou Yinggao Liu Chunlin Yang ^*

• Sichuan Agricultural University, Chengdu, China

Fishscale bamboo rhombic-spot caused by Neostagonosporella sichuanensis poses a significant threat to Phyllostachys heteroclada, mainly in Sichuan province. Cutinase, a crucial pathogenic factor for degrading plant epidermis, has not been explored in N. sichuanensis. In this study, four cutinase genes, NsCut1-NsCut4, were screened and cloned based on the genome analysis of this fungus, with functional validation. Bioinformatics analysis showed that all cutinase proteins were secreted, lacked transmembrane structural domains, possessed Cutinase structural domains, and were evolutionarily highly homologous to other fungal cutinases. Recombinant proteins were obtained by a prokaryotic expression system, and their optimal reaction conditions were measured to be 40°C and pH 8.0, showing strong hydrolytic activity against glycerol tributyrate and P. heteroclada bamboo white cream. Further secretory function and subcellular localisation analyses showed that the cutinase gene had an effective signal peptide secretion function and the protein was localised in the cell wall.The knockout strain ΔNsCut and complementary strain ΔNsCut+ of four cutinase genes were constructed by gene knockout. ΔNsCut1 and ΔNsCut3 were found to exhibit reduced pigmentation and decreased spore production in terms of colony morphology and spore production, as well as a significant increase in susceptibility to NaCl, H2O2, and Congo red, along with reduced pathogenicity.It is hypothesised that they are involved in metabolic and reproductive processes, oxidative stress response, maintenance of cell wall integrity, and pathogenicity. ΔNsCut2 and ΔNsCut4 were not significantly different from the wild-type.