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

## TL;DR

This study identifies and characterizes four cutinase genes in a fungus that causes disease in fishscale bamboo, revealing their roles in pathogenicity and stress response.

## Contribution

The functional characterization of NsCut1–NsCut4 cutinase genes in Neostagonosporella sichuanensis and their effects on pathogenicity and stress tolerance.

## Key findings

- NsCut1 and NsCut3 are essential for pigmentation, spore production, stress resistance, and pathogenicity.
- Recombinant cutinase proteins show strong hydrolytic activity against glycerol tributyrate and bamboo white cream.
- NsCut2 and NsCut4 deletions do not significantly affect fungal traits compared to the wild type.

## Abstract

Fishscale bamboo rhombic-spot, caused by Neostagonosporella sichuanensis, poses a significant threat to Phyllostachys heteroclada in Sichuan province. Based on genomic analysis, four cutinase genes, NsCut1–NsCut4, were identified, cloned, and functionally validated. Bioinformatics analyses revealed that the proteins encoded by these genes possess secretory functions, lack transmembrane domains, and contain conserved cutinase domains highly homologous to those in other fungi. Recombinant proteins expressed via a prokaryotic system exhibited strong hydrolytic activity against glycerol tributyrate and bamboo white cream at 40°C and pH 8.0, while signal peptide and subcellular localization analyses confirmed their secretory function and localization to the cell wall. Gene knockout experiments were performed to construct deletion strains ΔNsCut and corresponding complemented strains ΔNsCut+. Notably, ΔNsCut1 and ΔNsCut3 resulted in reduced pigmentation, decreased spore production, and increased sensitivity to NaCl, H2O2, and Congo red, along with reduced pathogenicity—indicating that these genes play key roles in metabolic and reproductive processes, oxidative stress responses, and the maintenance of cell wall integrity. In contrast, ΔNsCut2 and ΔNsCut4 did not exhibit significant differences compared to the wild type. This work advances our understanding of the role of cutinases in the pathogenic interaction between N. sichuanensis and P. heteroclada, providing a theoretical basis for further exploration of the pathogen’s underlying mechanisms.