Global characterization of GH11 family xylanases genes in Neostagonosporella sichuanensis and functional analysis of Nsxyn1 and Nsxyn2

Rhombic-spot disease, caused mainly by <i>Neostagonosporella sichuanensis</i>, significantly impacts the yield and quality of fishscale bamboo (<i>Phyllostachys heteroclada</i>). Xylanases are essential for pathogenic fungi infection, yet their specific functions in the physiology and pathogenicity of <i>N. sichuanensis</i> remain unclear. Here, we characterized three xylanase proteins with glycosyl hydrolase 11 domains from the <i>N. sichuanensis</i> SICAUCC 16-0001 genome and examined the function of Nsxyn1 and Nsxyn2. Purified Nsxyn1 and Nsxyn2 proteins displayed specific xylanase activity <i>in vitro</i> and induced cell death in <i>Nicotiana benthamiana</i>, independent of their enzymatic function. Both proteins possessed signal peptides and were confirmed as secreted proteins using a yeast secretion system. Subcellular localization revealed that Nsxyn1 and Nsxyn2 localized in both the cytoplasm and nucleus and can trigger cell death in <i>N. benthamiana</i> through <i>Agrobacterium tumefaciens</i>-mediated transient transformation. qRT-PCR results showed notable upregulation of <i>Nsxyn1</i> and <i>Nsxyn2</i> during infection, with <i>Nsxyn1</i> exhibiting an 80-fold increase at 15 days post-inoculation. Deletion of <i>Nsxyn1</i> and <i>Nsxyn2</i> in <i>N. sichuanensis</i> impaired xylan degradation, adaptation to osmotic and oxidative stress, and pathogenic full virulence. Deletion of <i>Nsxyn1</i> notably slowed fungal growth and reduced spore production, whereas only a reduction in microconidial production was observed in <i>Nsxyn2</i> mutants. Complementation of <i>Nsxyn1</i> and <i>Nsxyn2</i> only partially restored these phenotypic defects in the ∆<i>Nsxyn1</i> and ∆<i>Nsxyn2</i> mutants. These findings suggest that Nsxyn1 and Nsxyn2 contribute to <i>N. sichuanensis</i> virulence and induced plant defense responses, providing new insights into the function of xylanases in the interaction between fishscale bamboo and <i>N. sichuanensis</i>.