Identifying the key motif essential for enhancing plant defense against Botrytis cinerea by β-glucanase from Bacillus velezensis LJ02

The glycosyl hydrolase β-glucanase elicits plant immune responses against pathogens and enhances plant immunity by activating signaling pathways. The specific functional domains responsible for disease prevention remain unclear. In this study, transient expression of β-glucanase significantly increased leaves resistance to Botrytis cinerea in Nicotiana benthamiana systemic leaves. Through sequence alignment and similarity analysis, five conserved motifs in the amino acid sequence of β-glucanase were identified, and five deletion mutants were generated to investigate its essential regions further. Notably, the N-terminal amino acid sites 5-54 deletion mutation of β-glucanase decreased resistance to B. cinerea infection. These results indicate that N-terminal amino acids 5-54 (N54) are crucial for β-glucanase induced N. benthamiana defense response and for enhancing resistance to B. cinerea. Further analysis using real-time quantitative fluorescent PCR (qRT-PCR) revealed a significant reduction in gene expression within the N54 region compared to that of unmutated β-glucanase. Additionally, there was a notable reduction in the relative expression levels of FRK, CYP71D20, WRKY7, WRKY8, ACRE31 and PTI genes. Therefore, the first 50 amino acids at positions 5-54 within the N-terminal domain were essential for triggering plant defense responses and enhancing resistance against B. cinerea infection. This study provides an important theoretical foundation for systematic investigation into key functional domains within β-glucanase that trigger defensive responses in plants against B. cinerea.