Data_Sheet_1_LysM Proteins Regulate Fungal Development and Contribute to Hyphal Protection and Biocontrol Traits in Clonostachys rosea.PDF

Lysin motif (LysM) modules are approximately 50 amino acids long and bind to peptidoglycan, chitin and its derivatives. Certain LysM proteins in plant pathogenic and entomopathogenic fungi are shown to scavenge chitin oligosaccharides and thereby dampen host defense reactions. Other LysM proteins can protect the fungal cell wall against hydrolytic enzymes. In this study, we investigated the biological function of LysM proteins in the mycoparasitic fungus Clonostachys rosea. The C. rosea genome contained three genes coding for LysM-containing proteins and gene expression analysis revealed that lysm1 and lysm2 were induced during mycoparasitic interaction with Fusarium graminearum and during colonization of wheat roots. Lysm1 was suppressed in germinating conidia, while lysm2 was induced during growth in chitin or peptidoglycan-containing medium. Deletion of lysm1 and lysm2 resulted in mutants with increased levels of conidiation and conidial germination, but reduced ability to control plant diseases caused by F. graminearum and Botrytis cinerea. The Δlysm2 strain showed a distinct, accelerated mycelial disintegration phenotype accompanied by reduced biomass production and hyphal protection against hydrolytic enzymes including chitinases, suggesting a role of LYSM2 in hyphal protection against chitinases. The Δlysm2 and Δlysm1Δlysm2 strains displayed reduced ability to colonize wheat roots, while only Δlysm1Δlysm2 failed to suppress expression of the wheat defense response genes PR1 and PR4. Based on our data, we propose a role of LYSM1 as a regulator of fungal development and of LYSM2 in cell wall protection against endogenous hydrolytic enzymes, while both are required to suppress plant defense responses. Our findings expand the understanding of the role of LysM proteins in fungal-fungal interactions and biocontrol.

Lysin基序（Lysin motif, LysM）模块长度约为50个氨基酸，可结合肽聚糖（peptidoglycan）、几丁质（chitin）及其衍生物。植物病原真菌与昆虫病原真菌中的部分LysM蛋白已被证实可清除几丁寡糖，从而抑制宿主的防御反应；另有部分LysM蛋白能够保护真菌细胞壁免受水解酶的降解。本研究针对重寄生真菌粉红粘帚霉（Clonostachys rosea）中的LysM蛋白的生物学功能展开了系统探究。该菌株的基因组中存在3个编码含LysM结构域蛋白的基因，基因表达分析结果显示，lysM1与lysM2在与禾谷镰孢（Fusarium graminearum）的重寄生互作过程以及小麦根系定殖阶段均被诱导表达。其中，lysM1在萌发的分生孢子中表达受到抑制，而lysM2在以几丁质或肽聚糖为营养的培养基中生长时表达量显著上调。分别敲除lysM1与lysM2后获得的突变体，其分生孢子产生能力与分生孢子萌发率均有所提升，但抑制禾谷镰孢与灰葡萄孢（Botrytis cinerea）所致植物病害的能力显著下降。ΔlysM2突变体呈现出显著的菌丝解体加速表型，同时伴随生物量合成减少以及菌丝抵御包括几丁质酶在内的水解酶的能力降低，这表明LYSM2在菌丝抵御几丁质酶的过程中发挥关键作用。ΔlysM2单突变体与ΔlysM1ΔlysM2双突变体的小麦根系定殖能力均有所降低，而仅ΔlysM1ΔlysM2双突变体无法抑制小麦防御反应基因PR1（Pathogenesis-related protein 1）和PR4（Pathogenesis-related protein 4）的表达。基于本研究数据，我们提出：LYSM1作为真菌发育的调控因子，LYSM2则负责保护真菌细胞壁免受内源性水解酶的破坏，而二者均为抑制植物防御反应所必需。本研究结果进一步拓展了学界对LysM蛋白在真菌-真菌互作及生物防治领域中作用的认知。