A Mitogen-Activated Protein Kinase Tmk3 Participates in High Osmolarity Resistance, Cell Wall Integrity Maintenance and Cellulase Production Regulation in Trichoderma reesei

The mitogen-activated protein kinase (MAPK) pathways are important signal transduction pathways conserved in essentially all eukaryotes, but haven't been subjected to functional studies in the most important cellulase-producing filamentous fungus Trichoderma reesei. Previous reports suggested the presence of three MAPKs in T. reesei: Tmk1, Tmk2, and Tmk3. By exploring the phenotypic features of T. reesei Δtmk3, we first showed elevated NaCl sensitivity and repressed transcription of genes involved in glycerol/trehalose biosynthesis under higher osmolarity, suggesting Tmk3 participates in high osmolarity resistance via derepression of genes involved in osmotic stabilizer biosynthesis. We also showed significant downregulation of genes encoding chitin synthases and a β-1,3-glucan synthase, decreased chitin content, ‘budded’ hyphal appearance typical to cell wall defective strains, and increased sensitivity to calcofluor white/Congo red in the tmk3 deficient strain, suggesting Tmk3 is involved in cell wall integrity maintenance in T. reesei. We further observed the decrease of cellulase transcription and production in T. reesei Δtmk3 during submerged cultivation, as well as the presence of MAPK phosphorylation sites on known transcription factors involved in cellulase regulation, suggesting Tmk3 is also involved in the regulation of cellulase production. Finally, the expression of cell wall integrity related genes, the expression of cellulase coding genes, cellulase production and biomass accumulation were compared between T. reesei Δtmk3 grown in solid state media and submerged media, showing a strong restoration effect in solid state media from defects resulted from tmk3 deletion. These results showed novel physiological processes that fungal Hog1-type MAPKs are involved in, and present the first experimental investigation of MAPK signaling pathways in T. reesei. Our observations on the restoration effect during solid state cultivation suggest that T. reesei is evolved to favor solid state growth, bringing up the proposal that the submerged condition normally used during investigations on fungal physiology might be misleading.丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）信号通路是几乎所有真核生物中保守存在的重要信号转导通路，但在目前最为重要的产纤维素酶丝状真菌里氏木霉（Trichoderma reesei）中，尚未对其开展系统的功能学研究。已有研究报道显示，里氏木霉中存在3种MAPK：Tmk1、Tmk2与Tmk3。本研究通过解析里氏木霉tmk3基因缺失突变株（Δtmk3）的表型特征，首次发现该菌株在高渗环境下对氯化钠的敏感性显著升高，同时甘油/海藻糖生物合成相关基因的转录受到抑制，提示Tmk3可通过解除渗透稳定剂生物合成相关基因的转录阻遏，参与高渗胁迫抗性调控。本研究同时证实，里氏木霉tmk3缺失突变株中，几丁质合酶与β-1,3-葡聚糖合酶编码基因的转录水平显著下调，几丁质含量降低，呈现出细胞壁缺陷菌株典型的“出芽状”菌丝表型，且对荧光增白剂/刚果红的敏感性升高，表明Tmk3参与维持里氏木霉的细胞壁完整性。进一步研究发现，在液体深层培养过程中，里氏木霉tmk3缺失突变株的纤维素酶转录与合成水平均出现明显下降；同时，已知的纤维素酶调控转录因子上存在MAPK磷酸化位点，提示Tmk3还参与纤维素酶生产的调控过程。最后，本研究对比了固态培养基与液体培养基中培养的里氏木霉tmk3缺失突变株的细胞壁完整性相关基因表达、纤维素酶编码基因表达、纤维素酶产量以及生物量积累情况，发现固态培养基可显著修复tmk3缺失带来的各类缺陷。上述研究结果揭示了真菌Hog1型MAPK所参与的全新生理过程，首次完成了里氏木霉中MAPK信号通路的实验探究。我们在固态培养过程中观察到的修复效应提示，里氏木霉演化出了偏好固态生长的特性，这也提出了一项新的假说：当前真菌生理学研究中普遍采用的液体培养条件可能存在误导性。