Identification Of The Role Of A MAP Kinase Tmk2 In Hypocrea Jecorina (Trichoderma Reesei)

Hypocrea jecorina (syn. Trichoderma reesei) is one of the most important cellulase-producing industrial filamentous fungal species with strong glycoside hydrolase activities and cellulase hyper-secreting capability.The mitogen-activated protein kinase (MAP kinase, MAPK) pathways are among the most well-known signal transduction pathways in eukaryotes. These pathways involve three serine/threonine protein kinases:MAPKKK, MAPKK and MAPK. These three kinases collaborate to pass phosphorylation states from upstream sensors to MAPKKK, to MAPKK, to MAPK, and eventually to downstream elements such as transcription factors for the regulation of physiological functions.Despite the important role of MAPKs in signal transduction, their functions in the cellulase hyper-producing filamentous fungus Hypocrea jecorina haven’t been studied except for the Hog 1-like Tmk3. In this work, we constructed H. jecorina △tmk2 by homologous recombination and explored the features of to identify the role of this Slt2-homologous Tmk2.A reduction of growth on agar plates of different carbon sources was observed for H. jecorina △tmk2 in comparison to the TU-6. Sporulation levels of H. jecorina parent and △tmk2 strains were assayed by comparing the level of spore production after 6 days of growth on PDA plates. A significant smaller amount of spores was produced by △tmk2 than the parent strain. These results lead to the suggestion that deletion of Imk2 affect the growth of H. jecorina and results in reduced sporulationin.The sensitivity of TU-6 and △tmk2 strains to CR and CFW was tested, The TU-6 could tolerate at least 200 μg/ml CR or 80 μg/ml CFW, while the △tmk2 strain was unable to grow on plates containing 150 μg/ml CR or 40 μg/ml CFW. These results lead to the suggestion that the cell wall synthsis of △tmk2 is suppressed. Real-time PCR analysis of the transcriptional levels of chitin synthase coding genes showed they are clearly and significantly downregulated in △tmk2. Along with findings that deletion of tmk2 leads to increased sensitivity to cell wall interfering substances CR and CFW, these results suggest that Tmk2 is involved in cell wall integrity maintenance.We assayed extracellular cellulase production in both//. jecorina parent and △tmk2 strains. Higher cellulase production levels could be identified in the △tmk2 strain while the ATP level was not statistically different in submerged cultures used for cellulase production analysis. Therefore leading to the suggestion that H. jecorina △tmk2 produces more cellulase per unit of biomass than the parent strain. The transcriptional levels of cellulase coding genes and transcription factors that regulate cellulase expression were analyzed using qPCR, the difference is too minor to conclude a biologically significant difference A suggestion can be made that the modulation of Tmk2 on cellulase production is not on the transcriptional level.Both Tmk2 and Tmk3 are involved in cell wall integrity maintenance. This leads to a question:What is the relationship of Tmk2 and Tmk3 in cell wall integrity maintenance? Whether they work in the same MAPK pathway?We overexpressed Tmk3 in H. jecorina △tmk.2 to construct △tmk2::OEtmk3.TU-6、△tmk2、△tmk2::OEtmk3 were were grown on PDA plates with different concentration of CR and CFW. △tmk2 strain was unable to grow on plates containing 200 u.g/ml CR or 20μg/ml CFW, while △tmk2::OEtmk3 could. When the concentration of CFW reached 40△g/ml, only TU-6 could tolerate it. These results lead to the suggestion that overexpression of Tmk3 makes a certain degree of recovery of cell wall integrity. Tmk2 and Tmk3 work in different MAPK pathway to regulate cell wall integrity. Real-time PCR analysis of the transcriptional levels of chitin synthase coding genes also supportted this conclusion.We assayed extracellular cellulase production-. biomass and transcriptional levels of cellulase coding genes. These results suggest the different of extracellular cellulase production maybe caused by the change of cell wall integrity.These observations show more detailed function of Tmk2, the relationship between Tmk2 and Tmk3, help us to more clearly understand fungal physiology in H. jecorina, in particular cellulase production and regulation mechanisms.