Abstract
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.
Highlights
The systematic risk inherent to a fossil fuel based economy has drawn worldwide attention, leading to the proposal of replacing these non-renewable energy sources with renewable energy sources including energy from renewable biomass
It can be concluded that Tmk3 is homologous to Hog1-type mitogen-activated protein kinase (MAPK), which function in high osmolarity resistance in S. cerevisiae and other filamentous fungi [3]
Examination of the growth of parent and Dtmk3 strains on plates lead to several interesting findings: 1) The growth of T. reesei Dtmk3 on each minimal media plate containing a tested carbon source is significantly worse than the parent strain; 2) Only slightly slower growth was observed when T. reesei Dtmk3 was grown on complete media (PDA plates); 3) a significantly larger transparent zone was observed around the colony of T. reesei Dtmk3 in comparison to the parent strain, when the two strains were grown on double layer plates containing avicel (Fig. 1)
Summary
The systematic risk inherent to a fossil fuel based economy has drawn worldwide attention, leading to the proposal of replacing these non-renewable energy sources with renewable energy sources including energy from renewable biomass. The most widely used and important cellulase hyper-producing organism is Trichoderma reesei Physiological investigations of this so-called industrial cellulase-producing ‘workhorse’ are essential in both scientific and technological regards [2]. Three major classes of MAPKs are present, respectively homologous to yeast Hog1p, Slt2p, and Fus3p These MAPKs have been shown to function in a variety of physiological processes such as fruiting body development [6], polarized growth [7], biosynthesis [8], conidiation [9], pathogenicity [10], circadian rhythmicity [11], stress response [12,13], protein production [9,14] and cell wall integrity maintenance [15]
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