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.

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 Hog1-like Tmk3. In this work, we constructed and explored the features of H. jecorina Δtmk2 to identify the role of this Slt2-homologous Tmk2. It is suggested from the results that Tmk2 is involved in cell wall integrity, sporulation and cellulase production. Although bearing similar roles in cell wall integrity maintenance, Tmk2 and Tmk3 appear to also have distinct functions: Tmk3 participates in high osmolarity resistance while Tmk2 does not; Tmk2 participates in sporulation but not Tmk3; Tmk3 is involved in promoting cellulase production while Tmk2 is involved in repressing cellulase formation. These studies provide the first insight into the function of Tmk2 in H. jecorina and contribute to understanding the signal transduction processes leading to the regulation of cellulase production in this important cellulase hyper-producer.

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The biosynthetic pathway for hyaluronic acid (HA) has been proposed; however, a thorough genetic and functional analysis is required to further elucidate the roles of genes involved in HA production. Previously, we developed a markerless gene-deletion system for Streptococcus zooepidemicus and confirmed that hasA is essential for HA synthesis. Here, we constructed a comprehensive set of deletion mutants and investigated the roles of ten additional predicted genes in the HA synthetic pathway. Phenotypic assays revealed that all ten genes play a role in cell growth and/or HA synthesis. As expected, the deletion of hasA or hasB abolished HA production with little effect on growth, while the deletion of genes that are also required for peptidoglycan biosynthesis (hasE, glmM, and glmS) significantly reduced cell growth and HA production. Either of the glmU homologues (hasD and gcaD) was sufficient for optimal growth and the mucoid phenotype, while no double mutant could be isolated. Of the two UDP-glucose pyrophosphorylase (UGPase) paralogues, the operon-encoded hasC1 was responsible for 65 % of the activity, while hasC2 was responsible for the remaining 35 %. The deletion of hasC1 had no effect on cell growth and caused only a moderate decrease in the UDP-glucose level and HA production. The deletion of both hasC1 and hasC2 resulted in a severe growth defect and negligible UDP-glucose accumulation, HA production, and pyrophosphorylase activity. Of the two phosphoglucomutase paralogues, pgm1 and pgm2, the former is responsible for around 10 % of activity, while the latter is responsible for 90 %. The deletion of pgm1 showed no apparent effect on HA synthesis and growth, while the deletion of pgm2 resulted in the abolishment of HA synthesis and a significantly slower growth. These results should guide the metabolic engineering of S. zooepidemicus to improve HA productivity and quality.

Populus alba × Populus glandulosa (84K poplar) is model material with excellent genetic engineering resource and ornamental value. In our study, AmRosea1 (Antirrhinum majus) was overexpressed in 84K poplar, and the transgenic 84K (AM) poplar with high content of anthocyanin exhibited red pigmentation leaves. The transcriptome analysis between wild type (WT) and AM showed that 170 differentially expressed genes (DEGs) (86 up-regulated and 84 down-regulated) were found, and some DEGs were involved in flavone and flavonol biosynthesis, flavonoid biosynthesis and anthocyanin biosynthesis. The metabolome analysis showed that 13 anthocyanins-related differentially accumulated metabolites (DAMs) were detected in AM. The correlation analysis between DEGs and DAMs were performed, and the results revealed that 18 DEGs, including 11 MYB genes, two BZ1 genes, one FG2 gene, one ANS gene, and three IF7MAT genes, were negatively or positively correlated with 13 DAMs. The phylogenetic analysis demonstrated that there was high homology between AmRosea1 and PagMYB113, and MYB113 co-expressed with BZ1, ANS and DFR directly. Our results elucidated the molecular mechanism of plant color change mediated by anthocyanin biosynthesis pathway, which laid the foundation for the development and utilization of colorful woody plant.