Mitogen-activated protein kinase (MAPK) signaling pathways are evolutionarily conserved in eukaryotes and modulate responses to both internal and external stimuli. Pmk1 and Mps MAPK pathways regulate stress tolerance, vegetative growth and cell wall integrity in Saccharomyces cerevisiae and Pyricularia oryzae. Here, we deployed genetic and cell biology strategies to investigate the roles of the orthologs of Pmk1 and Mps1 in Sclerotiophoma versabilis (herein referred to as SvPmk1 and SvMps1, respectively). Our results showed that SvPmk1 and SvMps1 are involved in hyphal development, asexual reproduction and pathogenesis in S. versabilis. We found that ∆Svpmk1 and ∆Svmps1 mutants have significantly reduced vegetative growths on PDA supplemented with osmotic stress-inducing agents, compared to the wild type, with ∆Svpmps1 being hypersensitive to hydrogen peroxide. The two mutants failed to produce pycnidia and have reduced pathogenicity on Pseudostellaria heterophylla. Unlike SvPmk1, SvMps1 was found to be indispensable for the fungal cell wall integrity. Confocal microscopic analyses revealed that SvPmk1 and SvMps1 are ubiquitously expressed in the cytosol and nucleus. Taken together, we demonstrate here that SvPmk1 and SvMps1 play critical roles in the stress resistance, development and pathogenesis of S. versabilis.

Reversible protein phosphorylation is essential in cellular signal transduction. The rice blast fungus Magnaporthe oryzae contains six putative type 2C protein phosphatases, namely MoPtc1, MoPtc2, MoPtc5, MoPtc6, MoPtc7, and MoPtc8. The major functions of MoPtc1 and MoPtc2 have been reported recently. In this communication, we found that MoPtc1 and MoPtc2 were induced by calcium chloride. We also found that the deletion of both MoPtc1 and MoPtc2 resulted in the overstimulation of both the high-osmolarity glycerol (Hog1) and pathogenicity MAP kinase 1 (Pmk1) pathways in M. oryzae. MoPtc1 was recruited directly to Osm1 (the osmotic stress-sensitive mutant) by the adaptor protein MoNbp2 to inactivate the Osm1 during hypoosmotic stress, distinct from the budding yeast. Moreover, we showed that MoPtc1 and MoPtc2 were localized in different cellular compartments in the fungal development. Taken together, we added some new findings of type 2C protein phosphatases MoPtc1 and MoPtc2 functions to the current knowledge on the regulation of MAPK signaling pathways in M. oryzae.

Protein kinases and phosphatases catalyze the phosphorylation and dephosphorylation of their protein substrates, respectively, and these are important mechanisms in cellular signal transduction. The rice blast fungus Magnaporthe oryzae possesses 6 protein phosphatases of type 2C class, including MoPtc1, 2, 5, 6, 7 and 8. However, only very little is known about the roles of these phosphatases in filamentous fungi. Here in, we deployed genetics and molecular biology techniques to identify, characterize and establish the roles of MoPtc5 and MoPtc7 in M. oryzae development and pathogenicity. We found that during pathogen-host interaction, MoPTC7 is differentially expressed. Double deletion of MoPTC7 and MoPTC5 suppressed the fungal vegetative growth, altered its cell wall integrity and reduced its virulence. The two genes were found indispensable for stress tolerance in the phytopathogen. We also demonstrated that disruption of any of the two genes highly affected appressorium turgor generation and Mps1 and Osm1 phosphorylation levels. Lastly, we demonstrated that both MoPtc5 and MoPtc7 are localized to mitochondria of different cellular compartments in the blast fungus. Taken together, our study revealed synergistic coordination of M. oryzae development and pathogenesis by the type 2C protein phosphatases.

Oyster mushrooms are regarded as one of the most significant edible mushrooms in terms of commercial value because of their rich nutritional profile. Many bioactive extracts from Pleurotus species have been found to exhibit antitumor and antioxidant activities. However, to grow oyster mushrooms in this study, the pasteurized Cenchrus fungigraminus was used as culture material, a type of grass that proliferates and has a high root growth rate. It contains high levels of sugar and protein and yields a large amount of biomass. Because of these characteristics, it is considered an efficient and cost-effective energy crop with various applications, including phytoremediation and fodder production. A pasteurization technique for this grass that is suited for the simplest formulation is simple and cost-effective for growing oyster mushrooms on small farms. This study used pasteurized Cenchrus fungigraminus as a substrate to grow three mushroom species: Pleurotus ostreatus, Pleurotus pulmonarius and Pleurotus florida. The aim was to evaluate their enzyme activities, growth rate, and yields. The findings demonstrated that the average growth rate of three species grown in pasteurized C. fungigraminus was between 25 days and 36 days. Therefore, the mycelium growth rate of P. ostreatus was faster than other pleurotus species in this study. The highest biological efficiency was recorded with P. ostreatus at 78.23%, then P. pulmonarius at 59.88, and lastly, 39.66% P. florida. The changes in five enzyme activities in distinct developmental stages of three different pleurotus species were evaluated. Therefore, the laccase had the highest peak with 13.8 U/g on the 20th day during the growth phase and gradually decreased to the fruiting body stage of P. ostreatus. The expression of manganese peroxidase reached the highest activity of 3.6 U/g in P. ostreatus compared to P. florida and P. pulmonarius on the 10th day. The expression of other enzymes varied between species and developmental phases. The results indicate the usefulness of pasteurized C. fungigraminus for cultivating Pleurotus species and expression enzyme activity in different Pleurotus species.

Agrocybe cylindracea is an important mushroom highly valued as a functional food for its nutritional and medicinal benefits. Many bioactive extracts from A. cylindracea have been found to exhibit antitumor and antioxidant activities. This research investigated the distinct substrates that affected the physicochemical and biocomponent properties and biological efficiency of A. cylindracea. The substrates used were 48% giant juncao grass mixed with 30% Dicranopteris dichotoma grasses, 48% wasted tea leaves mixed with 30% sawdust, and 78% cottonseed hulls, all supplemented with 20% wheat bran and 2% lime. The findings indicated that A. cylindracea grown in the cotton seed hulls had a significant biological efficiency, at 35.8%, followed by the GD + DD (31.5%) and WTL + SD (28.7%). The ash content peaked in the fruiting bodies grown on giant juncao grass and D. dichotoma, while the fat content was highest in those grown on giant juncao grass and D. dichotoma, followed by wasted tea leaves and sawdust. The protein content was significantly higher in the fruiting bodies cultivated on wasted tea leaves and sawdust, followed by cottonseed hulls. The carbon dioxide emissions varied across substrates, with the highest emissions observed during the maturity stage of the fruiting bodies grown on giant juncao grass with D. dichotoma and wasted tea leaves and sawdust. Emissions decreased sharply 110 days after cultivation. Essential metabolites, such as dopamine and caffeine, were enriched in the fruiting bodies grown on wasted tea leaves, sawdust, and cottonseed hulls. In contrast, tyramine and uracil were enriched in those grown on cottonseed hulls.

Rice blast caused by Magnaporthe oryzae pathotype is the worst disease that leads to serious food insecurity globally. Understanding rice blast disease pathogenesis is therefore essential for the development of a blast disease mitigation strategy. Reverse phosphorylation mediated by phosphatases performs a vital function in the activation of diverse biological mechanisms within eukaryotic. However, little has been reported on the roles of PP2Cs in the virulence of blast fungus. In this current work, we deployed functional genomics and biochemical approaches to characterize type 2C protein phosphatase MoPtc6 in blast fungus. Deletion of MoPTC6 led to a drastic reduction in conidiophore development, conidia production, hyphal growth, and stress tolerance. Western blotting assay demonstrated that the phosphorylation level of MoOsm1 was decreased while MoMps1 was increased in the MoPtc6 deletion mutant, and comparative transcriptome assay revealed a higher number of expressed genes between mutant and wild type. Localization assay confirmed that MoPtc6 is sub-localized in the cytoplasm of mycelia, spores, and in the appressoria of M. oryzae. Furthermore, disruption of MoPTC6 impaired appressoria turgor pressure and glycogen utilization; more findings revealed attenuation of hyphal penetration and virulence upon deletion of MoPTC6. Generally, present findings suggested the role of MoPtc6 in the growth and virulence of M. oryzae.