Functional Analysis Of CmSnf1 In Cordyceps Militaris

Cordyceps militaris is an edible and medieinal fungus bearing various phannacological activities. It is also an important entomopathogen which can parasitize in many kinds of insects belonging to Lepidoptera, Diptera and Coleoptera. Many studies have found that carbon source has a great influence on growth of hyphae, sporocarp and the content of active ingredients of cordyceps militaris. SNF1 pathway is well conserved in eukaryotic cells, which is crucial to the maintenance of energy balance. In this study, based on the targeted-gene knockout strategy and phenotypic analyses, combined with qRT-PCR, we identified and characterized α subunits of SNF1 complex in Cordyceps militaris, to contribute a better understanding of how SNF1 pathway of Cordyceps militaris. The main results acquired are listed below: 1) CmSnf1 is involved in utilization of carbon sourcesInvestigation of growth situation of various strains cultured in PDA and solid MM supplemented with different sole carbon sources, including xylogen, sodium carboxymethyl cellulose, glycerinum, ethyl alcohol, glucose, sucrose, fructose, maltose and arabinose. Mycelial morphology showed varies in degree. When glucose, xylogen or arabinose was used as the sole carbon source, the growth of the ΔCmSnf1 was more significantly decreased than that of the WT, particularly arabinose. The result showed that CmSnf1 is involved in utilization of carbon sources. 2) CmSnf1 is involved in conidiation and conidial germinationThe conidial yield of WT was 13.43 times of the ΔCmSnf1 on PDA media. The germination rate of theΔCmSnf1 was significantly lower than that of WT. The result showed that CmSnf1 plays an important role in conidiation and conidial germination. 3) CmSnf1 deficiency disrupts ability in clearing up reactive oxygen speciesTo study the role of CmSnf1 in oxidative responses, staining assays were performed to investigate the oxidative sensitivity of various strains. ΔCmSnf1 strains declined sensitivity to oxidative stress(2.5 mM H2O2). NBT staining showed the ability in clearing up ROS of ΔCmSnf1 strains was badly damaged. Detecting the expression of PEX by q RT – PCR, the result show that compared with WT and Comp strains, transcription levels of PEX were induced in ΔCmSnf1 strains, especially PTS2, Pex13 and Pex19.These results suggest that variations in strain stability derive at least partly from differences in their ability to eliminate ROS, further establishing the link between oxidative stress and CmSnf1. 4) CmSnf1 disruption affects sporocarp on silkworm chrysalisΔCmSnf1 strains in the rice medium had good fruiting ability, but couldn’t on silkworm chrysalis. Compared with WT and Comp strains, transcription levels of proteolytic enzyme gene and chitinase gene were induced in ΔCmSnf1 strains. The ΔCmSnf1 without fruiting ability indicate that CmSnf1 is crucial to fruiting body on silkworm chrysalis, perhaps through regulating proteolytic enzyme and chitinase. 5) CmSnf1 disruption affects cell wall integrityPositioning signal GTP- Snf1 suggests that CmSnf1 is located in the cell walls. Transcription levels of XYL-1, XYL-2 and ExoPG was significantly induced in ΔCmSnf1 strains, compared with WT. ΔCmSnf1 strains declined sensitivity to Congo red and Calcofluor white. CmSnf1 is essential for cell wall integrity and this functionality is achieved by regulating cell wall synthesis related gene transcription. 6) CmSnf1 disruption affects the expression of SNF1 pathway genesDetecting the expression of SNF1 pathway genes by qRT – PCR, the result show that compared with WT, Comp strains, transcription levels of upstream kinases Tos3, Sak1 and downstream kinases Mig1, Sip4 and γ subunit Snf4 was significantly induced in ΔCmSnf1 strains. The expression quantity of SNF1 pathway genes in all strains were increase after starvation, and transcription levels of Mig1, γ subunit Snf4 and β subunit Gal83 in ΔCmSnf1 strains were even higher than that of WT strain. These results suggest that CmSnf1 disruption affects the expression of SNF1 pathway genes and γ subunit Snf4 and β subunit Gal83 can instead of α subunit Snf1 to some extent when α subunit Snf1 missing. 7) CmSnf1 disruption affects the expression of autophagy related genesDetecting the expression of autophagy related genes by qRT – PCR, the result show that compared with WT, Comp strains, transcription levels was significantly induced in ΔCmSnf1 strains, and Atg17 couldn’t even express. The expression quantity of autophagy related genes in all strains were increase after starvation, and the increment in ΔCmSnf1 strains were higher than that of WT strain. There’s synergy between CmSnf1 and autophagy related genes under starvation according to the experimental data.