The Function Of Transcription Factor Mavib-1 And Its Molecular Mechanism Affecting The Conidiation Of Metarhizium Acridum

Conidium is the main infection unit and reproductive unit of pathogenic fungi.The conidial yield,time to enter a stable conidiation period,and the proportion of mycelia in conidial product are restrictive conditions for large-scale production.Most fungi have two types of conidiation:normal conidiation(NC)and microcycle conidiation(MC).Normally,conidiophores arise from the vegetative mycelia and produce large amounts of conidia.In microcycle conidiation,the fungi can bypass the mycelia period,and develop secondary conidia on conidiophores produced from germ tubes or directly from conidial cells.Conidia from MC have more advantages than normal conidiation in terms of large-scale production and application,such as faster conidiation and more conidial yield.Therefore,clarification of the conidiation mechanisms of entomopathogenic fungi has important practical significance for the large-scale production of fungal insecticides in the future.This study mainly focused on the function of the transcription factor Mavib-1 and protein kinase Ma Rim15 in regulation of the conidiation in Metarhizium acridum.The main results are as follows:1.Transcriptional activation experiment showed that the C-terminal of Mavib-1had a strong transcriptional activation activity in yeast.Subcellular localization analysis with EGFP fusion expression showed that Mavib-1 was localized in the nucleus.2.Mavib-1 affects the conidia size and hyphal extension in M.acridum.Compared with WT,the?Mavib-1 conidia had shortened diameter and OE conidia had smaller size in both length and diameter.Both?Mavib-1 and OE had significantly shorter apical cell compartments(p<0.01)and the subapical cell(p<0.05)compared with WT.3.Mavib-1 affects the conidial germination,the tolerance of UV-B and heat stress in M.acridum.Compared with WT,conidial germination was delayed in?Mavib-1 and OE strains with significantly increased the half germination time.After UV-B or 46?heat treatment,the germination rate of?Mavib-1 strain was lower than WT with significantly decreased half-inhibition time(IT₅₀).4.Mavib-1 affects the cell wall integrity of M.acridum.The stress tolerance to CR and H₂O₂ was decreased in?Mavib-1 strain,and the stress resistance to CR and CFW was decreased in OE strain.Scanning electron microscope analysis of conidial surface showed that the conidial cell wall surface of wild-type conidia was rough and covered with the characteristic rodlets.Conidial surface of the?Mavib-1 was smoother compared to that of WT,indicating that Mavib-1 affected the conidial surface morphology in M.acridum.Moreover,index of hydrophobicity conidia was significantly decreased in?Mavib-1 and significantly increased in OE strain compared to WT,suggesting that Mavib-1 affected the conidial hydrophobicity in M.acridum.5.Mavib-1 was a transcription factor that positively regulated growth and negatively regulated conidiation of M.acridum.The transcription factor Mavib-1 has the NDT80?Pho G domain related to nutrition perception.The growth,conidiation pattern and conidial yield of WT,?Mavib-1,CP and OE strains on different carbon and nitrogen sources were determined and compared.Results showed that?Mavib-1 had light conidia color and smaller colony under the condition of nutrient deficiency,indicating a positive role of Mavib-1 gene in melanin formation and normal fungal growth.Conidia yield was significantly decreased when Mavib-1 was disrupted under either nutrient rich or deficient media.Mavib-1 played a positive role in regulation of the utilization of carbon sources,showing different growth phenotype with different carbon source.Mavib-1 also affected the utilization of different nitrogen sources including both dominant nitrogen sources and non-dominant nitrogen sources.The Mavib-1 played a positive role in the fungal growth and hyphal extension at the edge of the colony on media with different nitrogen sources.The?Mavib-1 strain performed microcycle conidiation earlier than WT on nutrient-rich medium 1/4 SDAY,and performed normal conidiation slightly earlier on nutrient-deficiency medium SYA-S(the?Mavib-1 strain produced spores at 24 h while WT strain just formed a conidiation structure at that time);Conidiation of OE strain on media containing different carbon source was delayed compared to WT and even no spore was produced at 36 h.6.Mavib-1 played important roles in regulation of the expression of carbohydrate active enzymes and carbon source metabolism of M.acridum.In order to clarify the mechanism that Mavib-1 affected conidiation in response to different carbon and nitrogen sources,RNA-seq was performed in WT,?Mavib-1 and OE strains cultured on SYA medium for 20 h.Results showed that,compared with WT,there were a total of291 differentially expressed genes(DEGs)(79 up-regulated and 212 down-regulated)in the?Mavib-1 strain and 486 DEGs(318 up-regulated and 168 down-regulated)in the OE strain.Among these DEGs,22 DEGs had opposite expression trend in?Mavib-1and OE(18 DEGs were down-regulated in?Mavib-1 and up-regulated in OE,4 DEGs were up-regulated in?Mavib-1 and down-regulated in OE).Among these DEGs,there were 9(291 in total)and 41(486 in total)carbohydrate-active enzymes in the?Mavib-1and OE respectively.7.Ma Rim15 was found as a downstream target gene regulated by Mavib-1 using RT-q PCR and yeast one-hybrid analysis.A C2H2 type DNA binding regulatory protein Amd X,zinc finger protein opl and crol gamma were possibly upstream regulators of Mavib-1 by bioinformatics analysis and yeast one-hybrid screening library of M.acridum.8.Mavib-1 affects the conidiation but not the growth through Ma Rim15 in M.acridum.Similar as Mavib-1,Ma Rim15 affected the conidiation in M.acridum,but played a positive regulatory role and did not affect the growth of M.acridum.