The Functions Of C2H2 Zinc Finger Protein MaNCP1 And Its Molecular Mechanism In Regulating Conidiation Pattern Shift Of Metarhizium Acridum

Fungal dimorphism is that most fungi commonly generate two cellular morphologies,the highly polarized multicellular hyphae and the unicellular yeast,namely fungal dimorphic.Most filamentous fungi have two conidiation patterns,normal conidiation and microcycle conidiation.Normal conidiation is a basic part in the life cycle of the fungus,which sporulates after proper mycelial growth.During microcycle conidiation,however,conidia can be directly generated from germinated conidia.The conidiation pattern shift,which exhibited the typical dimorphic switch.Microcycle conidiation is a survival mechanism for fungi to adapt to the adverse environments.It is influenced by many factors such as temperature,p H,nutrients etc.,of which nutrition is the most common influencing factor.Precise nutrient perception is extremely important for fungal growth and development.Fungi can uptake a variety of compounds as nitrogen sources,this utilization process is more complicated and involves in multiple parallel signals and regulation systems,of which the most widely known is the nitrogen catabolism repression（NCR）pathway.Entomopathogenic fungi are natural insect pathogenic microorganisms,can infect and kill host insects.So far,many species or subspecies of the insect pathogenic fungi have been developed as insecticides.Conidia are very important for the reproduction and persistence of fungi,meanwhile,they are not only the main infectors of insect pathogenic fungi,but also the effective components of insecticidal fungal pesticides.The conidia produced in microcycle conidiation with a higher conidial yield and a stronger resistance to adversity compared to that produced in normal conidiation in Metarhizium acridum.Therefore,elucidating the molecular mechanism of conidiation pattern shift in M.acridum is of great significance in improving the conidial yield,quality and the sustainable control of pests.MaNCP1,a novel C2H2 zinc finger protein,was screened from the differential expression library of the conidiation patterrn shift that regulated by nitrate in M.acridum.The whole gene knockout strain（ΔMaNCP1）,N-and/or C-terminal zinc fingers deletion mutants(MaNCP1^?N,MaNCP1^?C,and MaNCP1^?N+C)and complemented strain（CP）were obtained by homologous recombination technique.In this study,we focused on the biological control potential of MaNCP1 and its molecular mechanism in regulating the conidiation pattern shift.The main results are as follows:1.Bioinformatics analysis showed that MaNCP1 contained two zinc finger clusters（the C-terminal contained two C2H2 zinc fingers and the N-terminal contained three C2H2 zinc fingers）,and MaNCP1 was relatively close to the filamentous fungi in evolution.Moreover,MaNCP1 protein sequence contained two nuclear localization signals.Subsequently,the heterologous expression of MaNCP1 in Saccharomyces cerevisiae showed that MaNCP1 had the transcriptional activity.Meanwhile,the MaNCP1::e GFP fusion protein was located in the nucleus.In summary,these results indicated that MaNCP1 had the typical characteristics of transcription factors.2.Deletion of MaNCP1 delayed the conidial germination and decreased the conidial yield.Moreover,the N-terminal zinc fingers made a great effect on conidial germination,both the N-and C-terminal zinc fingers made great effects with no significant difference on the conidial yield.Loss of MaNCP1 weakened the tolerances to UV-B and heat-shock and the N-terminal zinc fingers made great effects on the tolerances to UV-B and heat-shock.In addition,disruption of MaNCP1 or the C2H2zinc fingers,whether they are at the N-or C-terminal,changed the conidial surface microstructures of the mutant strains,which became more smoother compared to the WT or CP strain,and reduced the conidial surface hydrophobicity.3.Deletion of MaNCP1 reduced the virulence,and the N-terminal C2H2 zinc fingers made a greater effect on the virulence than that in the C-terminal.The topical inoculation test showed that MaNCP1^?N,MaNCP1^?N+C and?MaNCP1 strains affected the development of appressoria and with a lower cuticle penetration ability,resulting in the virulence of these three strains was significantly decreased compared to the WT and CP strains.4.The ability of utilize nitrate,ammonium and glutamine were reduced in the absence of MaNCP1,it suggested that MaNCP1 was involved in regulating nitrogen utilization.In addition,the conidial yield of theΔMaNCP1 strain grown on SYA medium were significantly decreased,which could be restored or even higher than that of the WT strain when cultured on SYA+Nitrate medium for 15 days.Meanwhile,q RT-PCR analysis revealed that MaNCP1 affected the expression levels of genes involved in nitrate assimilation pathway.Analysis of the differentially expressed genes（DEGs）between WT andΔMaNCP1 strains via RNA-seq revealed that the conidial yield regulated by nitrate might be related to NCR path.Further study found that Ma Are A was a downstream target gene of MaNCP1,indicating that MaNCP1 may regulated nitrate metabolism through binding to Ma Are A.5.Deletion of MaNCP1 shifted the conidiation pattern of M.acridum from microcycle conidiation to normal conidiation when grown on SYA medium,and C2H2zinc fingers in the N-terminal of MaNCP1 protein made a greater effect on the conidiation pattern shift than that in the C-terminal.Meanwhile,we found a down-regulated Nmr A family gene（MAC＿02196）in the DEGs between WT andΔMaNCP1 strains,but it did not involved in the regulation of conidiation pattern shift.Subsequently,we demonstrated that Ma Nmr A（MAC＿00749）was a downstream target gene of MaNCP1,and disruption of Ma Nmr A also changed the conidiation pattern from the microcycle conidiation to normal conidiation.Further study revealed that MaNCP1and Ma Nmr A were involved in regulating the reduction pathway of NO synthesis,and the conidiation patterns ofΔMaNCP1 andΔMa Nmr A strains could be restored to microcycle conidiation when grown on SYA media supplementing with the NO donor SNP.In addition,the overexpression of Ma Nmr A in theΔMaNCP1 strain could increase the content of endogenous NO and change the conidiation pattern to microcycle conidiation.Taken together,we concluded that the MaNCP1 governed the conidiation pattern shift through affecting the NO content by regulating the reductive pathway of NO synthesis via directly targeting the Ma Nmr A gene.In conclusion,deletion of MaNCP1 delayed the conidial germination and hyphal growth,decreased the conidial yield,reduced the tolerances to UV-B irradiation and heat-shock,changed conidial surface structure and decreased the conidial surface hydrophobicity,which also reduced the virulence,and the N-terminal zinc fingers made a great effect on these traits.Moreover,we found that MaNCP1 governed conidiation pattern shift through regulating the reductive pathway of nitric oxide synthesis via directly targeting the Ma Nmr A gene in the model insect pathogenic fungus M.acridum.