Functional Analysis Of The Nitrogen Catabolite Repression Pathway Regulatory Protein MaNmrA In Metarhizium Acridium

Developing biopesticides is of great significance for reducing the pesticide residues in agricultural and sideline products and the pollution in the farmland ecological environment.As an important insecticidal microorganism,entomopathogenic fungi have some advantages,such as environmental friendliness,sustainable control and safety for human and livestock.However,some disadvantages have limited their large-scale applications,such as slow insecticidal speed,high production cost and being easily affected by the adverse environment.Therefore,it not only has important theoretical significance but also has considerable practical value to elucidate the molecular mechanism related to the biocontrol traits of entomopathogenic fungi.The regulation of nutrient metabolism is closely related to the growth and development of fungi and the ability of infection and pathogenicity in fungi.Previous studies have shown that Nmr A plays important roles in the regulation of nitrogen and carbon metabolism in fungal development and pathogenicity.To date,the functions of Nmr A in entomopathogenic fungi have not been reported.Therefore,the aim of this study is to clarify the functions of MaNmrA in Metarhizium acridum.Bioinformatics analysis showed that the MaNmrA gene encoded a protein with 461 amino acids.Domain analysis revealed that the MaNmrA protein has NADP binding site and NADB-Rossmann superfamily domain.This gene is highly conserved in fungi.The knockout and complemented strains were constructed respectively for the functional analysis of MaNmrA in M.acridium.The results showed that the conidial germination and the conidiation rate were significantly delayed,and the conidial yield was significantly reduced,when the MaNmrA was deleted.Compared with wild-type strains,the ΔMaNmrA exhibited the significantly reduced tolerances to UV irradation,heat,cell wall disturbing agents,oxidative and hypertonic stresses.In addition,on the medium with different single nitrogen sources or carbon sources,the aerial hyphae of theΔMaNmrA strain became more fluffy and denser.Especially when a non-preferred nitrogen source was added,the growth rates of the ΔMaNmrA strain were significantly increased,indicating that the ability of the ΔMaNmrA strain in nitrogen and carbon utilization had been changed.Moreover,the ΔMaNmrA mutant showed a decreased sensitivity to rapamycin,When the preference nitrogen source glutamine is added,the tolerance of the ΔMaNmrA strain to rapamycin is significantly increased.The results of bioassays showed that the disruption of MaNmrA significantly reduced the fungal virulence against locusts by both topical inoculation and intrahemocoel injection.Further research found that the conidial germination of the ΔMaNmrA strain on the hindwings of the migratory locust slowed down,as well as the appressorium formation rates of the ΔMaNmrA strain were significantly reduced.These results indicated that the absence of MaNmrA affected the ability of cuticle penetration in M.acridium.The above results indicate that MaNmrA is involved in the growth and development,the resistance to adversity,the pathogenicity by regulating the utilization of environmental nutrients in M.acridium.