Functional Analysis Of NADPH Oxidase Gene Family In Nomuraea Rileyi

Nomuraea rileyi?=Syn.Metarhizium rileyi?is a ubiquitous entomopathogenic fungus.It can infect many lepidopteran insectsmainly in the Noctuidae in the field and has received much attention because of its friendly natural environment and human health.After years of exploration,our laboratory has successfully induced the microsclerotia?MS?of N.rileyi with strong resistance,high stability and resistance to storage,which can effectively replace the conidia for the production of insecticides.The transcriptomic analysis of the developmental process of MS was performed in the previous period and it was found that its process was accompanied by the stimulation of oxygen stress.NADPH oxidase?NOX?is an important source of reactive oxygen species?ROS?production.It catalyzes the transformation of O₂ into highly reactive ROS,which is involved in transmission of cell signals or activating signaling pathways.And it plays a key role in the morphogenesis,growth and development of eukaryotes and in responding to adverse environmental conditions.However,the biological function of this gene family has not been reported in entomopathogenic fungi.In view of this,N.rileyi is used as the research object,and gene knockout methods are used to study the function of NOX family genes in this study.The main results are as follows:Sequence analysis and bioinformatics analysis of NADPH oxidase family genes of N.rileyiThe transcriptome and genomic data of N.rileyi were searched and it was found that the NADPH oxidase family of N.rileyi consists of NoxA,NoxB and NoxR genes.The alignment results of the full-length sequences of the three genes showed that the open reading frame?ORF?of NoxA is 1813bp,encoding 557 amino acids,with 2 introns.The ORF of NoxB is 3110bp,encoding 603 amino acids,and containing 2 introns.The ORF of NoxR is 1953bp,encoding 516 amino acids,with 5 introns.Bioinformatics analysis showed that the molecular weights of the three genes encoding proteins NoxA,NoxB and NoxR were 141.3KDa,148.6KDa and 126.6KDa,respectively,as well as the theoretical pI of 4.93,4.91 and4.96.The secondary structureis dominated by?-helices and random coils.NoxA,NoxB,NoxR have 5,8 and 0 transmembrane structures,respectively.NoxA and NoxB have NOX_DUOX_like_FAD_NADP domain and are involved in the electron transport on the membrane.The N-terminus of NoxR contains a TPRs site and the C-terminus contains a phoX+Bem1 domain?PB1?,which binds to small G protein family members and activates the NOX complex.After homologous sequence comparisons and phylogenetic analysis,it was found that the species is closely related to the species of Metarhizium.Single knockout of NOX family genes and acquisition of mutant strainsThe flanking sequences of about 2000bp upstream and downstream of the three genes'coding region were amplified by PCR and then inserted into Pzp-Ptrpc-Hph-Knockvector harboring a hph cassette to yield Pzp-Ptrpc-Hph-NoxA,Pzp-Ptrpc-Hph-NoxB and Pzp-Ptrpc-Hph-NoxR,respectively.The Agrobacterium tumefaciens mediated transformation?ATMT?system of N.rileyi CQNr01 was used.The knockout vectors were introduced into Agrobacterium strain AGL1,and co-cultured with blastospore of N.rileyi,using hygromycin as a screening marker,and the deletion mutant strains?NoxA,?NoxB and?NoxR were obtained.Phenotypic analysis of mutant strains of N.rileyiComparison of phenotypes of mutants with wild-type?WT?showed that the time of dimorphic transition of?NoxA and?NoxB was slightly longer,while the spore germination rate of?NoxR was increased,and significantly accelerate the transformation process of both types.The absence of NoxA resulted in a sharp decrease of spore production by 94%,hyphae branches were reduced,and the mycelium was shortened.There was no significant difference between?NoxB and WT,and it may only play a secondary role in the production of ROS in NOX.The conidia of?NoxR were strikingly larger than WT,and its yield has dropped by 84%,Moreover,the rapid elongation of the branched mycelium caused the increase of radiation area of the colony.In the salt stress,cell wall disrupting agent,osmotic pressure and oxygen stress experiments,?NoxA and?NoxB strains grew better under salt stress treatment,while?NoxR showed obvious decrease for the tolerance of salt,colony became smaller and sporulation decreased.Under osmotic stress,the?NoxR hardly produces spores and the mycelium grows thick and dense.Mutants showed no sensitivity to other stress.This shows that NoxR plays a major role in response to stress in NOX.In this study,?NoxA induced a significant reduction in the production of MS,and the size of MS is not uniform and pigment deposition decreased.MS of?NoxB becomes smaller,the number slightly increased.Deletion of NoxR severely affected the normal formation of MS and decreased the density of sclerotia,and most of the polar mycelium accumulated in the state of clusters,one of the most significant feature is that the culture fluid can not see the formation of red brown pigment.The above phenomenon indicates NOX is essential for the morphogenesis of strains,stress response,and MS formation.Expression levels of NOX family genes during MS developmentRT-qPCR results showed that all three genes were highly expressed at the early stage of MS induction.Up to the 84h,there was a sudden rise in the expression of NoxB and NoxR at the peak period of MS.The expression of NoxA has continued to increase during the entire development process,reaching the highest value in the maturation phase of MS.Analysis of the expression pattern of other NOX genes in the mutants showed that?NoxA had no significant effect on the expression of NoxB and NoxR,and the absence of NoxB or NoxR notably affected the expression levels of the other two genes during the formation of MS.This indicates that three genes regulate the formation of MS through complementary coordination.Virulence analysis of the mutans of N.rileyiThe virulence of the mutants was tested by in vivo injection and surface infection.In injection test results showed that the LT₅₀ of the WT was 8.24 days,and the LT₅₀ of?NoxA,?NoxBand?NoxR were delayed by 1.99d,1.31d and 0.95d,respectively.The delay of the?NoxA was more significant.The results of topical infection experiment showed that the LT₅₀ of WT was 9.11d,and the LT₅₀0 of?NoxA,?NoxB and?NoxR were1.93d,1.06d and 3.18d delayed compared with the WT strain,respectively.Among them,the virulence of?NoxR decreased more severely.From the pathogenicity of injected inoculation and surface infection,combined with the phenotypes of strains in solid plates and liquid cultures,it can be seen that?NoxA and?NoxB mainly affect the pathogenicity of strains in insects.The absence of the NoxR has a greater effect on the ability of the strain to penetrate the body wall of Spodoptera litura.This shows that NOX plays an important role in the infection of Spodoptera litura by N.rileyi.In summary,the results showed that the NADPH oxidase family genes play an important regulatory role in the morphological development,sporulation,germination,growth rate,and dimorphic transition,resistance to stress,MS formation and host virulence of N.rileyi strains,with mutual cooperation.