The Functional Analysis Of NoxA,NoxB And NoxR In Nomuraea Rileyi

Nomuraea rileyi has the characteristics of being a biological insecticide,that can infect a variety of Lepidoptera insects under natural conditions.At present,almost all of fungal pesticide use conidia as an active ingredient,but the problem is that the production of spores of N.rileyi requires special growth conditions,which affects its application in biocontrol.In 2012,the team of Professor Wang Zhongkang of Chongqing University successfully induced the N.rileyi to produce microsclerotia（MS）,which can be used as an effective ingredient of fungal insecticides.Since then,the formation mechanism of MS of N.rileyi has become a research hotspot.It is generally accepted that reactive oxygen species（ROS）and oxidative stress are key inducers that stimulate the formation of MS,and NADPH oxidase（Nox）is the main source of ROS.In this study,the NrNoxA,NrNoxB,and NrNoxR mutant strains were obtained to study the functions of NrNoxA,NrNoxB,and NrNoxR on the growth,the formation of MS and virulence of N.rileyi,and the following preliminary results were obtained.（1）Role of ROS in the formation of MSThe treatment of N.rileyi with oxidants or antioxidants showed that 10^-8 M H₂O₂ and10^-6 M H₂O₂ promoted the increase of microsclerotia yield by 26.37%and 8.23%,respectively,and 0.05g/L and 0.1g/L ascorbic acid cause the yield of microsclerotia was only 55.96%and 67.08%under normal conditions.（2）NrNoxA,NrNoxB and NrNoxR have a high expression level during microsclerotia formationIt was found that NrNoxA,NrNoxB and NrNoxR have a similar expression pattern,that the expression levels were not high in the early stage of formation,but kept increasing in the middle and late stage,by detecting the expression levels of those genes at various time points of microsclerotia formation.（3）Phenotypic analysis of mutant strainsROS tests showed that it is decreased in the mycelium of△NrNoxA,increased in△NrNoxR and changing not obviously in△NrNoxB.Compared with the wild type,the△NrNoxA mutants showed a slower germination rates and switching from yeast-cell to hyphae,increased number of mycelial branching and colony diameter,reduced spore production,and sensitivity to cell destroyers;knockout of NrNoxB has no obvious effect on the basic growth of the strain;knockout of NrNoxR resulted in accelerated spore germination rate,decreased mycelium branches,increased colony diameter,sharply decreased spore production,and increased sensitivity to cell wall destructors,Na Cl and KCl.In the microsclerotia induction experiment,the absence of NrNoxA or NrNoxR seriously affected the formation of microsclerotia,while the MS production and total biomass of△NrNoxB both increased,additionally,we found that the absence of NrNoxR seriously inhibits the formation of pigments in microsclerotia.（4）The NrNoxA and NrNoxB gene play a role in infection process,and NoxR play a role in infection process and the process in host bodyToxicity tests on Spodoptera litura show that△NrNoxR strains show less virulence in both topical infection and injection assay,and△NrNoxA and△NrNoxB strains show a significant decrease in virulence on topical infection assay,Compared to the wild type.（5）Comparative transcriptome analysis of the effect of NrNoxR knockout on MS development.The result showed that there were 739 significantly differentially expressed genes （DEGs）between the wild type and△NrNoxR on MS development.Enrichment analysis of Gene Ontology（GO）of DEGs shows that NrNoxR has an effect on cell membrane and cell membrane components,metabolic processes and cellular processes.The results of KEGG pathway enrichment indicate that NrNoxR has a greater influence on sugar metabolism,amino acid metabolism,secondary biomass metabolism and lipid metabolism.Futhermore,we found that a number of genes related to active oxygen production were up-regulated in△NrNoxR,including 43 oxidoreductases and 3dioxygenases.The above results indicate that NrNoxR and NrNoxA genes play a certain role in responding to external stress,the development of microsclerotia,and the virulence on S.litura,while NrNoxB gene seem play an unimportant role in the above aspects except in virulence and the development of microsclerotia.