Functional Study Of Pythium Spp.and Fungus.Effectors And Preliminary Analysis Of Secreted Protein From Pythium Guiyangense

The study of plant pathology shows that when pathogen infects host,a large number of effectors will be produced,which will interfere with the normal physiological and biochemical reactions of host and cause host disease.There are many pathogenic effectors,two of which can kill host cells directly as toxins.First,CRN class effectors(crinkling and necrosis proteins)exist widely in various pathogenic fungi,but they obviously expand in oomycetes,causing necrosis and shrinkage of host cells.The second is NLP effectors(necrosis and ethylene-inducing peptide 1-like protein),which have cell-killing activity through in vitro processing.In the early stage of our laboratory,we found that many pathogenic oomycetes and fungi also have these two types of effectors in their genomes.Therefore,we proposed the hypothesis that the effectors from pathogenic ovozymes and fungi may also have toxic effects on host insect cells,which is expected to be a new resource for the identification of insecticidal proteins.In 1994,professor Su Xiaoqing,professor of Guizhou medical college,discovered a pathogenic oozymete toxic to mosquito larvae.Researchers have been trying to understand the mechanism of the toxicity of Pythium guiyangense,providing a more effective and safe method for mosquito control.Protease,chitinase,lipase,enzyme or enzyme system that breaks down cellulose and phenolic compounds are all secreted by insect pathogenic fungi.Therefore,we believe that some enzymes or enzyme lines may be secreted by Pythium guiyangense to promote infection.Therefore,in this study,the effectors from insect-borne Pythium guiyangense,Aspergillus flavus and Fusarium oxysporum were selected.Based on the characteristics that cotton bollworm can feed on nicotiana benthamiana,these effectors were expressed in nicotiana benthamiana,and the effects of these effectors on cotton bollworm were investigated to obtain novel insecticidal proteins.The effects of these effectors on plant disease resistance and abiotic stress were also studied.And obtained the following main results:Transgenic tobacco strains with expressed effectors were obtained:bioinformatics analysis showed that the Pythium guiyangense that could infect mosquitoes contained 28 CRN effectors at the early stage.By using insect cell line toxicity test,6 effectors with cytotoxicity to insects were obtained in our laboratory(PgCRN19,PgCRN31,PgCRN33,PgCRN37,PgCRN40 and PgCRN44).At the same time,14 NLP effectors were identified from Aspergillus flavus and Fusarium oxysporum.In this study,a plant transgenic expression vector based on pbin-gfp-2 vector was constructed,and the genetic transformation experiment of these 20 effectors was conducted on tobacco.Finally,three transgenic tobacco plants expressing CRN 19 effector of rot mould in Pythium guiyangense,four transgenic tobacco plants expressing AfNLPl effector of Aspergillus flavus and four transgenic tobacco plants expressing FoNLP3 effector of Fusarium oxysporum were obtained.Phenotypic analysis of transgenic plants:transgenic plants inoculated the fungus found that Pythium guiyangense CRN19 effect,Aspergillus flavus AfNLP1 effect and Fusarium oxysporum FoNLP3 effect of transgenic plants to pepper the fungus resistance and negative control,there were no significant differences(expressing GFP gene)to illustrate the effect of three children don't affect the disease resistance of plants.Bollworm feeding three effects of transgenic plants experiment showed that the expression of Bt protein gene transfer tobacco plants(positive control)of cotton bollworm resistance is strong,and the three effects of transgenic plants and the expression of GFP gene negative control tobacco plant insect-resistant similar levels,to illustrate the effect of three children in the experiment system of cotton bollworm has no toxicity.However,abiotic stress experiments showed that transgenic plants with three effectors showed enhanced salt tolerance,while NLP effectors from Aspergillus flavus and Fusarium oxysporum could improve the drought tolerance of plants.Analysis of the secreted proteins of Pythium guiyangense:by studying the secreted proteins of Pythium guiyangense when it infected mosquitoes,we found that the expression levels of Prl(subtilase)and Pr2(trypsin)were increased,indicating that the two enzymes played a role in the infection process.Real-time fluorescence quantitative analysis of the homologous DNA showed that the expression of these two enzymes varied with time during the infection process,but generally showed a positive expression trend.In conclusion,this study proposes the hypothesis that the genome of entomogenous rot mold and fungus contains effectors homologous to the sequence of plant pathogens,which may be a new resource for identification of insecticidal proteins.After that,a batch of candidate effectors were identified,plant expression vectors were constructed,transgenic tobacco strains expressing three effectors were obtained,and phenotypes of transgenic materials were analyzed,which laid a material foundation for the further study of functional differentiation of effectors in plant pathogens and insect pathogens.