The Function Of Colletotrichum Fructicola Effector Protein CfCE92 And Its Interaction Targetscreening

The Colletotrichum fructicola causes diseases on a broad range of plant species.C.fructicola casuses Glomerella leaf spot?GLS?on Golden Delicious,inducing severe early defoliation and fruit spots and reducing the commercial quality of the apple fruit.Effector proteins of Colletotrichum fructicola as an important pathogenic factor affect the host's immune response to promote host disease,but the mechanisms of pathogenicity have remained elusive.In the early stage of our laboratory,an effector protein Cf CE92 was screened and identified from C.fructicola.In this study,Preliminary laboratory work It was found that an effector protein,Cf CE92,further studied its pathogenic function,the mainresults asstudy intends to conduct a systematic study on its pathogenic function and1. Leaes?cv.Gala?were inoculated with Cf CE92 mutant wild type strains of Colletotrichum fructicola,respectively.Extraction of RNA after 24 hours,transcriptome sequencing,gene prediction and annotation were performed.A total of 52.41 gigabytes were obtained.Total 45116 genes were annotated and 2370 differentially expressed genes were obtained from the mutant and the wild type.310 genes were up-regulated significantly.Pathway enrichment analysis showed that,glyoxylate and dicarboxylate metabolism,flavonoid biosynthesis,galactose metabolism,amino sugar and nucleotide sugar metabolism,glycerolipid metabolismand MAPK signaling pathway.2. MAPK signal pathway can mediate many physiological changes and such as cell growth,differentiation and pathological process.In the MAPK pathway,seven genes were enriched:abscisic acid receptor pyl4-like,pathogenesis-related protein 1-like?PR1a?,endochitinase-like precursor?PR3?,receptor-like serine threonine-protein kinase,ethylene-responsive transcription factor,wrky transcription factor 29,abscisic acid receptor.The PR1a,PR3 and WRKY29 were analyzed by q RT-PCR,the results showed that the gene expression level of?Cf CE92 mutant was significantly higher than that of the wild type.3. Flavonoid is an important secondary metabolite in plants.It is important in plant defensins.Three genes were enriched in the flavonoid biosynthesis pathway:chalcone synthase,chalcone-flavonone isomerase 3,shikimate o-hydroxycinnamoyltransferase-like.The three genes were analyzed by q RT-PCR,the results showed that the gene expression level of?Cf CE92 mutant was significantly higher than that of the wild type.4. Metabolome analysis:2',3,4,4',6'-Pentahydroxychalcone,7,4'-Dihydroxy-flavone,Liquiritigenin,Naringenin,Phloretin,Phlorizin,Pinocembrin flavonoid biosynthesis were significantly increased.Colletotrichum fructicola were inoculated in PDA medium containing 7 compounds respectively.The colony diameters were measured,which showed that all of the seven flavonoids could inhibit the growth of C.fructicola.The values of EC₅₀showed that 2',3,4,4',6'-pentahydroxychalcone had the most growth inhibition(EC₅₀₌36.6mg/L),naringenin?143.4 mg/L?and 7,4'-dihydroxy-flavone?150.9 mg/L?was similar in inhibitory effect.These results indicate that,Cf CE92 can inhibit the expression of defense-related genes in the process of infection,and then facilitate the infection.5. The effector protein Cf CE92 localized in the cytoplasm and nucleus after instantaneous expression in Nicotiana benthamiana.Screening of target proteins from c DNA of infected leaves of Colletotrichum fructicola with Yeast Two-hybrid,68 candidate target proteins were obtained.The interaction of Cf CE92 with nucleoside diphosphate protein kinase?NDPK?,Glutathione peroxidase?GPx?and Leucine zipper protein?GILZ?was demonstrated in yeast.Cf CE92 and NDPK was found to interact by bimolecular fluorescence assay?BIFC?.However,Cf CE92 could not interact with GPx and GILZ when co-injected into Nicotiana benthamiana.Cf CE92 interacts with the host target protein NDPK,suppressing plant host immune response to promote infection.This study confirmed that Cf CE92 could inhibit the expression of host-associated defense genes,and interact with the host NDPK protein.NDPK is related to plant defense response and effector protein Cf CE92 may contribute to the pathogenecity of C.fructicola through interacting with NDPK.In this study,the pathogenic mechanism of effector Cf EC92was preliminarily revealed,which provides a foundation for further study on the pathogenic mechanism of C.fructicola and a target for the development of new pesticides.