Function And Pathway Of Adhesin MAD2 In Metarhizium Anisopliae And Peanut Interaction

Metarhizium anisopliae(M.anisopliae)is an important entomopathogenic fungus used as a biopesticide in control of agricultural and forestry pests.The fungus evoluted differently trophic modes such as pathotroph,symbiotroph and saprotroph,to adapt to different environments and sustain populations.In recent years,the endogeneity of insect-pathogenic fungi has become interesting in the field of fungus-plant relationship.Metarhizium adhesion like protein 2(MAD2)is a specific adhesive protein in the interaction of M.anisopliae and plants,it plays an essential role in achieving persistence in adverse environment and selective recognition of hosts.But the interaction intermechanism is rarely studied.In this paper,based on the previous analysis of peanut root transcriptome treated with M.anisopliae,we compared the transcribed and protein expression response of key genes related to receptors and downstream pathways in peanut roots,with the treatments of the MAD2 protein and M.anisopliae spores.The host's induced response to MAD2 will provide useful data for explaining the endophytic mechanisms and environmental survivability of M.anisopliae.It may be great significant for guiding the development and application of M.anisopliae in comprehensive plant protection.The main results were as follows:1.Cloning,expression and sequence analysis of adhesion gene Mad2 of M.anisopliae.The gene Mad2 was cloned using the M.anisopliae c DNA as the template.After sequencing and NCBI comparison,the complete open reading frame was 921 bp,encoding 306 amino acids,and the theoretical molecular weight of the protein was 30.6 k Da.The eukaryotic expression system of Pichia pastoris was constructed,SDS-PAGE and Western blot showed that MAD2 protein was successfully expressed and carried a purified His tag,finally MAD2 protein was obtained.The prediction of hydrophobic amino acids and protein binding sites of MAD2 by Proteinpredict,it is showed there is a GPI binding site on the 290 th amino acid,and 24 hydrophobic amino acids exposed nearby indicates that MAD2 may bind on the cell membrane.2.Different responses of peanut receptors and related genes under treatment with the MAD2 and M.anisopliae.At first,the transcriptation-level expression of 9 candidate receptor genes based on the previous transcriptome analysis was verified by RT-q PCR in peanut roots treated with M.anisopliae conidium for 4 days.It indicated that immune-related receptors;stress related receptors;and the symbiotic receptor SYMRK showed up-regulated expression,which was consistent with the transcriptome results.Then we treated peanuts with M.anisopliae and MAD2,the results showed Lys M receptor kinases LYK3 and G type agglutinin S receptor-like serine/threonine protein kinase Gs SRK were significantly up-regulated when treated with MAD2 for 12 h,FLS2 receptor was significantly up-regulated after treatment with MAD2 protein,and down-regulated when treatment with M.anisopliae for 12 h.BAK1 and CERK1 were down regulated under the treatment with MAD2 and M.anisopliae.Moreover,the detection of disease-related,stress-related and symbiosis-related genes showed that the treatment of MAD2 inhibited the MAPK pathway and down-regulated the expression of stress-related and disease-related genes,and the genes involved in ion transport and lipid transfer were up-regulated.The results showed that LYK3 may respond to the induction of MAD2 and activate the downstream symbiosis related genes and inhibit the related resistance genes,thus promoting the establishment of symbiosis between metarhizium and peanut.3.Quantitative analysis of protein expression of peanut treated by M.anisopliae and MAD2.We detecting the different expression protein of peanut with the treatment of MAD2.By using the method of TMT protein quantification,total 2550 differentially expressed genes were obtained,and 1326 up-regulated protein were detected in KEGG.A total of 113 differentially expressed proteins were enriched in the ester metabolic pathway under the two treatments.Screening and founding that 15 difference expression proteins were the same under the treat with MAD2 and Metarhizium anisopliae.In view of the known that Flavonoids are involved in the expression of nodule genes in rhizobia and symbiotic genes in arbuscular mycorrhizal.Those 15 genes were involved in flavonoids and isoflavones synthesis such as pinocembrin,isoliquiritigenin,phloretin,xanthohumol,naringenin,eriodictyol and homoeriodictyol,it also plays an important role in the formation of formononetin and prunetrin from isoliquiritigenin and phloretin,This indicated that the lipid synthesis and metabolism of peanut were accelerated after MAD2 treatment,and it was speculated that MAD2 may induce the synthetic pathways of peanut flavonoids,accelerate the substances communication between metarhizium and peanut,and promote the colonization of Metarhizium in peanut.