Prediction Analysis And Pathogenicity Determination Of Effectors In Nimbya Alternantherae

Alternanthera philoxeroides is one of the first batch of invasive weed published in China,which can seriously damage the biological diversity and ecosystem of the lands invaded,and also has severe threat to the agriculture production and socioeconomic development.Nimbya alternantherae is the biocontrol fungi of the alligator weed possessing the potential value of development.A large number of researches have been carried out for N.alternantherae such as the weed control efficacy,herbicidal activity of metabolites and the mechanism of action and so on.So far,there is no articles about effector pathogenic function of N.alternantherae.This study took advantage of the bioinformatics analysis to predict effectors from the whole genome of N.alternantherae,and then employed Pichia pastoris to express a part of effectors,in addition,determined the pathogenicity of expression product.The results are as follows:1.Prediction analysis of effectors.889 secreted proteins were acquired from 10470 proteins sequences of Nimbya alternantherae whole genome by the procedure of Signal P?TMHMM?Target P ? Pro Comp,110 candidate effectors were obtained by the features of small molecular(<300 aa)and rich cysteine(? 6),48 effectors were achieved through the analysis of macro transcriptome expression date.Function classification and annotation of 48 effectors were carried out via blasting with PHI and Nr date base,Go,KOGand KEGG analysis.2.The gene clone and eukaryotic expression of seven effectors.The total of seven effectors whichg 121,g925,g4757,g5745,g7484,g7868 and g9900 were used for gene clone and eukaryotic expression.The sequences of genome and open reading frame belong to seven effectors were gained,and then the number of intron and the evolutionary relationship of seven effectors were analysed.Furthermore,the homologous gene comparison was draw between seven effectors and similar species.Rcombinant ecpression vectors were constructed by connecting the eukaryotic expression vector p PICZ?A with seven genetic megments,and then entered into Pichia pastoris GS115 by Gene Pulser Xcell,which induced to express through methyl alcohol.Expression products were isolated by SDS-PAGE.The results that the purpose bands isolateds from the expression products ofseven effectors all in accord with the size of forecast proteins illustrated the success of eukaryotic expressions for seven effectors.3.Pathogenicity determination of effectors.The coincident health-free alligator weed was chosen to detached leaves and plants to mensurate the pathogenicity by inoculating effectors expression products on the detached leaves extruded wound spots and living plants roots respectively,others cases inoculated with the expression product of null vector,the liquid culture medium of BMMY and filtered water as control.The wound spot of detached leaves inoculated the expression conducts of effectors become dark brown.The development of lesions was obvious.A substantial part of leaf area or the whole was hygrophanous and brown after 72 hours,yet the controls inoculated the expression conducts of null vector and BMMY liquid culture medium become only a little dark.There is no obvious lesions.The controls of inoculated filtered water become slightly yellow.The plants dealt with others six effectors expression products all appeared obvious sympton of wilts after 12 hours except the plants roots dealt with g4757 expression products whose action a bit slower,and the more time,the more serious.But the plants inoculated with the expresstion products of null vector,the liquid culture medium of BMMY and filtered water were still normal growth.The expression condfucts of seven effectors have turned out to be have a serious pathogenic impact on the alligator weed.