Negative Regulatory Function Study Of Hevea Brasiliensis LFG Gene In Inhibiting Fungal Infection

Natural rubber is widely used in industry,agriculture,national defense,transportation,machinery manufacturing and medical and health,it has important economic and strategic significance.Phytopathogenic fungi-powdery mildew,Colletorichuim gloeosporioides and Phytophthora will harm the Hevea brasiliensis.These pathogenic fungi will cause serious reduction of natural rubber and cause significant economic losses.At present,the research on disease-resistant breeding of Hevea brasiliensis is relatively lagging.This work initially explored the function of Hevea brasiliensis LFG gene in the infection of pathogenic fungi.in order to provide theoretical basis and technical paving for Hevea brasiliensis disease breeding.The main findings are as followsEleven LFG homologous proteins were identified in the rubber tree gene pool,by alignment with the AtLFG gene,the two genes with the highest homology were selected HbLFG1 and HbLFG2.HbLFG1 and HbLFG2,respectively.The bioinformatics analysis of these two genes showed that the proteins encoded by the two genes have seven transmembrane domains and do not contain signal peptides,all of which contain a BI-1-like superfamily conserved domain.However,there are 5 differences in 3 of the three conserved motifs of the two proteinsThe expression levels of HbLFG1 and HbLFG2 genes in the leaves of Hevea brasiliensis infested with rubber powder,0 h,3 h.6 h,12 h,24 h,48 h,and the results showed that the two genes increased with time in the first 24 h.However,the expression of HbLFG1 was more significant than that of HbLFG2 gene.The expression levels of HbLFG1 and HbLFG2 genes in leaves of different susceptible grades after infection by powdery mildew of rubber tree were determined.The results showed that with the increase of grades,the expression of two genes showed a trend of increasing first and then decreasing,and the expression of HbLFG1 was higher than that of HbLFG1.The results indicated that HbLFG1 and HbLFG2 genes may be involved in the regulation of Hevea brasiliensis resistance to powdery mildew,and HbLFG1 is more effective than HbLFG2 in response to pathogen infection.To determine whether the HbLFG1 gene has a regulatory effect on pathogen infection,knock out AtLFG in Arabidopsis wild type using CRISPR/Cas9 technology to obtain a stably inherited mutant,and then transiently express HbLFG1(?AtLFG/HbLFG1)to Arabidopsis LFG deletion mutant.Pathogenicity assays were performed on Arabidopsis wild type,AtLFG deletion mutants and ?AtLFG/HbLFG1 plants using Phytophthora capsici and Colletorichuim gloeosporioides.We divided the virulence results into three grades:grade 1.grade 2 and grade 3.which were:pathogens were not infected,pathogenic mycelial invasion is blocked and short.Pathogenic bacteria form reticular invading hyphae.The results showed that among the 50 infection sites of Phytophthora capsici and Colletorichuim gloeosporioides,the mycelial invasion of Arabidopsis wild type accounted for 92%and 96%;the mycelial invasion of LFG deletion mutant accounted for 32%and 30%,the mycelial invasion of ?AtLFG/HbLFG1 accounted for 70%and 78%.The results showed that AtLFG gene has a negative regulatory effect on plant disease resistance,HbLFG1 gene has a similar effect to AtLFG gene,and HbLFG1 is also a negative regulatory disease resistance gene.In summary,this study preliminarily proved that the HbLFG1 gene from the Hevea brasiliensis may play an important role in the disease resistance of fungal diseases,and provide important reference for the resistance breeding of Hevea brasiliensis.