Early Molecular Detection And Identification Of Differential Expressed Proteins Of Adzuki Bean Infected With Uromyces Vignae

Vigna angularis is a traditional coarse cereals crop in China,which play important roles in agricultural planting structure adjustment due to its characteristics of poor soil tolerance,stress resistance and short growth period.However,adzuki bean rust caused by the biotrophic fungus Uromyces vignae can cause the leaves necrosis and fall off early.Uromyces vignae can form multiple re-infection in a growing season with a long period of latent infection,which can cause a large-scale epidemic and seriously yield and quality losses.However,limited information about the early diagnosis techniques for adzuki bean rust and the molecular resistance mechanisms of adzuki bean result in lack of mature theory for guiding prevention practices.Therefore,in current study,a nested PCR based molecular detection system for adzuki bean rust was established and applied to clarify the infection after different time points of artificial inoculation.Furthermore,the inoculated leaves in different times according to the nested PCR were collected for differential expressed proteins identification using i TRAQ technology.Results in the present study can provide technical support for adzuki bean rust diagnosis,prevention,and further exploration of the molecular resistance mechanism of adzuki bean in response to rust.The research achieved the following important results:1.Specific primer UV-ITS for Uromyces vignae detecteion was screened based on normal PCR.Thereafter,a nested PCR primer UV-MX was designed according to the sequence of the PCR product of UV-ITS to construct nested PCR detection system.Sensitivity and specificity analysis showed that the amplification efficiency was 100,000 times than the normal PCR.Additionally,the nested PCR system was applified to detect the leaves of adzuki bean at different times post artificial inoculation.The results showed that the nested PCR can detect the rust fungus as early as the leaves at 12 hour post inoculation(hpi).In addition,comparing the brightness of agarose gel electrophoresis bands of PCR products at different times indicating an increased of biomass at 24,48,and 120 hpi.2.Based on the results of nested PCR,leaves of the resistant cultivar QH1 inoculated with U.vignae spores at 24,48 and 120 hpi with three replicates were collected for i TRAQ analysis,leaves inoculated with sterile water were collected as control.Eighteen samples for proteomic analysis were conducted and 6899 proteins were identified.Proteins with a threshold of log2 ratio > 1.2,p-value < 0.05 were considered as differential expressed proteins(DEPs).Results show that there were 36 DEPs at 24 hpi,in which 11 were up-regulated and 25 were down-regulated.While at 48 hpi,48 DEPs were identified with 23 up-regulated and 25 down-regulated proteins,respectively.In addition,70 DEPs were identified at 120 hpi,and there were 59 up-regulated proteins and 11 down-regulated proteins.These results indicated that the continuous enhanced defense response accompanied by the rust infection is a key factor for the high resistance of QH1.3.The functional annotation of the DEPs at 24 hpi showed that the reactive oxygen species metabolism-related enzymes,including SOD and POD were significantly up-regulated.At 48 hpi,numbers of pathogenesis related proteins,such as PR-2,PR-9 and PR-14,were significantly up-regulated.And at 120 hpi,numbers of pathogenesis related proteins,such as PR-2,PR-3,PR-4,PR-5 and PR-9,were significantly up-regulated.Additionally,GO and KEGG enrichment analysis of the DEPs at different stages of rust fungal infection showed that the reactive oxygen species(ROS)metabolism,and secondary metabolites biosynthetic pathway were significantly enriched at 24 hpi.While the enrichment analysis of the DEPs of 48 showed that gene expression regulation and flavonoid biosynthesis pathways were significantly enriched.And the enrichment analysis of the DEPs of 120 hpi showed that defense response,innate immune response,flavonoid biosynthesis pathways,isoflavonoid biosynthesis pathways and phenylpropanoid biosynthesis pathways were significantly enriched.These results indicted that the variation of the ROS homestasis in adzuki bean at the early stage of U.vignae infection activating the downstream defense responses,including PRs,proteins involved in phytotoxins and innate immunity.4.Based on the functional annotation of the DEPs,we found that the members of the DIR protein family were significantly up-regulated among the different samples.To gain insight into the role of the DIR proteins of adzuki bean in response to the rust fungal resistance,expression profiling of the genes encoding DIR proteins were analyzed by using the q RT-PCR.Results showed that,compared with the susceptible cultivar,6 selected DIR proteins were significantly induced at the early stage during the U.vignae infection in resistant cultivar.Furthermore,different DIR proteins have different expression patterns in response to U.vignae infection.Results suggested that the DIR proteins of adzuki bean might played an important role in response to adzuki bean rust resistance and the different members of this gene family might play different roles in response to U.vignae infection.