Transcriptome Analysis And Differentially Expressed Genes Identification Of Adzuki Bean-Uromyces Vignae Interaction

Heilongjiang province is the major area in China for adzuki bean（Vigna angularis）production,and the adzuki bean is one of the important economic sources of the local farmers.With adjustment of agricultural planting structure,the planting area of adzuki bean increased year by year in Heilongjiang.However,the adzuki bean rust caused by the fungus Uromyces vignae has been seriously affect the adzuki bean production.Additionally,there is no mature theoretical and technical achievements for the disease prevention due to limited information about the interaction between V.angularis and U.vignae were reported.Therefore,RNA-Seq technology was used for analyzing the genes expression of adzuki bean in response to U.vignae infection for screaning genes involved in rust resistance.Subsequently,the internal reference genes were screaned and the real-time quantitative PCR（qRT-PCR）technology system was established for expression analysis of adzuki bean genes.Based on qRT-PCR and the reference genes,the expression analysis of the genes that predicted by RNA-Seq associated with rust resistance was conducted to revealing the function of the genes in response to U.vignae infection.The results of the present study can lay a foundation for further exploration of the molecular mechanism of anti-rust disease and the management of adzuki bean rust.The current study achieved the following important results:1.Using transcriptome sequencing technology,the whole genes expression profilling of the resistant variety ‘QH1’ at different hours post inoculation（hpi）with U.vignae were analyzed.The sterile water was inoculated in the same time as control.Twelve samples including 4treatments with 3 biological repeats were sequenced,and a total of 0.38 to 0.54 Gb clean reads were collected.After mapping to the reference genome of V.angularis,the differential expressed genes（DEGs）were identified.Compared with control,a total of 2973 DEGs were identified at24 h post inoculation（24_hpi）,while 856 DEGs were identified at 48_hpi.Fewer number of DEGs found at 48_hpi indicating that the defense of the adzuki bean was significantly induced at the eraly stage of the fungal invasion.Functional annotation of the DEGs showed that multiple pathogenesis relataed（PR）genes were significantly up-regualted at 24_hpi.The subsequent GO and KEGG enrichment analysis revealed that after the rust fungal infection,multiple key genes involved in ethylene（ET）biosynthesis（ACS and ACO）and ET signaling transduction（EBF1,ERF1 b,ERF096,ERF110,ERF113）were up-regualted.These results indicated that the rust infection may induce the change of endogenous ET content of adzuki bean,which activates the ET disease resistance signaling pathway and the subsequent PR genes expression.This may be an important factor for the high resistance against rust infection of the adzuki bean variety QH1.2.To confirm the resistance genes from a large number of DEGs obtained by RNA-Seq,qRT-PCR can be used to analyze the expression of candidate genes in response to U.vignae infection,a suitable expressed qRT-PCR system and stable internal reference genes for adzuki bean were needed.Therefore,9 commonly used housekeeping genes as candidate internal reference were selected and evaluated under different conditions.The results showed that among different varieties,ACT or PTB can be used as internal reference.Among different tissues and organs of the same variety,the EF or UBN is the most stable and can be used as internal reference.Under inoculation with U.vignae and drought stress,ACT or ZMPP can be used as internal reference gene.Under alkaline stress,UBC or Fbox can be used as internal reference.Under waterlogging stress,the PTB or Fbox was the most stable that can be used as internal reference.To evaluate the reliability of the internal reference genes screened previously,ACT,combination of UNC+UBN,UBC+UBN+EF and PP2 A were used for 3 disease resistance related genes（CAT,CHI and GLU）expression analysis in response to U.vignae infection.The results shwoed that the expression levels of the selected genes could be accurately assessed by using ACT as internal referece.It is indicated that the internal reference genes obtained by this study were more reliable and can be used as internal reference for adzuki bean genes expression analysis.3.Using the qRT-PCR analysis system established and the internal reference genes obtained previously,leave samples from resistant and susceptible cultivars after inoculation with rust were colleted for two resistant related genes（VaNATA1 and VaEG45）,predicted by RNA-Seq,expression analysis were conducted.Compared with the expression pattern in susceptible cultivar,the two genes were significantly induced in the resistant variety.The expression of VaNATA1 was significantly up-regulated both at the early（12-24 hpi）and late（120 hpi）stages of fungal infection,while VaEG45 was significantly up-regulated mainly at the late（48-120 hpi）stage of the fungal infection.The results indicating that VaNATA1 was involved in resistance duiring the fungal invasion and expansion,while VaEG45 is only associated with inhibition offungal spreading.These results suggested that the expression levels of VaNATA1 and VaEG45 were positively correlated with disease resistance,but the molecular mechanisms of the two genes involved in disease resistance still needs further experimental verification.4.The resistance gene VaEG45 was cloned by RT-PCR,and the analysis of its amino acid sequence showed that VaEG45 belongs to the plant natriuretic peptide（PNP）family gene and contains a DPBB₁ domain.It is a secreted extracellular protein.After transient expression in tobacco,the diameter of the lesions of tobacco leaves transiently expressing VaEG45 was significantly reduced,which was nearly 40% lower than that of the control,indicating that the gene significantly increased the resistance of tobacco to Botrytis cinerea infection.Further analysis of the callose staining technique revealed that the transient expression of VaEG45 could induce callose deposition in tobacco mesophyll cells.It is speculated that VaEG45 may enhance the resistance of tobacco to B.cinerea by enhancing the mechanical strength of host cell wall.