Identification Of Wheat Exogenous Resistance-related Genes And Pathogen-derived Effector Proteins Based On Transcriptomes Under Powdery Mildew Stress

Wheat powdery mildew(PM)is a fungal leaf disease caused by Blumeria graminis f.sp.tritici(Bgt)which severely reduces the grain yield and quality.The pathogen forms haustorium through appressorium in the epidermal cells of the host plant by secreting a set of effector proteins and establishes a close parasitic relationship with its host.At the same time,the pathogen exhibits high degree of genetic variability,and the race-specific resistance of wheat is easily overcome by new virulence mutants.Therefore,there is dire need to cultivate varieties with ideal PM-resistance for wheat safety production and environmental protection.In this study,the characteristics and differences of cytological responses of host-pathogen between compatible and incompatible interactions were firstly investigated.Based on these information,transcriptomes obtained from dynamic host-pathogen bi-directional sequencing of leaf samples of Pm97033,a Triticum aestivum-Dasypyrum villosum 6DL·6V#4S translocation line,and its susceptible background wheat Wan7107,both of which were challenged with the pathogen,were used to screen and identify genes related to PM resistance from 6V#4S and the candidate pathogenic effector genes from the pathogen by molecular biology method.The study could lay a foundation for further identification of the PM resistance gene(s)from D.villosum 6V#4S and comprehending of its broad spectrum resistance mechanism.The main findings are as follows:1.The percentages of haustoria,papillae and hypersensitive reaction(HR)are measured in both verities Pm97033(resistant)and Wan7107(susceptible).Firstly,haustoria with haustorial body and lobes were observed at 24 h after inoculation.In Wan7107,the haustoria index grown rapidly and reached its peak at 48 h,which is about 80%.While in Pm97033,development of haustoria was much slower and reached its peak at 36 h,which is about 10%.Secondly,the papillae formation on host cell walls beneath the pathogen attempt infecting sites reached its peak at 24 h after inoculation in both wheat lines.At this time,the percentage of papillae formation in Wan7107 is 17% while in Pm97033 is 25%.The incidence of HR in epidermal cells infected by pathogen spores increased rapidly at 36 h after inoculation.In both wheat lines,HR reached its peak at 60 h after inoculation but Pm97033 shows 32% while Wan7107 shows 12%.2.A total of 196 differentially expressed genes between Wan7107 and Pm97033 were screened from the leaves of high-throughput transcriptional sequencing at 0 h,20 h and 48 h after the inoculation with Bgt.By using collinearity analysis,33 genes were preliminarily located on the short arm of wheat 6 homologous group.Further,19 genes from 6V#4S were identified and confirmed by PCR amplification of genomic DNA of Dasypyrum villosum and target product sequencing.19 genes were analyzed by qRT-PCR,and 12 genes showed up-regulated expression in Pm97033 after inoculated with Bgt.Among them,the defense functions of four genes,P106302,P116577,P35896 and P25583,were analyzed by virus-induced gene silencing(VIGS)for their PM resistance.The results showed that BSMV:C1-25583,C2-35896 and C3-116577,respectively,was silenced on Pm97033,and the appressorium invasion rates on the leaves were increased to some extent,and the formation of secondary hypha was significantly different from the control BSMV:EV,which could be disease resistance related genes.At the same time,nine genes located on 6V#4S were developed into specific molecular markers that could track the 6VS or 6V#4S chromosome arm of Dasypyrum villosum in different wheat genetic backgrounds.Among them,the primer P461-5a can be used as the specific molecular markers to distinguish the 6V#2S and 6V#4S chromosome arms of Dasypyrum villosum.3.Of 393 genes annotated as effector proteins from the transcriptome of Bgt,15 genes with high expression in compatible interaction were selected.Based on the results of semi-quantitative analysis,three candidate important genes BGT96224_E3119,NOVE100590 and BGT96224_E5891,which were differentially expressed between compatible and incompatible interaction,togather with a gene NOVE100718 that was described as a glycosyl hydrolase effector protein gene were further identified.qRT-PCR analysis confirmed the expression patterns of four genes.However,no significant virulence phenotype was observed when single-cell transient analysis of four genes.Using BGT96224_E3119 as bait,Yeast two-hybrid(Y2H)technique was used to screen the cDNA library of compatible interaction.Eight possible host-interacting proteins were preliminarily captured.Among them,six genes were presented in the host transcriptome data afer tBLASTx and compared with the host transcriptome data.