Isolation And Functional Analysis Of A Key Gene TiRB Of BYDV Resistance In Wheat-Thinopyrum Intermedium Translocation Line

Barley yellow dwarf virus (BYDV) is the most serious virus disease of wheat (Triticum aestivum)worldwide. No effective resistant source was found in common wheat besides one tolerable gene in fewWheat germplasm. However, efficient resistant sources were identified in common wheat relativespecies Thinopyrum intermedium. The efficient resistant source from the long arm chromosome of7Ai-1L of Thinopyrum intermedium was introgressed into common wheat genome, breedingtranslocations YW642and YW443, widely in agriculture. No resistance gene or the mechanism ofdefense to BYDV has been cloned or elucidated so far. It is very important to identify resistance geneand elucidate the mechanism of defense to BYDV.The wheat-Thinopyrum intermedium translocation line YW642which carries BYDV resistancegene Bdv2and common wheat Zhong8601(susceptible parent Zhong8601) were main materials in thisstudy. Affymetrix GeneChip Wheat Genome Arrays were used to analyze the transcriptomes ofYW642and Zhong8601for profiling genes and pathways response to BYDV infection. One key generesistance to BYDV was identified by molecular markers, comparative genomics, virus induced genesilence combining transgene analysis, named TiRB. Overexpressing TiRB exhibited well resistance toBYDV in Zhong8601and knockouting TiRB showed poor symptom to BYDV in YW642. Somecharacters of TiRB were also assayed.This study made the following progresses:1. The transcriptomes of YW642and its susceptible parent Zhong8601were analyzed byAffymetrix GeneChip Wheat Genome Arrays. Firstly,12and72h were identified as crucial timepoints by quantification of BYDV contents in YW642and Zhong8601under differentBYDV-inoculated time respectively. Secondly, thousands of differentialy expression transcriptsincluding some R (resistance) like genes were identified by transcriptomes.335resistance related genesresponse to BYDV invloved in ROS (reactive oxygen species), JA (jasmonic acid) and PA(phospholipid acid) signalings pathways which participate in defense BYDV in seedling YW642.Hydrogen peroxide and photosynthesis were key factors of resistance to BYDV in mature YW642.2. TiRB, a candidate gene resistance to BYDV, was identified by comparative genomics. Astheory of comparative genomics, one candidate gene to resistance to BYDV was cloned byBrachypodium distachyon genome database, Oryza sative genome database, Sorghum bicolor genomedatabase and molecular markers (especially one marker form a resistance related gene TiDPK1,locateing on7Ai-1L) traced Bdv2, named TiRB. TiRB encondes a NBS-LRR protein (consist of967AA)and was expressing only in wheat-Thinopyrum intermedium translocations and Thinopyrumintermedium. Association studies show TiRB maybe participates in resistance to BYDV.3. TiRB was verified as an important gene to defese BYDV by VIGS and transgene assays.Flagments of TiRB were inserted into BSMV-γVIGS vector and generated recombined vectors.Subsequently, recombined vectors were digested and transcribed into RNA in vitro. Leaves of two-weeks YW642were infected with recombined virus RNA, inducing TiRB silencing. AfterBYDV-inoculation35d, Zhong8601, TiRB-silencing YW642were chlorotic and dwarfed, and BYDVcontents increasing obviously. Other plants, including YW642and γYW642were immune to BYDVnearly. An overexpressing vector and a RNA interfering vector of TiRB were constructed forgain-of-function and loss-of-function assays respectively. Comparing to the susceptible wheatZhong8601(transgenic receptor), transgenic wheat lines overexpressing TiRB exhibited well resistanceto BYDV, likely to the gene donor YW642; However, symptom of transgenic wheat silencing TiRBplants (YW642as transgenic receptor) were similar to the Zhong8601. Based on these results, TiRBshould be a key gene resistance to BYDV in YW642.4. Expressions of TiRB were analysed. TiRB was upregulated by BYDV, and the top expression atBYDV-inoculation12-24h. TiRB transcript was tissue-specific; the transcript level was highest inleaves, moderate in stems, and lowest in roots. The transcript level of TiRB in the seedling wassignificantly upregulated by treatment for12-24h with exogenous SA and BR hormones.5. TiRB distributes in cytoplasm and nucleus, and interacts with BYDV CP. ORF of TiRBinserted into163hGFP vector and generated an expression vector163hTiRB-GFP, and transientexpression in onion and wheat epidermis single-cell following the instructions of the PDS1000/HeParticle Delivery System (Bio-Rad). Under35S promoter, TiRB was observed at cytoplasm and nucleusby a confocal microscope, the result was validated by Western assay with CTAPi–3×HA-TiRB. TiRBinteracts with BYDV CP were validated by yeast two-hybrid and bimolecular fluorescencecomplementation assays.