Identification And BSMV-VIGS Analysis Of Candidate Genes Resistant To Fusarium Head Blight

Fusarium head blight (FHB) was mainly caused by Fusarium graminearum Schwabe. Itwas prevailed mainly in the warm humid and subhumid regions around the world. In China,FHB had been occurred in Yangtse River Valley and along with the global warming it had atendency of spreading to Huanghuaihai region. However, the resistant resources of FHB werenarrow, Sumai3and its derived lines were major resistant resources applied in wheat genomicsstudy. Also, because of their strong and stable resistance to FHB, the cultivars were widely usedto locate major QTLs and for wheat breeding. With the development of biochemical,sequencing and gene manipulating technology, identification and functional analysis of theFHB resistance genes had achieved great progress in genomics and proteomics research.In this article, we combined transcriptome sequencing and bioinformatics analysis toexplore the resistance relevant genes in scab sensitive and resistance near-isogenic linesApogee and Apogee73S2infected with Fusarium graminearum. Barely stripe mosic virusinduced by gene silencing (BSMV-VIGS) was practiced to verify the candidate genesdiscovered in the transcriptome database and their biochemical characteristics were ensured inthe present study. The results were as follows:1. BSMV-VIGS system had been successfully established in wheat leaves and spikes usingTaPDS as a report gene. The photobleaching phenotype could last more than21days in wheatleaf and30to40days in spike, which made a good establishment to study the target genesresistance to other diseases. Meanwhile, we also established the detached leaf culture systemwhich made the identification of FHB easier and faster.2. Three FHB resistance candidate genes, i.e. TaPGIP3encoding a polygalacturonaseinhibiting protein, TaPDR7encoding a pleiotropic drug resistance protein and TaCBRLKencoding a CaM binding protein kinase were identified in FHB sensitive and resistancenear-isogenic lines Apogee and Apogee73S2. Real time PCR was used to analyze theirexpression patterns under the treatment of Fusarium graminearum and trichothecenedeoxynivalenol (DON), the results showed that all of the three genes were up-regulated after treatments of either Fusarium graminearum or DON, which indicated that they might be thecandidate genes in the FHB resistance.3. The FHB resistance of the three target genes had been verified using the detached leafculture after silencing each of the genes by BSMV-VIGS appraoch. The results showed thatgrowth and spread of Fusarium graminearum were much faster compared to the leaves infectedwith BSMV::00and wild type after silencing the genes in wheat leaves. Typan blue wasadopted to check the scab spots make by the invasion of Fusarium graminearum, it was testedthat, scab spots produced in the gene silencing leaves were much larger than those in the leavesinfected with BSMV::00and wild type. All of the findings accounted for the FHB resistance ofthe three genes. In the experiment of silencing TaPGIP in wheat spikes, after infecting withFusarium graminearum, the hypha grew faster in the TaPGIP silencing spike comparing to thespikes inoculated with BSMV::00and wild type, besides, the dried-up spikelets were more thanspikelets inoculated with BSMV::00and wild type. All of the results conferred that TaPGIPmight be a candidate FHB resistance gene in wheat.4. Sub-cellular location with tobacco and onion epidermis cells showed that TaPGIP waslocated on the cell wall of tobacco and onion epidermis cells, while TaPDR7and TaCBRLKwere located on the plasmid membrane of tobacco epidermis cells. According to thephysiological function of the three genes and their locations in plant cells, we drew a model onhow the three genes worked during the invasion of Fusarium graminearum: TaPGIP which waslocated on the cell wall was able to percept stimulus of Fusarium graminearum and then washighly expressed to translate plenty of PGIP proteins to combine with PGs produced byFusarium graminearum in order to inhibit the invasion of Fusarium graminearum; whenFusarium graminearum reached to the membrane, however, TaPDR7could be up-regulted byFusarium graminearum and translated enough ABC transporters to transport DON outside ofthe cell to keep the stage of cell health, then mitigated damage caused by Fusariumgraminearum invasion; in the mean time, TaCBRLK could be up-regulated to translate huge ofCaM binding protein kinases to transduce signals inside plant cells to activate the antiviraldefense systems in plants to defend against Fusarium graminearum. Real time PCR waspracticed to analysis the expression patterns of the three genes under the treatment of planthormones, the results implied that all of the three genes were up-regulated by indole acetic acid(IAA).