Functional Analysis Of Wheat Stripe Rust Resistance Genes Yr10and Defense-related Gene TaMDHAR4

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the mostdamaging diseases of wheat (Triticum aestivum L.) worldwide, particularly in cool and wetregions. It has been proven that the deployment of resistant cultivars was considered as themost economical, safest and effective method to control wheat stripe rust. To exploit thewheat stripe rust resistance resource becomes the primary problem for phytopathologists andwheat breeder. Therefore, functional analysis of wheat stripe rust resistance genes anddefense-related genes not only to probe mechanismof wheat and Pst. interaction and alsoenrich wheat resistance resources. These studies are very important for reasonable utilizationof disease-resistant varieties and durable control of wheat stripe rust.BSMV-VIGS method was employed to identify gene function. To improve this system inwheat can provide technological support for future study.First of all, on the basis of successfulisolations of genomic clone4B and its Pseudogene gene4E, functional analysis wascharacterized via bioinformatics, transgenic and virus-induced gene silencing methods.Second, a defense-related gene TaMDHAR4EST was functionally characterized.1. Wheat PDS was employed as a report gene in BSMV-VIGS system. Comparison with189bp and476bp inserts,316bp inserts showed best silencing effect. And PDS inserts insense or antisense, sense and antisense directions showed different silencing effect. Noobvious difference was apparent between PDS insert in sense and antisense direction. PDSinsert in antisense was a little better than that in sense for silencing. However, the PDSfragment inserted into viral vector in both sense and antisense direction can improve thesilencing frequency. The profile of PDS transcript in silenced wheat shows that expressionof PDS gene down-regulated. At10dpi, the gene expression was lowest and a second lowerexpression was apparent at14dpi～18dpi. Subsequently, PDS gene up-regulated andrecovered the initial level. It suggested PDS gene silencing can last for three weeks at least.Besides, PDS was silenced in wheat spikes and immature grains.2. Two linked genomic clones4B and its pseudogene4E. These two clones were84% identical and a similar level of conservation was observed between intron and exons for thetwo clones.The absence of a TSS and putative TATA-box and CAAT-box in clone4Esuggeststhat this clone represents a non-expressed pseudogene. Furthermore, comparison to otherCC-NBS-LRR sequences, Yr10was unique. The most identity with reported NBS-LRR genewasLr10, the similarity was only40%at most. However, highly conserved homologues (E=0.0) were identified in Aegilops tauschii and in numerous monocots including Hordeumvulgare and Brachypodium distachyon. Related sequences were also identified in genomicdatabases of maize (Zea mays), rice (Oryza sativa) and in sorghum (Sorghum bicolor). Itsuggested Yr10is an evolutionary-conserved gene. The stripe rust susceptible cultivar Fielderwas transformed with clone4Bunder control of its native promoter. After transgene, Fieldercarrying Yr10become resistant from susceptible to Pst. race SRC84.Yr10was successfullysilenced by BSMV-VIGS method. After silencing, Moro become susceptible to resistant to Pst.race SRC84. These results indicated Yr10gene played a key role in Moro resistance to Pst.3. TaMDHAR4was firstly isolated from wheat cultivar Suwon11, and this proteinexhibits high similarity to MDHAR proteins from other plant species. Bioinformatics analysesindicated that TaMDHAR4contains typical structural features, such as mPTS-like sequencesin the C-terminal extension and trans-membrane domain followed by five basic arginineresidues (-RKRRR), which predicted that this protein may be localized in the peroxisome.qRT-PCR analyses demonstrated that TaMDHAR4could be induced by various exogenoushormones, such as ABA, MeJA, and ETH. TaMDHAR4is sharply downregulated at12and18hpi only in wheat leaves challenged with Puccinia striiformis f. sp. tritici (Pst) race CYR23and induced at48hpi with both Pst races CYR23and CYR31. SOD and APX injectionanalyses demonstrated that TaMDHAR4may be involved in the interaction between wheatand Pst through the regulation of its expression. Moreover, the knockdown of TaMDHAR4through virus-induced gene silencing (VIGS) enhanced the wheat resistance to Pst byinhibiting sporulation in the compatible interaction. Histological observations alsodemonstrated that silenced wheat resulted in an increased proportion of necrotic area at theinfection sites and suppressed Pst hypha elongation. The study provided that TaMDHAR4participate in HR respond. It was speculated that TaMDHAR4regulates the resistance ofplants through the pathogen-induced ROS metabolism.