Isolation, Functional Identification Of Anti-rhizoctonia Cerealis Gene RS33and Freezing Tolerance Analysis Of TaPIE1in Wheat

The disease wheat sharp eyespot, mainly caused by Rhizoctonia cerealis, has become one of the mostserious and devastating diseases of wheat in China. Breeding and utilization of disease-resistant wheatcultivars were the most economical, effective and safety measures to control this disease. However,wheat cultivars resistant to sharp eyespot were very scarce, so it is important to explore anti-R. cerealisgenes and unravel their defense mechanisms. In this study, the resistant wheat line CI12633andsusceptible cultivar Wenmai6were used as experimental materials and conducted to RNA-Seqencing(RNA-Seq). A NBS-LRR gene RS33highly expressing in the resistance line was isolated fromdifferential expressed genes screened via the RNA-Seq analysis. The function of RS33in wheat defenseresponse to R. cerealis was primarily studied. The main results are as follows:(1) The expression of theNBS-LRR gene RS33was induced by R. cerealis. The analysis on RS33expression indicated that theexpression of RS33was induced by R. cerealis, and its expression level in Shanhongmai and CI12633was higher than that in Wenmai6.(2) The full-length cDNA and genomic DNA sequences of RS33wereisolated from CI12633. Comparison on these cDNA and genomic DNA sequences of RS33showed thatRS33cantained3exons and2introns, encoded a NB-LRR protein consisting of945amino acids.Conserved domain sequence analysis indicated that the RS33protein possesses the typical domains ofNBS-type proteins: Kinase1a (P-loop), Kinase-2(Walker B), Kinase3a, hydrophobicity section (GLPL),NBS-D, and13leucine-rich repeat (LRR) domains. Phylogenetic analysis showed that RS33wasclosely related to rice Pikp-2and Pi1-6, and barley Mla1, Mla7and Mla10proteins.(3) Theexpression characteristics of RS33were analyzed.(q)RT-PCR analysis suggested that the expression ofRS33was induced after inoculation with R. cerealis, and exogenous hormones. SA induction was thefastest (the expression level of RS33reached the peak1h after SA treatment), while induction washysteretic (the expression level of RS33reached the peak12h after ABA treatment), ET restrainedslightly transcription of RS33.(4) The RS33gene was located on chromosome3B of wheat. Usinggenomic DNAs of nullisomic lines of wheat cultivar Chinese Spring as templates, the specificamplificationwith RS33-specific primers indicated that RS33gene was located on chromosome3B.(5)The fusion protein of RS33-GFP was targeted to nucleus and cytomembrane.(6) The function of RS33in wheat defense response to Rhizoctonia cerealis was analyzed via virus-induced gene silencing (VIGS)developed with barley stripe mosaic virus (BSMV). Compared with the BSMV:00-infected CI12633plants, the RS33-underexpressing CI12633plants (BSMV: RS33-infected CI12633plants) showed moresusceptive to R. cerealis, suggesting that RS33was required in wheat defense response to R. cerealisinfection.(7) The defense function of RS33was confirmed by molecular detection and resistanceidentification of plants overexpressing RS33gene. The RS33expressing vector was constructured andintroduced into wheat cultivars Kenong199and Yangmai16via particle bombardment transformation.Q-RT-PCR analysis results indicated that RS33was over-expressed in the RS33transgenic wheat plants,in the crowns of which, there were fewer R. cerealis mycelia colonized. Low temperature stresses, especially “late spring coldness”, affect the growth of wheat seriously.Increasing studies show that ERF transcription factors play an important role in stress signaltransduction, expression regulation of stress-related genes, adaption to stress environment. Previously,we isolated an ERF gene TaPIE1from winter wheat Shannong0431, and introduced it into the springwheat cultivar Yangmai12. In this study, we researched the function of TaPIE1resistance to freezingstresses, and obtained the following results:(1) The expression of TaPIE1was induced by freezingtreatment. qRT-PCR analysis showed that the accumulation of TaPIE1transcripts in wheat cultivarYangmai12reached the peak12h after cold treatment.(2) TaPIE1positively regulated the resistanceof wheat to freezing stress. Compared with Yangmai12, the transgenic lines exhibited enhancedresistance to freezing, whereas, the TaPIE1-underexpression wheat plants showed more susceptible.(3)TaPIE1activated the expression of stress-responsive genes. Wheat GeneChip Array and qRT-PCRanalysis of stress-responsive genes in transgenic and Yangmai12plants indicated that TaPIE1canincrease the transcriptional levels of these genes.(4) ET and JA biosynthesis positively modulateTaPIE1-mediated resistance to freeze in wheat. The transcription of TaPIE1and stress-esponsive geneswere up-regulated by ET and JA but down-regulated by CoCl2(ET biosynthesis inhibitor) and DIECA(JA biosynthesis inhibitor). Treatment with ET biosynthesis precursor ACC signifcantly increasedfreezing tolerance mediated by TaPIE1overexpression in wheat, whereas inhibition of ET and JAbiosynthesis reduced the freezing resistance, which suggested that resistance responses to freezingmediated by TaPIE1were modulated mainly by ET and JA biosynthesis.