| Rice (Oryza saliva L.) is one of the most important grain crops in China, even in the world. Sheath blight (SB) is one of the three most serious diseases, which leads to sever loss each year, having become the most destructive disease in many regions of southern China. The resistance to rice sheath blight is a typical quantitative trait. As the resistance mechanism is unknown, breeding progress of resistance to sheath blight of rice has been slow. In this study, we found it was feasible to improve rice resistance against Rhizoctonia solani by transgenic strategies regulating expression of its own OsOSM1 gene of rice or increasing rice endogenous cytokinin content. Consequently, we also focused on the functional analysis of resistant QTL qSB-11LE. The main results of the study were:1. Increasment of expression of OsOSM1 gene enhanced resistance to rice sheath blight in transgenic riceIn this study, we found that overexpression of the OsOSM1 gene, encoding an osmotin protein belonging to the pathogenesis-related protein 5 family, was able to improve rice resistance to SB in field. There were two osmotin genes in rice, OsOSM1 was the one mainly expressed in leaf sheath at the booting stage, coinciding with the critical stage of SB development in field. In addition, OsOSM1 expression was strongly induced by R. solani in SB resistant rice variety YSBR1, but not in susceptible varieties, suggesting its involvement in SB resistance. Overexpression of OsOSM1 (OsOSMlox) in susceptible variety Xudao 3 significantly increased resistance to SB in transgenic rice. The OsOSM1 mRNA levels in different transgenic lines were found positively correlated with their SB resistance levels. Intriguingly, although extremely high levels of OsOSM1 were detrimental to rice development, appropriately elevated levels of OsSOM1 were obtained that enhanced rice SB resistance without affecting rice development or grain yield. The OsSOM1 protein was localized on plasma membrane. OsOSMl was upregulated by jasmonic acid (JA); furthermore, JA responsive marker genes were induced in OsOSM1ox lines. These results suggest that the activation of JA signaling pathway may account for the increased resistance in transgenic OsOSM1ox lines. Taken together, our results demonstrate that OsOSMl plays an important role in defense against rice SB disease and have laid an important foundation for further development of new materials with resistance to rice sheath blight by transgenic strategies regulating expression of its own gene of rice and screening germplasm with high expression of OsOSM1 gene from natural resources.2. Increasment of CK content was able to enhance rice resistance to sheath blight caused by necrotrophic pathogen Rhizoctonia solaniRhizoctonia solani Kuhn (R. Solani), is a soil borne fungus with strong saprophytism. Based on the nature of necrotrophic pathogen, it should kill the cell first, which may provide easy access to nutrients during the necrotrophic phase of the fungal life cycle. The previous studies showed that transgenic plants with increased endogenous CK level could reduce cell death and delay senescence. In this stdy, we found that exogenous application of kinetin could reduce R. Solani-induced symptom development. And then, by the method of Agrobacterium mediated transformation, the vector from a CK synthesis gene IPT from Agrobacterium under the control of a rice senescence-specific promoter PSAG39 (PSAG39::IPT), was introduced into the susceptible variety Xudao3 (Japonica). Finally, we obtained transgenic plants with increased endogenous CK and chlorophyll concentration and delayed senescence. Compared to wild type, the transgenic plants showed significantly higher resistance to SB both in adult inoculation assay in field and detached leaf inoculation assay in laboratory and had much more strong ability to suppress cell death induced by R.solani. In order to further confirm the relationship between CK content and sheath blight resistance, the CKX4 overexpressing (CKX4ox) transgenic plants were introduced. Compared to wild type, the CKX4ox plants did show significantly lower chlorophyll and CK content in leaf, reduced resistance to SB and reduced ability of plants to suppress cell death caused by SB fungus. Moreover, we found that a natural rice mutant YD8-sgr with stay-green morphology also had the ability to suppress cell death caused by R.solani and therefore increased the resistance to SB and significantly higher chlorophyll and CK content than that of WT. Further,10 of 11 OsCKXs genes in the mutant presented significantly lower expression levels than that in the WT, which indicated that the resistance to sheath blight improved in mutant was mainly due to maintain higher CK content through reducing the expression of OsCKXs, decelerating the degradation of CK and suppressing cell death caused by R.solani. These results firstly elucidate that CK plays an important role in the regulation of resistance against rice sheath blight, laid an important foundation for further mechanism of CK in plant defense response signaling, and provide a new idea for rice molecular breeding of resistance to sheath blight.3. Cloning and functional analysis of resistant QTL qSB-11LEThe resistant allele of qSB-11 came from a relatively susceptible rice cultivar Lemont, named qSB-11LE. In our previous study, qSB-11LE had been mapped into a 78kb region on chromosome 11 of Lemont. The four candidate genes were identified by sequencing and expression analysis, including ORF1 (Transmembrane amino acid transporter protein), PAD4 (Phytoalexin deficient 4), ORF11 (Receptor-like protein kinase 5 precursor) and ORF10 (Phytosulfokine LRR receptor kinase). Based on the previous work, in this study, each candidate gene was first studied by RNA interference (RNAi) in the background of Lemont. The results showed that compared with Lemont, the susceptibility of ORF1-RNAi, PAD4-RNAi and ORF11-RNAi plants was not aggravated, while that of the ORF10-RNAi plants was significantly increased, which suggested that ORF10 was the most likely to be the candidate gene involved in the regulation of rice resistance to sheath blight. We further overexpressed ORF10 and ORF11 in the background of susceptible NIL-qsb11TQ. The results showed that compared with NIL-qsb11TQ, the resistance levels of ORF10-OX plants was significantly increased, while that of the ORF11-OX plants was no obvious change, which further indicated that ORF10 was the most likely candidate gene for qSB-llLE. Subsequently, we further found that NIL-qsb11TQ transformed with the complementary construct containing full-length ORF10 gene could almost restore the resistance, which confirmed that the ORF10 gene was qSB-11LE. By sequencing, we identified that there were 7 allelic variations in the coding region of 82 rice natural varieties and made a preliminary study on the resistance of some alleles.By qRT-PCR, we found that ORF10 was expressed highly in leaf sheath at the booting stage, coinciding with the critical stage of SB development in field. In addition, ORF10 expression was induced by Rhizoctonia solani, not Xanthomonas oryzae. Nicotiana benthamiana transient expression assays and rice protoplast transfection indicated that ORF10 was localized predominantly in the cytomembrane and nucleus. We also used the RNA-Seq technique to analysis the transcriptomes of transgenic ORF10-RNAi plants (10Ri-1) and Lemont, and preliminarily found that ORF10 may be involved in the regulation of ethylene (ET) signaling. By qRT-PCR, we futher found that OsACO7 (ET synthesis-related gene) and OsEBP89 (marker gene in ET signaling pathway) were significantly enhanced expression after the infection of Rhizoctonia solani in Lemont, while that in NIL-qsbl 1TQ and 10Ri-1 were no no obvious change. Then we found that ethylene production in Lemont was significantly higher than that in NIL-qsb11TQ,10Ri-1 and 10Ri-2 at 24 and 48h after inoculation with R. solani. These results suggested that ORF10 involved in ET-mediated defense response through the regulation of ET synthesis pathway induced by the infection of Rhizoctonia solani.Because of ORF10 encoding receptor-like protein kinase (RLK), we speculated that it may be involved in the regulation of PTI Signaling or early immune response in rice as the receptor protein had been cloned. To examine whether ORFIO affected ROS generation after PAMP elicitor treatment, we collected leaves from Lemont, NIL-qsb11TQ and 10Ri-1 transgenic plants and measured the ROS level after chitin and flg22 treatments. The results showed that chitin-induced ROS was quickly and largely accumulated in Lemont, followed by 10Ri-1, while ROS in NIL-qsbllTQ was almost not induced. Flg22-induced ROS accumulation was all increased in Lemont, NIL-qsb11TQ and 10Ri-1, but the amount was higher in Lemont than others. These results indicated that ORFIO was indeed involved in the early immune response in rice.Based on the above results, we speculate that the possible resistant mechanism of ORFIO is: ORFIO regulates basal resistance through the involvement of early immune signaling in rice after infection of the pathogen, and then involves in ET-mediated defense response through the regulation of ET synthesis pathway induced by the infection of Rhizoctonia solani, finally improves the resistance to sheath blight. |
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