Map-Based Cloning And Functional Analysis Of Rice Stripe Virue Resistance Gene In Rice (Oryza Sativa L.)

Rice stripe virus disease caused by rice stripe virus (RSV) and transmitted by small brown planthopper [Laodelphax striatellus Fallen (Homoptera:Delphacidae), SBPH], is one of the most serious viral diseases in rice (Oryza sativa L.), to which most of japonica varieties are susceptible; while most of indica varieties are generally resistant. Development of resistant varieties offers a more economical, efficient and environmentally safe way to control this disease. However, none of RSV resistance gene has been identified, so our understanding of RSV resistance remains limited. Isolation rice stripe virus resistance gene would shed new insight into plant viral defense mechanisms and suggest effective means of breeding RSV resistant crops using molecular marker-assisted selection or genetic engineering.Small brown planthopper [Laodelphax striatellus Fallen (Homoptera:Delphacidae), small brown planthopper, SBPH] and rice virus disease, such as RSV, and so on, transmitted by SBPH cause serious damage to rice. Screening rice accessions for resistance to SBPH and quantitative trait locus (QTL) analysis of resistance to SBPH have great significance for plant resistance breeding and the study on disease resistance mechanism. The results of this study divided into two aspects were as follows:1. Mapp-based cloning and functional analysis of STV11 gene conferred resistance to rice stripe virus in rice.(1) Kasalath harboring STV11 shows strong RSV resistance. KK34 is a near isogenic line carrying the STV11 from Kasalath (indica) in the genetic background of Koshihikari (Kos, japonica), a susceptible commercial variety. When infested with RSV, Kos showed diseased stripe leaves and the plants finally withered, whereas there was no visible symptom in KK34. There was no difference between KK34 and Kos in response to SBPH attack, other two viruses, rice black stripe dwarf virus (RBSDV) and rice dwarf virus (RDV) infection. KK34 and Kos were showed susceptible to SBPH, RBSDV and RDV. Transcript levels of the coat protein (CP) gene in plants and protoplast following RSV infection, respectively, was measured, and showed that the resistance to RSV in KK34 was achieved through inhibition of the viral replication. Taken together, these results suggest that the resistance to RSV in KK34 was RSV-specific and was achieved through inhibition of the viral replication.(2) Map-based cloning and transgenic complementation assay revealed that STV11 is predicted to encode a sulfotransferase domain-containing protein included 376 amino acids with unique exon and a transcript length of 1,131 bp. Furthermore, expression of STV11 was induced by RSV infection. STV11 protein was co-localized with the ER, cis-Golgi and trans-Golgi.(3) Interaction of STV11-R or STV11-S with each of the 7 RSV-encoding proteins using the yeast two hybrid assay revealed that neither STV11-R nor STV11-S interacts with any the RSV proteins. Vitro enzymatic assay showed that STV11-R, not STV11-S, was able sulphonate salicylic acid (SA) to sulphonated SA (SSA). We measured SA content in KK34, Kos and a transgenic NIP line, and found that accumulation of SA was induced by RSV infection.(4) In order to investigate whether SA or SSA is associated with RSV resistance, leaf protoplasts of Kos were infected with RSV RNP particles in presence or absence of SA and SSA, respectively. Both SA and SSA effectively inhibited replication of RSV, but the inhabitation by SSA was more substantial. Further more, the nature of STV11 gene, sulphonated SA may function as a signal molecule for activation of SA, activation of the SA-mediated defense signaling pathway, such as SAR, the salicylhydroxamic acid (SHAM) sensitive pathway and RNA-mediated gene silencing pathway against virus infection.(5) Sequenced analysis the STV11 gene revealed that a total of seven STV11 haplotypes were identified in Oryza rufipogon, but just three of the seven haplotypes(STV11-R, STV11-R’and STV11-S) were found in the analyzed cultivars. Interestingly, the majority of Chinese O. rufipogon carries STV11-S, whereas most of the South or Southeast O. rufipogon carries either STV11-R or STV11-R’, suggesting that the STV11-R/R’and STV11-S alleles pre-existed in different populations of the O. rufipogon ancestor. Further, sequence analysis revealed that the majority of indica cultivars carries either the STV11-R or the STV11-R’ resistance alleles. On the contrary, the majority of japonica accessions have the susceptible STV11-S allele, providing a molecular explanation for the differential resistance of indica and japonica varieties to RSV. These results suggest that STV11-R/R’ and STV11-S are derived from distinct geographic gene pools of a common wild ancestor, which is consistent with the proposition that indica and japonica rice arose from wild ancestors, Oryza rufipogon distributed in South/Southeastern Asia and South China, respectively. STV11-R allele has been increasingly introgressed into more japonica cultivars from indica varieties during recent breeding programs to combat RSV.2. Screening rice accessions for resistance to SBPH and detection of quantitative trait loci (QTLs) for resistance to SBPH and rice stripe virus in rice.(1) We screened 312 rice accessions for resistance to SBPH by the modified seedbox screening test (MSST). Among the 312 landraces and commercial varieties from different rice growing regions,13 were highly resistant to SBPH,66 were resistant,52 were moderately resistance,64 were susceptible and 117 were highly susceptible. The Indian landrace, N22, was highly resistant, and the Japanese elite japonica variety, USSR5, was highly susceptible.(2) One hundred and eighty two recombinant inbred lines (RELs) derived from a cross of N22 and the highly susceptible variety, USSR5,were used for quantitative trait locus (QTL) analysis of resistances to SBPH. In a modified seedbox screening test, three QTLs for SBPH resistance, qSBPH2, qSBPH3 and qSBPH7.1, were mapped on chromosomes 2,3 and 7, a total explaining 35.1% of the phenotypic variance. qSBPH7.2 and qSBPH11.2, conferring antibiosis against SBPH, were detected on chromosomes 7 and 11 and accounted for 20.7% of the total phenotypic variance. In addition, qSBPH5 and qSBPH7.3, expressing antixenosis to SBPH, were detected on chromosomes 5 and 7, explaining 23.9% of the phenotypic variance. qSBPH7.1, qSBPH7.2 and qSBPH7.3, located in the same region between RM234 and RM429 on chromosome 7, using three different phenotyping methods indicate that the locus or region plays a major role in conferring resistance to SBPH in N22.(3) Using the field test (FT) and seedling test (ST) for RSV resistance, three QTLs, qSTV4, qSTV11.1 and qSTV11.2, were detected on chromosomes 4 and 11. The former was detected only in FT conditions, with a LOD score of 5.20, explaining 13.4% of the phenotypic variance. qSTV11.1 and qSTV11.2 are located in the same region between RM287 and RM209 on chromosome 11, where it explained 30.2% and 28.9% of the phenotypic variance, indicated that this QTL is a major-effect gene for resistance to RSV in N22.