Zhihui Xia

Testifying the rice bacterial blight resistance gene xa5 by genetic complementation and further analyzing xa5 (Xa5) in comparison with its homolog TFIIAγ1

The recessive gene xa5 for resistance to bacterial blight resistance of rice is located on chromosome 5, and evidence based on genetic recombination has been shown to encode a small subunit of the basal transcription factor IIA (Iyer and McCouch in MPMI 17(12):1348–1354, 2004). However, xa5 has not been demonstrated by a complementation test. In this study, we introduced the dominant allele Xa5 into a homozygous xa5-line, which was developed from a cross between IRBB5 (an indica variety with xa5) and Nipponbare (a japonica variety with Xa5). Transformation of Xa5 and subsequent segregation analysis confirmed that xa5 is a V39E substitution variant of the gene for TFIIAγ on chromosome 5 (TFIIAγ5 or Xa5). The rice has an addition gene for TFIIAγ exists on chromosome 1 (TFIIAγ1). Analysis of the expression patterns of Xa5 (TFIIAγ5)/xa5 and TFIIAγ1 revealed that both the genes are constitutively expressed in different rice organs. However, no expression of TFIIAγ1 could be detected in the panicle by reverse transcriptase-polymerase chain reaction. To compare the structural difference between the Xa5/xa5 and TFIIAγ1 proteins, 3-D structures were predicted using computer-aided modeling techniques. The modeled structures of Xa5 (xa5) and TFIIAγ1 fit well with the structure of TFIIA small subunit from human, suggesting that they may all act as a small subunit of TFIIA. The E39V substitution in the xa5 protein occurs in the α-helix domain, a supposed conservative substitutable site, which should not affect the basal transcription function of TFIIAγ. The structural analysis indicates that xa5 and Xa5 potentially retain their basic transcription factor function, which, in turn, may mediate the novel pathway for bacterial blight resistance and susceptibility, respectively.

Do transgenesis and marker-assisted backcross breeding produce substantially equivalent plants? A comparative study of transgenic and backcross rice carrying bacterial blight resistant gene Xa21.

BackgroundThe potential impact of genetically modified (GM) plants on human health has attracted much attention worldwide, and the issue remains controversial. This is in sharp contrast to the broad acceptance of plants produced by breeding through Marker Assisted Backcrossing (MAB).ResultsFocusing on transcriptome variation and perturbation to signaling pathways, we assessed the molecular and biological aspects of substantial equivalence, a general principle for food safety endorsed by the Food and Agricultural Organization and the World Health Organization, between a transgenic crop and a plant from MAB breeding. We compared a transgenic rice line (DXT) and a MAB rice line (DXB), both of which contain the gene Xa21 providing resistance to bacterial leaf blight. By using Next-Generation sequencing data of DXT, DXB and their parental line (D62B), we compared the transcriptome variation of DXT and DXB. Remarkably, DXT had 43% fewer differentially expressed genes (DEGs) than DXB. The genes exclusively expressed in DXT and in DXB have pathogen and stress defense functions. Functional categories of DEGs in DXT were comparable to that in DXB, and seven of the eleven pathways significantly affected by transgenesis were also perturbed by MAB breeding.ConclusionsThese results indicated that the transgenic rice and rice from MAB breeding are substantial equivalent at the transcriptome level, and paved a way for further study of transgenic rice, e.g., understanding the chemical and nutritional properties of the DEGs identified in the current study.

Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection

Rice bacterial blight (BB) is a devastating rice disease. The Xa21 gene confers a broad and persistent resistance against BB. We introduced Xa21 into Oryza sativa L ssp indica (rice 9311), through multi-generation backcrossing, and generated a nearly isogenic, blight-resistant 9311/Xa21 rice. Using next-generation sequencing, we profiled the transcriptomes of both varieties before and within four days after infection of bacterium Xanthomonas oryzae pv. oryzae. The identified differentially expressed (DE) genes and signaling pathways revealed insights into the functions of Xa21. Surprisingly, before infection 1,889 genes on 135 of the 316 signaling pathways were DE between the 9311/Xa21 and 9311 plants. These Xa21-mediated basal pathways included mainly those related to the basic material and energy metabolisms and many related to phytohormones such as cytokinin, suggesting that Xa21 triggered redistribution of energy, phytohormones and resources among essential cellular activities before invasion. Counter-intuitively, after infection, the DE genes between the two plants were only one third of that before the infection; other than a few stress-related pathways, the affected pathways after infection constituted a small subset of the Xa21-mediated basal pathways. These results suggested that Xa21 primed critically important genes and signaling pathways, enhancing its resistance against bacterial infection.

Identification and gene prediction of a 24 kb region containingxa5, a recessive bacterial blight resistance gene in rice (Oryza sativa L.)

Ricexa5 gene provides recessive, race-specific resistance to bacterial blight disease caused by the pathogenXanthomonas oryzae pv.oryzae and has great value for research and breeding. In an effort to clonexa5, an F2 population of 4892 individuals was developed from thexa5 near isogenic lines, IR24 and IRBB5. A fine mapping procedure was conducted and tightly linked RFLP markers were used to screen a BAC library of IRBB56, a resistant rice line containing thexa5 gene. A 213 kb contig covering thexa5 locus was constructed. According to the sequences from the International Rice Genome Sequening Project (IRGSP), the Chinese Superhybrid Rice Genome Project (SRGP) and some sub-clones of the contig, twelve SSLP and CAPS markers were developed for fine mapping. Thexa5 gene was mapped to a 0.3 cM interval between markers K5 and T4, which spanned an interval of approximately 24 kb, co-segregating with marker T2. Sequence analysis of the 24 kb region revealed that an ABC transporter and a basal transcription factor (TFIIa) were potential candidates for thexa5 resistance gene product. The molecular mechanism by which thexa5 gene provides recessive, race-specific resistance to bacterial blight will be elucidated by the functional tests of the 24 kb DNA and the candidate genes.

Construction of Double Right-Border Binary Vector Carrying Non-Host Gene Rxo1 Resistant to Bacterial Leaf Streak of Rice

Rxo1 cloned from maize is a non-host gene resistant to bacterial leaf streak of rice. pCAMBIA1305-1 with Rxo1 was digested with Sca I and NgoM IV and the double right-border binary vector pMNDRBBin6 was digested with Hpa I and Xma I. pMNDRBBin6 carrying the gene Rxo1 was acquired by ligation of blunt-end and cohesive end. The results of PCR, restriction enzyme analysis and sequencing indicated that the Rxo1 gene had been cloned into pMNDRBBin6. This double right-border binary vector, named as pMNDRBBin6-Rxo1, will play a role in breeding marker-free plants resistant to bacterial leaf streak of rice by genetic transformation.

Application of functional markers to identify genes for bacterial blight resistance in Oryza rufipogon.

Abstract Field resistances of nine accessions of common wild rice (Oryza rufipogon Griff.) and one rice variety (IR24) were evaluated by using nine strains of bacterial blight pathogen (Xanthomonas oryzae pv. oryzae) from the Philippines. IR24 was highly susceptible to all the strains, and six common wild rice accessions resisted all the nine strains, with a resistance frequency of 67%. The accessions Yulin and Wanning were only susceptible to PXO280 and PXO71, respectively. The accession Gaozhou was susceptible to the three strains PXO79, PXO99 and PXO339, whereas resistant to the other six strains. It could be concluded that there is at least one resistance gene in each common wild rice accession. The functional markers of the genes xa5, xa13, Xa21 and Xa27 were used to detect the presence of these resistance genes in the nine tested wild rice accessions, and it was found that four wild rice accessions contained heterozygous xa13. Among the nine common wild rice accessions, five were homozygous for Xa27 and three homozygous for xa27, and the accession Laibin contained neither xa27 nor Xa27. In addition, there were no xa5 and Xa21 in all of these accessions.