Bacterial Blight Resistance Genes Xa21 Research Articles

Molecular Breeding for Incorporation of Submergence Tolerance and Durable Bacterial Blight Resistance into the Popular Rice Variety 'Ranidhan'.

Ranidhan is a popular late-maturing rice variety of Odisha state, India. The farmers of the state suffer heavy loss in years with flash floods as the variety is sensitive to submergence. Bacterial blight (BB) disease is a major yield-limiting factor, and the variety is susceptible to the disease. BB resistance genes Xa21, xa13, and xa5, along with the Sub1 QTL, for submergence stress tolerance were transferred into the variety using marker-assisted backcross breeding approach. Foreground selection using direct and closely linked markers detected the progenies carrying all four target genes in the BC1F1, BC2F1, and BC3F1 generations, and the positive progenies carrying these genes with maximum similarity to the recipient parent, Ranidhan, were backcrossed into each segregating generation. Foreground selection in the BC1F1 generation progenies detected all target genes in 11 progenies. The progeny carrying all target genes and similar to the recipient parent in terms of phenotype was backcrossed, and a total of 321 BC2F1 seeds were produced. Ten progenies carried all target genes/QTL in the BC2F1 generation. Screening of the BC3F1 progenies using markers detected 12 plants carrying the target genes. A total of 1270 BC3F2 seeds were obtained from the best BC3F1 progeny. Foreground selection in the BC3F2 progenies detected four plants carrying the target genes in the homozygous condition. The bioassay of the pyramided lines conferred very high levels of resistance to the predominant isolates of bacterial blight pathogen. These BB pyramided lines were submergence-tolerant and similar to Ranidhan in 13 agro-morphologic and grain quality traits; hence, they are likely to be adopted by farmers.

Molecular Marker Assisted Breeding of a New <i>Japonica</i> Hybrid Rice

The development of Japonica  hybrid rice is of great significance to ensure supply of good quality Japonica  rice and food security in China. Aiming at the problems of late growth period, unstable yield, and the need to improve rice quality and seed production yield of Japonica  hybrid rice, a new Japonica  hybrid rice combination 'Shenyou28' with early maturity, good quality, blast resistance, stable and high yield and easy seed production was developed by using molecular breeding techniques including high-density rice gene chip. 'Shenyou28', bred by Shanghai Academy of Agricultural Sciences, is a three-line Japonica  hybrid rice with 'Shen21A' as sterile line and 'Shenhui26-28' as restorer line. For two-year regional trials from 2018 to 2019, the average output were 10 588.5 kg/hm 2 , which was 11.6% higher than the control variety 'Huayou14'. For the one-year production trial, the output reached 10 473.0 kg/hm 2 , which was 4.8% higher than the control variety 'Huayou14'. 'Shenyou28' has good quality, reaching the second grade of national standard, and has fragrance because it carries fragrance gene badh2-p-5 ’ UTR . It also has good resistance to rice blast and bacterial blight because it aggregates the rice blast resistance genes Pi2 , Pita , Pib , Pi9 , Pi54 , Pikm , Pit , and bacterial blight resistance gene Xa21 . It was approved by the Crop Variety Certification Committee of Shanghai in 2020. The breeding process, variety characteristics and key points of high yield mechanized seed production technology of 'Shenyou28' are introduced in this study.

Marker-assisted introgression of bacterial blight resistance gene xa13 into improved CO43

Marker-assisted introgression of bacterial blight resistance gene xa13 into improved CO43

Analysis of immune response-related LRR-RLKs differential genes in the rice blast resistance near-isogenic lines of <italic>Japonica</italic> Variety Yunyin

Rice ( Oryza sativa L.) is a staple food in the world as well as an important monocotyledonous model plant for molecular biology, which has a long history of cultivation and domestication. However, many diseases will break out during rice production. For example, the rice blast is the most destructive disease, which can cause the heavy loss or even total crop failure. Therefore, there are constant demands to cultivate durably blast-resistant varieties and identify more broad-spectrum blast resistance genes. Besides, it’s also very significant for agricultural production to elucidate the process and mechanism of immune response during the rice blast infection. The whole-genome sequencing of the resistant and susceptible near-isogenic lines derived from the broad-spectrum blast resistance Japonica Variety Yunyin and the universally blast-susceptible variety Lijiangxintuanheigu (LTH) was completed. Eight differential genes were screened from the Go terms that were associated with the immune response based on the whole-genome sequence data. We analyzed their gene structure, evolution and sequence variability between the resistant and susceptible near-isogenic lines. The results indicated that the homology of these protein sequences was between 55% and 80%, and the conserved protein domain analysis showed that most of them contained different numbers and types of leucine-rich repeat domains, a transmembrane domain and a C-terminal kinase domain, which were typical leucine-rich repeat receptor-like protein kinases. Moreover, they are all located within a cluster of chromosome 11 and were homologous to the bacterial blight resistance gene Xa21 . The leucine-rich repeat receptor-like protein kinases (LRR-RLKs) genes are a large conserved superfamily during the plant evolution. In Arabidopsis , there are 14 subfamilies and more than 200 members of LRR-RLKs were identified, whereas the rice genome contained more than 900 LRR-RLKs genes. The perception and recognition of pathogen infection are mainly mediated by the plant cells surface pattern-recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors. PRRs contain many receptor kinases including the leucine-rich repeat receptor-like protein kinases, which play an important role in the signal transduction during the plant development, immune response and sense of the environmental changes. In order to further explore the potential function of these genes during the rice blast disease response, the rice seedlings were inoculated with Magnaporthe oryzae by spray and punch methods and then the expression pattern of these genes during the fungal infection were analyzed by qRT-PCR. The results showed that some genes response to the blast disease. Thus, we speculated that some of them might be involved in the rice blast disease response. This study laid a foundation for further studying genes related to the rice blast disease response.

A novel intragenic marker targeting the ectodomain of bacterial blight-resistance gene Xa21 for marker-assisted selection in rice

ABSTRACT The rice (Oryza sativa L.) Xa21 gene is known to confer resistance against Xanthomonas oryzae pv. oryzae, the causal organism of bacterial blight (BB) through its involvement in pathogen recognition and immunity responses. The closely linked sequence-tagged site marker pTA248 is frequently used for marker-assisted selection (MAS) of Xa21. However, lower precision and linkage drag of linked-markers hinders its reliability and effectiveness in MAS compared to flanking/intergenic markers. In the current study, a diagnostic intragenic marker was developed for MAS of Xa21 in rice. The new marker ABUOP0001 targets a 19-bp insertion/deletion on the ectodomain of Xa21 and amplifies a 200-bp amplicon from rice line IRBB 62 (known to carry the “resistance allele”), and a 181-bp amplicon from IRBB 7 (known to carry the “susceptible allele”). The ABUOP0001 marker assay identified the resistance allele in 15 rice lines/accessions. In a field study, 14 of these lines/accessions had highly resistant BB responses, and one had an intermediate response. Further, Xa21 resistance allele was identified in 1,675 rice accessions through an in-silico genomic sequence analysis. The marker ABUOP0001 performs better than the previously used linked-marker pTA248 (anchored to a gene 225 kbp upstream of Xa21) as ABUOP0001 is an intragenic marker targeting the functionally important ectodomain of Xa21. Further, ABUOP0001 is compatible for high-throughput screening with high-resolution melting and can be multiplexed effectively with an intragenic marker of BB resistance gene Xa4. Therefore, ABUOP0001 can be recommended as a high-throughput diagnostic intragenic marker for MAS of Xa21 in rice.

TaXa21, a Leucine-Rich Repeat Receptor-Like Kinase Gene Associated with TaWRKY76 and TaWRKY62, Plays Positive Roles in Wheat High-Temperature Seedling Plant Resistance to Puccinia striiformis f. sp. tritici.

Puccinia striiformis f. sp. tritici causes wheat stripe rust, one of most important diseases of wheat worldwide. High-temperature seedling plant (HTSP) resistance of wheat to P. striiformis f. sp. tritici is one specific type of host resistance, induced by high temperature (HT). Receptor-like kinases (RLKs) play key roles in regulating plant development and signaling networks, but there have been no reports on possible roles played by RLKs in wheat HTSP to P. striiformis f. sp. tritici. In the present study, a leucine rich repeat (LRR)-RLK gene, TaXa21, with a high homology with rice bacterial blight resistance gene Xa21, was cloned from wheat cultivar Xiaoyan 6 (XY 6). TaXa21 expression was up-regulated by the exposure to HT (20°C) for 24 h at 8 days postinoculation with P. striiformis f. sp. tritici and was induced by ethylene (ET) and hydrogen peroxide (H2O2). Knocking down TaXa21 using virus-induced gene silencing reduced HTSP resistance to P. striiformis f. sp. tritici compared with the control plants. In addition, the expression level of TaCAT in the H2O2 pathway was induced and TaACO in the ET signal pathway was reduced in the HT-treated TaXa21-silenced plants. Transient expression of TaXa21 in tobacco leaves confirmed its subcellular localization in plasma membrane, consistent with the prediction from bioinformatics analysis. The transmembrane and kinase domain of TaXa21 can interact with TaWRKY76 in the nucleus and cell membrane, which is different from the localization of Xa21 in rice. The interaction between TaWRKY76 and TaWRKY62 (positively involved in the HTSP resistance of XY 6) were observed. Together, these results indicated that TaXa21 is a RLK associated with TaWRKY76 and TaWRKY62 and functions as a positive regulator of wheat HTSP resistance to P. striiformis f. sp. tritici. Furthermore, the host defense is mediated by the H2O2 and ET signal pathways.

A Strain of an Emerging Indian Xanthomonas oryzae pv. oryzae Pathotype Defeats the Rice Bacterial Blight Resistance Gene xa13 Without Inducing a Clade III SWEET Gene and Is Nearly Identical to a Recent Thai Isolate.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) injects transcription activator-like effectors (TALEs) that bind and activate host “susceptibility” (S) genes important for disease. Clade III SWEET genes are major S genes for bacterial blight. The resistance genes xa5, which reduces TALE activity generally, and xa13, a SWEET11 allele not recognized by the cognate TALE, have been effectively deployed. However, strains that defeat both resistance genes individually were recently reported in India and Thailand. To gain insight into the mechanism(s), we completely sequenced the genome of one such strain from each country and examined the encoded TALEs. Strikingly, the two strains are clones, sharing nearly identical TALE repertoires, including a TALE known to activate SWEET11 strongly enough to be effective even when diminished by xa5. We next investigated SWEET gene induction by the Indian strain. The Indian strain induced no clade III SWEET in plants harboring xa13, indicating a pathogen adaptation that relieves dependence on these genes for susceptibility. The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective disease resistance.

Virulence profiling of Xanthomonas oryzae pv. oryzae isolates, causing bacterial blight of rice in India

Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo), remains a major production constraint in rice cultivation especially in irrigated and rainfed lowland ecosystems in India. The pathogen is highly dynamic in nature and knowledge on pathotype composition among the Xoo population is imperative for designing a scientific resistance breeding program. In this study, four hundred isolates of Xoo collected from diverse rice growing regions of India were analyzed for their virulence and genetic composition. Virulence profiling was carried out on a set of differentials consisting of 22 near isogenic lines (NILs) of IR24 possessing different BB resistance genes and their combinations along with the checks. It was observed that different NILs possessing single BB resistance gene were susceptible to about 59–94% of the Xoo isolates except IRBB 13 (containing BB resistance gene xa13), which showed susceptibility to about 35% of the isolates. Based on the reaction of the Xoo isolates on the differentials, they were categorized into 22 pathotypes. Among the 22 pathotypes, IXoPt-1 and IXoPt-2 were least virulent and IXoPt # 18–22 were highly virulent. Pathotype IXoPt-19 which was virulent on all single BB resistance genes except xa13 constituted the major pathotype (22.5% isolates) and was widely distributed throughout India (16 states). This was followed by pathotype IXoPt-22 (17.25%) which was virulent on all the NILs possessing single BB resistance genes. Molecular analysis was carried out using two outwardly directed primers complementary to sequence of IS1112, a repetitive element of Xoo. A high level of genetic polymorphism was detected among these isolates and the isolates were grouped into 12 major clusters. The data indicated complex nature of evolution of the Xoo pathotypes and there was no strong correlation between pathotypes and genetic clusters as each genetic cluster was composed of Xoo isolates belonging to different pathotypes. The study indicated that none of the single BB resistance genes can provide broad spectrum resistance in India. However, two-gene combinations like xa5 + xa13 and different 3 or 4 genes combination like Xa4 + xa5 + xa13, Xa4 + xa13 + Xa21, xa5 + xa13 + Xa21 and Xa4 + xa5 + xa13 + Xa21 are broadly effective throughout India.

The expression and binding properties of the rice WRKY68 protein in the Xa21-mediated resistance response to Xanthomonas oryzae pv. Oryzae

Plant WRKY transcription factors are involved in various physiological processes, including biotic and abiotic stress responses, as well as developmental processes. In this study, the expression patterns of the WRKY68 protein during interactions between rice 4021 containing the bacterial blight resistance gene Xa21 and Xanthomonas oryzae pv. oryzae (Xoo) were investigated. A possible modified form of the WRKY68 protein appeared in the Xa21-mediated disease resistance response, and its expression levels were similar in compatible and incompatible responses, but differed significantly from that of the mock control treatment, suggesting that WRKY68 may be involved in the bacterial blight response in rice. To further understand WRKY68’s roles in the resistance signaling pathway, WRKY68 recombinant protein was expressed in Escherichia coli and a microscale thermophoresis analysis was performed to investigate the interactions between WRKY68 and cis-elements in crucial pathogenesis-related (PR) genes. The results showed that the WRKY68 protein binds to W-boxes in the PR1b promoter region, with an apparent dissociation constant of 25 nmol L−1, while the binding between WRKY68 and PR10a was W-box independent. The results suggested that a possible modified form of the WRKY68 protein was induced during the interaction between rice and Xoo, which then regulated the activity of the downstream PR genes by binding with the W-boxes in the PR1b gene’s promoter region. Moreover, the constitutive transcription of the WRKY68 gene in dozens of rice tissues and the expression of the WRKY68 protein in leaves during all growth stages suggests that WRKY68 plays important roles in rice during normal growth processes.

Integration and expression of Xa21 in transgenic rice CX8621

Agrobacterium tumefaciens-mediated transformation system has been widely applied. However, the function of target gene is affected by multiple factors. With this system, we obtained a transgenic rice line CX8621 carrying the bacterial blight resistance gene Xa21. In previous work, we have confirmed that it was selectable maker-free and vector backbone-free. And after 16 generations of breeding, it still maintained perfect resistance to bacterial blight disease. On this basis, we analyzed the integration and expression of Xa21 in CX8621 at the present study. First, based on the border sequences of plasmid pBXa21 and Xa21, we designed nested primers and assured the integrity of Xa21 in CX8621. Second, we cloned the flanking sequences and located Xa21 on chromosome 2 using improved Tail-PCR. Then we analyzed the expression pattern of Xa21 in several tissues and at different developmental stages by RT-PCR. The results show that Xa21 can be stably expressed in CX8621, agreeing well with the disease resistance response as reported previously. In addition, we detected the protein levels of XA21 in CX8621 with antibody of natural XA21 protein. Surprisingly, no XA21 protein was detected in the seeds of CX8621. Thus, the integration and expression analysis of Xa21 in CX8621 provided a part of scientific evidences for the safety assessment of genetically modified rice.

Identification of bacterial leaf blight resistance genes in wild rice of eastern India

An experiment was conducted during the 2013 monsoon season to screen 35 wild rice accessions against the BX043 strain of Xanthomonas oryzae pv. Oryzae and identify the presence of bacterial blight resistance genes Xa21, xa13, xa5, Xa4, and Xa2. Among the accessions the area under the disease progress curve ranged from 174.88 (NKSWR-65) to 680.54 (NKSWR-34) compared with resistant controls RP bio-226 (93.92), CRMAS 2231-37 (097.28), CRMAS 2232-71 (098.58), and Tetep (178.62) and susceptible control PB-1 (1065.56). On the basis of disease severity 11 accessions showed moderate resistance, 21 were moderately susceptible, and 3 accessions showed susceptible response to the BX043 strain of Xanthomonas oryzae pv. Oryzae, while none of the accessions were found to be resistant. The genetic frequency of the 5 resistance genes varied from 00.00% to 45.71%. The accession NKSWR-25 harbored 3 resistance genes, xa5, Xa4, and Xa2, while accessions NKSWR-16, NKSWR-32, NKSWR-36, NKSWR-41, NKSWR-42, NKSWR-53, NKSWR-64, NKSWR-97, and NKSWR-99 each possessed 2 resistance genes of those 3 (xa5, Xa4, and Xa2). Therefore, these accessions could be used for the transfer of specific bacterial leaf blight resistance genes into well-adapted high-yielding rice cultivars.

Marker-assisted breeding of Thai fragrance rice for semi-dwarf phenotype, submergence tolerance and disease resistance to rice blast and bacterial blight

Rice is a staple food crop for more than half of the world’s population. However, rice production is affected by many types of abiotic and biotic stress. Genetic breeding by utilizing natural resistance or tolerance genes is the most economic and efficient way to combat or adapt to these stresses. Khao Dawk Mali 105 (KDML 105) is an elite cultivar of aromatic rice mainly grown in Thailand. However, the production of KDML 105 is affected by lodging problems due to its tall plant type, regular flash floods or short-term submergence during the monsoon season, and diseases such as blast and bacterial blight. Here we report the pyramiding of semi-dwarf gene sd1, submergence tolerance gene Sub1A, blast resistance gene Pi9 and bacterial blight resistance genes Xa21 and Xa27 in KDML 105 by marker-assisted selection. The improved line, designated T5105, has a semi-dwarf phenotype with improved lodging resistance and a greater harvest index. T5105 survives after 2 weeks of complete submergence without significant loss of viability. T5105 confers high resistance to all five Magnaporthe oryzae isolates tested and provides resistance or moderate resistance to 25 of the 27 Xanthomonas oryzae pv. oryzae strains tested. In addition, T5105 produced higher yield than KDML 105 in two field trials and retains similar good grain quality to KDML 105. The development of T5105 provides a new line to boost the production of high-quality aromatic rice in tropical regions.

Gene silencing using the recessive rice bacterial blight resistance gene xa13 as a new paradigm in plant breeding

Resistant germplasm resources are valuable for developing resistant varieties in agricultural production. However, recessive resistance genes are usually overlooked in hybrid breeding. Compared with dominant traits, however, they may confer resistance to different pathogenic races or pest biotypes with different mechanisms of action. The recessive rice bacterial blight resistance gene xa13, also involved in pollen development, has been cloned and its resistance mechanism has been recently characterized. This report describes the conversion of bacterial blight resistance mediated by the recessive xa13 gene into a dominant trait to facilitate its use in a breeding program. This was achieved by knockdown of the corresponding dominant allele Xa13 in transgenic rice using recently developed artificial microRNA technology. Tissue-specific promoters were used to exclude most of the expression of artificial microRNA in the anther to ensure that Xa13 functioned normally during pollen development. A battery of highly bacterial blight resistant transgenic plants with normal seed setting rates were acquired, indicating that highly specific gene silencing had been achieved. Our success with xa13 provides a paradigm that can be adapted to other recessive resistance genes.

Bacterial leaf blight resistance genes (Xa21, xa13 and xa5) pyramiding through molecular marker assisted selection into rice cultivars

Marker assisted selection was employed to pyramid three bacterial blight resistance genes Xa21, xa13 and xa5 into high yielding susceptible rice cultivars ADT43 and ADT47. With the assistance of PCR markers, homozygous and heterozygous genotypes were identified in F2 generation of two crosses (ADT43 × IRBB60 and ADT47 × IRBB60) and goodness of fit was tested. Eighty nine plants from F3 generation of ADT43 × IRBB60 were also screened for resistance genes. The genotypes carrying resistance genes in different combinations were identified. The pyramided lines showed a wider spectrum and higher level of resistance against two Xoo isolates under field conditions.

Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in Indica rice (cv. CO39)

Based on blast pathogen population dynamics and lineage exclusion assays, we found that the major blast resistance genes Pi-1 and Piz-5 confer resistance against most Magnaporthe grisea lineages. Near-isogenic rice lines C101LAC and C101A51 carrying these two major genes for blast resistance in the background of a most blast-susceptible genotype were used for developing the pyramids. The closely linked RFLP marker RZ536 and NBS-LRR r10 marker for Pi-1 and a PCR-based SAP marker RG64 for Piz-5 were used to identify the genes in the parents and in marker-assisted breeding of the pyramided populations. To achieve multiple resistance against blast and blight in this cultivar, these blast-resistant pyramids were transformed with the cloned bacterial blight resistance gene Xa21 known to confer resistance to all races of Xanthomonas oryzae pv. oryzae (Xoo). Bioassays with six independent transformants showed that transgenic CO39 plants were resistant to both pathogens, M. grisea and Xoo. We report here the stacking of three major genes (Pi-1 + Piz-5 + Xa21) into rice using two different approaches of molecular breeding: marker-assisted selection (MAS) and genetic transformation.

Breeding bacterial blight-resistant hybrid rice with the cloned bacterial blight resistance gene Xa21

The cloned bacterial blight (BB) resistance gene Xa21 was transferred into Minghui63, a widely used restorer line of indica hybrid rice in China, through an Agrobacterium-mediated system. Molecular and resistance analyses revealed that the Xa21 gene was integrated in the genomes of transgenic plants and their progeny inherited resistance stably. For the purpose of hybrid breeding, Xa21 transgenic homozygous restorer lines were selected through `within-lane' dosage comparison of hybridization signal in combination with PCR and resistance analyses. The selected transgenic restorer lines were then crossed with a commonly used sterile line, Zhenshan97A, to produce Xa21 transgenic hybrid rice, Shanyou63-Xa21. The hybrid rice plants with Xa21 displayed high broad-spectrum resistance to Xanthomonas oryzae pv. oryzae (Xoo) races and maintained elite agronomic characters of Shanyou63. The propagation of this BB-resistant hybrid variety with Xa21 will benefit rice production.

RAPD and RFLP mapping of the bacterial blight resistance gene xa-13 in rice

RAPD and RFLP mapping of the bacterial blight resistance gene xa-13 in rice

RAPD and RFLP mapping of the bacterial blight resistance gene xa-13 in rice

Bacterial blight (BB) caused by Xanthomonas oryzae pv oryzae (Xoo) is one of the most serious diseases of rice. The recessive gene xa-13 confers resistance to Philippine race 6 of Xoo. To tag xa-13 with molecular markers, RAPD analysis was conducted with the combined use of near-isogenic lines and bulked segregant analysis. From the survey of 260 arbitrary 10-nucleotide primers, one primer (OPAC05) was detected to amplify specifically a 0.9-kb band from the DNA of susceptible plants. The distance between the RAPD marker OPAC05-900 and xa-13 was estimated to be 5.3 cM. The RAPD marker was then mapped on chromosome 8 using a mapping population of doubled haploid lines derived from the cross of IR64/Azucena. The linkage between RFLP markers and the RAPD marker was analyzed using an F2 population of 135 plants derived from a cross between a near-isogenic line for xa-13, IR66699-5-5-4-2, and IR24. No recombinants were found between RZ28 and CDO116 and their distance from xa-13 was estimated to be 4.8 cM. RG136 was located at 3.7 cM on the other side of xa-13. The mapping of xa-13 with closely linked DNA markers provides the basis for marker-aided selection for rice improvement.

Bacterial blight of rice and its control in Nepal

Research on Xanthomonas oryzae pv. oryzae, the bacterial blight of rice pathogen, was initiated at the Institute of Agriculture and Animal Science (IAAS) with the main objective of assessing the population structure of X. o. pv. oryzae through the use of both conventional and molecular markers in combination with virulence typing. A high DNA polymorphism was detected in the pathogen populations using different DNA probes and rep-PCR primers. Most strains were avirulent to cultivars containing the bacterial blight resistance gene Xa-21, which suggested the strategy that targets gene deployment is feasible in Nepal.