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[Construction of mutant population of differential race of Xa23 resistant to rice bacterial blight and avirulence activity identification of mutants].

The mutant population of Xanthomonas oryzae pv oryzae strain differential to rice bacterial blight resistance gene Xa23 has been constructed mediated by transposon in vivo . The results of PCR amplification with specific primers and analysis of flanking sequence of mutants indicated that the foreign DNA has been integrated into X. oryzae pv oryzae genome. Four mutants with changed avirulent activity to Xa23 gene have been identified by artificial inoculation. It is possible to clone genes that are required for AvrXa23 avirulence activity using this new strategy.

Bacterial Proteins↗

Biochemical characterization of the kinase domain of the rice disease resistance receptor-like kinase XA21.

The rice disease resistance gene, Xa21, encodes a receptor kinase-like protein consisting of leucine-rich repeats in the putative extracellular domain and a serine/threonine kinase in the putative intracellular domain. The putative XA21 kinase domain was expressed as maltose-binding and glutathione S-transferase fusion proteins in Escherichia coli. The fusion proteins are capable of autophosphorylation. Phosphoamino acid analysis of the glutathione S-transferase fusion protein indicates that only serine and threonine residues are phosphorylated. The relative phosphorylation rate of the XA21 kinase against increasing enzyme concentrations follows a first-order rather than second-order kinetics, indicating an intramolecular phosphorylation mechanism. Moreover, the active XA21 kinase cannot phosphorylate a kinase-deficient mutant of XA21 kinase. The enzymatic activity of the XA21 kinase in a buffer containing Mn(2+) is at least 15 times higher than that with Mg(2+). The K(m) and V(max) of XA21 kinase for ATP are 0.3 microm and 8.4 nmol/mg/min, respectively. Tryptic phosphopeptide mapping reveals that multiple sites on the XA21 kinase are phosphorylated. Finally, our data suggest that the region of XA21 kinase corresponding to the RD kinase activation domain is not phosphorylated, revealing a distinct mode of action compared with the tomato Pto serine/threonine kinase conferring disease resistance.

Amino Acid Sequence↗

Development of PCR-based allele-specific and InDel marker sets for nine rice blast resistance genes.

Blast resistance is one of the most important traits in rice breeding, and application of molecular markers for blast resistance breeding is likely to allow the rapid screening for the trait during early growth stages, without the need for inoculation of pathogen and phenotyping. Allele-specific PCR markers and insertion/deletion (InDel) markers, which genotype single-nucleotide polymorphisms and InDel polymorphisms, respectively, are useful tools for marker-assisted selections. We developed sets of allele-specific PCR and InDel markers for nine rice blast resistance genes -- Piz, Piz-t, Pit, Pik, Pik-m, Pik-p, Pita, Pita-2, and Pib -- which are commonly used in Japanese blast resistance rice breeding programs. For each resistance gene, we used the segregation information from thousands of progeny in several crosses or published gene locations to generate a marker that cosegregated with the gene and markers that closely flanked the gene on either side. The developed cosegregating markers uniquely discriminated among each of the lines with the individual resistance genes (except for Pita and Pita-2). Therefore, these markers will likely facilitate the development of multiline cultivars carrying one or a combination of these nine blast resistance genes. In addition, the systems we developed may be valuable tools in the quality control of seed production from blast-resistant multiline cultivars.

Alleles↗

Mapping of quantitative trait loci associated with ultraviolet-B resistance in rice (Oryza sativa L.).

The detection of quantitative trait loci (QTLs) associated with UV-B resistance in rice should allow their practical application in breeding for such a complex trait, and may lead to the identification of gene characteristics and functions. Considerable variation in UV-B resistance exists within cultivated rice (Oryza sativa L.), but its detailed genetic control mechanism has not been well elucidated. We detected putative QTLs associated with the resistance to enhanced UV-B radiation in rice, using 98 BC(1)F(5) (backcross inbred lines; BILs) derived from a cross between Nipponbare (a resistant japonica rice variety) and Kasalath (a sensitive indica rice variety). We used 245 RFLP markers to construct a framework linkage map. BILs and both parents were grown under visible light with or without supplemental UV-B radiation in a growth chamber. In order to evaluate UV-B resistance, we used the relative fresh weight of aerial parts (RFW) and the relative chlorophyll content of leaf blades (RCC). The BIL population exhibited a wide range of variation in RFW and RCC. Using composite interval mapping with a LOD threshold of 2.9, three putative QTLs associated with both RFW and RCC were detected on chromosomes 1, 3 and 10. Nipponbare alleles at the QTLs on chromosome 1 and 10 increased the RFW and RCC, while the Kasalath allele at the QTL on chromosome 3 increased both traits. Furthermore, the existence of both QTLs on chromosomes 1 and 10 for UV-B resistance was confirmed using chromosome segment substitution lines. Plants with Kasalath alleles at the QTL on chromosome 10 were more sensitive to UV-B radiation than plants with them on chromosome 1. These results also provide the information not only for the improvement of UV-B resistance in rice though marker-associated selection, but also for the identification of UV-B resistance mechanisms by using near-isogenic lines.

Chromosome Mapping↗

Molecular Bases and Genetic Design of Rice Disease Resistance for Optimized Yield and Sustainable Agriculture.

Rice diseases continue to undermine yield stability and threaten the sustainability of rice production. The central challenge is therefore not simply to maximize immune activation, but to identify genetic interventions that remain effective across diverse pathogen races and environmental conditions without imposing excessive penalties on growth or yield. Here, we synthesize the molecular basis of rice immunity from a design-oriented perspective. We first examine cell-surface pattern-recognition receptors and intracellular nucleotide-binding leucine-rich repeat receptors, and then assess the shared signaling hubs and defence outputs that connect pathogen perception to antimicrobial responses. Rather than treating these components as equivalent breeding targets, we compare their translational potential according to resistance spectrum, anticipated durability, tunability, pleiotropic risk, and the strength of field evidence. We further discuss breeding strategies based on receptor engineering, editing of susceptibility genes and cis-regulatory elements, post-translational motif engineering, pathogen-inducible and upstream open reading frame-mediated regulation, resistance-gene stacking and artificial intelligence-assisted prediction. We argue that rational resistance design in rice should move beyond constitutive immune activation toward allele-specific, quantitative, spatially restricted and infection-responsive regulation. Integrating mechanistic insights with precision genome editing, accelerated breeding and responsible deployment offers a practical route to durable, yield-compatible disease resistance while reducing dependence on chemical control.

breeding strategy↗

Genetic and physical mapping of a rice blast resistance locus, Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea.

We have identified, genetically mapped and physically delineated the chromosomal location of a new rice blast resistance locus, designated Pi-CO39(t). This locus confers resistance to Magnaporthe grisea isolates carrying the AVR1-CO39 avirulence locus. The AVR1-CO39 locus is conserved in non-rice (cereals and grasses)-infecting isolates of M. grisea, making Pi-CO39(t) useful for engineering M. grisea resistance in rice and other cereals. The resistance in the rice line CO39 was inherited as a single dominant locus in segregating populations derived from F(2) and F(3) crosses between disease-resistant (CO39) and susceptible (51583) rice genotypes. Microsatellite, RFLP and resistance gene analog (RGA) markers were used to map the Pi-CO39(t) locus to a 1.2-cM interval between the probenazole-responsive ( RPR1) gene (0.2 cM) and RFLP marker S2712 (1.0 cM) on the short arm of rice chromosome 11. RFLP markers G320 and F5003, and resistance gene analogs RGA8, RGA38 and RGACO39 were tightly linked to the Pi-CO39(t) locus (no recombination detected in a sample of ~2400 gametes). A large-insert genomic library of CO39 was constructed in the binary plant transformation vector pCLD04541. A library screen using RGA8, RGA38 and probes derived from the ends of CO39 clones, as well as BAC end probes from the corresponding locus in the rice cv. Nipponbare, resulted in the assembly of three CO39 contigs of 180 kb, 110 kb and 145 kb linked to the Pi-CO39(t) locus. A 650-kb contig was also constructed representing the susceptible locus, pi-CO39(t), in the Nipponbare genome. The two genomes are highly divergent with respect to additions, deletions and translocations at the Pi-CO39(t) locus, as revealed by the presence or absence of mapping markers.

Blotting, Southern↗

[Effects of nutrient and water regimes on lodging resistance of rice].

A field experiment was conducted to study the effects of nutrient and water regimes on lodging resistance of rice plant by measuring and comparing the morphological and physical traits and the K2O and SiO2 contents of rice basal stems. The results indicated that a combined application of chemical fertilizers and organic materials, especially and rice straw (CS), could markedly increase the diameter, wall thickness and weight of basal stems, but reduce plant height, which resulted in the effective increase of snapping resistance (RS) and marked decrease of lodging index (LI). The effectiveness of the combined application of chemical fertilizers and rice straw (CS) on the increase of lodging resistance of rice plant was greater in alternate wetting and drying (AWD) and controlled irrigation (DRA), compared to continuous waterlogging (CWL). In addition, the combined application of chemical fertilizers and organic sources, especially with rice straw (CS), greatly increased the K2O and SiO2 contents in rice basal stems, especially in alternate wetting and drying (AWD) and controlled irrigation (DRA). Correlation analysis showed that the wall thickness, weight and snapping resistance of basal stems were significantly positively correlated to the K2O and SiO2 contents in rice basal stems (P<0.05 or P<0.01) in alternate wetting and drying (AWD) and controlled irrigation (DRA).

Agriculture↗

Identification and functional analysis of a locus for improvement of lodging resistance in rice.

We identified a new locus responsible for increased pushing resistance of the lower part of rice (Oryza sativa) and analyzed its physiological function to understand how to improve lodging resistance in rice. Quantitative trait loci (QTLs) controlling pushing resistance of the lower part were analyzed in a population of backcross inbred lines of japonica Nipponbare x indica Kasalath plants cut out at 40 cm to exclude the effect of the weight of the upper parts. Five QTLs for pushing resistance were detected; only one QTL from Kasalath on chromosome 5 (prl5) had a positive effect. The likelihood odds ratio curve of prl5 echoed that for lodging resistance by typhoon. We selected three near-isogenic lines (NILs) in which the chromosomal region of prl5 was substituted with that of Kasalath in the Nipponbare background. The dry weights and densities and the contents of accumulated carbohydrate in stems below 40 cm (lower stems) in each NIL were significantly higher than those of Nipponbare. There was no difference between Nipponbare and the NILs in yield, root characteristics, or the weights of the upper parts. Pushing resistance of the lower part and lodging resistance in the NILs were up to twice as high as in Nipponbare. These results suggest that prl5 might affect the characteristics of the lower stems of the NILs, thus increasing lodging resistance.

Carbohydrate Metabolism↗

Genetic and physical mapping of Pi5(t), a locus associated with broad-spectrum resistance to rice blast.

To gain an understanding of the molecular basis for resistance to rice blast (Magnaporthe grisea), we have initiated a project to clone Pi5(t), a locus associated with broad-spectrum resistance to diverse blast isolates. AFLP-derived markers linked to Pi5(t)-mediated resistance were isolated using bulked segregant analysis of F(2) populations generated by crossing three recombinant inbred lines (RILs), RIL125, RIL249, and RIL260 with the susceptible line CO39. The most tightly linked AFLP marker, S04G03, was positioned on chromosome 9 of the fingerprint-based physical map of Nipponbare, a well-characterized rice genotype. Flanking BAC-based Nipponbare markers were generated for saturation mapping using four populations, the three initial RILs and an additional one derived from a cross between M202 and RIL260. A BIBAC (binary BAC) library was constructed from RIL260. Using these resources Pi5(t) was mapped to a 170-kb interval, and a contiguous set of BIBAC clones spanning this region was constructed. It had previously been suggested that Pi3(t) and Pi5(t) might be allelic, due to their identical resistance spectrum and tight linkage. We therefore compared genomic regions for lines containing Pi3(t) using the Pi5(t)-linked markers. DNA gel-blot analyses indicated that the region around Pi3(t) is identical to that of Pi5(t), suggesting that Pi3(t) and Pi5(t) are the same resistance gene.

Alleles↗

Genetic analysis for drought resistance of rice at reproductive stage in field with different types of soil.

Drought resistance of rice is a complex trait and is mainly determined by mechanisms of drought avoidance and drought tolerance. The present study was conducted to characterize the genetic basis of drought resistance at reproductive stage in field by analyzing the QTLs for drought response index (DRI, normalized by potential yield and flowering time), relative yield, relative spikelet fertility, and four traits of plant water status and their relationships with root traits using a recombinant inbred population derived from a cross between an indica rice and upland rice. A total of 39 QTLs for these traits were detected with individual QTL explained 5.1-32.1% of phenotypic variation. Only two QTLs for plant water status were commonly detected in two environments, suggesting different mechanisms might exist in two types of soil conditions. DRI has no correlation with potential yield and flowering time under control, suggesting that it can be used as a good drought resistance index in field conditions. The co-location of QTLs for canopy temperature and delaying in flowering time suggested a usefulness of these two traits as indexes in drought resistance screening. Correlation and QTL congruence between root traits and putative drought tolerance traits revealed that drought avoidance (via thick and deep root traits) was the main genetic basis of drought resistance in sandy soil condition, while drought tolerance may play more role in the genetic basis of drought resistance in paddy soil condition. Therefore, both drought mechanisms and soil textures must be considered in the improvement of drought resistance at reproductive stage in rice.

Adaptation, Physiological↗

[Secondary compounds in rice varieties resistant to Nilaparvata lugens].

By the method of high-performance liquid chromatography (HPLC), this paper studied the contents of 13 secondary compounds from 130 samples of 26 rice varieties resistant to brown planthopper (BPH) Nilaparvata lugens biotype II. A prediction model was established through principal component analysis and multiple regression analysis: Y = 3.4593 - 0.02491 X1 + 0.08475 X2 - 0.04227 X8 + 0.1174 X12. The relationships between the BPH-resistant level of rice varieties and the area values of chromatographic peaks were significant (r2 = 0.84, P < 0.01). The results demonstrated that Peak 1, Peak 2, Peak 8 and Peak 12 were the major secondary compounds affecting the resistance to BPH biotype II, which suggested that the BPH resistance of the varieties came from the action of several secondary compounds that varied in contribution to the resistance.

Animals↗

RFLP-facilitated investigation of the quantitative resistance of rice to brown planthopper ( Nilaparvata lugens).

Quantitative trait loci (QTLs), conferring quantitative resistance to rice brown planthopper (BPH), were investigated using 160 F(11) recombinant inbred lines (RILs) from the Lemont/Teqing cross, a complete RFLP map, and replicated phenotyping of seedbox inoculation. The paternal indica parent, Teqing, was more-resistant to BPH than the maternal japonica parent, Lemont. The RILs showed transgressive segregation for resistance to BPH. Seven main-effect QTLs and many epistatic QTL pairs were identified and mapped on the 12 rice chromosomes. Collectively, the main-effect and epistatic QTLs accounted for over 70% of the total variation in damage scores. Teqing has the resistance allele at four main-effect QTLs, and the Lemont allele resulted in resistance at the other three. Of the main-effect QTLs identified, QBphr5b was mapped to the vicinity of gl1, a major gene controlling leaf and stem pubescence. The Teqing allele controlling leaf and stem pubescence was associated with resistance, while the Lemont allele for glabrous stem and leaves was associated with susceptibility, indicating that this gene may have contributed to resistance through antixenosis. Similar to the reported BPH resistance genes, the other six detected main-effect QTLs were all mapped to regions where major disease resistance genes locate, suggesting they might have contributed either to antibiosis or tolerance. Our results indicated that marker-aided pyramiding of major resistance genes and QTLs should provide effective and stable control over this devastating pest.

Journal Article↗

[Utilization of a microsatellite marker to identify rice blast resistance gene in two segregating populations].

The microsatellite (SSLP) marker RM262, which is tightly linked to the blast resistance gene Pi-d(t) in rice, provides means to conduct marker-aided selection in a rice breeding program. The objective of this study was to investigate the ability of this marker to select the blast resistance gene, Pi-d(t), in two crosses for rice blast resistance breeding. The products with amplified the microsatellite primers were polymorphic between the three varieties examined. To examine the power of the identified microsatellite marker in predicting the Pi-d(t) locus, we determined the genotypes of the two F2 populations at the Pi-d(t) locus by performing progeny testing for the disease respones. The results indicated an accuracy of more than 98% in identifying the resistant plants in both populations. Therefore the microsatellite marker can be utilized in marker-assisted selection and breeding for new varieties with blast resistance.

Microsatellite Repeats↗

[Effect of rice variety resistance on population dynamics of Nilaparvata lugens and Sogatella furcifera].

The effect of rice varieties with different insect-resistance on dynamics of Nilaparvata lugens and Sogatella furcifera population was examined, based on the investigation of their inhabiting ratio, egg amount, survival rate of egg and nymph, nymph development, and the amount of their offspring population. Four rice varieties, TN1, N22, AsD7 and Ptb33 were investigated. Ptb33 had a significantly lower inhabiting ratio, egg amount, and survival ratio of eggs and nymphs of both parasitic populations, a prolonged development time from nymphs to adults, and an inhibited offspring population, indicating an evident resistance of this host variety. N22 and AsD7 inhibited the development of offspring S. furcifera and N. lugens, respectively. The parasitic populations of S. furcifera and N. lugens developed more rapidly on TN1, compared with N. lugens population on N22 and S. furcifera on AsD7. The relationship between the quantitative dynamics of both population and rice variety replacement was discussed, and the possibility of using resistant rice varieties to control these two insect populations was also analyzed.

Animals↗

Expression and inheritance of hypersensitive resistance to rice hoja blanca virus mediated by the viral nucleocapsid protein gene in transgenic rice.

Rice hoja blanca virus (RHBV) is a major virus disease of economic importance affecting rice in northern South America, Central America and the Caribbean. This is the first report of transgenic resistance to RHBV and the transformation of an indica rice variety from Latin America. Rice transformed with the RHBV nucleocapsid protein ( N) gene had a significant reduction in disease development. Several reactions were observed that ranged from susceptible to completely resistant plants (immunity). The resistant reactions were characterized by the production of local lesions like a hypersensitive reaction or a recovery phenotype with the emergence of symptom-less new leaves. These transgenic RHBV-resistant rice lines expressed the N gene RNA at low levels that were below the detection limit by Northern blots and only resolved by RT-PCR. The nucleocapsid protein could not be detected in any of the transgenic plants either by Western or ELISA tests. These results suggest that the resistance encoded by the N gene in these plants appears to be mediated by RNA. When challenged with RHBV, the resistant transgenic lines showed a significant increased performance for important agronomic traits including the number of tillers, the number of grains per plant and the yield as compared to the susceptible control. Furthermore, upon inoculation some of the most-resistant transgenic lines showed agronomic traits similar to the uninoculated non-transgenic Cica 8 control. Using both agronomic traits and disease severity as criteria, several of the most-resistant lines were followed through the R(4) generation and demonstrated that the N gene and RHBV resistance was inherited in a stable manner. These transgenic rice lines could become a new genetic resource in developing RHBV-resistant cultivars.

Capsid Proteins↗

Xa3, conferring resistance for rice bacterial blight and encoding a receptor kinase-like protein, is the same as Xa26.

Xa3-mediated resistance for rice bacterial blight, one of the most devastating rice diseases worldwide, is influenced by genetic background. Xa3 is genetically tightly linked to Xa26, another gene for bacterial blight resistance. Xa26 belongs to a clustered multigene family encoding leucine-rich repeat (LRR) receptor kinase-like proteins. To characterize Xa3, we fine mapped it using a population segregating for only one resistance gene and markers developed from Xa26 family. Genetic analysis showed that Xa3 co-segregated with the marker of Xa26 gene and segregated from the markers of other members of Xa26 family. DNA fingerprinting revealed that rice line IRBB3 carrying Xa3 had the same copy numbers of Xa26 family members as rice line Minghui 63 carrying Xa26. Phenotypic comparison showed that all the rice lines carrying either Xa3 or Xa26 developed dark brown deposition at the border between the lesion caused by incompatible-pathogen infection and health leaf tissue, while other rice lines did not show this dark brown deposition in either incompatible or compatible interactions. These results suggest that Xa3 and Xa26 is the same gene. We name it Xa3/Xa26 to indicate the relationship between the two gene symbols. The putative encoding products of Xa3/Xa26 and its susceptible allele xa3/xa26 shared 92% sequence identity. The sequence difference occurred in the LRR domains, specifically at the solvent-exposed amino acid residues, might be the major cause that differentiates the resistant and susceptible proteins.

Amino Acid Sequence↗

Mutations in the eIF(iso)4G translation initiation factor confer high resistance of rice to Rice yellow mottle virus.

We report here evidence of the role that the isoform of the eukaryotic translation initiation factor 4G (eIF(iso)4G) plays in naturally occurring resistance in plant/virus interactions. A genetic and physical mapping approach was developed to isolate the Rymv1 locus controlling the high recessive resistance to Rice yellow mottle virus (RYMV) in the rice (Oryza sativa) variety Gigante. The locus was mapped to a 160-kb interval containing a gene from the eIF(iso)4G family. The stable transformation of a resistant line with the cDNA of this gene, derived from a susceptible variety, resulted in the loss of resistance in transgenic plants. The allelic variability of this gene was analysed in three resistant and 17 susceptible varieties from different cultivated rice species or subspecies. Compared with susceptible varieties, resistant varieties present specific alleles, characterized by either amino acid substitutions or short amino-acid deletions in the middle domain of the protein. The structure of this domain was modelled and showed that the substitutions were clustered on a small surface patch. This suggests that this domain may be involved in an interaction with the virus.

Alleles↗

Functional characterization of OsRacB GTPase--a potentially negative regulator of basal disease resistance in rice.

The rice genome contains at least seven expressed Rop small GTPase genes. Of these Rops, OsRac1 is the only characterized gene that has been implicated in disease resistance as a positive regulator. To our interest in finding a negative ROP regulator of disease resistance in rice, we applied a "phylogeny of function" approach to rice Rops, and identified OsRacB based on its close genetic orthologous relationship with the barley HvRacB gene, a known negative regulator of disease resistance. To determine the function of OsRacB, we isolated the OsRacB cDNA and conducted gene expression and transgenic studies. OsRacB, a single copy gene in the genome of rice, shared 98% identity with HvRacB at the amino acid level. Its mRNA was strongly expressed in leaf sheath (LS) and in panicles, but was very weakly expressed in young and mature leaves. The basal mRNA level of OsRacB in LS of two-week-old seedlings was strongly down-regulated upon wounding by cut and treatment with jasmonic acid. A dramatic down-regulation in the OsRacB transcripts was also found in plants inoculated with the blast pathogen, Magnaporthe grisea. Interestingly, transgenic rice plants over-expressing OsRacB showed increased symptom development in response to rice blast pathogens. Additionally, fluorescence microscopy of green fluorescent protein (GFP):OsRacB-transformed onion cells and Arabidopsis protoplasts revealed OsRacB association with plasma membrane (PM), suggesting that PM localization is required for proper function of OsRacB. Based on these results, we suggest that OsRacB functions as a potential regulator for a basal disease resistance pathway in rice.

Amino Acid Sequence↗