The rice (Oryza sativa) spotted leaf11 (spl11) mutant was identified from an ethyl methanesulfonate-mutagenized indica cultivar IR68 population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice fungal and bacterial pathogens. Here, we have isolated Spl11 via a map-based cloning strategy. The isolation of the Spl11 gene was facilitated by the identification of three additional spl11 alleles from an IR64 mutant collection. The predicted SPL11 protein contains both a U-box domain and an armadillo (ARM) repeat domain, which were demonstrated in yeast and mammalian systems to be involved in ubiquitination and protein-protein interactions, respectively. Amino acid sequence comparison indicated that the similarity between SPL11 and other plant U-box-ARM proteins is mostly restricted to the U-box and ARM repeat regions. A single base substitution was detected in spl11, which results in a premature stop codon in the SPL11 protein. Expression analysis indicated that Spl11 is induced in both incompatible and compatible riceblast interactions. In vitro ubiquitination assay indicated that the SPL11 protein possesses E3 ubiquitin ligase activity that is dependent on an intact U-box domain, suggesting a role of the ubiquitination system in the control of plant cell death and defense.

IR64, the most widely grown indica rice in South and Southeast Asia, possesses many positive agronomic characteristics (e.g., wide adaptability, high yield potential, tolerance to multiple diseases and pests, and good eating quality,) that make it an ideal genotype for identifying mutational changes in traits of agronomic importance. We have produced a large collection of chemical and irradiation-induced IR64 mutants with different genetic lesions that are amenable to both forward and reverse genetics. About 60,000 IR64 mutants have been generated by mutagenesis using chemicals (diepoxybutane and ethylmethanesulfonate) and irradiation (fast neutron and gamma ray). More than 38,000 independent lines have been advanced to M4 generation enabling evaluation of quantitative traits by replicated trials. Morphological variations at vegetative and reproductive stages, including plant architecture, growth habit, pigmentation and various physiological characters, are commonly observed in the four mutagenized populations. Conditional mutants such as gain or loss of resistance to blast, bacterial blight, and tungro disease have been identified at frequencies ranging from 0.01% to 0.1%. Results from pilot experiments indicate that the mutant collections are suitable for reverse genetics through PCR-detection of deletions and TILLING. Furthermore, deletions can be detected using oligomer chips suggesting a general technique to pinpoint deletions when genome-wide oligomer chips are broadly available. M4 mutant seeds are available for users for screening of altered response to multiple stresses. So far, more than 15,000 mutant lines have been distributed. To facilitate broad usage of the mutants, a mutant database has been constructed in the International Rice Information System (IRIS; http: //www.iris.irri.org) to document the phenotypes and gene function discovered by users.

Candidate genes involved in both recognition (resistance gene analogs [RGAs]) and general plant defense (putative defense response [DR]) were used as molecular markers to test for association with resistance in rice to blast, bacterial blight (BB), sheath blight, and brown plant-hopper (BPH). The 118 marker loci were either polymerase chain reaction-based RGA markers or restriction fragment length polymorphism (RFLP) markers that included RGAs or putative DR genes from rice, barley, and maize. The markers were placed on an existing RFLP map generated from a mapping population of 116 doubled haploid (DH) lines derived from a cross between an improved indica rice cultivar, IR64, and a traditional japonica cultivar, Azucena. Most of the RGAs and DR genes detected a single locus with variable copy number and mapped on different chromosomes. Clusters of RGAs were observed, most notably on chromosome 11 where many known blast and BB resistance genes and quantitative trait loci (QTL) for blast, BB, sheath blight, and BPH were located. Major resistance genes and QTL for blast and BB resistance located on different chromosomes were associated with several candidate genes. Six putative QTL for BB were located on chromosomes 2, 3, 5, 7, and 8 and nine QTL for BPH resistance were located to chromosomes 3, 4, 6, 11, and 12. The alleles of QTL for BPH resistance were mostly from IR64 and each explained between 11.3 and 20.6% of the phenotypic variance. The alleles for BB resistance were only from the Azucena parent and each explained at least 8.4% of the variation. Several candidate RGA and DR gene markers were associated with QTL from the pathogens and pest. Several RGAs were mapped to BB QTL. Dihydrofolate reductase thymidylate synthase co-localized with two BPH QTL associated with plant response to feeding and also to blast QTL. Blast QTL also were associated with aldose reductase, oxalate oxidase, JAMyb (a jasmonic acid-induced Myb transcription factor), and peroxidase markers. The frame map provides reference points to select candidate genes for cosegregation analysis using other mapping populations, isogenic lines, and mutants.

Most agronomically important traits, including resistance against pathogens, are governed by quantitative trait loci (QTL). QTL-mediated resistance shows promise of being effective and long-lasting against diverse pathogens. Identification of genes controlling QTL-based disease resistance contributes to breeding for cultivars that exhibit high and stable resistance. Several defense response genes have been successfully used as good predictors and contributors to QTL-based resistance against several devastating rice diseases. In this study, we identified and characterized a rice (Oryza sativa) mutant line containing a 750 bp deletion in the second exon of OsPAL4, a member of the phenylalanine ammonia-lyase gene family. OsPAL4 clusters with three additional OsPAL genes that co-localize with QTL for bacterial blight and sheath blight disease resistance on rice chromosome 2. Self-pollination of heterozygous ospal4 mutant lines produced no homozygous progeny, suggesting that homozygosity for the mutation is lethal. The heterozygous ospal4 mutant line exhibited increased susceptibility to three distinct rice diseases, bacterial blight, sheath blight, and rice blast. Mutation of OsPAL4 increased expression of the OsPAL2 gene and decreased the expression of the unlinked OsPAL6 gene. OsPAL2 function is not redundant because the changes in expression did not compensate for loss of disease resistance. OsPAL6 co-localizes with a QTL for rice blast resistance, and is down-regulated in the ospal4 mutant line; this may explain enhanced susceptibility to Magnoporthe oryzae. Overall, these results suggest that OsPAL4 and possibly OsPAL6 are key contributors to resistance governed by QTL and are potential breeding targets for improved broad-spectrum disease resistance in rice.

Four genes of rice, Oryza sativa L., conditioning resistance to the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae), were tagged by restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. No recombinants were observed between xa-5 and RFLP marker loci RZ390, RG556 or RG207 on chromosome 5. Xa-3 and Xa-4 were linked to RFLP locus XNpbl81 at the top of chromosome 11, at distances of 2.3 cM and 1.7 cM, respectively. The nearest marker to Xa-lO, also located on chromosome 11, was the RAPD locus 0072o00 at a distance of 5.3 cM. From this study, the conventional map [19,28] and two RFLP linkage maps of chromosome 11 [14,26] were partially integrated. Using the RFLP and RAPD markers linked to the resistance genes, we selected rice lines homozygous for pairs of resistance genes, Xa-4 + xa-5 and Xa-4 + Xa-lO. Lines carrying Xa-4 + xa-5 and Xa-4 + Xa-lO were evaluated for reaction to eight strains of the bacterial blight pathogen, representing eight pathotypes and three genetic lineages. As expected, the lines carrying pairs &genes were resistant to more of the isolates than their single-gene parental lines. Lines carrying Xa-4 + xa-5 were more resistant to isolates of race 4 than were either of the parental lines ('quantitative complementation'). No such effects were seen for Xa-4 + Xa-lO. Thus, combinations of resistance genes provide broader spectra of resistance through both ordinary gene action expected and quantitative complementation.

Lesion mimic mutants are characterized by the formation of necrotic lesions in the absence of pathogens. Such genetic defects often result in enhanced resistance to pathogen infection and constitutive expression of defense response genes. To understand the genetic mechanisms leading to these mutations, we characterized 21 lesion mimic mutants isolated from IR64 rice mutant populations produced by mutagenesis with diepoxybutane (D), gamma rays (G), and fast neutrons (F). Four mutations are controlled by single dominant genes, one of which is inherited maternally. Five lesion mimics are allelic to known spotted leaf (spl) mutants spl1, spl2, spl3, or spl6. In total, 11 new lesion mimic mutations, named spl16, spl17, and spl19 through Spl27, were established based on allelism tests. Two lesion mimics, spl17 and Spl26 showed enhanced resistance to multiple strains of Magnaporthe oryzae, the rice blast pathogen, and Xanthomonas oryzae pv. oryzae, the bacterial blight (BB) pathogen. Co-segregation analyses of blast and BB resistance and lesion mimic phenotypes in segregating populations of spl17 and Spl26 indicate that enhanced resistance to the two diseases is conferred by mutations in the lesion mimic genes. A double mutant produced from two independent lesion mimics showed more severe lesions and higher level of resistance to X. o. pv. oryzae than their single mutant parents indicating a synergistic effect of the two mutations. In mutants that exhibit enhanced disease resistance to both pathogens, increases in expression of defense response genes PR-10a, POX22.3, and PO-C1 were correlated with lesion mimic development and enhancement of resistance. These lesion mimic mutants may provide essential materials for a comprehensive dissection of the disease resistance pathways in rice.

Several transposable elements were isolated from the genome of Xanthomonas oryzae pv. oryzae. These elements and an avirulence gene isolated from X. oryzae pv. oryzae were used as hybridization probes for a collection of X. oryzae pv. oryzae strains from the Philippines. Each of the sequences was present in multiple copies in all strains examined and showed distinct patterns of hybridizing bands. Phenograms were derived from the restriction fragment length polymorphism data obtained for each of the individual probes and for pooled data from multiple probes. The phenograms derived from the different probes differed in topology and, on the basis of bootstrap analysis, were not equally robust. For all of the probes, including the avirulence gene, some groups (even some haplotypes) consisted of multiple races. The strains were grouped into four major clusters on the basis of the two probes giving the highest bootstrap values. These groups were inferred to represent phylogenetic lineages. Three of the six races ofX. oryzae pv. oryzae appeared in more than one of the lineages, and another was present in two sublineages. For three of the races, strains representing different

To ensure food security in the face of population growth, decreasing water and land for agriculture, and increasing climate variability, crop yields must increase faster than the current rates. Increased yields will require implementing novel approaches in genetic discovery and breeding. Here we demonstrate the potential of field-based high throughput phenotyping (HTP) on a large recombinant population of rice to identify genetic variation underlying important traits. We find that detecting quantitative trait loci (QTL) with HTP phenotyping is as accurate and effective as traditional labor-intensive measures of flowering time, height, biomass, grain yield, and harvest index. Genetic mapping in this population, derived from a cross of an modern cultivar (IR64) with a landrace (Aswina), identified four alleles with negative effect on grain yield that are fixed in IR64, demonstrating the potential for HTP of large populations as a strategy for the second green revolution.