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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.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.

The avrXa10 gene of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight of rice, is a member of the avrBs3 avirulence gene family and directs the elicitation of resistance in a gene-for-gene manner on rice lines carrying the resistance gene Xa10. The carboxyl (C) terminus of AvrXa10 has a previously undescribed domain that is structurally similar to the acidic activation domain of many eukaryotic transcription factors in addition to three nuclear localization signal (NLS) sequences. Removal of the C-terminal 38 codons containing the putative activation domain, but retaining the NLS sequences, was concomitant with the loss of avirulence activity. The C-terminal coding regions of avrBs3 and avrXa7 can be replaced by the corresponding region of avrXa10, and the genes retained specificity for the resistance genes Bs3 in pepper and Xa7 in rice, respectively. The avrBs3 and avrXa7 avirulence activities of the hybrid genes were also lost upon removal of the terminal 38 codons. When fused to the coding sequence of the Gal4 DNA binding domain, AvrXa10 activated transcription in yeast and Arabidopsis thaliana. Removal of the carboxyl region severely reduced transcriptional activation. AvrXa10 would have to be localized to the host cell nucleus to function autonomously in transcriptional activation. Consistent with this requirement, mutations in all three NLS sequences of avrXa10 caused a loss in avirulence activity. The findings demonstrate the requirement of the C terminus for AvrXa10 function and the potential for the members of this family of avirulence gene products to enter the host nucleus and alter host transcription.

Induction of peroxidase has been correlated with resistant interactions between rice and Xanthomonas oryzae pv. oryzae. To assist in analysis of the role of rice peroxidases in plant defense against the bacterial pathogen, three peroxidase genes, POX22.3, POX8.1, and POX5.1, were identified from a rice cDNA library that was constructed from leaves of plants undergoing a resistant reaction. These genes were highly similar in nucleic acid and amino acid sequences and belonged to a gene family. The three genes showed differential expression in infiltrated rice leaves during pathogen interactions and mechanical stress. Only two peroxidase genes, POX8.1 and POX22.3, were predominantly expressed during resistant interactions. These two genes also were expressed during susceptible interactions, but induction was delayed compared with resistant interactions. POXgX9, a fourth peroxidase gene that was isolated from a genomic library, is adjacent to POX22.3 in the rice genome and has greater than 90% similarity in nucleotide and amino acid sequence identity to POX22.3. Interestingly, POXgX9 was expressed only in the roots of rice plants. While POX22.3 was expressed in both leaves and roots, POX8.1 and POX5.1 were not detected in roots but were induced in leaves by mechanical wounding at different times after treatment. POX22.3, POX8.1, and POX5.1 were estimated to be present in single copies in rice haploid genome. These results indicate that different members of the rice peroxidase gene family are distinctly regulated in response to various environmental cues.