The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3-Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar 'Sasanishiki' harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3-Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type 'Sasanishiki' allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar 'Himenomochi' lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection.

Previous analysis of transcriptional changes after elicitation of Cf-9 transgenic tobacco (Nicotiana tabacum) by Avr9 peptide revealed a rapidly upregulated gene, ACRE276. We show that ACRE276 is transiently induced in wounded leaves within 15 min, but upon Avr9 elicitor treatment, this upregulation is enhanced and maintained until cell death onset in Cf-9 tobacco. ACRE276 RNA interference (RNAi) silencing in tobacco results in loss of hypersensitive response (HR) specified by Cf resistance genes. ACRE276 RNAi plants are also compromised for HR mediated by the tobacco mosaic virus defense elicitor p50. Silencing tomato (Lycopersicon esculentum) ACRE276 leads to breakdown of Cf-9-specified resistance against Cladosporium fulvum leaf mold. We confirmed that tobacco ACRE276 is an E3 ubiquitin ligase requiring an intact U-box domain. Bioinformatic analyses revealed Arabidopsis thaliana PLANT U-BOX17 (PUB17) and Brassica napus ARC1 as the closest homologs of tobacco ACRE276. Transiently expressing PUB17 in Cf-9 tobacco silenced for ACRE276 restores HR, while mutant PUB17 lacking E3 ligase activity fails to do so, demonstrating that PUB17 ligase activity is crucial for defense signaling. Arabidopsis PUB17 knockout plants are compromised in RPM1-and RPS4-mediated resistance against Pseudomonas syringae pv tomato containing avirulence genes AvrB and AvrRPS4, respectively. We identify a conserved class of U-box ARMADILLO repeat E3 ligases that are positive regulators of cell death and defense across the Solanaceae and Brassicaceae.

SUMMARYPlants frequently possess operon-like gene clusters for specialized metabolism. Cultivated rice, Oryza sativa, produces antimicrobial diterpene phytoalexins represented by phytocassanes and momilactones, and the majority of their biosynthetic genes are clustered on chromosomes 2 and 4, respectively. These labdanerelated diterpene phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent-copalyl diphosphate or syn-copalyl diphosphate. The two gene clusters consist of genes encoding diterpene synthases and chemical-modification enzymes including P450s. In contrast, genes for the biosynthesis of gibberellins, which are labdane-related phytohormones, are scattered throughout the rice genome similar to other plant genomes. The mechanism of operon-like gene cluster formation remains undefined despite previous studies in other plant species. Here we show an evolutionary insight into the rice gene clusters by a comparison with wild Oryza species. Comparative genomics and biochemical studies using wild rice species from the AA genome lineage, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis and the progenitor of Asian cultivated rice Oryza rufipogon indicate that gene clustering for biosynthesis of momilactones and phytocassanes had already been accomplished before the domestication of rice. Similar studies using the species Oryza punctata from the BB genome lineage, the distant FF genome lineage species Oryza brachyantha and an outgroup species Leersia perrieri suggest that the phytocassane biosynthetic gene cluster was present in the common ancestor of the Oryza species despite the different locations, directions and numbers of their member genes. However, the momilactone biosynthetic gene cluster evolved within Oryza before the divergence of the BB genome via assembly of ancestral genes.

SummaryUV-B, UV-A and blue light control a variety of aspects of plant development via distinct photoreceptors and signalling pathways. The known photoreceptors for UV-A/ blue light are cryptochrome (cry)1 and cry2, and the phototropism photoreceptor, phototropin. Redox processes are important in cry and phototropin signal transduction. A specific photoreceptor for UV-B has not been identified and there appear to be several possible UV-B signalling pathways. We are investigating the UV and blue light regulation of transcription of the chalcone synthase gene ( CHS ) in Arabidopsis . Experiments with photoreceptor mutants show that distinct UV-A/blue (cry mediated) and UV-B photoreception systems control CHS expression. Experiments with an Arabidopsis cell suspension culture show that the UV-B and cry1 signalling pathways differ kinetically and pharmacologically. In contrast to some other UV-B responses, the UV-B induction of CHS does not appear to involve oxidative stress signalling. Promoter elements and candidate transcription factors that effect CHS induction have been identified. Interactions within a network of UV-B, cry and phytochrome signalling pathways regulate CHS expression . Synergistic interactions between the UV-B pathway and distinct UV-A and blue-light pathways maximize the response. In addition, specific phytochromes positively control the cry1 pathway via distinct potentiation and coaction effects, and negatively regulate the UV-B pathway.

Genebanks provide access to diverse materials for crop improvement. To utilize and evaluate them effectively, core collections, such as the World Rice Core Collection (WRC) in the Genebank at the National Agriculture and Food Research Organization, have been developed. Because the WRC consists of 69 accessions with a high degree of genetic diversity, it has been used for >300 projects. To allow deeper investigation of existing WRC data and to further promote research using Genebank rice accessions, we performed whole-genome resequencing of these 69 accessions, examining their sequence variation by mapping against the Oryza sativa ssp. japonica Nipponbare genome. We obtained a total of 2,805,329 single nucleotide polymorphisms (SNPs) and 357,639 insertion–deletions. Based on the principal component analysis and population structure analysis of these data, the WRC can be classified into three major groups. We applied TASUKE, a multiple genome browser to visualize the different WRC genome sequences, and classified haplotype groups of genes affecting seed characteristics and heading date. TASUKE thus provides access to WRC genotypes as a tool for reverse genetics. We examined the suitability of the compact WRC population for genome-wide association studies (GWASs). Heading date, affected by a large number of quantitative trait loci (QTLs), was not associated with known genes, but several seed-related phenotypes were associated with known genes. Thus, for QTLs of strong effect, the compact WRC performed well in GWAS. This information enables us to understand genetic diversity in 37,000 rice accessions maintained in the Genebank and to find genes associated with different phenotypes. The sequence data have been deposited in DNA Data Bank of Japan Sequence Read Archive (DRA) (Supplementary Table S1).