Adding Research Strength to IRFGC

To accelerate gene discovery, IRRI has invested USAID Linkage Program funds into seven collaborative projects with eight US research laboratories. These projects bring together a range of expertise to advance IRFGC goal of characterizing the function of all agronmically important rice genes. Pending on signing of research agreements, these projects will be implemented in the second half of 2004. To know more about the contents of the research, see abstracts of projects below.

Project Abstracts

1. Elucidating the function of novel ubiquitination-related E3 ligase genes in the defense response to rice pathogens

PI: Guo-Liang Wang, Ohio State University

Ubiquitination is emerging as a common regulatory mechanism that controls a range of cellular processes in plants. Recent exciting discoveries from several laboratories suggest that ubiquitination may also play an important role in plant disease resistance. In our lab, we have recently identified several rice E3 ligase genes that were induced in the resistance response to rice blast. The expression patterns of three E3 genes were confirmed in Northern analyses and showed high correlation with the microarray experiments. These results suggest that the identified E3 genes are most likely involved in plant defense signal transduction. To elucidate the functions of these genes in disease resistance, we propose 1) to isolate the full-length cDNAs of these E3 genes; 2) to screen for their interacting proteins using the yeast two-hybrid system and 3) to evaluate the disease reaction of BMV or RNAi silenced and over-expressed transgenic plants. The results from this proposal will help us understand the role of protein degradation in defense response and may provide a new strategy to engineer durable resistant rice cultivars in the future.

2. Use and Optimization of Virus-Induced Gene Silencing in Rice

PI: Richard S. Nelson, Noble Foundation

Virus induced-gene silencing (VIGS) is a powerful technique for the study of gene function through transcript knock-out. The technique is based on the use of a virus vector to over-express RNA, which is recognized as aberrant and targeted for sequence-specific destruction. VIGS has been used successfully in both reverse and forward genetic screens to determine the function of genes from dicotyledonous plants. To date VIGS has not been used to study gene function in rice because no rice-infecting virus amenable to this procedure was known. We recently identified a strain of Brome mosaic virus, F-BMV, which infects multiple monocot species, including rice. The virus was cloned and altered to serve as a vector during VIGS studies. Expression of host genes such as phytoene desaturase (PDS) and actin by the virus vector led to unique visual phenotypes on infected plants and to the destruction of the specific host transcript in the affected tissue. The range of biological systems in which this vector can provide useful information to researchers now must be explored: for example, in the study of biotic resistance genes in rice. In addition, since the silencing phenotype is transient and penetration throughout the affected tissue is incomplete, studies to optimize silencing also must be conducted. Research proposed in this document is directed toward a) exploring the usefulness of the current BMV silencing vector in determining the function of representative biotic stress and resistance genes during pathogen challenge and b) optimizing the host, environment and vector to allow an enhanced VIGS response.

3. Validating Functions of Candidate Defense Genes for Broad-Spectrum Disease Resistance

PI: Jan E. Leach, Kansas State University

Genetic improvement for disease resistance is a continuing process to minimize the capacity of plant pathogens to overcome host resistance. In developing countries where control measures for diseases are limited, broad-spectrum resistance (BSR), defined as disease resistance against multiple races of a pathogen and multiple diseases, is particularly important. We have accumulated evidence supporting the hypothesis that there are common genetic loci that can contribute to BSR. The availability of the rice genomic sequence further positions us to identify candidate genes that may account for BSR. We established and continue to build upon a set of candidate genes controlling BSR in rice based on multiple criteria: (1) homology to genes known or suspected to play a role in defense based on biochemical/physiological studies in rice and other plant species, (2) chromosomal position in relation to disease-resistance QTL, (3) altered gene expression under pathogen challenge, and (4) altered phenotypes in mutant lines. Using virus- induced gene silencing (VIGS), we will study the gene function of these candidate genes through transcript suppression. Our objectives are to (1) refine the assays for using VIGS to validate rice gene function in rice-Xanthomonas oryzae pv. oryzae (bacterial blight, BB) and rice-Magnaporthe grisea (blast) interactions, (2) determine if candidate NBS-LRR recognition and downstream defense pathway genes contribute to broad-spectrum, durable disease resistance traits by suppression of their expression using VIGS, and (3) for a few select candidate genes, initiate development of constructs to determine if their overexpression genes leads to broad-spectrum, durable resistance. If the defense genes are involved in BSR, then suppression of their expression should make plants more susceptible to bacterial blight and blast, and their overexpression should enhance resistance to these two pathogens. The project will produce a rich set of data with information on the molecular basis for BSR.

4. Allelic function in wild, indica and japonica rices

PI: Scott Jackson, Purdue University

The importance of rice as a staple crop cannot be understated. However, rice is beset by many biotic and abiotic stresses such as disease causing pathogens and metal toxicities. Although, naturally occurring variation within the cultivated germplasm pool for many of these problems may be available, many times it is necessary to find genetic variation from wild relatives of rice in order to overcome these stresses. For example, advanced breeding lines and released varieties have been produced from interspecific introgression from Oryza rufipogon for tungro resistance/tolerance and acid sulphate (aluminum) tolerance, The goal of this project is to use existing resources, genomic libraries for 12 wild Oryza species, to 1) find genetic markers to follow introgression of wild alleles into cultivated rice, 2) understand the molecular basis of this variation to more efficiently capture it in improvement programs, in particular, using tungro and Al tolerance as test traits , and 3) make this data public and integrate our results with ongoing rice improvement programs at IRRI. To accomplish these goals, we will first find genomic clones orthologous to regions of the rice genome where genes conferring resistance are located. Once these clones are found they will then be used to develop DNA-based genetic markers for following introgression in improvement populations, and in several instances the DNA sequence will be determined so that the underlying molecular basis of variation can be determined. This research should result in a more fundamental understanding of allelic variation and how better to capture and manipulate it in breeding programs as well as enhance the collaboration of Purdue University-based scientists with their IRRI counterparts.

5. Molecular and genomic characterization of submergence tolerance in rice

PIs: Julia Bailey-Serres, UC-Riverside Pam Ronald, UC-Davis

The goal of the proposed research is to elucidate the mechanisms that underlie submergence tolerance in rice (Oryza sativa). The two University of California labs are collaborating on a complementary study of the molecular-genetic nature of this important phenotype. The Ronald lab will focus on confirmation of the submergence1 candidate gene, which has been tentatively identified as a plant-specific transcription factor. SUB1 transcripts show increased accumulation in response to submergence. The proposed research includes gene complementation in transgenic plants, evaluation of the significance of alternatively spliced transcripts generated from Sub1, and DNA microarray-based identification of gene transcripts regulated by the Sub1 genotype. The Bailey-Serres lab will focus on the role of a monomeric RHO-like G-protein (Rop) in the response to rice to oxygen deprivation. The regulation of the activity of this G-protein was shown to underlie tolerance of transient oxygen deprivation in Arabidopsis. The hypothesis to be tested is that Rop signaling is integral to adjustments of gene expression that control the level and duration of sucrose breakdown and ethanolic fermentation under oxygen deprivation. The proposed experiments will evaluate if activation and inactivation of Rop signaling is differentially regulated in submergence tolerant and intolerant lines. Together with the Ronald lab, DNA microarray analyses will be performed to elucidate the genes regulated by Sub1 and Rop-mediated signal transduction. This research should reveal whether manipulation of Sub1 genotype and Rop signaling can be used to enhance submergence tolerance.

6. Genomic comparisons between barley and rice in relation to salt adaptation and heritable salt tolerance.

PI: Timothy J. Close, UC-Riverside

Barley (Hordeum vulgare) and rice (Oryza sativa) share similarities in the development of their vegetative and reproductive structures. Barley is among the most salt tolerant crop plants and grows well with NaCl concentrations in the range of 100 mM. In addition, relatively more salt tolerant cultivars of barley have been developed with enhanced sodium ion partitioning and more stable grain production under saline conditions. We hypothesize that useful parallels exist between barley and rice which can be revealed by global gene expression analyses. We will test this hypothesis by examining pertinent barley and rice genotypes during vegetative and early reproductive growth under non-stressed and mildly saline stressed conditions. Genes whose expression is associated with physiological adaptation and/or heritable tolerance will be compared across and within the two species. Barley genotypes will include Morex, Golden Promise and Maythorpe. Morex has a 6.3 genome coverage BAC library, and several mapping populations include Morex as a parent. Golden Promise is the standard for transformation and is a salt-tolerant semi-dwarf cultivar isogenic to salt-sensitive Maythorpe. Rice genotypes will include IR29, FL478, Pokkali and possibly IR633 or other salt tolerant genotypes. IR29 is susceptible to salinity at vegetative and reproductive stages. Pokkali is a landrace tolerant at both stages. FL478 is the best RIL from a mapping population developed from a cross of IR29 x Pokkali and used for mapping a major QTL for Na uptake. IR633 is a salt tolerant genotype that derived its tolerance from a somaclonal variant of Pokkali. The Affymetrix "Barley1" GeneChip will be used for barley gene expression analyses in Riverside. Rice genotypes will be grown and RNA extracted from them in Riverside. IRRI will perform the rice microarray work. Data comparisons will be facilitated by knowledge of orthologous gene relationships and genome synteny between barley and rice.

7. Functional analysis of drought-associated regulatory genes and high throughput RNA interference in rice

PI: Yinong Yang, University of Arkansas

Drought stress is a major constraint for rice production worldwide. Although significant progress has been made towards the understanding of abiotic stress signaling in Arabidopsis, little is known about the gene function and regulation during drought stress in rice and other cereal crops. Among various signaling pathways, mitogen-activated protein (MAP) kinase cascades play a pivotal role in regulating biotic and abiotic stress responses in plants. Recently, we have identified and characterized an abscisic acid (ABA)-responsive rice MAP kinase that positively regulates drought, salt and cold tolerance. In the proposed project, we plan to further characterize this rice MAP kinase and its upstream MAP kinase kinase and to determine the role of the ABA-responsive MAP kinase cascade in regulating drought tolerance in rice. Furthermore, a series of RNAi vectors will be constructed and tested for high throughput cloning and efficient gene silencing in rice. These RNAi vectors and related protocols will be a iimportant tool for functional genomic analysis of drought-associated rice genes in the near future.

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