| Title |
Investigators | Department | Objectives | Approach Keywords | Progress Reports | Impact Statements | Publications | |
Project * COL00750 | |
| Title | Molecular Basis of Broad Spectrum, Durable Disease Resistance in Crop Plants |
| Investigator(s) | Leach, JE; |
| Department | Bioagricultural Sciences and Pest Mgmt. |
| Objectives | Our long-term goals are to understand the genetic and molecular basis for broad-spectrum, durable resistance in crop plants using rice as a model system. The specific objectives are to: 1) Refine and optimize traditional (RNAi) and new high throughput (Virus induced gene silencing, VIGS) assays to validate gene function in disease resistance in cereals. 2) Determine the contribution of candidate resistance gene analogs and downstream defense response pathway genes to broad spectrum resistance by using (a) the refined suppression technologies and (b) overexpression in transgenic plants. |
| Approach | Several experimental strategies are available to confirm functions of candidate genes in durable resistance. These include scoring for the phenotype of interest in plants where the candidate gene is overexpressed or mutated (then complemented), or where the expression of the gene is suppressed (RNA interference, RNAi, or Virus Induced Gene Silencing, VIGS). In this study we focus on suppression to validate gene function. For RNAi suppression, we will develop constructs using a Ubi-ds-G construct that will direct synthesis of a RNA composed of antisense-GUS- sense (P. Ronald, pers. commun.). We will produce about 20 independently transformed lines for each RNAi construct. T0 plants will be evaluated for reduced expression of the target gene (semi-quantitative PCR) and for altered Xanthomonas oryzae pv . oryzae (Xoo) and Magnaporthe grisea (Mg) resistance. We will test the utility of the VIGS assays for these studies using two easily scored disease resistance genes, Pita and Xa21. After introduction into the BMV-vector, we will test the ability of these constructs to suppress endogenous Pita and Xa21 expression; successful suppression will lead to susceptibility of the VIGS-rice to Mg and Xoo, respectively. These interactions will be used to determine optimal light, temperature, etc. to measure VIGS in these interactions. The VIGS assay will be used to identify functional resistance genes among the NBS-LRR family members. The NBS-LRR recognition gene family members that co-localize with R genes predicted to confer durable resistance will be cloned into the BMV-VIGS vector and the infectious recombinant BMV virus will be used for inoculation of rice with Xa7 (IRBB7) and Xa4 (IRBB4). A semi-quantitative PCR will be used to monitor for target gene silencing in newly developing leaves. Leaves showing silencing will be inoculated with Xoo strains by infiltration. The loss of resistance to Xoo will be determined as a loss of hypersensitive response. VIGS will be used to study suppression of candidate defense response (DR) genes that co-localize with the disease QTL. We will determine if suppression of these DR genes enhances susceptibility to Xoo and Mg. Amplified fragments of the genes that correspond to family members will be introduced into the BMV-VIGS vector, and the infectious virus will be inoculated to rice cultivars with and without the QTL. A semi-quantitative PCR will be used to monitor target gene silencing in newly developing leaves. Leaves showing the expected silencing will be inoculated with Xoo and Mg strains. If the genes contribute to quantitative resistance, reduced disease should be observed after silencing. For over-expression, full length cDNAs of NBS-LRR and candidate DR genes will be inserted into the pCAMBIA vector with the ubiquitin gene promoter for constitutive expression of the genes. Constructs will be delivered into the japonica cultivar by Agrobacterium-mediated. Expression of the genes in plants will be tested by RT-PCR. Reaction of T2 transgenic plants to Xoo and Mg will be evaluated. |
| Keywords | quantitative trait loci, QTL, disease resistance, RNAi, Virus induced gene silencing, rice |
| Progress Reports | |
| 2005 | The need for durable genetic resistance against a broad spectrum of diseases is more critical as we move to less dependence on chemicals for crop production. This type of resistance is considered to be controlled by many genes (quantitative resistance). To manipulate these traits to sustain crop productivity, we need to know the function and role of particular genes in resistance. Our long-term goals are to investigate this fundamental question in crop protection using rice as a model system. We have identified gene candidates, based on association of candidate genes with QTL in mapping studies. To prove that these gene candidates function in disease resistance, we are developing constructs to interfere with their expression. We are using stable (RNAi) and transient (virus induced gene silencing, VIGS) assay systems. The demonstrated use of VIGS in rice is recent; thus, we are optimizing this technique. The silencing constructs, once developed, will be introduced into rice plants that contain the wild-type genes. After suppression, the plants will be screened for increased disease susceptibility to two important rice diseases, bacterial blight and blast. As this project started in July, we are still in developing constructs. |
| 2006 | Genetic improvement for disease resistance is a continuing process to minimize the capacity of plant pathogens to overcome host resistance. To that end, we are studying durable and broad-spectrum resistance (DBSR), defined as disease resistance that lasts a long time over a large area, and is effective against multiple races of a pathogen and multiple diseases. We have accumulated evidence supporting the hypothesis that there are common genetic loci that can contribute to DBSR. The approach in this proposal is to determine if virus- induced gene silencing (VIGS) will provide a needed tool to study gene function of candidate genes for DBSR through transcript suppression. One of our objectives was to refine the VIGS technology developed at the Noble Foundation for use to validate rice gene function in rice-Xanthomonas oryzae pv. oryzae (bacterial blight, BB) and rice-Magnaporthe grisea (blast) interactions. We used VIGS vectors refined by the Noble Foundation to develop constructs to suppress a known lesion mimic gene, Spl11. The VIGS construct was inoculated to rice, and a lesion format was observed within 2 weeks after inoculation. In addition, the suppression of the transcript was verified by semi-quantitative RT-PCR. With this success in hand, we began screening other candidates to discover defense response pathway genes that have been identified from lesion mimic mutants (spl1 and spl3) using VIGS. In addition, we asked if, after suppression of gene expression, we can screen with pathogen inoculation for reduced resistance. Based on the success with Spl11, we developed VIGS constructs containing Rxo1, which is an NBS-LRR gene that recognizes the avrRxo1 gene in bacteria and signals the elicitation of a defensive hypersensitive response (HR). Suppression of Rxo1 should lead to susceptibility after inoculation with X. oryzae pv. oryzae containing avrRxo1. That is, if the Rxo1 gene is suppressed, the plants will not show an HR in 24-48 h following bacterial infiltration, but instead will show a water-soaked phenotype. Simultaneously, this approach is being followed to test the ability to suppress a blast resistance gene (Pita), and screen for the phenotype of susceptibility. However, after multiple attempts at refining the technique, suppression by VIGS did not provide sufficiently clear results to be useful in further studies. Therefore, we have moved completely to RNAi silencing of the gene candidates. Although a lengthy process , this method is tried-and-true. We have now silenced several BSDR gene candidates, and confirmed that they are silenced by RT-PCR. Screening of those lines with pathogens is now in progress. |
| Impact | |
| 2005 | The project will provide important insights into the molecular basis of quantitative disease resistance, which is fundamental to broad spectrum, durable resistance. From a practical perspective, knowledge of the effects of the targeted candidate genes will guide breeding programs to solve disease problems in a sustainable way. |
| 2006 | The project will provide important insights into the molecular basis of quantitative disease resistance, which is fundamental to broad spectrum, durable resistance. From a practical perspective, knowledge of the effects of the targeted candidate genes will guide breeding programs to solve disease problems in a sustainable way. For example, this information will provide the basis for which genes to pyramid for quantitative resistance in rice and other cereals. |
| Publications | |
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