Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Agricultural Biology
Non Technical Summary
Warming in the climate system, an abiotic stress, impacts plant biology in many ways, including how plants respond to pathogens. Many plant diseases are predicted to intensify as environmental temperatures increase, and many widely used single gene sources of disease resistance (R genes) are less effective at high temperatures. In this project, we seek to understand the generality and mechanisms behind increased disease and break down of most resistance sources at high temperatures. We hypothesize that high temperatures increase disease by affecting the regulation of host factors involved in susceptibility and resistance. Based on our preliminary data, we propose that the plant hormone abscisic acid (ABA) plays a critical role in regulating the responses to combined abiotic and biotic stresses. Here, we will test the role of ABA in regulating plant responses to combined stresses of Xoo and high temperatures by determining the generality of the involvement of ABA responsive pathways, and by using a biochemical approach to test if ABA regulates the responses. We will computationally identify conserved transcriptional regulators in the promoters of genes that contribute to tolerance of these combined stresses. This information will be incorporated in the training modules for a short course we will co-organize to train a next generation of crop plant biologists. Our research is proposed in rice, but the approach described could be readily applied to improving resilience to climate and other stresses in other important crop species, thereby providing critical solutions to enhance the sustainable production of nutritious food for a growing global population.
Animal Health Component
35%
Research Effort Categories
Basic
65%
Applied
35%
Developmental
(N/A)
Goals / Objectives
Increasing temperatures associated with climate change impact plants in many ways, including how plants respond to pathogens. Many plant diseases intensify as environmental temperatures increase, and many widely used single gene sources of disease resistance (R genes) are less effective at high temperatures. While the phenomenon of temperature-induced susceptibility is well documented, the mechanisms are not known. For example, high temperatures are conducive to bacterial blight (BB) disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo), and most R genes are less effective at controlling disease at high relative to low temperature regimes [1,2]. The increase in disease susceptibility and reduced efficacy of most R genes at high temperatures provide an excellent model to study the impacts of climate change-associated temperature changes on plant-bacterial interactions in an important crop plant.Our long-term goal is to understand the generality and mechanisms behind increased disease and break down of most resistance sources at high temperatures. We hypothesize that high temperatures increase disease by affecting the regulation of host factors involved in susceptibility and resistance. Our preliminary data suggest that high temperatures cause an increased expression of genes that mediate disease susceptibility and decreased expression of genes that mediate disease resistance, and that these changes might contribute to increased BB disease. Specifically, we found increased expression of genes in abscisic acid (ABA) biosynthesis and downstream ABA regulated genes during susceptible interactions at high temperatures [3]. In contrast, when R mediated resistance was effective at high temperatures (as in interactions governed by BB R gene, Xa7), ABA biosynthetic and responsive genes were down-regulated. In the proposed studies, we will explore further the role of ABA in resistant and susceptible interactions involving the combined stresses of Xoo and high temperatures. Towards translation of this information to crop improvement, we will computationally identify conserved transcriptional regulators in the promoters of genes that contribute to tolerance of these combined stresses. This information will be incorporated in the training modules for a short course we will co-organize to train a next generation of crop plant biologists. Our specific objectives are to:Obj 1. Determine the generality of the ABA pathway gene responses during combined stresses (Xoo and high temperatures) in interactions involving various rice BB resistance (R) genes.Justification: We found that resistance responses mediated by the BB R gene Xa7 under heat stress caused significant downregulation of ABA-responsive genes, while the genes were up-regulated by high temperature stress alone as well as combined high temperatures and susceptible interactions [3]. It is not yet known how general these responses are across other BB R genes. That is, while we know those genes are less effective at high temperatures, we do not know if ABA responsive genes are activated similar to the susceptible/high temperature response, i.e. with no R gene present or if they respond intermediate to the combinations of high temperature and susceptible or Xa7-mediated responses. Here, we characterize the phenotypic responses and expression of the ABA responsive genes in combined stresses of high temperature, Xoo and rice with different BB R genes.Obj 2. Determine if ABA contributes to the responses of rice to combined stresses of heat and BB. Justification: Despite advances in mutant development and analysis, limitations such as gene redundancy and lethality or developmental defects in mutations still hamper the studies of gene function. Thus, highly specific, chemical analogs that block plant hormone biosynthesis, metabolism, transport, and signal transduction are invaluable resources for studying the roles of hormones in plant responses [38]. Indeed, a number of chemical inhibitors have been used to decipher the auxin biosynthesis, transport, and signaling pathways (reviewed in [38]. We have collaborated with a synthetic organic chemist (Suzanne Abrams, University of Saskatchewan) who developed inhibitors of various steps in ABA pathways [39-42] to characterize a new class of ABA antagonists that prevent folding of the ABA analog-bound receptor required for ABA signaling [42], and have demonstrated the effect of one inhibitor (A1019) in this group in rice [43]. Dr. Abrams has kindly made this inhibitor available to our analysis of the importance of ABA to rice responses to combined heat and pathogen stress.Obj 3. Identify transcriptional regulators that control the responses to the combined stresses of high temperature and BB in rice. Justification. Tolerance in plants to stresses, such as high temperature and disease, involves changes in expression patterns of many genes. These changes are controlled by short sequences in the gene promoters, specifically cis-regulatory elements (CRE) or combinations of CRE organized as modules (cis-regulatory modules, CRM). CRE/CRM are in promoters of many genes co-activated by a single stress, and are common to genes co-activated in plants with enhanced tolerance to multiple stresses. We previously identified several conserved regulatory motifs in the promoters of differentially expressed genes, including CRE that respond to ABA [3,25,33]. We suggest a role of ABA as a regulator of plant defense responses at high-temperature, and, in this objective, will determine if CRE/CRM that respond to ABA, high temperature, and disease are enriched in promoters of genes that respond to the combined stresses. CRE related to ABA responses may ultimately be good targets for markers to simultaneously enhance tolerance to both heat and disease in crop breeding programs.Obj 4. Co-organize and teach an international short course, Crop Improvement for Resistance to Combined Stresses, to train a new generation of plant scientists to understand the importance of innovative plant science in addressing global problems.
Project Methods
Objective 1. Determine the generality of the ABA pathway gene responses during combined stresses (Xoo and high temperatures) in interactions involving various rice BB R genes. Approach: For evaluation of combined stresses (high temperature and interactions with Xoo), we have adapted a high-throughput plant assay, where rice seed are planted in a 96-well dish format (~48 plants per dish), and where the roots can extend into hydroponic solution. Rice near-isogenic lines carrying the Xa4, xa5, Xa7, Xa10, and Xa21 R genes will be infiltrated with Xoo strains with and without the corresponding effector genes using the scissor clip method [34]. Disease and resistance will be assessed by measuring bacterial numbers, lesion expansion [35-37]. Expression of ABA responsive genes will be monitored by RT-PCR [3].Objective 2. Determine if ABA contributes to the responses of rice to combined stresses of heat and BB.Approach: We will use a biochemical approach to determine the requirement for ABA signaling in rice during combined pathogen and high temperature stresses, taking advantage of a number of the chemical antagonists of ABA activities. For evaluation of interactions with Xoo, we will use the high-throughput plant assay format (~48 plants per dish). Exogenous ABA and/or ABA antagonists are gently sprayed onto fully-extended rice leaves 12-24 h prior to pathogen inoculation by the scissor-clip method [34]. Effects of the antagonists and exogenous ABA on disease and resistance will be assessed by measuring bacterial numbers, lesion expansion [35-37], and expression of ABA responsive genes by RT-PCR [3].Objective 3. Identify transcriptional regulators that control the responses to the combined stresses of high temperatures and BB in rice.Approach: Using our transcriptomics data [3] as well as published data from rice undergoing single heat, disease, and resistant interactions [25,33], we will identify and compare CRE/CRM that are over-represented in promoters of ABA-, disease-, resistance-, and thermotolerance genes. The literature suggests that there are common genes and pathways to abiotic and biotic stresses [25]. We will create a bioinformatics pipeline from an input of genes of interest (in our case ABA responsive (including thermotolerance genes), disease susceptibility, and defense response genes), where promoter regions are extracted from a genome of interest (rice Japonica and Indica cultivars) and the sequence composition of the set of interest is automatically compared with all other genes in the genome. A first step of this pipeline will involve integrating different prediction software to identify both de novo or known motifs (CREs) in the input set and the whole genome [44-46]. Similar or overlapping motifs identified by multiple software will be merged, and the overall architecture (grouping and arrangement of CREs) of each promoter will be used to identify candidate modules (CRMs) as shown before [33,47]. Different statistical measures (Hypergeometric test and Fisher's exact test) will then be used to determine enrichment of CREs and CRMs in the set of interest compared with the rest of the genome. To ensure robustness, multiple permutations of the analyses will be made where random samples of the input and control set of genes are used for the analyses, and each motif is then given a score based on how many permutations they were classified as enriched.We will re-run the pipeline using as an input a combination of disease/defense and ABA-responsive genes. This will allow us to identify CRE/CRM that are common to both heat- and disease/defense-response gene promoters, indicating that these particular genes are activated by both stresses. For our purposes, we will also conduct re-sampling by excluding or including certain transcriptomic datasets from our previous work, to ensure the results are unbiased. This pipeline will be made available in a dedicated github repository and will be available for use with any plant genome and any set of genes of interest.Objective 4. Co-organize and teach in a short course located at international CGIAR centers. Over the past 10 years, Leach has co-organized and offered international training courses that provide young scientists hands-on experiences in modern crop production and improvement techniques and that help them develop a broader appreciation for international agriculture, collaborative approaches to science, and the possibilities for putting modern plant sciences into practice. Previous courses were held at CGIAR centers around the world, including the International Rice Research Institute (IRRI) in the Philippines, at AfricaRice in Senegal, and at CIAT in Colombia. We will offer a new training course at CIAT in Colombia that builds on the prior courses, but that expands beyond rice as a model, and brings the importance of looking at combined stresses in crop improvement. The course, Crop Improvement for Resistance to Combined Stresses, will focus on strategies for crop improvement that emphasize the importance of considering combined biotic and abiotic stresses. Classroom, lab, and field-based learning will be augmented by topical discussions on how advanced technologies and novel resources can be incorporated into crop improvement for combined stresses, and the sociological and economic issues for the acceptance of these improved crops by farmers and consumers. A team class project will produce a podcast of interviews, perhaps including consumers, growers and scientists on issues related to the adoption of crops improved by various approaches. The short course will train a new generation of crop scientists to think broadly about crop improvement strategies, bringing the complexity of improving crops for resistance to combined stresses to the forefront. We will also help the participants understand and rationally discuss challenging science issues related to international food security with a broad and diverse audience. The participants will be well networked into the international crop improvement community and will understand the importance of innovative plant science in addressing global problems.