Development of Genomic Tools to Dissect Regulatory Gene Networks in Wheat
Summary: Jorge Dubcovsky's goal is to develop functional genomics resources to empower the wheat research community and to use those resources to answer basic questions on wheat development and disease resistance.
My main research contributions have been in the area of wheat development and wheat improvement. In collaboration with colleagues and students in my laboratory, I have worked on three developmental processes that have major impacts on wheat productivity: the transition between vegetative and reproductive stages (which affects adaptation), the regulation of spike development (which affects yield), and the regulation of senescence (which affects nutrient remobilization). During the past 15 years, I have developed genomic tools that were missing in wheat because of its large and complex genome. Our wheat molecular maps, large insert libraries, expressed sequence tag resources, single-nucleotide polymorphism markers, and mutant populations are publicly available and are used extensively worldwide.
In the area of reproductive development, we used these new genomic tools to clone and validate the three main vernalization genes in wheat: Vrn1, Vrn2, and Vrn3. Our work showed that the vernalization pathway in temperate cereals differs from that in the model species Arabidopsis. We also developed molecular markers for the different natural alleles of these three major regulatory genes. These markers are being used extensively by wheat-breeding programs worldwide. The precise regulation of the reproductive phase is essential for wheat adaptation to changing environments.
A second developmental process that is critical for wheat productivity is the regulation of spike formation, since it determines the maximum number of grains per spike. We discovered Eps1, a locus that increases spikelet number by up to 30 percent in diploid wheat. We sequenced the complete Eps1 region and identified Mot1 as the most likely candidate gene to explain the differences observed at the Eps1 locus. Allelic variants at this gene are associated with a longer spike development period and a significant increase in spikelet number.
Our group is also interested in terminal senescence, a highly regulated process during which nutrients are remobilized from the leaf to the wheat grain. With colleagues from my laboratory and the University of Haifa, we cloned the high–grain protein content (GPC) gene Gpc1 from wild wheat. This gene accelerates nutrient remobilization, increases grain protein and iron concentrations by 10 percent, and increases zinc concentrations by 5 percent. We have incorporated this Gpc1 allele into our wheat varieties and distributed them to breeding programs worldwide. The Gpc1 gene is an early regulator of senescence, and the transgenic and mutant lines developed in my laboratory are helping to dissect the complex senescence regulatory network.
My laboratory is also involved in the characterization of the gene networks involved in wheat resistance to rusts, a devastating group of pathogens that produce large yield losses all over the world. My laboratory cloned Yr36, a slow rusting gene that delays the progression of stripe rust in wheat. This gene encodes a protein with a novel architecture not found before in any other known protein. The combination of a lipid-binding domain with a kinase domain provides a molecule with the potential to sense the alteration of lipids during pathogen infection and to trigger a signal cascade through its kinase domain. We are investigating the gene network involved in this slow rusting response.
To advance the previous areas of wheat research, more efficient reverse genetic tools are required. In collaboration with Luca Comai (University of California, Davis), we have recently developed mutant tetraploid and hexaploid wheat populations with an extremely high density of mutations. This high rate of mutation is possible because of the polyploid nature of wheat, which has redundant copies for most genes. Once mutants are generated, pooled DNAs and CelI assays are used to screen for mutations in the desired genes (TILLING). The technical demands of this process have limited the access to the benefits of these populations to a few laboratories. We plan to sequence a large proportion of the genes in our tetraploid mutant population. We will use next-generation sequencing and gene capture to sequence ~30,000 genes in 1,000 lines from our tetraploid mutant population. We will develop Web tools to enable the search for mutations in the desired genes and will increase the mutant seed stocks to make them publicly available. Access to these mutant populations will change the paradigm of what can be done in wheat functional genomics.
In my laboratory, we will use these mutants to dissect wheat gene networks that regulate developmental and disease resistance. In the area of reproductive development we have recently discovered that the competitive interactions between vernalization and photoperiod pathways are mediated by NF-Y transcription factors. These transcription factors include three proteins that form trimeric complexes that interact with the promoters of several genes. There are approximately 10 different genes coding for each of these three units, which complicates the characterization of these transcription factors. We will use our mutant populations to understand the functions of the different members of these complexes and their contributions to the regulation of flowering.
In the area of spike development we will use our mutant populations to validate the role of Mot1 on the differences in spikelet number. We also plan to transfer the diploid wheat allele for increased spikelet number to polyploid wheat and to combine this natural diploid allele with available mutations in the B and D genome copies of Mot1. We hypothesize that this will enhance the effect of the diploid wheat natural mutation in polyploid wheat. To better understand the role of Mot1 in spike development, we will complement natural mutations with transgenic approaches.
In the area of senescence, we will use our available Gpc1 mutants and RNAi (RNA interference) transgenic line to identify the effect of this gene on the senescence transcriptome. These studies will be complemented by yeast two-hybrid studies to identify the proteins that directly interact with the Gpc1 protein. We then plan to use our mutant populations to perturb critical nodes of this regulatory network to understand better their functions. We hope to use this knowledge to optimize nutrient remobilization in wheat. Finally, we will use our mutant population to dissect the rust resistance pathway and to study the function of the proteins that interact with the Yr36 stripe rust resistance protein.
Wheat provides 20 percent of the calories consumed by humans and plays a central role in human nutrition. Better knowledge of the major developmental switches and disease resistance gene networks that affect wheat productivity is needed to engineer more productive and nutritious wheat varieties.
Grants from the United States Department of Agriculture National Institute of Food and Agriculture, the National Science Foundation, and the United States–Israel Binational Agricultural Research and Development (BARD) provided partial support for these projects.
As of May 30, 2012