PI: Gurmukh (Guri) S. Johal, Purdue University Co-PI: Clifford F. Weil, Purdue University Co-PI: James. W. Moyer, North Carolina State University Co-PI: Peter J. Balint-Kurti, North Carolina State University
Plant disease resistance (R) genes encode proteins that respond to pathogen invasion by triggering a rapid, localized "hypersensitive reaction" (HR), in which host tissue immediately adjacent to the site of infection undergoes programmed cell death. Although, HR is a fundamental part of the immune response in plants, very little is known about the genes that trigger, signal, execute, and then contain it. Exhaustive mutagenesis screens have been conducted in Arabidopsis, but have turned up only a few genes linked to HR, possibly because of genetic redundancy or lethality of HR mutants. An alternate and potentially huge resource of HR variation is available in natural germplasm. If effectively tapped and delineated, this resource has the potential to advance considerably our understanding of HR . By taking advantage of a constitutive-active allele of the Rp1 gene, which confers resistance to the common rust disease of maize, we have demonstrated that the maize germplasm contains vast amounts of variation capable of impacting HR.
This project will test the broad use of an innovative approach, which looks for naturally occurring alleles that suppress mutant phenotypes to rapidly detect and explore natural variation underlying HR. Using this approach, one HR-modulating locus (Hrml1) has already been identified and mapped in a preliminary test using the IBM population of maize; this QTL will be cloned. In addition, many more QTL will be identified and characterized by broadening the screen to the 5000-line Nested Association Mapping (NAM) population currently available to the maize community.
The broader impacts of the project are many-fold. First, the approach of discovering and harnessing genetic diversity is likely to be broadly applicable to a large number of important traits in most plant species. Second, an outreach component will bring the uses of biodiversity to middle school curricula (Grades 6-8) and the topics of DNA sequence analysis and quantitative trait analysis to high school curricula, with the development and test of teaching materials that can be widely distributed. Third, these teaching modules will be developed for and with students from a wide range of cultural, racial and educational backgrounds, and their teachers. This approach will allow development of several versions of these modules, using different examples, different assessments and different forms of presentation so that they can be applied effectively in a variety of settings. The goal is to overturn the stereotype that these subjects are too complex for middle and high school students to grasp, and to prime student to investigate plant diversity and genetics in greater detail later in their education so that they are better able to understand the issues. These topics are not generally covered in most science curricula at these levels, despite their increasing importance scientifically and socially.
The outcomes and biological resources generated by this project will be available through the project website: www.Rp1-MAGIC.purdue.edu.
A unique feature of the project was that we exploited the natural variation between different maize varieties to uncover components of the HR response. We used a novel genetic strategy relying on HR initiated not by a pathogen but by a mutant R gene (called Rp1-D21) that spontaneously triggers HR; in other words a type of plant autoimmunity. The response caused by Rp1-D21 is very much exaggerated and much more easily measured compared to normal HR (which is barely visible to the naked eye) and its severity changes depending on what maize variety the gene is in, being enhanced to lethal levels in some and suppressed almost completely in others. These different levels of HR caused by Rp1-D21 are due to the presence of different genes or versions of genes that affect the HR in the different varieties. Genes that affect any easily-measured trait can be ‘mapped’ in a fairly straightforward manner and that is what we did in this project: We mapped genes that affect this aberrant immune response on the assumption that they would also affect the normal immune response (which is much harder to measure). Since, in this approach we use an exaggerated, mutant version of our trait of interest as a ‘reporter’ for the trait, this strategy has been named Mutant-Assisted Gene Identification and Characterization, or MAGIC for short. Using this strategy we were able to efficiently assess dozens of varieties of maize for versions of genes that either boosted or dampened the HR response. Rp1-D21 is an aberrant form of the maize Rp1 disease resistance gene, which normally confers HR-based immunity to the common rust disease caused by a fungal pathogen. The diverse germplasm was derived from two public sources, the maize diversity panel and the NAM association mapping panel, the development and genetic characterization (genotyping) of which was made possible by funds from NSF previously to other groups. The two major initial goals for this project were: 1) A comprehensive screen for genes that contribute naturally-occurring variation in the hypersensitive defense response, 2) Proof-of-concept for the MAGIC approach. Both of these goals were largely achieved. Our study demonstrated that MAGIC is a very effective approach in exploring the genetic basis of a complex trait using natural variation. It provided the first system-wide analysis of natural variation that modulates the HR response in plants, and identified the location of 44 genes that play a role in controlling the HR response. We have now taken this work further. In two cases we have identified the specific genes associated with modulating HR. One of these genes is involved in lignin production, an important component of the cell wall, the other in protein degradation. We have shown that some of the genes that control HR also control disease resistance and a type of low level, constant defense response naturally found in some plants that makes them more disease resistant but also stunts their growth. This project provided training for 17 postdoctoral scholars, 7 graduate students, and more than 30 undergraduate and high school students in the science of genetics and genomics. Half of these trainees were women. In our outreach efforts we collaborated with the North Carolina Museum of Natural Sciences (http://naturalsciences.org/) in a number of activities. We co-developed, financially supported and participated in two classes and a teacher workshop that have already reached more than 1000 students and 50 teachers and have been incorporated into the standard offerings of the museum. We organized plant genetics demonstrations at several local elementary and middle schools, gave several presentations on plant genetics to the public. Six publications have already resulted from this work, and several others are in review for publication or are in preparation.
