Functional Dissection of the PTO Resistance Gene Using DNA Shuffling

University Of California-Davis, Davis CA

Investigators

Abstract

Understanding the early events in disease resistance has both fundamental and practical importance. Molecular dissection of disease resistance is currently one of the most intensively studied areas of plant biology and interesting parallels are being established with the animal-pathogen interactions. Although considerable progress has been made over the past ten years, early recognition events that lead to resistance are only just beginning to be understood. In the long term, understanding the molecular basis of specificity in plant-pathogen interactions and the molecular events resulting in resistance will provide new options for developing disease resistant plants. Our long-term goals are to determine the molecular basis of recognition and signaling in plant disease resistance and to engineer new disease resistance specificities. The specific goal of this proposal is to dissect the functional regions of the Pto protein that confer the ability to bind different pathogen products and to initiate physiological pathways (downstream signaling) necessary for defense against the pathogens in diverse plant species. DNA shuffling is a powerful and novel approach for dissecting protein function that makes few a priori assumptions about function yet provides great resolution to dissect individual regions of the protein. DNA shuffling involves the generation of chimeric genes (recombinations from two gene sources) in the test-tube from fragments of naturally occurring versions of a gene. Our proposed studies will further develop DNA shuffling as an experimental tool and provide statistical approaches for interpreting the resulting data. The interaction between the pathogenic bacterium Pseudomonas syringae and Solanaceous plants has become one of the best-characterized plant-pathogen interactions at the molecular level and the Pto gene is ideally suited to DNA shuffling experiments. We have been studying this interaction for several years and have all the necessary biological materials and methodologies in hand to make rapid progress. The first round of DNA shuffling the Pto gene has demonstrated the power and feasibility of the DNA shuffling approach and revealed several previously unrecognized, potentially important domains for binding to the pathogen derived avirulence protein, AvrPto. In this research program we will conduct three parallel lines of investigation that will provide detailed and complementary data on the domains and amino acid sequences of Pto which are required for binding pathogen derived proteins and for downstream signaling: 1) We will conduct a second generation of DNA shuffling as well as specific amino acid substitutions to test the structure - function inferences derived from our first DNA shuffling experiment. 2) We will screen a shuffled library of chimeric genes to identify variants of Pto with new binding specificities. 3) We will shuffle Pto with genes encoding similar proteins from Arabidopsis to identify chimeras that confer the ability to recognize additional avirulence proteins. The proposed experiments are highly multidisciplinary. They are a combination of molecular biology and statistics that draws on plant breeding strategies as well as making and testing structure-function inferences. The postdoc, graduate student, and undergraduates will receive training in both molecular biology and statistics. In addition, they will gain experience in plant-pathogen interactions.