C. MICHIGANENSIS SUBSP. INSIDIOSUS AND M. TRUNCATULA - MECHANISMS OF VIRULENCE AND RESISTANCE

Many kinds of bacteria can cause diseases of plants, which reduce crop yields. Bacterial diseases caused by one type of bacteria, Gram-negative bacteria, have been well studied. However, Gram-positive bacteria can also cause plant diseases. These bacteria are very different from Gram-positive bacteria, and cause disease in different ways. Not much is known about how these bacteria cause disease, or about why some plants are disease-resistant. In this project, we will study these questions using bacteria called Clavibacter michiganensis subsp. insidiosus and the plant Medicago truncatula, which is a close relative of alfalfa, an important forage crop. Our project has three parts. In part 1, we will use the offspring from a genetic cross between two Medicago truncatula parent lines, one which is susceptible to Cmi, and one which is resistant. This collection of offspring is called a set of recombinant inbred (RI) lines. We will measure the extent of Cmi growth in the RI lines. By combining this information with existing genetic map information for the RI lines, we will be able to determine the location of genes that are important for resistance to Cmi. In part 2, we will study plant defense responses that are activated when Cmi infects roots. We will compare resistant and susceptible plant varieties to look for responses that are stronger in resistant plants, and may contribute to resistance. We will measure responses that are known to occur in other plant-pathogen interactions, including deposition of callose, a carbohydrate polymer that strengthens plant cell walls, activation of a type of protein called a MAP kinase, known to be important in plant defense signaling, production of reactive oxygen compounds associated with plant defense, and activation of plant defense genes. In part 3, we will study pathogen genes important for virulence. First, we will compare the genomic DNA sequence of Cmi to sequences from related pathogens, and identify some genes likely to be important for resistance. We will then make mutations in those genes and test the bacteria for lost virulence. If they have lost virulence, then the mutant gene must have been important for virulence. We will also do similar experiments using bacteria in which random genes have been mutated.