2012 Annual Report
2: Develop and evaluate the use of site-specific recombination to break the linkage drag associated with the introgression of transgenes from laboratory lines to field cultivars. [NP 301, C 4, PS 4C]
For objective 2, a construct will be made by recombinant DNA methods with sets of recombination sites designed for interchromosomal recombination and harboring a first marker gene. This construct will be transformed into line #1. The transgenic locus of the line #1 will be introduced into another genetic background such that the flanking DNA of the new line #2 differs from that of line #1. The transgenic locus of line #1 will be further modified through site-specific insertion of a second marker gene to form line #3. Line #3, with first and second marker genes, will be crossed to line #2 containing the first marker gene. The efficiency of exchange by site-specific recombination between the two transgenic loci will be examined in progeny, as determined by linkage to polymorphic flanking DNA.
Progress was made on all objectives, which fall under National Program 301 Plant Genetic Resources, Genomics and Genetic Improvement, Problem Statement 3A Fundamental knowledge of plant biological and molecular processes.
The first objective of our project was to understand the virulence mechanisms of the YopJ/HopZ family of type III secreted effector proteins. To achieve this goal, we comprehensively synthesized literature on the YopJ superfamily of type III effector proteins in plant-associated bacteria. As well, we demonstrated that HopZ2 specifically targets MLO2, a plant susceptibility factor.
The second objective of our project was to develop methodology to elucidate protein-protein interactions in a high-throughput fashion. To accomplish this, we developed a high-throughput yeast two-hybrid screening platform coupled with Illumina sequencing, which allows us to create quantitative interaction maps. This is a powerful technique that can be applied to many areas of investigation.
Lewis, J.D., Wan, J., Ford, R., Gong, Y., Fung, P., Wang, P.W., Desveaux, D., Guttman, D.S. 2012. Quantitative Interactor Screening with next-generation Sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Psuedomonas syringe type III effector HopZ2. Biomed Central (BMC) Genomics. 13:8.
Lewis, J.D., Lee, A.H., Ma, W.B., Zhou, H., Guttman, D.S., Desveaux, D. 2011. The YopJ superfamily of type III efforts in plant-associated bacteria. Molecular Plant Pathology. 12(9):928-937.