Location: Plant Gene Expression Center2012 Annual Report
1a. Objectives (from AD-416):
1: Develop and evaluate transformation vectors that use site-specific recombination systems for more efficient and precise integration of DNA into the host genome. [NP 301, C 4, PS 4C] 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]
1b. Approach (from AD-416):
For objective 1, vectors will be constructed by recombinant DNA methods using as much as possible publicly available genetic components. Vectors will be designed for use in direct DNA transformation. Model dicot and monocot plant systems will be transformed with the initial empty vector harboring a reporter gene. The initial transformants will be screened for single copy insertion and acceptable expression level of the reporter construct. Several of the initial transformants that meet the above criteria will be propagated as "target lines". Progeny of these target lines will be used to test the site-specific integration of additional DNA into the same locus, mediated by the transient co-introduction of the appropriate recombinase gene. Site-specific integrants will be subjected to further introduction of a second recombinase gene to remove unneeded DNA. Characterization of the structure and expression of the introduced DNA will be performed by standard molecular analysis. 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.
3. Progress Report:
New scientist hired November 6, 2011. The new project is presently under OSQR review, and has adjusted objectives and approaches. 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.
1. Development of a high-throughput quantitative protein interaction platform. Determining how proteins function often requires knowing what proteins they interact with. ARS scientists in the Plant Gene Expression Center in Albany, California, developed a high-throughput method to identify interacting proteins in a quantitative fashion. The application of Illumina next-generation sequencing technology to yeast two-hybrid analysis allows results to be quantitative and easily compared among laboratories, allows assessment of cDNA library quality and completeness, and enables high-throughput analysis by eliminating the need to individually sequence the cDNA in each interacting colony. This work also identified a plant susceptibility factor MLO2 as being targeted by bacterial effector proteins. This accomplishment will facilitate the identification of defense-related genes in crop plants, in order to protect against plant diseases.
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.