2008 Annual Report 1a.Objectives (from AD-416) Develop an in planta expression system for functional analysis of spiroplasma and phytoplasma genes. Identify pathogen and host genes that are involved in disease induction and symptom expression. The ultimate goal of this project is to exploit virus-based constructs to identify potential molecular targets for spiroplasmal and phytoplasmal disease control. 1b.Approach (from AD-416) Analyze spiroplasma and phytoplasma genome sequences using bioinformatics tools. Establish clone libraries of selected spiroplasma and phytoplasma genes. Engineer gene constructs for expression of individual pathogen genes. Functional test of cloned spiroplasma and phytoplasma genes will be carried out in experimental host plant Nicotiana benthamiana using a Potato virus X-based vector. Inoculated plants will be examined for abnormal phenotypes caused by expression of the introduced pathogen genes. Once such abnormal phenotypes are observed, further studies will be performed on two fronts (i) mutational studies on the pathogen genes that elicited the phenotypes, and (ii) dissection of signal transduction events that lead to the host responses. 3.Progress Report The cooperative research was initiated to gain new knowledge of pathogenesis of phytoplasmas and spiroplasmas, cell wall-less bacteria that cause economically important plant diseases worldwide. Biotechnological approaches and bioinformatics tools have been used to identify pathogen and host genes involved in disease induction and symptom expression. Comparative sequence analyses were conducted on genomes of two ‘Candidatus Phytoplasma asteris’-related strains, aster yellows witches’-broom phytoplasma (AYWB) and onion yellows phytoplasma mild strain (OYM). As a result, a sophisticated molecular regulatory component, simple sequence repeats (SSRs), was found present in the genomes of the two phytoplasma strains. Several SSR tract-bearing genes were identified as potential contingency genes that may play important roles in phytoplasma-host interactions and in phytoplasmas’ rapid adaptation to vastly different intracellular environments during sequential transkingdom host-switch. Findings from computational analyses were complimented by examinations of SSRs in different phytoplasma strains that were maintained in experimental plants. Genome information was used to design primers for amplification and molecular cloning of selected putative contingency genes from several phytoplasma strains. Results revealed that SSRs in these putative contingency genes are polymorphic among different strains, reinforcing the notation that SSRs function as a regulator, controlling the expression of phytoplasma contingency genes. The potential impact of these SSRs on associated genes is currently under investigation. The findings from this cooperative research provide new knowledge on regulation of phytoplasma gene expression and phytoplasma-host interactions, which are important to a better understanding of phytoplasma pathogenesis. This project was monitored by conference calls, regularly scheduled meetings, and exchanges of ideas, materials, and data.