2010 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. In this reporting period, our research has progressed significantly in three areas. On the pathogen side, we continued to focus on identification and characterization of putative phytoplasma phase-variable or contingency genes that bear simple sequence repeats (SSRs). We searched SSR loci in additional phytoplasma genomes and narrowed our focus to SSR loci that encode secreted and cell-surface localized proteins. Findings from computational analyses were complimented by examinations of selected SSR loci in different phytoplasma strains that were maintained in experimental plants. Results revealed that SSRs in some putative contingency genes were polymorphic among several closely-related aster yellows phytolasma strains, supporting the notation that SSRs may function as a regulator of these genes. Functional assays of these SSR-associated genes are being undertaken to assess potential roles of the regulatory loci. On the host side, we examined involvement of plant growth regulators in phytoplasmal disease symptom modulation. Phytoplasma infections of plants induce symptoms including excessive shoot proliferation, witches’-broom growths, general stunting, rapid senescence, and abnormal floral development. These symptoms indicate that hormonal balance may be disrupted in affected plants. The lack of plant hormone biosynthesis genes in all completely-sequenced phytoplasma genomes implies that the presumed hormonal imbalance in phytoplasma-infected plants may be caused either by changes in endogenous hormone levels or by alterations in sensitivity to hormones. Tissue sections prepared from potato purple top (PPT) phytoplasma-infected and healthy tomato plants were subjected to comparative immunohistochemical analyses using antibodies against auxin, cytokinins, abscisic acid, and gibberellic acid. Our results revealed notable changes in levels and distribution patterns of plant hormones in PPT-infected vs healthy plants. Microarray and qRT-PCR results indicated that expression profiles of several genes responsible for growth regulator metabolism and signaling were altered in phytoplasma-infected plants. We also found that exogenous applications of growth regulators were able to modify PPT disease symptoms in tomato. The findings provide a clue to understanding mechanisms of phytoplasma pathogenesis and symptom expression. In this reporting period, we also established and maintained a aster leafhopper colony in preparation for developing an insect transformation system for functional analysis of phytoplasma pathogenesis-related genes. This project was monitored by conference calls, regularly scheduled meetings, and exchanges of ideas, materials, and data.