2011 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, we have made significant progress along two fronts. On the pathogen side, we continued to search phytoplasmal genes and regulatory elements that bear simple sequence repeats (SSRs). We found additional SSR loci that are associated with genes encoding putative secretory and cell-surface localized proteins. On the host side, we focused on investigating the involvement of plant growth regulators in phytoplasmal disease symptom modulation. Plants infected by phytoplasmas often exhibit an array of 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. We developed a semi-quantitative immunohistochemical assay protocol to evaluate endogenous levels of four major growth regulators in potato purple top (PPT) phytoplasma-infected tomato plants. We found that, in response to PPT phytoplasma infection, the levels of abscisic acid (ABA) and cytokinins (N6-benzyladenosine, trans-zeatin riboside, and cis-zeatin riboside) were elevated, and the levels of auxin (IAA) and gibberellin acid (GA3) were decreased. Quantitative RT-PCR results indicated that expression profiles of several genes responsible for growth regulator metabolism and signaling were also altered in PPT phytoplasma-infected tomato plants. Our findings provide a clue to understanding mechanisms of phytoplasma pathogenesis and symptom expression. This project was monitored by conference calls, regularly scheduled meetings, and exchanges of ideas, materials, and data.