INHIBITION OF XYLELLA FASTIDIOSA POLYGALACTURONASE TO PRODUCE PIERCE’S DISEASE RESISTANT GRAPEVINES Project Leader:

Polygalacturonases (PG) (EC 3.2.1.15), catalyze the random hydrolysis of 1, 4-alpha-D-galactosiduronic linkages in pectate and other galacturonans. Xylella fastidiosa (Xf) possesses a single PG gene, pglA (PD1485) and Xf mutants deficient in the production of PG result in lost pathogenicity and a compromised ability to systemically infect grapevines. We have cloned the pglA gene into a number of protein expression vectors and a small amount of active recombinant PG has been recovered, unfortunately most of the protein expressed is found in inclusion bodies in an inactive form. The goal of this project is to use phage panning to identify peptides that can bind to and inhibit Xf PG. Once peptides are discovered that can inhibit PG activity in vitro these peptides will be expressed in grapevine root stock to determine if the peptides can provide protection to the plant from Pierce’s disease. INTRODUCTION Polygalacturonases (PG) have been shown to be virulence factors of a number of plant pathogenic bacteria including Ralstonia solanacearum, Xanthomonas campestris, and Erwinia carotova (Huang and Allen 2000; Dow et al. 1989; Lei et al. 1985). Xylella fastidiosa (Xf) possesses a single PG gene pglA (PD1485), and mutation of this gene results in lost pathogenicity and reduced ability to systemically infect grapevines (Roper et al. 2007). In order for Xf to systemically infect a grapevine it must break down the pit membranes that separate individual xylem elements. Pectic polymers determine the porosity of the pit membrane (Baron-Epel, et al. 1988; Buchanan et al. 2000) and Xf PG allows the bacterium to breakdown the pectin in these membranes. The premise of this research is to identify a peptide that can be expressed in the xylem of a grapevine that can suppress Xf PG activity thus limiting the ability of Xf to spread systemically through grapevines and cause Pierce’s disease (PD). To accomplish this we will use phage display of a random dodecapeptide library attached to the coat protein gp38 of M13 phage in a phage panning experiment using active recombinant Xf PG as the target. After three rounds of panning, phage that show a high binding affinity for Xf PG will be screened for their ability to inactivate PG activity in vitro in reducing sugar assays. Once a suitable inhibitory peptide is discovered it will be cloned into an Agrobacterium binary vector and used to transform tobacco and grapevines by the UCD Plant Transformation Facility. These transgenic plants will then be inoculated with Xf and compared to non transgenic plants in PD symptom progression. If significant disease inhibition is shown we will use these transgenic grapevines as rootstock and see if they can also provide resistance to grafted scions. OBJECTIVES 1. Isolate a sufficient amount of biologically active Xf polygalacturonase enzyme to conduct phage panning and PGinhibition assays. 2. Isolate M13 phage that possess high binding affinities to Xf PG from a M13 random peptide library. 3. Determine if selected M13 phage and the gp38 M13 protein which mediates phage binding to Xf PG can inactivate PG activity in vitro. 4. Clone anti-Xf PG gp38 protein into an Agrobacterium binary vector and provide this construct to the UCD Plant Transformation facility to produce transgenic SR1 tobacco and Thompson Seedless grapevine. 5. Determine if anti-Xf PG gp38 protein is present in xylem sap of transgenic plants. 6. Mechanically inoculate transgenic plants with Xf and compare PD development with inoculated, non-transgenic control plants. RESULTS Objective 1.Currently we have obtained a small amount of active recombinant Xf PG, however, most of the expressed protein is in the form of insoluble and inactive inclusion bodies. Fortunately it is possible to measure the activity of the small amount of active PG that we have obtained by using visible spectrum reducing sugar assays such as the dinitrosalicylic acid (Figure 1) (Wang et al. 1997, Sumner 1921) and 3-Methyl-2-benzothiazolinonehydrazone methods (Anthon and Barrett 2002, Honda et al. 1981). This is a significant improvement over the tedious High-performance liquid chromatography (HPLC) assays that were previously used to demonstrate Xf PG activity (Roper et al. 2007). These methods should also be appropriate for the PG-inhibition assays once we have determined a suitable candidate peptide, however, we have yet to