A SCREEN FOR XYLELLA FASTIDIOSA GENES INVOLVED IN TRANSMISSION BY INSECT VECTORS

• Author(s): Lindow, Steven; Purcell, Alexander; Baccari, Clelia;
• Abstract: A strategy is being pursued that will identify genes involved in insect transmission of Xylella fastidiosa (Xf) by both gain of function and loss of function mutagenesis of a Xf mutant that is unable to be transmitted by sharpshooters. KLN61 is an rpfF mutant strain of Xf (Newman et al. 2004) that cannot be transmitted to plants by the sharpshooter leafhopper, Graphocephala atropunctata, a common vector of the wild-type strain of this bacterium. This mutant, which did not form biofilm and was not retained in the vector mouthparts (Figure 1), was hypervirulent and formed biofilm when mechanically inoculated into grapevines (Newman et al. 2004). In this work, we are further investigating the rpfF mutant non-transmissible phenotype. A molecular approach is being used to further mutate this mutant using a transposome-mediated mutagenesis technique. We created a mutant library compatible with the KLN61 mutant background in order to identify other Xf genes involved in the complex process of transmission. We designed and successfully constructed a Streptomycin EZ::TN custom transposome mutagenesis system in order to further mutate the Xf rpfF mutant strain, KLN61. We introduced our transposome into KLN61 by electroporation, yielding 5 X 10(3) mutants per g of DNA. To date we have mechanically inoculated grapevines with about a thousand mutants. In complementary studies we are examining the process of colonization of plants by Xf and determining the extent to which cell-cell communication via signal molecule production occurs in planta in Xf. We are testing whether production of a signaling molecule in the plant by the wild-type Xf strain would restore the transmissibility of the mutant. The finding that co-inoculations with two different Xf strains results in infections in which each stain is equally likely to be found as the dominant strain suggests that Pierces disease (PD) is characterized by a process by which many sequential occurrences of movement of a few cells to neighboring xylem vessels occurs in the process of colonizing grape plants.
• Publication Date: Dec 2005
• Journal: 2005 Pierce's Disease Research Symposium