Development of experimental assays for screening pathogenicity and adhesion of Xylella fastidiosa mutants

Abstract

Our objectives are to take advantage of the new Xylella fastidiosa (Xf) genome data base and recent developments in Xf research at Universidade de São Paulo (Piracicaba), Universidade Estadual Paulista (Jaboticabal) and University of California (Berkeley and Davis). The scientists involved have complementary interests and expertise to develop molecular methods to address the question of how Xf attaches to insect vector and plant substrates. This is probably an important feature of how vectors transmit Xf and how the bacterium moves systemically within plants causing diseases such as citrus variegated chlorosis (CVC) and Pierce's Disease (PO). Specific goals and activities proposed are directly related to the main objectives of FAPESP's Functional Genomics Program (2nd Edital) and involve: 1) development of experimental assays to screen Xf mutants for pathogenicity in citrus, with optimization of techniques for mechanical inoculation and expression of CVC symptoms, as well as adaptation of a method for culture and quantification of viable bacterial populations in inoculated plants; 2) adaptation of in vitro systems (membrane feeding and flow cell) to study Xylella-vector interactions, particularly bacteriaI adhesion to the mouthparts and transmission, as well as development of new methodologies for microscopy studies of Xf in plants and insect vectors; and 3) development of transposon mutagenesis for production of Xf mutants. Mutants with disrupted type-IV fimbriae genes will be used in preliminary tests with these techniques to assess attachment in CVC vectors and multiplication in citrus. Natural mutants derived from a PO strain of Xf that are mildly or not virulent will be used to study attachment of Xf in host plants and vectors. (AU)