Identification and characterization of Xylella fastidiosa genes expressed in infected citrus plants and regulated by quorum sensors

Abstract

The causal agent of citrus variegated chlorosis (CVC) has been identified as Xylella fastidiosa, a gram-negative, xylem-limited bacterium. CVC became a major problem for citrus growers in São Paulo State and has been subject of intensive research. The genome of X. fastidiosa is being totally sequenced and several genes involved in the regulation of plant-bacteria interaction may be identified by homology to other bacterial sequences. However, many important genes may not have homology to known avirulence/virulence genes from plant pathogenic bacteria. This indirect approach for identifying biotechnologically important genes has its drawbacks and should be complemented with additional strategies. Based on the fact that citrus plants may normally show disease symptoms a long time after infection, disease development may depend on the differential expression of cell density regulated genes, and quorum sensing is a common feature of gram-negative bacteria we hypothesize that X. fastidiosa proteins in the intercellular fluid of infected plants may be involved in pathogenicity and that the expression of pathogenesis-related genes may involve an autoinducer and an autoinducer-biding transcription factor, homologous to LuxR/Luxl. To test these hypotheses, the following approaches will be used: 1.Identify and characterize proteins from the intercellular fluid of citrus plants that are induced in infected plants showing disease symptoms, compared to infected plants without symptoms and non-infected plants, using 2D-gel electrophoresis; 2. Identify and clone cell density regulated genes using representational differences analysis (RDA) coupled to microarray hybridization; 3. Determine whether X. fastidiosa synthesize an acyl homoserine lactone-autoinducer using bioassays; 4. Identify possible signal molecules (autoinducers) in the intercellular fluid of infected plants, comparing the reversed phase HPLC profiles of infected plants showing disease symptoms, infected plants without symptoms and non-infected plants. Using these approaches we expect to identify proteins/genes regulated by quorum sensors which may be involved in the control of disease development in citrus infected with X. fastidiosa. Disruption of the signal transduction pathways leading to pathogenesis-related gene expression in X. fastidiosa, e.g. using analogs of autoinducers, might be an efficient strategy to inhibit disease development. (AU)