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Comparative analysis of ESTs involved in grape responses to Xylella fastidiosa infection

USDA-ARS, 9611 S, Riverbend Avenue, Parlier, California 93648, USA.
BMC Plant Biology (Impact Factor: 3.94). 02/2007; 7:8. DOI: 10.1186/1471-2229-7-8
Source: PubMed

ABSTRACT The gram-negative bacterium Xylella fastidiosa (Xf) is the causal agent of Pierce's disease (PD) in grape as well as diseases of many fruit and ornamental plants. The current molecular breeding efforts have identified genetic basis of PD resistance in grapes. However, the transcriptome level characterization of the host response to this pathogen is lacking.
Twelve tissue specific subtractive suppression hybridization (SSH) cDNA libraries derived from a time course sampling scheme were constructed from stems, leaves and shoots of PD resistant and susceptible sibling genotypes (V. rupestris x V. arizonica) in response to Xf infection. A total of 5,794 sequences were obtained from these cDNA libraries from which 993 contigs and 949 singletons were derived. Using Gene Ontology (GO) hierarchy, the non-redundant sequences were classified into the three principal categories: molecular function (30%), cellular components (9%) and biological processes (7%). Comparative analysis found variations in EST expression pattern between infected and non-infected PD resistant and PD susceptible grape genotypes. Among the three tissues, libraries from stem tissues showed significant differences in transcript quality suggesting their important role in grape-Xylella interaction.
This study constitutes the first attempt to characterize the Vitis differential transcriptome associated with host-pathogen interactions from different explants and genotypes. All the generated ESTs have been submitted to GenBank and are also available through our website for further functional studies.

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    • "Transcript profiling has shown that grape plant response to Xf infection is different among species, tissues and between resistant and susceptible siblings, and the stages of infection. While broad spectrum and presumably non-specific plant responses were observed in V. vinifera species, including the induction of transcripts such as WRKY transcription factor 30, CBF like transcription factor, NDR-1 like protein, and phi-1 (an AvrPto-Pto, or AP responsive gene), a majority did not overlap with the response of the resistant genotype (Lin et al., 2007). "
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    ABSTRACT: The xylem-limited bacterium Xylella fastidiosa (Xf) causes Pierce's disease (PD) with the symptoms pri- marily due to the result of xylem vessel blockage in susceptible grapevines. Grapevine genotypes differ in their susceptibility/tolerance to Pierce's disease (PD). This may be related to the concentration and presence or absence of chemical compounds in the xylem sap and/or due to anatomical features of the xylem. Experimental as well as anecdotal information indicates that a considerable range in tolerance to PD exists among grapevine genotypes. It appears that a number of Vitis as well as Muscadinia species have evolved mechanisms that allow them to tolerate infection by Xf. However, while it is often thought that many wild genotypes evolved tolerance mechanisms, it is also possible that it is the induction of a deleterious response by V. vinifera genotypes that renders them more susceptible than tolerant geno- types which may not respond to a challenge by Xf. Therefore, understanding the mechanisms underlying differential sensitivity is critical for the development of PD resistant grapes. The rich diversity of grapevine genotypes tolerant to PD is currently being utilized to serve as a source for PD resistance for breeders. However, while PD resistant species have been identified (Mortensen et al., 1977; Krivanek and Walker, 2004; Fritschi et al., 2007), the molecular mechanisms of resistance have not been identified. Breeding of resistant genotypes is likely the most sustainable means of combating PD. In order to generate highly PD tolerant grape cultivars, knowledge of the kind and function of resistance mechanisms is paramount. Therefore, our research focuses on • Host-pathogen interactions using comparative analyses of Xf population dynamics among a group of grapevine genotypes. • Examination of xylem anatomical factors using microscopic approaches. • Evaluation of the effects of xylem saps from resistant and susceptible grapevines on planktonic growth, biofilm formation and virulence-related gene expression in Xf. • Microarray global gene expression analysis of PD resistant and susceptible grapes in response to Xf infection.
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    • "It does this by overcoming sample bias that can occur based on relative differences in abundance in mRNA through a hybridization step that normalizes sequence abundance (Diatchenko et al. 1996). This method has been successfully used for examining the genetic response to plant/pathogen interactions (Kong, Anderson, and Ohm 2005, Lin et al. 2007, Lu et al. 2005) and to uncover contributors to quantitative resistance (Han et al. 2005). Puthoff and Smigocki (2007) recently outlined the utility of SSH for studies in sugar beet, which has limited genomic data available and large varietal variability. "
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    ABSTRACT: Purified cell-wall constituents or grape xylem sap added to media affected Xylella fastidiosa (Xf) in vitro growth, biofilm formation, cell aggregation and gene expression. Media containing xylem sap from Pierce's disease (PD)-susceptible plants provided better support for bacterial growth and biofilm formation than media supplemented with xylem sap from PD-resistant plants. Culturing Xf on media containing various purified cell-wall constituents demonstrated that CM-cellulose, xylan, β-D-glucan, k-carrageenan, cello-oligosaccharide and laminarin promoted bacterial growth whereas lichenan strongly suppressed growth. However, only laminarin, xylan, and k-carrageenan promoted biofilm formation in vitro. Lichenan, oligosaccharide, k-carrageenan, laminarin, xylan and β-D-glucan all significantly decreased bacterial cell aggregation in vitro. Quantitative real-time PCR assays revealed that expression of genes encoding extracellular endoglucanase, endo-1,4-beta-glucanase, and periplasm protease were differentially regulated in response to amendment of media with xylem sap from PD-resistant and PD-susceptible grapevines. This study indicates that composition of xylem sap and cell walls may influence the interaction of Xf with grape hosts in planta and may account for differences in pathogenesis of Xf on PD-resistant and -susceptible grapevines.
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