Role of calcium during plant infection by Xylella fastidiosa
Type of DegreePhD Dissertation
Entomology and Plant Pathology
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Xylella fastidiosa (Xf) is a Gram-negative plant pathogenic bacterium that lives in the xylem vessels of infected plants and the foregut of sharpshooter insect vectors. X. fastidiosa causes diseases in many economically important crops worldwide. Virulence mechanisms of X. fastidiosa involve colonization of host plants through twitching movement, and development of biofilm leading to vessel occlusion and water stress. Essential micro- and macronutrients including calcium (Ca) are required for a myriad of functions within cells and are under tight homeostatic control. Previous results from our group show that: 1) X. fastidiosa uses Ca to enhance virulence traits, and 2) X. fastidiosa infection triggers a host response that leads to accumulation of Ca in plant tissue. In X. fastidiosa, Ca has been demonstrated to increase biofilm formation, surface attachment, cell-to-cell aggregation, and twitching motility. In tobacco plants a significant component of the defense response includes numerous genes involved in Ca signaling. These observations suggest that interactions between plant hosts and the bacterium are affected by Ca. This led us to hypothesize that X. fastidiosa is hijacking the Ca-defense response of the plant host to increase its virulence. Previous studies showed that Xf uses Ca to enhance virulence traits, such as twitching motility, while Xf infection triggers a host response that leads to accumulation of Ca in the plant tissue. Among genes transcriptionally regulated by Ca, PD0913 was selected for functional analysis because it has putative Ca-binding motifs in its sequence and is part of a putative genomic island absent in a non-virulent strain, as revealed by comparative genomics. For my first objective, PD0913-X. fastidiosa mutant was generated by site-directed mutagenesis. Analysis in vitro and in planta were performed to determine the role of this gene in bacterial virulence under different Ca concentrations. Our results showed that the absence of PD0913 increased twitching motility, surface attachment at 2mM Ca concentration, and virulence at 8mM Ca concentration, compared to the WT. This suggests that the PD0913 gene negatively regulates twitching motility and virulence of X. fastidiosa under different levels of Ca concentrations. For my second objective, tobacco plant inoculation in greenhouse and virulence assessments over time were performed in 41 selected X. fastidiosa isolates obtained from vineyards in California. For virulence assessments, area under disease progression curve (AUDPC), percentage of leaf scorch, and severity were determined. Isolates Je32, Je86, Je96 and Je121 showed the highest numbers in virulence assays when compared to TemeculaL and WM1-1 (reference controls). Contrastingly, Je98, Je111 and Je118 showed the lowest values on AUDPC, incidence and severity. No correlation was found between virulence and host cultivar or geographical location of the isolates. These results indicate that virulence differences between isolates were apparent, and have potential implications for understanding the context of genomic variability, host adaptation and geographic location on the identification and selection of resistant cultivars for the development and improvement of disease management on this bacterium. For my third objective, to define the role of CAX3 in the interaction with Xf and prove that more Ca flowing through the xylem will cause higher disease severity, an overexpression of CAX3 was made using the Gateway system. Two different markers were used: GUS and GFP, and tobacco plants were transformed by Agrobacterium. Plants could not develop from the calli generated by Agrobacterium- mediated transformation. We hypothesized that CAX3 can alter the phenotype and development of the plants and that is why no plant was grown, and we propose that Ca application in the medium can restore the phenotype. In summary, during my research I contributed to the understanding of the molecular basis of the role of Ca in the X. fastidiosa infection process, using the knowledge acquired by the interaction of the bacterium with different concentrations of Ca in vitro and in planta along with the analysis of a group of proteins that export cations of the cytosol to maintain optimal ionic concentrations in the cell and how different isolates can influence the virulence in tobacco plants.