Molecular Basis of the Role of Calcium in Xylella fastidiosa Infection Process

Abstract

Xylella fastidiosa, a xylem-limited bacterial phytopathogen, is the causal agent of devastating diseases on many economically important plants worldwide. During plant infection, X. fastidiosa modifies the mineral content (viz., ionome) of symptomatic hosts. Specifically, calcium (Ca) concentration in infected leaves is significantly higher than healthy ones. In vitro, Ca affects virulent traits of X. fastidiosa via interaction with Ca-binding proteins and regulation of gene expression. We propose that Ca is critical for the relationships between this pathogen and its host plants. During my dissertation, I conducted studies towards understanding the molecular basis of the role of Ca during the X. fastidiosa infection process. Firstly, a microfluidic chamber (MC) system, which mimics the natural habitat of X. fastidiosa, was adapted for whole transcriptome analysis of this pathogen growing under different Ca concentration conditions. The data indicates that Ca transcriptionally regulates the machinery of type IV pili (TFP), and other genes related to pathogenicity and host adaptation of X. fastidiosa. The data were compared with our previous assessment in biofilm cells in batch culture suggesting the regulatory role of Ca in X. fastidiosa acts differently depending on the stage of the infection process. In addition, phenotypic assessment revealed that Ca enhanced natural transformation of X. fastidiosa. Secondly, a hybrid histidine kinase (HyHK) in X. fastidiosa encoded by the PD0576 gene was identified and characterized. In X. fastidiosa WM1-1, the mutation of PD0576 gene impaired cell adhesion, enhanced twitching motility, accelerated disease development, and affected gene expression including Ca-regulated genes. These results suggest that the HyHK encoded by PD0576 may interact with Ca, and is part of a regulatory cascade that influences cell adhesion, twitching motility, and virulence of X. fastidiosa. Lastly, leaf ionomes of X. fastidiosa-infected plants with different responses in terms of colonization and symptomatology were characterized. We found that the weakly virulent strain EB92-1 of X. fastidiosa cannot extensively colonize tobacco as opposed to other virulent WT strains. On the other hand, strain Temecula1 can establish a successful asymptomatic colonization in tomato, but cannot colonize sunflower nor ragweed. Results of the ionome analysis indicated that the X. fastidiosa infection-triggered Ca concentration increase in host leaves is associated only with pathogenic interactions. Overall, I found that MC is an ideal system for whole transcriptome analysis of vascular bacteria, identified molecular components of the Ca regulatory network in X. fastidiosa, and demonstrated that the X. fastidiosa infection-triggered Ca concentration increase of host leaves is associated only with pathogenic interactions. All of these findings contribute to the understanding of the molecular basis of the role of Ca in the X. fastidiosa infection process.