Role of zinc in biofilm production and virulence of Xylella fastidiosa
Type of Degreethesis
Entomology and Plant Pathology
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Xylella fastidiosa is a Gram-negative plant pathogenic bacterium that exclusively colonizes the xylem vessels of the host plant and the foreguts of sharpshooter insect vectors (Cicadelidae, Cercopidae, Machaerotidae, and Cicadidae). X. fastidiosa infects a wide range of hosts, most of them of great economic value, including grape, almond, pecan, blueberry, peach, coffee and citrus trees. Diseases caused by this bacterium are mainly restricted to the Americas, and in the United States, they are restricted to the southern states and California, where the bacterium is endemic. Currently, there is no effective control strategy for X. fastidiosa. X. fastidiosa-infected plants can show several symptoms depending on the host, including marginal leaf scorch and chlorosis, berry mummification, dwarfing, shortened internodes and in extreme cases plant death. These symptoms have been associated with water deficits caused by xylem blockage due to the formation of bacterial biofilms and products of plant defense response such as gums and tyloses. Xylem sap chemistry has a great impact on the expression of virulence traits by this bacterium, which includes twitching motility, surface and cell-to-cell attachment and biofilm production. Heavy metals, such as Zn have been shown to reduce biofilm production in vitro; however, there are contradictory reports on the role of this metal element during infection. Thus, the objectives of this work were to address the effects of Zn on the physiology and virulence traits of X. fastidiosa in vitro and to evaluate its role during disease development. Batch cultures supplemented with Zn showed that this metal reduces growth and biofilm production; however, it induces an increase in the production of exopolysaccharides (EPS). In microfluidic chambers, under flow condition and with constant bacterial supplementation (closer to conditions inside the host), a dramatic increase in biofilm aggregates was seen when Zn was supplemented to the basal medium. This phenomenon was attributed to the increased EPS production induced by Zn. Additionally; viability analyses suggest that X. fastidiosa may be able to enter the viable but non-culturable state in vitro, and that Zn can hasten the onset of this state. The effects of Zn on virulence of X. fastidiosa and disease development were assessed by the construction and evaluation of two mutant strains defective in zur and czcD, two genes involved in Zn homeostasis. When grown in synthetic media both mutants accumulated higher intracellular levels of Zn than the wild type (WT), showing that these genes are involved in the efflux of this metal. The mutant strains were also affected in their abilities to grow in media supplemented with Zn and in xylem sap, compared to the WT. Biofilm production was increased or not affected, in the zur and czcD mutant; respectively, however this effect was dependent on the concentration of Zn in the medium. EPS production was reduced in both mutant strains only in media supplemented with Zn. Plants inoculated with either zur or czcD mutants had reduced bacterial populations and reduced symptoms compared to plants inoculated with the WT. Additionally, both mutants failed to produce an extensive alteration to the leaf ionome of inoculated plants, as was seen in plants inoculated with the WT. Together, these results show that Zn acts as a stress factor that reduces the growth, viability and culturability of X. fastidiosa, and suggests that the increased EPS production observed may be a response to counteract the effects of stress. Thus, detoxification of detrimental Zn levels in the host xylem vessels, constitutes an important virulence and survival strategy, which allows a more efficient colonization and alteration of the host physiological status.