The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

Brittany K Pierce,Tanja Voegel,Bruce C Kirkpatrick

doi:10.1371/journal.pone.0113504

Brittany K Pierce, Tanja Voegel + Show 1 more

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https://doi.org/10.1371/journal.pone.0113504

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Abstract

Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa.

Highlights

Xylella fastidiosa (Xf) is a gram-negative, xylem-limited plant pathogenic bacterium and the causal agent of a variety of economically important diseases including Pierce’s disease (PD) of grapevines, almond leaf scorch and citrus variegated chlorosis [1,2,3,4]
Our studies indicate that PD1063 plays a role in cell-cell aggregation in vitro but does not support a role for PD1063 in regulation of biofilm formation or as pathogenicity factor
PD1063 was PCR amplified from the Xf wild type Fetzer genome using primer pairs PD1063for (59-TCAGCGCTCTAAAACAATGGCGA-39) and PD1063rev (59-CACAGCCCGCCTATGGCACA) and the 1015 bp product was cloned into pCR2.1-TOPO vector to generate pCR2.1-PD1063 (Table 1)

Summary

Introduction

Xylella fastidiosa (Xf) is a gram-negative, xylem-limited plant pathogenic bacterium and the causal agent of a variety of economically important diseases including Pierce’s disease (PD) of grapevines, almond leaf scorch and citrus variegated chlorosis [1,2,3,4]. Once transmitted to the host plant, Xf forms biofilms within the xylem vessels, allowing the pathogen to form a protected niche in which the bacteria can multiply. Bacteria within these protected niches may form large aggregates that effectively plug the xylem element, impede or block transpiration and induce scorching symptoms, similar to what occurs when plants are under water stress. Some plant hosts, such as Vitis vinifera grapevines, often die from Xf infection [2]

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