UC Riverside

Xylella fastidiosa is a re-emerging, Gram-negative bacterium that causes disease in many economically important crops including grape, olive, and citrus. X. fastidiosa subsp. fastidiosa, the causal agent of Pierce’s disease of grapevine, remains a significant problem for grape growers in California. This plant-pathogenic bacterium resides exclusively in the foregut of its insect vectors and in the plant host xylem. Bacterial lipopolysaccharide (LPS) covers most of the cell surface of Gram-negative bacteria, like X. fastidiosa. This molecule is a well-described microbe-associated molecular pattern that elicits immune responses in mammals and plants. Vitis vinifera grapevines pre-treated with the X. fastidiosa MAMP, lipopolysaccharide (LPS), as a priming stimulus had a significant decrease in both external and internal symptoms of PD as well as the rate of overall disease progression indicating that LPS primes the grapevine immune response to better defend itself against future challenge with the X. fastidiosa pathogen. This enhanced defense was phenotypically manifested in a suppression of both internal and external symptoms. Differential gene expression analysis revealed major transcriptomic reprogramming in primed vines in response to pathogen challenge at the point of inoculation and 20 nodes distal to the point of inoculation when compared to naive, untreated vines. Furthermore, a weighted gene co-expression analysis identified modules of co-expressed genes common to the point inoculation and 20 nodes above indicating that primed vines mount a temporally and spatially synchronous response to initial pathogen challenge. These responses included genes involved in signal perception, signal transduction, as well as auxin-related pathways.X. fastidiosa produces exopolysaccharides (EPS) and forms robust biofilms in the plant host xylem and in the mouthparts of its insect vectors. Here we demonstrate that the X. fastidiosa EPS is a β-1,4-glucan backbone with alternating 3-linked side chains, one with terminal β-glucuronic acid and α-1,2-mannose residues, and the other with a terminal β-glucose and α-1,2-mannose residues. An endoglucanase mutant, ΔengXCA2, has a hyper mucoid colony morphotype in vitro suggesting that EngXCA2 is involved in EPS processing and/or turnover. Additionally, ΔengXCA2 was severely impaired in several key steps in biofilm development that included cell-cell aggregation, attachment to surface substrata and biofilm maturation. In vitro, ΔengXCA2 biofilms were structurally compromised compared to wildtype biofilms. In planta, ΔengXCA2 biofilms were encased in copious amounts of EPS compared to wild type biofilms which corroborates our in vitro findings. Recombinant EngXCA2 digested X. fastidiosa EPS confirming that EngXCA2 utilizes the EPS as a substrate. These results demonstrate that EngXCA2 plays an important role in regulating X. fastidiosa biofilm formation through enzymatic degradation of the β-1,4-glucan backbone of EPS. X. fastidiosa is nonflagellated and relies upon its type IV pili (T4P) for movement. T4P are filamentous appendages that quickly polymerize and depolymerize to pull and drag the cell along a surface. These structures are also involved in biofilm formation by linking cells to each other. Finally, to explore the role of T4P in X. fastidiosa pathogenesis in grapevines, we created three mutant strains in genes involved in the T4P machinery: pilB, pilA1, and pilA2. ΔpilB and ΔpilA1 strains were deficient in cell-cell aggregation compared to wildtype and ΔpilA2 strains. Moreover, ΔpilB- and ΔpilA1-inoculated grapevines had significantly less overall disease and lower X. fastidiosa titer while ΔpilA2 behaved similarly to wildtype. Our results indicate that PilB, a putative ATPase that drives pili extension, and PilA1, a putative pilin subunit, are necessary for PD symptom development and grapevine colonization.