Complete Genome Sequence of Xanthomonas arboricola pv. juglandis 417, a Copper-Resistant Strain Isolated from Juglans regia L.

Here, we report the complete genome sequence of Xanthomonas arboricola pv. juglandis 417, a copper-resistant strain isolated from a blighted walnut fruit (Juglans regia L. cv. Chandler). The genome consists of a single chromosome (5,218 kb)

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction

Molecular Profiling of Pierce’s Disease Outlines the Response Circuitry of Vitis vinifera to Xylella fastidiosa Infection

Pierce’s disease is a major threat to grapevines caused by the bacterium Xylella fastidiosa. Although devoid of a type 3 secretion system commonly employed by bacterial pathogens to deliver effectors inside host cells, this pathogen is able to influence host parenchymal cells from the xylem lumen by secreting a battery of hydrolytic enzymes. Defining the cellular and biochemical changes induced during disease can foster the development of novel therapeutic strategies aimed at reducing the pathogen fitness and increasing plant health. To this end, we investigated the transcriptional, proteomic, and metabolomic responses of diseased Vitis vinifera compared to healthy plants. We found that several antioxidant strategies were induced, including the accumulation of gamma-aminobutyric acid (GABA) and polyamine metabolism, as well as iron and copper chelation, but these were insufficient to protect the plant from chronic oxidative stress and disease symptom development. Notable upregulation of phytoalexins, pathogenesis-related proteins, and various aromatic acid metabolites was part of the host responses observed. Moreover, upregulation of various cell wall modification enzymes followed the proliferation of the pathogen within xylem vessels, consistent with the intensive thickening of vessels’ secondary walls observed by magnetic resonance imaging. By interpreting the molecular profile changes taking place in symptomatic tissues, we report a set of molecular markers that can be further explored to aid in disease detection, breeding for resistance, and developing therapeutics

Trans-Graft Protection Against Pierce’s Disease Mediated by Transgenic Grapevine Rootstocks

A field study showed that transgenic grapevine rootstocks can provide trans-graft-mediated protection to a wild type scion against Pierce’s disease (PD) development. We individually field-tested two distinct strategies. The first expressed a chimeric antimicrobial protein (CAP) that targeted the functionality of the lipopolysaccharide (LPS) surface of Xylella fastidiosa (Xf), the causative agent of PD. The second expressed a plant polygalacturonase inhibitory protein (PGIP) that prevents PD by inhibiting breakdown of pectin present in primary cell walls. Both proteins are secreted to the apoplast and then into the xylem, where they migrate past the graft union, transiting into the xylem of the grafted scion. Transgenic Vitis vinifera cv. Thompson Seedless (TS) expressing ether CAP or PGIP were tested in the greenhouse and those lines that showed resistance to PD were grafted with wild type TS scions. Grafted grapevines were introduced into the field and tested over 7 years. Here we present data on the field evaluation of trans-graft protection using four CAP and four PGIP independent rootstock lines, compared to an untransformed rootstock. There was 30 to 95% reduction in vine mortality among CAP- and PGIP-expressing lines after three successive yearly infections with virulent Xf. Shoot tissues grafted to either CAP or PGIP transgenic rootstocks supported lower pathogen titers and showed fewer disease symptoms. Grafted plants on transgenic rootstocks also had more spring bud break following infection, more shoots, and more vigorous growth compared to those grafted to wild type rootstocks. No yield penalty was observed in the transgenic lines and some PGIP-expressing vines had enhanced yield potential. Trans-graft protection is an efficient way to protect grape scions against PD while preserving their valuable varietal genotypes and clonal properties

Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors of Xylella fastidiosa (LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we present in vivo validation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein from Xanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providing in silico validation before proceeding to the laborious in vivo work. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction

Complete Genome Sequence of Xanthomonas arboricola pv. juglandis 417, a Copper-Resistant Strain Isolated from Juglans regia L.

Here, we report the complete genome sequence of Xanthomonas arboricola pv. juglandis 417, a copper-resistant strain isolated from a blighted walnut fruit (Juglans regia L. cv. Chandler). The genome consists of a single chromosome (5,218 kb)

Sequence/structural analysis of xylem proteome emphasizes pathogenesis-related proteins, chitinases and β-1, 3-glucanases as key players in grapevine defense against Xylella fastidiosa

Background. Xylella fastidiosa, the causative agent of various plant diseases including Pierce’s disease in the US, and Citrus Variegated Chlorosis in Brazil, remains a continual source of concern and economic losses, especially since almost all commercial varieties are sensitive to this Gammaproteobacteria. Differential expression of proteins in infected tissue is an established methodology to identify key elements involved in plant defense pathways. Methods. In the current work, we developed a methodology named CHURNER that emphasizes relevant protein functions from proteomic data, based on identification of proteins with similar structures that do not necessarily have sequence homology. Such clustering emphasizes protein functions which have multiple copies that are up/down-regulated, and highlights similar proteins which are differentially regulated. As a working example we present proteomic data enumerating differentially expressed proteins in xylem sap from grapevines that were infected with X. fastidiosa. Results. Analysis of this data by CHURNER highlighted pathogenesis related PR-1 proteins, reinforcing this as the foremost protein function in xylem sap involved in the grapevine defense response to X. fastidiosa. β-1, 3-glucanase, which has both anti-microbial and anti-fungal activities, is also up-regulated. Simultaneously, chitinases are found to be both up and down-regulated by CHURNER, and thus the net gain of this protein function loses its significance in the defense response. Discussion. We demonstrate how structural data can be incorporated in the pipeline of proteomic data analysis prior to making inferences on the importance of individual proteins to plant defense mechanisms. We expect CHURNER to be applicable to any proteomic data set