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

A suberized exodermis is required for tomato drought tolerance.

Plant roots integrate environmental signals with development using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates flow of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation but is absent in the tomato endodermis. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here we demonstrate that the suberin regulatory network has the same parts driving suberin production in the tomato exodermis and the Arabidopsis endodermis. Despite this co-option of network components, the network has undergone rewiring to drive distinct spatial expression and with distinct contributions of specific genes. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response and that the exodermal barrier serves an equivalent function to that of the endodermis and can act in its place

A Systematic Review of Argumentation Related to the Engineering-Designed World

Background Across academic disciplines, researchers have found that argumentation‐based pedagogies increase learners\u27 achievement and engagement. Engineering educational researchers and teachers of engineering may benefit from knowledge regarding how argumentation related to engineering has been practiced and studied. Purpose/Hypothesis Drawing from terms and concepts used in national standards for K‐12 education and accreditation requirements for undergraduate engineering education, this study was designed to identify how arguments and argumentation related to the engineering‐designed world were operationalized in relevant literature. Methodology Specified search terms and inclusion criteria were used to identify 117 empirical studies related to engineering argumentation and educational research. A qualitative content analysis was used to identify trends across these studies. Findings Overall, engineering‐related argumentation was associated with a variety of positive learner outcomes. Across many studies, arguments were operationalized in practice as statements regarding whether an existing technology should be adopted in a given context, usually with a limited number of supports (e.g., costs and ethics) provided for each claim. Relatively few studies mentioned empirical practices, such as tests. Most studies did not name the race/ethnicity of participants nor report engineering‐specific outcomes. Conclusions Engineering educators in K‐12 and undergraduate settings can create learning environments in which learners use a range of epistemic practices, including empirical practices, to support a range of claims. Researchers can study engineering‐specific outcomes while specifying relevant demographics of their research participants

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