M. Caroline Roper

Xylella fastidiosa requires polygalacturonase for colonization and pathogenicity in Vitis vinifera grapevines.

Xylella fastidiosa is the causal agent of Pierces disease of grape, an economically significant disease for the grape industry. X. fastidiosa systemically colonizes the xylem elements of grapevines and is able to breach the pit pore membranes separating xylem vessels by unknown mechanisms. We hypothesized that X. fastidiosa utilizes cell wall degrading enzymes to break down pit membranes, based on the presence of genes involved in plant cell wall degradation in the X. fastidiosa genome. These genes include several beta-1,4 endoglucanases, several xylanases, several xylosidases, and one polygalacturonase (PG). In this study, we demonstrated that the pglA gene encodes a functional PG. A mutant in pglA lost pathogenicity and was compromised in its ability to systemically colonize Vitis vinifera grapevines. The results indicate that PG is required for X. fastidiosa to successfully infect grapevines and is a critical virulence factor for X. fastidiosa pathogenesis in grapevine.

Cell Wall-Degrading Enzymes Enlarge the Pore Size of Intervessel Pit Membranes in Healthy and Xylella fastidiosa-Infected Grapevines

The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits, limiting the spread of xylem-localized pathogens and air embolisms from one conduit to the next. This paper provides a characterization of the size of the pores in the PMs of grapevine (Vitis vinifera). The PM porosity (PMP) of stems infected with the bacterium Xylella fastidiosa was compared with the PMP of healthy stems. Stems were infused with pressurized water and flow rates were determined; gold particles of known size were introduced with the water to assist in determining the size of PM pores. The effect of introducing trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), oligogalacturonides, and polygalacturonic acid into stems on water flux via the xylem was also measured. The possibility that cell wall-degrading enzymes could alter the pore sizes, thus facilitating the ability of X. fastidiosa to cross the PMs, was tested. Two cell wall-degrading enzymes likely to be produced by X. fastidiosa (polygalactuoronase and endo-1,4- β -glucanase) were infused into stems, and particle passage tests were performed to check for changes in PMP. Scanning electron microscopy of control and enzyme-infused stem segments revealed that the combination of enzymes opened holes in PMs, probably explaining enzyme impacts on PMP and how a small X. fastidiosa population, introduced into grapevines by insect vectors, can multiply and spread throughout the vine and cause Pierces disease.

Pantoea stewartii subsp. stewartii: lessons learned from a xylem-dwelling pathogen of sweet corn

UNLABELLED Pantoea stewartii subsp. stewartii is a Gram-negative enteric bacterium that primarily infects sweet corn. Studies of this bacterium have provided useful insight into how xylem-dwelling bacteria establish themselves and incite disease in their hosts. Pantoea stewartii subsp. stewartii is a remarkable bacterial system for laboratory studies because of its relative ease of propagation and genetic manipulation, and the fact that it appears to employ a minimal number of pathogenicity mechanisms. In addition, P. stewartii subsp. stewartii produces copious amounts of its quorum sensing (QS) signal, acyl-homoserine lactone (AHL), making it an excellent organism for studying QS-controlled gene regulation in a plant-pathogenic bacterium. In fact, P. stewartii subsp. stewartii has become the microbial paradigm for QS control of gene expression by both repression and activation via a QS regulator that binds DNA in the absence and dissociates in the presence of the signal ligand. Moreover, P. stewartii subsp. stewartii is a member of the Enterobacteriaceae, and lessons learned from its interaction with plants may be extrapolated to other plant-associated enterics, such as Erwinia, Dickeya and Pectobacterium spp., or enteric human pathogens associated with plants, such as Escherichia coli and Salmonella spp. TAXONOMY Bacteria; Gammaproteobacteria; family Enterobacteriaceae; genus Pantoea; species stewartii (Mergaert et al., 1993). MICROBIOLOGICAL PROPERTIES Gram-negative, motile, yellow pigmented, mucoid, facultative anaerobe. HOST RANGE Pantoea stewartii subsp. stewartii (Smith, 1898) Dye causes Stewarts wilt of corn (Zea mays). Early-maturing sweet corn varieties and some elite inbred maize lines are particularly susceptible. DISEASE SYMPTOMS There are two major phases of Stewarts wilt disease: (i) wilt and (ii) leaf blight. The wilt phase occurs when young seedlings are infected with P. stewartii subsp. stewartii (Fig. 1A). Water-soaked lesions first appear on the young expanding leaves and, later, seedlings may become severely wilted (Fig. 1B). The plants usually die when infected at the seedling stage. The leaf blight phase occurs when mature plants are infected (Fig. 1C). The bacteria enter the xylem and cause long linear yellow-grey lesions with a wavy margin that run parallel to the leaf veins. These lesions later turn necrotic and dark in colour. The leaf blight phase is most apparent after tasselling and does not generally cause death of the plant. In addition, the bacteria can sometimes break out of the xylem and cause pith rot in mature sweet corn plants. In resistant varieties, lesions are usually limited to only a few centimetres depending on the level of resistance of the particular hybrid (Claflin, 2000; Pataky, 2003). USEFUL WEBSITES http://www.apsnet.org/publications/apsnetfeatures/Pages/StewartsWilt.aspx.

Detection and visualization of an exopolysaccharide produced by Xylella fastidiosa in vitro and in planta.

ABSTRACT Many phytopathogenic bacteria, such as Ralstonia solanacearum, Pantoea stewartii, and Xanthomonas campestris, produce exopolysaccharides (EPSs) that aid in virulence, colonization, and survival. EPS can also contribute to host xylem vessel blockage. The genome of Xylella fastidiosa, the causal agent of Pierces disease (PD) of grapevine, contains an operon that is strikingly similar to the X. campestris gum operon, which is responsible for the production of xanthan gum. Based on this information, it has been hypothesized that X. fastidiosa is capable of producing an EPS similar in structure and composition to xanthan gum but lacking the terminal mannose residue. In this study, we raised polyclonal antibodies against a modified xanthan gum polymer similar to the predicted X. fastidiosa EPS polymer. We used enzyme-linked immunosorbent assay to quantify production of EPS from X. fastidiosa cells grown in vitro and immunolocalization microscopy to examine the distribution of X. fastidiosa EPS in biofilms formed in vitro and in planta and assessed the contribution of X. fastidiosa EPS to the vascular occlusions seen in PD-infected grapevines.

Biological Role of Pigment Production for the Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

ABSTRACT Pantoea stewartii subsp. stewartii, the causal agent of Stewarts wilt of sweet corn, produces a yellow carotenoid pigment. A nonpigmented mutant was selected from a bank of mutants generated by random transposon mutagenesis. The transposon insertion site was mapped to the crtB gene, encoding a putative phytoene synthase, an enzyme involved in the early steps of carotenoid biosynthesis. We demonstrate here that the carotenoid pigment imparts protection against UV radiation and also contributes to the complete antioxidant pathway of P. stewartii. Moreover, production of this pigment is regulated by the EsaI/EsaR quorum-sensing system and significantly contributes to the virulence of the pathogen in planta.

A Rhamnose-Rich O-Antigen Mediates Adhesion, Virulence, and Host Colonization for the Xylem-Limited Phytopathogen Xylella fastidiosa

Xylella fastidiosa is a gram-negative, xylem-limited bacterium that causes a lethal disease of grapevine called Pierces disease. Lipopolysaccharide (LPS) composes approximately 75% of the outer membrane of gram-negative bacteria and, because it is largely displayed on the cell surface, it mediates interactions between the bacterial cell and its surrounding environment. LPS is composed of a conserved lipid A-core oligosaccharide component and a variable O-antigen portion. By targeting a key O-antigen biosynthetic gene, we demonstrate the contribution of the rhamnose-rich O-antigen to surface attachment, cell-cell aggregation, and biofilm maturation: critical steps for successful infection of the host xylem tissue. Moreover, we have demonstrated that a fully formed O-antigen moiety is an important virulence factor for Pierces disease development in grape and that depletion of the O-antigen compromises its ability to colonize the host. It has long been speculated that cell-surface polysaccharides play a role in X. fastidiosa virulence and this study confirms that LPS is a major virulence factor for this important agricultural pathogen.

Siderophore-mediated iron acquisition influences motility and is required for full virulence of the xylem-dwelling bacterial phytopathogen Pantoea stewartii subsp. stewartii.

ABSTRACT Iron is a key micronutrient for microbial growth but is often present in low concentrations or in biologically unavailable forms. Many microorganisms overcome this challenge by producing siderophores, which are ferric-iron chelating compounds that enable the solubilization and acquisition of iron in a bioactive form. Pantoea stewartii subsp. stewartii, the causal agent of Stewarts wilt of sweet corn, produces a siderophore under iron-limiting conditions. The proteins involved in the biosynthesis and export of this siderophore are encoded by the iucABCD-iutA operon, which is homologous to the aerobactin biosynthetic gene cluster found in a number of enteric pathogens. Mutations in iucA and iutA resulted in a decrease in surface-based motility that P. stewartii utilizes during the early stages of biofilm formation, indicating that active iron acquisition impacts surface motility for P. stewartii. Furthermore, bacterial movement in planta is also dependent on a functional siderophore biosynthesis and uptake pathway. Most notably, siderophore-mediated iron acquisition is required for full virulence in the sweet corn host, indicating that active iron acquisition is essential for pathogenic fitness for this important xylem-dwelling bacterial pathogen.

OxyR and SoxR Modulate the Inducible Oxidative Stress Response and Are Implicated During Different Stages of Infection for the Bacterial Phytopathogen Pantoea stewartii subsp. stewartii

Reactive oxygen species (ROS) from a variety of sources are often encountered by invading plant pathogens during the infection process. Pantoea stewartii subsp. stewartii, the etiological agent of Stewarts wilt, is a serious bacterial pathogen of sweet corn that colonizes both the apoplast and xylem tissues in which ROS are produced. The P. stewartii genome predicts the presence of two redox-sensing transcriptional regulators, OxyR and SoxR, which both activate gene expression in response to oxidative stress. ROS exposure in the form of hydrogen peroxide and the superoxide-generating compound paraquat initiates an induced stress response through OxyR and SoxR that includes activation of the ROS-detoxifying enzymes alkyl hydroperoxide reductase and superoxide dismutase. P. stewartii ΔsoxR was more sensitive to paraquat and was compromised in the ability to form water-soaked lesions, while ΔoxyR was more sensitive to hydrogen peroxide treatment and was deficient in exopolysaccharide production and the elicitation of wilting symptoms. This demonstrates that both SoxR and OxyR play an important role in virulence in the different niches that P. stewartii colonize during the infection process.

Identification of a response regulator involved in surface attachment, cell–cell aggregation, exopolysaccharide production and virulence in the plant pathogen Xylella fastidiosa

Xylella fastidiosa, the causal agent of Pierces disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators (RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide (EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins.

Repertoire of novel sequence signatures for the detection of Candidatus Liberibacter asiaticus by quantitative real-time PCR

BackgroundHuanglongbing (HLB) or citrus greening is a devastating disease of citrus. The gram-negative bacterium Candidatus Liberibacter asiaticus (Las) belonging to the α-proteobacteria is responsible for HLB in North America as well as in Asia. Currently, there is no cure for this disease. Early detection and quarantine of Las-infected trees are important management strategies used to prevent HLB from invading HLB-free citrus producing regions. Quantitative real-time PCR (qRT-PCR) based molecular diagnostic assays have been routinely used in the detection and diagnosis of Las. The oligonucleotide primer pairs based on conserved genes or regions, which include 16S rDNA and the β-operon, have been widely employed in the detection of Las by qRT-PCR. The availability of whole genome sequence of Las now allows the design of primers beyond the conserved regions for the detection of Las explicitly.ResultsWe took a complimentary approach by systematically screening the genes in a genome-wide fashion, to identify the unique signatures that are only present in Las by an exhaustive sequence based similarity search against the nucleotide sequence database. Our search resulted in 34 probable unique signatures. Furthermore, by designing the primer pair specific to the identified signatures, we showed that most of our primer sets are able to detect Las from the infected plant and psyllid materials collected from the USA and China by qRT-PCR. Overall, 18 primer pairs of the 34 are found to be highly specific to Las with no cross reactivity to the closely related species Ca. L. americanus (Lam) and Ca. L. africanus (Laf).ConclusionsWe have designed qRT-PCR primers based on Las specific genes. Among them, 18 are suitable for the detection of Las from Las-infected plant and psyllid samples. The repertoire of primers that we have developed and characterized in this study enhanced the qRT-PCR based molecular diagnosis of HLB.