Polysaccharide compositions of intervessel pit membranes contribute to Pierce's disease resistance of grapevines.

Symptom development of Pierce's disease (PD) in grapevine (Vitis vinifera) depends largely on the ability of the bacterium Xylella fastidiosa to use cell wall-degrading enzymes (CWDEs) to break up intervessel pit membranes (PMs) and spread through the vessel system. In this study, an immunohistochemical technique was developed to analyze pectic and hemicellulosic polysaccharides of intervessel PMs. Our results indicate that PMs of grapevine genotypes with different PD resistance differed in the composition and structure of homogalacturonans (HGs) and xyloglucans (XyGs), the potential targets of the pathogen's CWDEs. The PMs of PD-resistant grapevine genotypes lacked fucosylated XyGs and weakly methyl-esterified HGs (ME-HGs), and contained a small amount of heavily ME-HGs. In contrast, PMs of PD-susceptible genotypes all had substantial amounts of fucosylated XyGs and weakly ME-HGs, but lacked heavily ME-HGs. The intervessel PM integrity and the pathogen's distribution in Xylella-infected grapevines also showed differences among the genotypes. In pathogen-inoculated, PD-resistant genotypes PM integrity was well maintained and Xylella cells were only found close to the inoculation site. However, in inoculated PD-susceptible genotypes, PMs in the vessels associated with bacteria lost their integrity and the systemic presence of the X. fastidiosa pathogen was confirmed. Our analysis also provided a relatively clear understanding of the process by which intervessel PMs are degraded. All of these observations support the conclusion that weakly ME-HGs and fucosylated XyGs are substrates of the pathogen's CWDEs and their presence in or absence from PMs may contribute to grapevine's PD susceptibility.

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- beta -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 Pierce's disease.

Characterization of the cell wall of the ubiquitous plant pathogen Botrytis cinerea.

The ascomycete Botrytis cinerea is a destructive and ubiquitous plant pathogen and represents a model organism for the study of necrotrophic fungal pathogens. Higher fungi possess a complex and dynamic multilayer cell wall involved in crucial aspects of fungal development, growth and pathogenicity. Plant resistance to microbial pathogens is determined often by the capacity of the plant to recognize molecular patterns associated with the surface of an interacting microbe. Here we report the chemical characterization of cell walls from B. cinerea during axenic growth. Neutral sugars and proteins constituted most of the mass of the B. cinerea cell walls, although chitin and uronic acids were detected. Glucose was the most abundant neutral sugar, but arabinose, galactose, xylose and mannose also were present. Changes in cell wall composition during culture were observed. As the culture developed, protein levels declined, while chitin and neutral sugars increased. Growth of B. cinerea was associated with a remarkable decline in the fraction of its cell wall material that was soluble in hot alkali. These results suggest that the cell wall of B. cinerea undergoes significant modifications during growth, possibly becoming more extensively covalently cross-linked, as a result of aging of mycelia or in response to decreasing nutrient supply or as a consequence of increasing culture density.

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

Xylella fastidiosa is the causal agent of Pierce's 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.

Temporal sequence of cell wall disassembly events in developing fruits. 1. Analysis of raspberry (Rubus idaeus).

Raspberry fruits were harvested at five developmental stages, from green to red ripe, and the changes in cell wall composition, pectin and hemicellulose solubilization, and depolymerization were analyzed. Fruit softening at intermediate stages of ripening was associated with increased pectin solubilization, which occurred without depolymerization. Arabinose was found to be the most abundant noncellulosic neutral sugar in the cell wall and showed dramatic solubilization late in ripening. No changes in pectin molecular size were observed even at the 100% red stage. Subsequently, as fruit became fully ripe a dramatic depolymerization occurred. In contrast, the hemicellulosic fractions showed no significant changes in content or polymer size during ripening. The paper discusses the sequence of events leading to cell wall disassembly in raspberry fruit.

Temporal sequence of cell wall disassembly events in developing fruits. 2. Analysis of blueberry (Vaccinium species).

Softening and pathogen susceptibility are the major factors limiting the marketing of blueberries as fresh fruits, and these traits are associated with fruit cell wall structure. However, few studies that characterize wall modifications occurring during development and ripening have been reported for this fruit. In this study the ripening-associated modifications of blueberry fruit cell walls (composition, pectin and hemicellulose solubilization, and depolymerization) at five stages of ripeness have been analyzed. Xylose was found to be the most abundant noncellulosic neutral sugar associated with fruit walls, and the observed high Xyl/Glc ratio suggested that xylans, which are usually a minor hemicellulosic fruit wall component, are abundant in blueberry. The pectic matrix showed increased solubilization at early and intermediate stages of ripening, but no changes were detected in late ripening. Furthermore, little reduction in pectin polymer size occurred during blueberry ripening. In contrast, hemicellulose levels decreased as ripening progressed, and a clear depolymerization of these components was observed. A model for cell wall degradation in this fruit is discussed.

Detection of uronic oxidase activity in ripening peaches.

Uronic acid oxidase activity was found in an extract from harvested peaches that was incubated with citrus pectin at pH 8.5. The product of this reaction was identified by GC-MS analysis to be galactaric acid. The reaction was linear at 37 degrees C for up to 20 h, and the pH optimum was 8.5. The activity found in firm peaches one day after harvest did not change as the peaches softened over 5 days to eating softness. The incubation conditions were those suitable for monitoring the activity of pectate lyase, but instead of finding an increase in galacturonosyl residue reducing groups due to generation of pectin-derived oligosaccharides, uronic acid oxidase catalyzed the oxidation of the aldehyde reducing functions to carboxyl groups.

Properties of a polygalacturonase-inhibiting protein isolated from 'Oroblanco' grapefruit.

Polygalacturonase inhibiting protein (PGIP) was extracted from 'Oroblanco' grapefruit type (triploid pummelo-grapefruit) albedo tissue, purified and partially characterized. Extraction was carried out at 4 degrees C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration the extract was chromatographed on concanavalin A-Sepharose. The PGIP activity was bound by the lectin and then eluted using 250 mM alpha-methyl mannopyranoside, resulting in a 17-fold purification of the PGIP and demonstrating its glycoprotein nature. The anion-exchange and size-exclusion chromatography steps that followed gave a PGIP that was 857-fold purified relative to the initial tissue extract, and having a 44 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibition activity was tested with endo-polygalacturonase (EC 3.2.1.15) produced by Penicillium italicum and Botrytis cinerea. The radial diffusion and reducing sugar assays showed that P. italicum and B. cinerea endo-PGs were affected by PGIP, whereas no endo-PG activity was detected in the culture filtrate of P. digitatum. In vitro tests revealed that PGIP inhibited P. italicum and B. cinerea growth. By contrast, the influence of PGIP on P. digitatum, growth was negligible, perhaps because this fungus does not produce endo-PG. Following heating for 10 min at 65 degrees C the inhibitory activity of PGIP was reduced by 43%. PGIP activity decreased further as heating temperature increased, and was completely suppressed after heating at 100 degrees C for 10 min.

Changes in cell wall pectins accompanying tomato (Lycopersicon esculentum Mill.) paste manufacture.

The texture of processed tomato products is influenced by the size and solubility characteristics of soluble and particle-bound cell wall polysaccharides they contain. The acidic (pectin) polysaccharides are important contributors to texture because of their gel-forming capability and the fact that they can form aggregates. The present work describes the pectins in ripe tomato fruits and then follows changes in several classes of pectins as the fruits are subjected to hot break and the juice is subsequently concentrated to a 30 degrees Brix paste. Continued processing leads to progressive solubilization and depolymerization of polysaccharides so that the ionically and covalently bound materials that are the major pectin classes of ripe fruit are substantially reduced in amount with the concomitant increase in the more soluble water-soluble pectins of the paste product. Juice content of soluble solids ( degrees Brix) rose steadily as water content was reduced during processing. Juice content of polymeric uronic acids (i.e., pectin) also rose with concentration, but to a lesser degree than the increase in soluble solids. This indicates that processing leads to almost complete pectin depolymerization and/or the alteration of uronic acid structures so that this assay could no longer detect them. It was concluded that reductions in heat input during processing would lead to pastes with greater pectin integrity and enhanced textural characteristics.

Xylella fastidiosa infection and ethylene exposure result in xylem and water movement disruption in grapevine shoots.

It is conventionally thought that multiplication of the xylem-limited bacterium Xylella fastidiosa (Xf) within xylem vessels is the sole factor responsible for the blockage of water movement in grapevines (Vitis vinifera) affected by Pierce's disease. However, results from our studies have provided substantial support for the idea that vessel obstructions, and likely other aspects of the Pierce's disease syndrome, result from the grapevine's active responses to the presence of Xf, rather than to the direct action of the bacterium. The use of magnetic resonance imaging (MRI) to observe the distribution of water within the xylem has allowed us to follow nondestructively the development of vascular system obstructions subsequent to inoculation of grapevines with Xf. Because we have hypothesized a role for ethylene produced in vines following infection, the impact of vine ethylene exposure on obstruction development was also followed using MRI. In both infected and ethylene-exposed plants, MRI shows that an important proportion of the xylem vessels become progressively air embolized after the treatments. The loss of xylem water-transporting function, assessed by MRI, has been also correlated with a decrease in stem-specific hydraulic conductivity (K(S)) and the presence of tyloses in the lumens of obstructed water conduits. We have observed that the ethylene production of leaves from infected grapevines is greater than that from healthy vines and, therefore, propose that ethylene may be involved in a series of cellular events that coordinates the vine's response to the pathogen.

Cell wall disassembly events in boysenberry (Rubus idaeus L. × Rubus ursinus Cham. & Schldl.) fruit development.

Boysenberry fruit was harvested at five developmental stages, from green to purple, and changes in pectin and hemicellulose solubilisation and depolymerisation, polymer neutral sugar contents, and the activities of cell wall degrading enzymes were analysed. The high xylose to glucose ratio in the 4% KOH-soluble hemicellulose fraction suggests that xylans are abundant in the boysenberry cell wall. Although the cell wall changes associated with fruit development do not proceed in discrete stages and the cell wall disassembly is a consequence of highly regulated changes occurring in a continuum, the results suggest that the temporal changes in cell wall degradation in boysenberry account for at least three stages: an early stage (green to 75% red colour), associated with metabolism of cellulose and cross-linking glycans; an intermediate period (75 to 100% red colour), characterised by substantial pectin solubilisation without depolymerisation in which α-arabinofuranosidase increases markedly and 50% of the wall arabinose is lost; and a final stage (100% red colour to purple), characterised mainly by a reduction of pectic galactose content and a dramatic increase in pectin depolymerisation associated with higher polygalacturonase, pectin methylesterase, acetyl esterase and ß-galactosidase activities. From a biotechnological perspective enzymes involved in pectin matrix disassembly seem to be the better candidates to affect boysenberry fruit late-softening by genetic intervention. A model for cell wall disassembly in boysenberry fruit is proposed.

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

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 Pierce's 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.

Determination of pathogen-related enzyme action by mass spectrometry analysis of pectin breakdown products of plant cell walls.

An analytical approach using matrix-assisted laser desorption/ionization mass spectrometry for the structural characterization and assessment of the degree of polymerization of cell wall pectin-derived oligosaccharides (PDOs) in three regions of Botrytis cinerea-infected tomato fruit tissue is described. The PDOs were isolated from lesion centers (extensively macerated tissue), the area just beyond visible lesion margins, and healthy and intact tissue of an inoculated fruit, sampled at a distance from developing lesions. PDO mixtures were directly analyzed by mass spectrometry without chromatographic separation, after minimum cleanup by membrane drop dialysis. The structures identified implied the action of three different pathogen pectin-modifying enzymes. Modifications such as methyl esterification were identified by determination of exact PDO molecular masses and tandem mass spectrometry via collision-induced dissociation. We have identified four PDO series that were generated through the breakdown of homogalacturonan pectins. The decayed and lesion edge areas had fewer and less diverse PDOs than healthy tissues, possibly due to metabolic by-products of the pathogen. This analytical technique provides a simple and rapid method to characterize the pectin-derived oligosaccharides produced by in vivo digestion during pathogen infection.

Micro-injection of Lygus salivary gland proteins to simulate feeding damage in alfalfa and cotton flowers.

Alfalfa and cotton flowers were pierced with small glass capillaries of an overall size and shape similar to that of Lygus stylets, and injected with small quantities (6 to 100 nL) of solutions that contained Lygus salivary enzymes. Crude and partially purified protein solutions from Lygus heads and isolated salivary glands showed substantial polygalacturonase (PG) activity, as has been previously reported. Following injection with both crude and partially purified protein solutions, as well as with pure fungal and bacterial PGs, flowers of both alfalfa and cotton exhibited damage similar to that caused by Lygus feeding. Injection with the same volume of a buffer control as well as a buffer control containing BSA at a comparable protein concentration (approximately 6 microg/mL) showed no symptoms. These results are consistent with a previously suggested hypothesis that the extensive tissue damage caused by Lygus feeding is primarily due to the action of the PG enzyme on the host tissue, rather than to mechanical damage caused by the insect stylet. Substantial genotypic variation for a PG inhibiting protein (PGIP) exists in alfalfa and cotton. We, therefore, suggest that breeding and selection for increased native PGIP levels, or transformation with genes encoding PGIP from other plant species, may be of value in obtaining alfalfa and cotton varieties that are more resistant to Lygus feeding damage.

Gibberellic acid, synthetic auxins, and ethylene differentially modulate alpha-L-Arabinofuranosidase activities in antisense 1-aminocyclopropane-1-carboxylic acid synthase tomato pericarp discs.

Alpha-L-Arabinofuranosidases (alpha-Afs) are plant enzymes capable of releasing terminal arabinofuranosyl residues from cell wall matrix polymers, as well as from different glycoconjugates. Three different alpha-Af isoforms were distinguished by size exclusion chromatography of protein extracts from control tomatoes (Lycopersicon esculentum) and an ethylene synthesis-suppressed (ESS) line expressing an antisense 1-aminocyclopropane-1-carboxylic synthase transgene. alpha-Af I and II are active throughout fruit ontogeny. alpha-Af I is the first Zn-dependent cell wall enzyme isolated from tomato pericarp tissues, thus suggesting the involvement of zinc in fruit cell wall metabolism. This isoform is inhibited by 1,10-phenanthroline, but remains stable in the presence of NaCl and sucrose. alpha-Af II activity accounts for over 80% of the total alpha-Af activity in 10-d-old fruit, but activity drops during ripening. In contrast, alpha-Af III is ethylene dependent and specifically active during ripening. alpha-Af I released monosaccharide arabinose from KOH-soluble polysaccharides from tomato cell walls, whereas alpha-Af II and III acted on Na(2)CO(3)-soluble pectins. Different alpha-Af isoform responses to gibberellic acid, synthetic auxins, and ethylene were followed by using a novel ESS mature-green tomato pericarp disc system. alpha-Af I and II activity increased when gibberellic acid or 2,4-dichlorophenoxyacetic acid was applied, whereas ethylene treatment enhanced only alpha-Af III activity. Results suggest that tomato alpha-Afs are encoded by a gene family under differential hormonal controls, and probably have different in vivo functions. The ESS pericarp explant system allows comprehensive studies involving effects of physiological levels of different growth regulators on gene expression and enzyme activity with negligible wound-induced ethylene production.