Possible Mechanisms Limiting Movement of Ralstonia solanacearum in Resistant Tomato Tissues

The distribution and appearance of Ralstonia solanacearum in the upper hypocotyl tissues of root‐inoculated tomato seedlings of resistant rootstock cultivar LS‐89 (a selection from Hawaii 7998) and susceptible cultivar Ponderosa were compared to clarify the mechanism that limits the movement of the bacterial pathogen in resistant tomato tissues. In stems of wilted Ponderosa plants, bacteria colonized both the primary and the secondary xylem tissues. Bacteria were abundant in vessels, of which the pit membranes were often degenerated. All parenchyma cells adjacent to vessels with bacteria were necrotic and some of them were colonized with bacteria. In stems of LS‐89 plants showing no discernible wilting symptoms, bacteria were observed in the primary xylem tissues but not in the secondary xylem tissues. Necrosis of parenchyma cells adjacent to vessels with bacteria was observed occasionally. The pit membranes were often thicker with high electron density. The inner electron‐dense layer of cell wall of parenchyma cells and vessels was thicker and more conspicuous in xylem tissues of infected LS‐89 than in xylem of infected Ponderosa or mock‐inoculated plants. Electron‐dense materials accumulated in or around pit cavities in parenchyma cells next to vessels with bacteria, and in vessels with bacteria. Many bacterial cells appeared normal in vessels, except for those in contact with the pit membranes. These results indicate that R. solanacearum moves from vessel to vessel in infected tissues through degenerated pit membranes and that restricted movement in xylem tissues was the characteristic feature in LS‐89. The limitation in bacterial movement may be related to the thickening of the pit membranes and/or the accumulations of electron‐dense materials in vessels and parenchyma cells.     

Nitrate Assimilation Contributes to Ralstonia solanacearum Root Attachment,Stem Colonization,and Virulence

Ralstonia solanacearum, an economically important plant pathogen, must attach, grow, and produce virulence factors to colonize plant xylem vessels and cause disease. Little is known about the bacterial metabolism that drives these processes. Nitrate is present in both tomato xylem fluid and agricultural soils, and the bacterium''s gene expression profile suggests that it assimilates nitrate during pathogenesis. A nasA mutant, which lacks the gene encoding the catalytic subunit of R. solanacearum''s sole assimilatory nitrate reductase, did not grow on nitrate as a sole nitrogen source. This nasA mutant exhibited reduced virulence and delayed stem colonization after soil soak inoculation of tomato plants. The nasA virulence defect was more severe following a period of soil survival between hosts. Unexpectedly, once bacteria reached xylem tissue, nitrate assimilation was dispensable for growth, virulence, and competitive fitness. However, nasA-dependent nitrate assimilation was required for normal production of extracellular polysaccharide (EPS), a major virulence factor. Quantitative analyses revealed that EPS production was significantly influenced by nitrate assimilation when nitrate was not required for growth. The plant colonization delay of the nasA mutant was externally complemented by coinoculation with wild-type bacteria but not by coinoculation with an EPS-deficient epsB mutant. The nasA mutant and epsB mutant did not attach to tomato roots as well as wild-type strain UW551. However, adding either wild-type cells or cell-free EPS improved the root attachment of these mutants. These data collectively suggest that nitrate assimilation promotes R. solanacearum virulence by enhancing root attachment, the initial stage of infection, possibly by modulating EPS production.     

Histopathology of Susceptible and Resistant Capsicum annuum Cultivars Infected with Ralstonia solanacearum

The vascular colonisation of resistant and susceptible hot chilli (Capsicum annuum) cultivars by Ralstonia solanacearum was examined using transmission electron microscopy. Tap roots of artificially-inoculated plants, grown in sterilised soil were investigated to observe the morphological barriers involved in the restriction of bacterial spread. In the resistant cultivar, several responses induced in response to bacterial infection, were observed. First, a cell wall coating material developed together with swelling of the primary wall of the xylem vessels, limiting the bacterial spread. Second, formation of various types of vesicles in the vascular parenchyma cells, which enveloped the bacterial mass and also partly restricted the pathogen spread. Third, induction of hypersensitive reaction in the xylem vessels resulted in the distortion and lysis of the bacteria. In the susceptible cultivar, vascular coating, production of vesicle and induction of hypersensitive reaction were not observed and bacterial spread was not limited. Rapid vascular colonisation of the susceptible cultivar seemed to be generalised which resulted in the rapid wilting of affected plants. Other reactions involved in both resistant and susceptible cultivars include disorganisation of cytoplasm of parenchyma cells, disintegration of nuclei, and rupturing of xylem vessel walls. The restriction of pathogen spread associated with the resistance in C. annuum to bacterial wilt was mainly attributed to some induced, morphological and physical barriers.     

Real Time Live Imaging of Phytopathogenic Bacteria Xanthomonas campestris pv. campestris MAFF106712 in ‘Plant Sweet Home’

Xanthomonas is one of the most widespread phytobacteria, causing diseases on a variety of agricultural plants. To develop novel control techniques, knowledge of bacterial behavior inside plant cells is essential. Xanthomonas campestris pv. campestris, a vascular pathogen, is the causal agent of black rot on leaves of Brassicaceae, including Arabidopsis thaliana. Among the X. campestris pv. campestris stocks in the MAFF collection, we selected XccMAFF106712 as a model compatible pathogen for the A. thaliana reference ecotype Columbia (Col-0). Using modified green fluorescent protein (AcGFP) as a reporter, we observed real time XccMAFF106712 colonization in planta with confocal microscopy. AcGFP-expressing bacteria colonized the inside of epidermal cells and the apoplast, as well as the xylem vessels of the vasculature. In the case of the type III mutant, bacteria colonization was never detected in the xylem vessel or apoplast, though they freely enter the xylem vessel through the wound. After 9 days post inoculation with XccMAFF106712, the xylem vessel became filled with bacterial aggregates. This suggests that Xcc colonization can be divided into main four steps, (1) movement in the xylem vessel, (2) movement to the next cell, (3) adhesion to the host plant cells, and (4) formation of bacterial aggregates. The type III mutant abolished at least steps (1) and (2). Better understanding of Xcc colonization is essential for development of novel control techniques for black rot.     

Unravelling physiological signatures of tomato bacterial wilt and xylem metabolites exploited by Ralstonia solanacearum

The plant pathogen Ralstonia solanacearum uses plant resources to intensely proliferate in xylem vessels and provoke plant wilting. We combined automatic phenotyping and tissue/xylem quantitative metabolomics of infected tomato plants to decipher the dynamics of bacterial wilt. Daily acquisition of physiological parameters such as transpiration and growth were performed. Measurements allowed us to identify a tipping point in bacterial wilt dynamics. At this tipping point, the reached bacterial density brutally disrupts plant physiology and rapidly induces its death. We compared the metabolic and physiological signatures of the infection with drought stress, and found that similar changes occur. Quantitative dynamics of xylem content enabled us to identify glutamine (and asparagine) as primary resources R. solanacearum consumed during its colonization phase. An abundant production of putrescine was also observed during the infection process and was strongly correlated with in planta bacterial growth. Dynamic profiling of xylem metabolites confirmed that glutamine is the favoured substrate of R. solanacearum. On the other hand, a triple mutant strain unable to metabolize glucose, sucrose and fructose appears to be only weakly reduced for in planta growth and pathogenicity.     

Endophytic Colonization of Vitis vinifera L. by Plant Growth-Promoting Bacterium Burkholderia sp. Strain PsJN

Patterns of colonization of Vitis vinifera L. cv. Chardonnay plantlets by a plant growth-promoting bacterium, Burkholderia sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with gfp (PsJN::gfp2x) or gusA (PsJN::gusA11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that Burkholderia sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.     

DoesAcetobacter diazotrophicusLive and Move in the Xylem of Sugarcane Stems? Anatomical and Physiological Data

We have previously shown that the nitrogen-fixing endophyteof sugarcane,Acetobacter diazotrophicuslives in the sugar solutionin the intercellular-space apoplast of the stem cortex. Variousauthors have claimed that it inhabits the xylem apoplast. Thispossibility has been investigated in the clone Ja 60-5 and shownto be most unlikely for the following reasons: (1) an adequatecarbon source is lacking in the xylem sap, and cannot be suppliedfrom the intercellular-space apoplast of the cortex. Diffusionof solutes into and out of the vascular bundles is preventedby complete lignification and suberization of the bundle sheathcell walls except at the nodes. (2) Longitudinal movement ofparticles as large as bacteria is severely limited at the nodes.Vessel end walls were found in 90% of vessels at each node,and only 1% of open vessels extended through two nodes. Noneextended as far as three nodes. In addition to vessel end walls,vessel continuity at nodes was interrupted by living cells.Dye solution in the transpiration stream in metaxylem vesselsdid not pass through these living cells, but accumulated incrystals (sump formation) in the vessels below the node. Onlyin some protoxylem vessels and cavities did dye solution movethrough many nodes. It is likely that selection of sugarcaneclones such as Ja 60-5 for resistance to bacterial wilt diseaseshave selected for clones that have limited vessel continuity.(3) When culturedA. diazotrophicuswas introduced into the transpirationstream, the xylem parenchyma reacted by secreting a bright redpolymer which killed the bacteria and blocked the movement ofwater. We conclude that the xylem flow-apoplast of this cloneof sugarcane is an unsuitable habitat forA. diazotrophicusandthat additional habitats to those of the intercellular-spaceapoplast should be sought elsewhere. Acetobacter diazotrophicus; endophytic bacteria; nitrogen fixation; sugarcane; vessel end walls; xylem apoplast; xylem bacteria; xylem segmentation     

Localization of cell wall proteins in relation to the developmental anatomy of the carrot root apex

A panel of monoclonal antibodies that recognize a class of cell wall proteins, related to the hydroxyproline-rich glycoproteins, has been assembled and characterized in relation to their restricted patterns of binding amongst the cells comprising the carrot root apex. The occurrence of the epitopes at the surface of cells and intercellular spaces in the region of the apex between the meristematic initials and the region of cell expansion indicates dynamic patterns that reflect aspects of the development of the anatomical pattern. The monoclonal antibody JIM11 reacts with the surface of cells in the central root cap and the region of the meristem. As the cortex/stele boundary becomes established the reactivity is seen in the inner cortical layers and finally in the whole cortex. Later in development the JIM11 epitope is also expressed by two pairs of pericycle cell files adjacent to the phloem region and also by the epidermis. The JIM12 monoclonal antibody is unreactive with cells in the region of the root cap and the meristem but is reactive with intercellular spaces formed at the junction of the oblique and radial walls in the double-layered sectors of the pericycle opposite the xylem poles. This epitope is also transiently expressed by the two phloem sieve tube element mother cells. Later in development JIM12 recognizes the future metaxylem cells. The antibody JIM20 recognizes all the cells and intercellular spaces recognized by JIM11 and JIM12. Immuno-chemical analyses indicate cross-reactivity with carrot taproot extensin and Solanaceous lectins.     

Dissection of Bacterial Wilt on Medicago truncatula Revealed Two Type III Secretion System Effectors Acting on Root Infection Process and Disease Development

Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease, which colonizes susceptible Medicago truncatula via the intact root tip. Infection involves four steps: appearance of root tip symptoms, root tip cortical cell invasion, vessel colonization, and foliar wilting. We examined this pathosystem by in vitro inoculation of intact roots of susceptible or resistant M. truncatula with the pathogenic strain GMI1000. The infection process was type III secretion system dependent and required two type III effectors, Gala7 and AvrA, which were shown to be involved at different stages of infection. Both effectors were involved in development of root tip symptoms, and Gala7 was the main determinant for bacterial invasion of cortical cells. Vessel invasion depended on the host genetic background and was never observed in the resistant line. The invasion of the root tip vasculature in the susceptible line caused foliar wilting. The avrA mutant showed reduced aggressiveness in all steps of the infection process, suggesting a global role in R. solanacearum pathogenicity. The roles of these two effectors in subsequent stages were studied using an assay that bypassed the penetration step; with this assay, the avrA mutant showed no effect compared with the GMI1000 strain, indicating that AvrA is important in early stages of infection. However, later disease symptoms were reduced in the gala7 mutant, indicating a key role in later stages of infection.     

Localized and Systemic Increase of Phenols in Tomato Roots Induced by Glomus versiforme Inhibits Ralstonia solanacearum

Ralstonia solanacearum is an important plant pathogen in tropical and subtropical countries. Here, we describe the inhibition of R. solanacearum as a result of increased phenols induced locally or systemically by an arbuscular mycorrhizal (AM) fungus. In pot cultures, R. solanacearum populations in the rhizosphere, on root surfaces and in the xylem were decreased by 26.7, 79.3 and 81.7%, respectively, following inoculation of tomato plants (Lycopersicon esculentum Mill.) with Glomus versiforme Berch. Colonization of the plants by both R. solanacearum and G. versiforme increased the contents of soluble phenols and cell‐wall bound phenols in root tissue, but with different patterns. Whereas R. solanacearum preferably promoted the cell‐wall bound phenol content, G. versiforme preferably enhanced the soluble phenol content. Split root experiments revealed that R. Solanacearum was inhibited by G. versiforme, and that G. versiforme also increased the phenol content systemically, but to a lesser extent than locally.     

Arabinogalactan protein-rich cell walls,paramural deposits and ergastic globules define the hyaline bodies of rhinanthoid Orobanchaceae haustoria

Background and Aims

Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae.

Methods

Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs).

Key Results

Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem.

Conclusions

The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra- and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.     

Distribution of pectic epitopes in cell walls of the sugar beet root

Immunolabelling techniques with antibodies specific to partially methyl-esterified homogalacturonan (JIM5: unesterified residues flanked by methylesterified residues. JIM7: methyl-esterified residues flanked by unesterified residues), a blockwise de-esterified homogalacturonan (2F4), 1,4-galactan (LM5) and 1,5-arabinan (LM6) were used to map the distribution of pectin motifs in cell walls of sugar beet root (Beta vulgaris). PME and alkali treatments of sections were used in conjunction with JIM5-7 and 2F4. The JIM7 epitope was abundant and equally distributed in all cells. In storage parenchyma, the JIM5 epitope was restricted to some cell junctions and the lining of intercellular spaces while in vascular tissues it occurred at cell junctions in some phloem walls and in xylem derivatives. After secondary wall formation, the JIM5 epitope was restricted to inner cell wall regions between secondary thickenings. The 2F4 epitope was not detected without de-esterification treatment. PME treatments prior to the use of 2F4 indicated that HG at cell corners was not acetylated. The LM5 epitope was mainly present in the cambial zone and when present in storage parenchyma, it was restricted to the wall region closest to the plasma membrane. The LM6 epitope was widely distributed throughout primary walls but was more abundant in bundles than in medullar ray tissue and storage parenchyma. These data show that the occurrence of oligosaccharide motifs of pectic polysaccharides are spatially regulated in sugar beet root cell walls and that the spatial patterns vary between cell types suggesting that structural variants of pectic polymers are involved in the modulation of cell wall properties.     

Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices

Monoclonal antibodies recognizing un-esterified (JIM5) and methyl-esterified (JIM7) epitopes of pectin have been used to locate these epitopes by indirect immunofluorescence and immunogold electron microscopy in the root apex of carrot (Daucus carota L.). Both antibodies labelled the walls of cells in all tissues of the developing root apex. Immunogold labelling observed at the level of the electron microscope indicated differential location of the pectin epitopes within the cell walls. The un-esterified epitope was located to the inner surface of the primary cell walls adjacent to the plasma membrane, in the middle lamella and abundantly to the outer surface at intercellular spaces. In contrast, the epitope containing methyl-esterified pectin was located evenly throughout the cell wall. In root apices of certain other species the JIM5 and JIM7 epitopes were found to be restricted to distinct tissues of the developing roots. In the root apex of oat (Avena sativa L.), JIM5 was most abundantly reactive with cell walls at the region of intercellular spaces of the cortical cells. JIM7 was reactive with cells of the cortex and the stele. Neither epitope occurred in walls of the epidermal or root-cap cells. These pattern of expression were observed to derive from the very earliest stages of the development of these tissues in the oat root meristem and were maintained in the mature root. In the coleoptile and leaf tissues of oat seedlings, JIM5 labelled all cells abundantly whereas JIM7 was unreactive. Other members of the Gramineae and also the Chenopodiaceae are shown to express similar restricted spatial patterns of distribution of these pectin epitopes in root apices.Abbreviations CDTA 1,2-diaminocyclohexane tetraacetic acid - RG rhamnogalacturonan J.P.K. was supported by the Agricultural and Food Research Council Cell Signalling and Recognition Programme. We thank J. Cooke and N. Stacey for technical assistance, H.A. Schols, Drs. P. Albersheim and A. Darvill for pectic polysaccharides, and Dr. R.R. Selvendran and M. McCann for useful discussions.     

Cinnamic,myristic and fumaric acids in tobacco root exudates induce the infection of plants by Ralstonia solanacearum

Aim

The secretion of allelochemicals from plant roots plays a key role in soil sickness and soil-borne disease. The goal of this study was to investigate the role of allelopathic chemicals in Ralstonia solanacearum-infected tobacco roots.

Methods

The organic acids investigated in the present study are major components of tobacco root exudates. Through a swarming assay, we assessed the chemotaxis and colonization of R. solanacearum in response to organic acids.

Results

Fumaric acid was detected, and the results showed that this acid could serve as a semiochemical for attracting R. solanacearum and inducing the formation of biofilms of this species. The results also revealed that cinnamic and myristic acids play significant roles on swarming motility and chemotaxis. In addition, cinnamic, myristic and fumaric acids could enhance the expression of chemotaxis- and motility-related genes in R. solanacearum cultured in minimal medium. Furthermore, these three acids promote R. solanacearum colonization and accelerate disease progression in tobacco.

Conclusion

Cinnamic, myristic and fumaric acids could serve as semiochemical attractants to induce the colonization and infection of R. solanacearum. The results of the present study enhance our understanding of the ecological effects of plant root exudates in plant-microbe interactions and help to reveal the relationship between tobacco bacterial wilt and the autotoxins and allelochemicals that accumulate from root exudates.

     

Ralstonia solanacearum Dps Contributes to Oxidative Stress Tolerance and to Colonization of and Virulence on Tomato Plants

Ralstonia solanacearum, an economically important soilborne plant pathogen, infects host roots to cause bacterial wilt disease. However, little is known about this pathogen''s behavior in the rhizosphere and early in pathogenesis. In response to root exudates from tomato, R. solanacearum strain UW551 upregulated a gene resembling Dps, a nonspecific DNA binding protein from starved cells that is critical for stress survival in other bacteria. An R. solanacearum dps mutant had increased hydrogen peroxide sensitivity and mutation rate under starvation. Furthermore, dps expression was positively regulated by the oxidative stress response regulator OxyR. These functional results are consistent with a Dps annotation. The dps mutant caused slightly delayed bacterial wilt disease in tomato after a naturalistic soil soak inoculation. However, the dps mutant had a more pronounced reduction in virulence when bacteria were inoculated directly into host stems, suggesting that Dps helps R. solanacearum adapt to conditions inside plants. Passage through a tomato plant conferred transient increased hydrogen peroxide tolerance on both wild-type and dps mutant strains, demonstrating that R. solanacearum acquires Dps-independent oxidative stress tolerance during adaptation to the host environment. The dps mutant strain was also reduced in adhesion to tomato roots and tomato stem colonization. These results indicate that Dps is important when cells are starved or in stationary phase and that Dps contributes quantitatively to host plant colonization and bacterial wilt virulence. They further suggest that R. solanacearum must overcome oxidative stress during the bacterial wilt disease cycle.Bacterial wilt caused by Ralstonia solanacearum is a lethal disease affecting diverse economically important crops worldwide (20). The pathogen attacks over 200 species in more than 50 plant families (21). Although known primarily as a soilborne plant pathogen, R. solanacearum also survives in soil, water, and latently infected plants (20). The bacterium typically invades its host through natural or mechanical root wounds, multiplies in the root cortex, and then rapidly colonizes the xylem, where it reaches high cell densities. Once wilt symptoms develop, plants usually die, releasing the pathogen back into the soil (42).R. solanacearum is a tropical bacterium adapted to warmer climates, with the exception of a clonal group belonging to phylotype II, sequevar 1, of the R. solanacearum species complex (13). This group, historically and for regulatory purposes known as race 3 biovar 2 (R3bv2), causes brown rot of potato and bacterial wilt of tomato in tropical highlands and some temperate zones (11, 41, 45, 46). Because of its virulence at relatively cool temperatures, R3bv2 is a quarantine pest in Europe and Canada and a select agent pathogen in the United States (27).R. solanacearum virulence is quantitative and complex, with many contributing factors such as type II-secreted proteins, type III-secreted effectors, extracellular polysaccharide, and several plant cell wall-degrading enzymes (16, 17, 36, 38). Much of what is known about R. solanacearum comes from studies focusing on mid- or end-stage disease caused by tropical or warm-temperate strains (8, 30). A few virulence factors are known to function early in disease development: motility, energy taxis, and type IV pili, which collectively direct the bacterium toward and facilitate attachment to the host root (26, 44, 49, 50). However, R. solanacearum traits that contribute to fitness and virulence in the rhizosphere are not well understood for either tropical or R3bv2 strains.In soil, bacteria experience environmental stressors, such as pH and temperature extremes and water and oxygen limitation, as well as competition for nutrients (47). Plant roots release exudates and sloughed-off cells, supplying sufficient energy to sustain large microbial communities, provided other nutrients such as N, P, and Fe are present (19, 47). While rhizosphere bacteria can enjoy rapid growth in this relatively rich environment, fluctuating nutrient availability means that soil-dwelling microbes must survive periods of starvation (47).R. solanacearum also encounters oxidative stress in the rhizosphere. Plant roots produce reactive oxygen species (ROS) in response to many stimuli (25, 32). Several studies implicate ROS in root development and in interactions between roots and microbes (5, 24). We previously found that during plant colonization R. solanacearum is exposed to host-derived ROS, which triggers a bacterial oxidative stress response that adapts the pathogen to the xylem environment and is necessary for full virulence (8, 14).We previously described an in vivo expression technology (IVET)-like screen that identified R. solanacearum genes upregulated in the tomato rhizosphere (12). These genes encoded several known bacterial wilt virulence factors, such as the type 3 secretion regulator HrpG, the type IV pilus assembly protein PilP, global virulence regulator VsrA, and early virulence regulator PehR. The screen further identified a high-affinity cytochrome c oxidase necessary for R. solanacearum growth in microaerobic conditions (12). This paper presents our analysis of another rhizosphere-induced gene that encodes Dps, a DNA binding protein from starved cells originally described in Escherichia coli (2). Dps belongs to a family of ferritin-like stress-induced proteins that bind nonspecifically to DNA in stationary-phase bacteria (2, 29, 40). In E. coli, Dps helps maintain DNA integrity under environmentally challenging conditions, including starvation, oxidative damage, pH shock, and thermal stress (2, 10, 18, 29, 33). Dps also protects the soilborne plant-associated bacteria Agrobacterium tumefaciens and Pseudomonas putida from oxidative stress (9, 37).Traits that adapt R. solanacearum to detrimental conditions in the rhizosphere are likely to be important for pathogenic success. We hypothesized that Dps is required for adaptation to nutrient and oxidative stress and, thus, for bacterial wilt disease development. We found that Dps was highly expressed after starvation and contributed to oxidative stress tolerance in starved R. solanacearum cells. Furthermore, this protein was necessary for wild-type bacterial wilt disease development and for colonization of tomato xylem, suggesting that the bacterium must overcome a nutrient-poor and/or oxidative environment in the rhizosphere and xylem of host plants.     

Colonization of Vitis vinifera by a Green Fluorescence Protein-Labeled, gfp-Marked Strain of Xylophilus ampelinus, the Causal Agent of Bacterial Necrosis of Grapevine

The dynamics of Xylophilus ampelinus were studied in Vitis vinifera cv. Ugni blanc using gfp-marked bacterial strains to evaluate the relative importance of epiphytic and endophytic phases of plant colonization in disease development. Currently, bacterial necrosis of grapevine is of economic importance in vineyards in three regions in France: the Cognac, Armagnac, and Die areas. This disease is responsible for progressive destruction of vine shoots, leading to their death. We constructed gfp-marked strains of the CFBP2098 strain of X. ampelinus for histological studies. We studied the colonization of young plants of V. vinifera cv. Ugni blanc by X. ampelinus after three types of artificial contamination in a growth chamber and in a greenhouse. (i) After wounding of the stem and inoculation, the bacteria progressed down to the crown through the xylem vessels, where they organized into biofilms. (ii) When the bacteria were forced into woody cuttings, they rarely colonized the emerging plantlets. Xylem vessels could play a key role in the multiplication and conservation of the bacteria, rather than being a route for plant colonization. (iii) When bacterial suspensions were sprayed onto the plants, bacteria progressed in two directions: both in emerging organs and down to the crown, thus displaying the importance of epiphytic colonization in disease development.     

Immunogold localization of pectins and glycoproteins in tissues of peach with reference to deep supercooling

Living xylem tissues and floral buds of several species of woody plants survive exposure to freezing temperatures by deep supercooling. A barrier to water loss and the growth of ice crystals into cells is considered necessary for deep supercooling to occur. Pectins, as a constituent of the cell wall, have been implicated in the formation of this barrier. The present study examined the distribution of pectin in xylem and floral bud tissues of peach (Prunus persica). Two monoclonal antibodies (JIM5 and JIM7) that recognize homogalacturonic sequences with varying degrees of esterification were utilized in conjunction with immunogold electron microscopy. Results indicate that highly esterified epitopes of pectin, recognized by JIM7, were the predominant types of pectin in peach and were uniformly distributed throughout the pit membrane and primary cell walls of xylem and floral bud tissues. In contrast, un-esterified epitopes of pectin, recognized by JIM5, were confined to the outer surface of the pit membrane in xylem tissues. In floral buds, these epitopes were localized in middle lamellae, along the outer margin of the cell wall lining empty intercellular spaces, and within filled intercellular spaces. JIM5 labeling was more pronounced in December samples than in July/August samples. Additionally, epitopes of an arabinogalactan protein, recognized by JIM14, were confined to the amorphous layer of the pit membrane. The role of pectins in freezing response is discussed in the context of present theory and it is suggested that pectins may influence both water movement and intrusive growth of ice crystals at freezing temperatures.