Influence of Pathogenic and Non-pathogenic Bacteria on Soybean Suspension Cells

Co-cultivations of plant suspension cultures of soybean ( Glycine max ) with compatible phytopathogenic ( Pseudomonas syringae pv. glycinea ), incompatible phytopathogenic ( Pseudomonas syringae pv. tomato ), and different non-pathogenic ( Erwinia herbicola, Escherichia coli ) bacteria were carried out. Growth and viability (triphenyltetrazolium chloride activity) of plant cells and bacteria as well as enzyme activities of peroxidase (PO), polyphenoloxidase (PPO), and phenylalanine ammonialyase (PAL) within the plant cells were investigated over an incubation period of 7 days. The compatible pathogen inhibited growth and viability of the plant cells after 1 day and led to the death of the majority of the plant cells by the seventh day. In contrast, the incompatible pathogen directly reduced growth and viability of the soybean cells and caused a strong induction of enzyme activities of PO and PAL to more than 4 times of the untreated control by the seventh day. The epiphytic bacterium Erwinia herbicola caused a slight inhibition of growth and viability of the plant cells after the second day of co-cultivation. The PO activity increased in the same manner as in the incompatible interaction. The saprophytic Escherichia coli strain had a negligible influence on soybean suspension cells. All the bacteria tested except for Escherichia coli multiplied rapidly during co-cultivation and reached populations of 10⁸-10⁹ colonyfoming units/ml in the stationary phase. The results from this study demonstrate that the soybean suspension cells react differently to compatible, incompatible and saprophytic bacteria.     

Evidence for the involvement of an oxidative stress in the initiation of infection of pear by Erwinia amylovora

Involvement of an oxidative burst, usually related to incompatible plant/pathogen interactions leading to hypersensitive reactions, was investigated with Erwinia amylovora, the causal agent of fire blight of Maloideae subfamily of Rosaceae, in interaction with pear (Pyrus communis; compatible situation) and tobacco (Nicotiana tabacum; incompatible situation). As expected, this necrogenic bacterium induced in tobacco a sustained production of superoxide anion, lipid peroxidation, electrolyte leakage, and concomitant increases of several antioxidative enzymes (ascorbate peroxidases, glutathion reductases, glutathion-S-transferases, and peroxidases), in contrast to the compatible pathogen Pseudomonas syringae pv tabaci, which did not cause such reactions. In pear leaves, however, inoculations with both the disease- and the hypersensitive reaction-inducing bacteria (E. amylovora and P. syringae pv tabaci, respectively) resulted in superoxide accumulation, lipid peroxidation, electrolyte leakage, and enzyme induction at similar rates and according to equivalent time courses. The unexpected ability of E. amylovora to generate an oxidative stress even in compatible situation was linked to its functional hrp (for hypersensitive reaction and pathogenicity) cluster because an Hrp secretion mutant of the bacteria did not induce any plant response. It is suggested that E. amylovora uses the production of reactive oxygen species as a tool to provoke host cell death during pathogenesis to invade plant tissues. The bacterial exopolysaccharide could protect this pathogen against the toxic effects of oxygen species since a non-capsular mutant of E. amylovora induced locally the same responses than the wild type but was unable to further colonize the plant.     

Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae

We performed large-scale mRNA expression profiling using an Affymetrix GeneChip to study Arabidopsis responses to the bacterial pathogen Pseudomonas syringae. The interactions were compatible (virulent bacteria) or incompatible (avirulent bacteria), including a nonhost interaction and interactions mediated by two different avirulence gene-resistance (R) gene combinations. Approximately 2000 of the approximately 8000 genes monitored showed reproducible significant expression level changes in at least one of the interactions. Analysis of biological variation suggested that the system behavior of the plant response in an incompatible interaction was robust but that of a compatible interaction was not. A large part of the difference between incompatible and compatible interactions can be explained quantitatively. Despite high similarity between responses mediated by the R genes RPS2 and RPM1 in wild-type plants, RPS2-mediated responses were strongly suppressed by the ndr1 mutation and the NahG transgene, whereas RPM1-mediated responses were not. This finding is consistent with the resistance phenotypes of these plants. We propose a simple quantitative model with a saturating response curve that approximates the overall behavior of this plant-pathogen system.     

hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria.

The majority of bacterial plant diseases are caused by members of three bacterial genera, Pseudomonas, Xanthomonas, and Erwinia. The identification and characterization of mutants that have lost the abilities to provoke disease symptoms on a compatible host and to induce a defensive hypersensitive reaction (HR) on an incompatible host have led to the discovery of clusters of hrp genes (hypersensitive reaction and pathogenicity) in phytopathogenic bacteria from each of these genera. Here, we report that predicted protein sequences of three hrp genes from Pseudomonas solanacearum show remarkable sequence similarity to key virulence determinants of animal pathogenic bacteria of the genus Yersinia. We also demonstrate DNA homologies between P. solanacearum hrp genes and hrp gene clusters of P. syringae pv. phaseolicola, Xanthomonas campestris pv. campestris, and Erwinia amylovora. By comparing the role of the Yersinia determinants in the control of the extracellular production of proteins required for pathogenicity, we propose that hrp genes code for an export system that might be conserved among many diverse bacterial pathogens of plants and animals but that is distinct from the general export pathway.     

Sugar-specific Attachment of Pseudomonas syringae pv. glycinea to Isolated Single Leaf Cells of Resistant Soybean Cultivars

Phase-contrast and scanning electron microscopy showed races of P. synngae pv. glycinea uniformly distributed over and attached to the whole surface of isolated single leaf cells of resistant soybean cultivars, as early as 30 to 180 min after inoculation. On the contrary, attachment in the compatible interaction did not occur within 10—15 h. In a later period, compatibility was characterized by the formation of adherent bacterial clusters. Early attachment of races 1 and 6 to cv. Harosoy and that of race 5 to cv. Flambeau leaf cells, each representing incompatible interaction, could be inhibited by L-rhamnose and D-glucose, respectively. Furthermore, the lack of Mn²⁺ and Fe²⁺ and heat-treatment of plant cells also affected the early attachment in incompatible combinations and resulted in cluster formation, suggesting incompatibility rather than compatibility to be the active phenomenon. Pre-inoculation of cells with an incompatible race induced changes that caused compatible bacteria also to distributively attach to plant cell surface indicating that a transfer of information or surface alterations occur upon attachment in incompatible interaction.     

Alteration of Response of Bean Leaves to Compatible and Incompatible Bacteria

Injection of Red Mexican bean leaves with Pseudomonas phascolicolaRace 2 (compatible, 18 h before P. mors-prunorum or P. phaseolicolaRace 1 (incompatible), or simultaneous inoculation with compatibleand incompatible bacteria (3:1) greatly delayed the appearanceof hypersensitive responses. When compatible bacteria were inoculated12 h or less before incompatible bacteria, or when the ratioof these bacteria was 1:1 or 0.3:1 in simultaneous inoculation,hypersensitive responses did develop. Earlier inoculation withincompatible bacteria delayed the appearance and severity ofdisease symptoms following later inoculation with compatiblebacteria. When selected areas of leaves were inoculated with compatiblebacteria, effects on hypersensitive responses were confinedto these areas when the whole leaf was inoculated later withincompatible bacteria. Inoculation through the upper leaf surfacewith incompatible bacteria did not affect susceptible responseswhen compatible bacteria were inoculated 24 h later throughthe lower surface. Treatment of leaves with heat-killed bacteria or live bacteriain numbers insufficient to cause hypersensitive responses didnot prevent development of these responses following later inoculationwith incompatible bacteria. Use of heat-killed bacteria didsuppress hypersensitive responses in tobacco leaves. Injectionof leaves with cycloheximide (20 p.p.m.) 30 min before inoculationwith incompatible bacteria suppressed leakage of electrolytesand browning of tissues associated with hypersensitive responses.Cycloheximide had little effect on leakage of electrolytes fromleaves inoculated with compatible bacteria or with the developmentof susceptible responses. Exposure of leaves to chloroform vapourdelayed hypersensitive responses by 24 h; treatment with peroxidasehad no effect.     

No Evidence of Direct Superoxide Anion Effect in Hypersensitive Death of Pseudomonas syringae Van Hall in Tobacco Leaf Tissue1

The hypothesis of superoxide anion generation as a direct bactericidal mechanism exercised by hypersensitive tobacco leaf tissue against an incompatible phytopathogenic pseudomonad was tested. The detection of O₂^? was made directly by reducing Cytochrome c and by producing nitrite from hydroxylamine chloride and indirectly by measuring the death of endophytic bacteria in the presence of superoxide dismutase. At 4 h and 6 h the low net specific reduction of Cytochrome c in the hypersensitive tissue was not confirmed by the specific production of nitrite. At the same time active superoxide dismutase did not give significant protection to the incompatible bacteria as compared to inactive superoxide dismutase. The protection provided by active superoxide dismutase as compared with the controls (bovine serum albumine treated tissue and non-treated tissue) at 4 h and at 6 h was not correlated to its superoxide anion scavenger activity. The results indicated that the death of the incompatible pseudomonad during the hypersensitive reaction was not directly linked to O₂^? generation.     

Rapid activation of a novel plant defense gene is strictly dependent on the Arabidopsis RPM1 disease resistance locus.

We cloned and sequenced cDNAs encoded by a novel plant defense gene, ELI3, from parsley and Arabidopsis thaliana. The predicted product shares no homology to known sequences. ELI3 mRNA accumulates in A. thaliana leaves in response to challenge with phytopathogenic Pseudomonas syringae strains. The timing and magnitude of this response are dictated by the genetics of the plant-pathogen interaction being analyzed. During incompatible interactions, where resistance in the plant genotype Col-0 is dictated by the dominant RPM1 locus, ELI3 mRNA accumulates to high levels 5-10 h post-inoculation. This kinetic behavior is also generated by the presence of a cloned bacterial avirulence gene, in otherwise virulent bacteria, which triggers resistance mediated via RPM1 action. The phenotypic outcome is a hypersensitive resistance reaction visible 8-15 h post-infiltration. Thus, the induction kinetics of ELI3 mRNA accumulation are consistent with a functional role for the ELI3 gene product in establishing the resistant phenotype. In contrast, during compatible interactions with the susceptible plant genotype Nd-0, which is homozygous recessive at the rpm1 locus, ELI3 mRNA accumulates significantly only after 15 h. We show genetically that ELI3 activation is strictly dependent on the presence of dominant alleles at RPM1 using an assay generalizable to any pathogen induced plant defense phenomena.     

Xanthan is not essential for pathogenicity in citrus canker but contributes to<Emphasis Type="Italic"> Xanthomonas</Emphasis> epiphytic survival

Xanthan-deficient mutants of Xanthomonas axonopodis pv. citri, the bacterium responsible for citrus canker, were generated by deletion and marker exchange of the region encoding the carboxy-terminal end of the first glycosyltransferase, GumD. Mutants of gumD did not produce xanthan and remained pathogenic in citrus plants to the same extent as wild-type bacteria. The kinetics of appearance of initial symptoms, areas of plant material affected, and growth of bacteria inside plant tissue throughout the disease process were similar for both wild-type and mutant inoculations. Moreover, exopolysaccharide deficiency did not impair the ability of the bacteria to induce hypersensitive response on non-host plants. Apart from variations in phenotypic aspects, no differences in growth or survival under different stress conditions were observed between the xanthan-deficient mutant and wild-type bacteria. However, gumD mutants displayed impaired survival under oxidative stress during stationary phase as well as impaired epiphytic survival on citrus leaves. Our results suggest that xanthan does not play an essential role in citrus canker at the initial stages of infection or in the incompatible interactions between X. axonopodis pv. citri and non-host plants, but facilitates the maintenance of bacteria on the host plant, possibly improving the efficiency of colonization of distant tissue.     

Intra- and Interspecific Variation in Plant Response to Inoculation with Deleterious Rhizosphere Pseudomonads

Accessions of wheat, spinach, lettuce and different Brassica species were tested in greenhouse experiments for reaction to inoculation with two isolates of growth-inhibitory rhizosphere bacteria. Seedlings grown in non-sterile soil were inoculated with bacterial suspension and shoot dry weight was measured after five weeks. Large differences were found between the plant species tested in their average sensitivity to each bacterial isolate, and in the majority of plant species, significant differences were also found between accessions in the response to one or both isolates. These findings suggest that, in addition to the variation between plant species, intraspecific variation in the reaction to deleterious bacteria is a common feature in plants. This supports the hypothesis that plant reaction to rhizosphere bacteria is under genetic control. The results further indicate specificity in the interactions between plants and bacterial isolates, both at the plant species level and at the accession level.     

植物与病原菌互作的蛋白质组学研究进展

蛋白质组学作为功能基因组学研究的主要内容之一,在阐述基因功能、了解生命现象和本质的分子机制等方面发挥着重要作用。植物蛋白质组学作为蛋白质组学的一个分支,研究应用也越来越广泛,尤其是探索植物与病原菌互作机制是其中的一个研究热点。本文就多年来植物与真菌、病毒、细菌互作的蛋白质组学研究做一综述,并对当前该领域今后的研究方向进行展望,以期为相关研究提供一些参考和理论基础。     

Activation of defence reactions in Solanaceae: where is the specificity?

When a potential pathogen attempts to infect a plant, biochemical and molecular communication takes place and leads to the induction of plant defence mechanisms. In the case of efficient defence, visible symptoms are restricted and the pathogen does not multiply (incompatible interaction); when defence is inefficient, the plant becomes rapidly infected (compatible interaction). During the last 30 years, a growing body of knowledge on plant-pathogen interactions has been gathered, and a large number of studies investigate the induction of various plant defence reactions by pathogens or by pathogen-derived compounds. However, as most papers focus on incompatible interactions, there is still a lack of understanding about the similarities and differences between compatible and incompatible situations. This review targets the question of specificity in Solanaceae-pathogen interactions, by comparing defence patterns in plants challenged with virulent or avirulent pathogens (or with pathogen-associated molecular patterns from these). A special emphasis is made on analysing whether defence reactions in Solanaceae depend primarily on the type of elicitor, on the plant genotype/species, or on the type of interaction (compatible or incompatible).     

Influence ofGlycine max nodulation on the persistence in soil of a genetically markedBradyrhizobium japonicum strain

Since competition with indigenous strains limits nodule occupancy by bacteria applied to seeds, the ecology of Bradyrhizobium inoculum strains used for soybean is of concern. A genetically marked strain,B. japonicum I-110 ARS, was directly enumerated from soil on selective medium. A clear long-term positive influence of even limitedGlycine max nodulation was shown by comparisons of population densities obtained with or without plant removal prior to nodule senescence in the first year and with an incompatible as well as a compatible soybean variety after 5 years.     

受稻瘟病原真菌侵染的水稻早期表达基因的cDNA克隆

以亲和性与非亲和性两个稻瘟病原真菌小种（Ｍａｇｎａｐｏｒｔｈｅ ｇｒｉｓｅａ（Ｈｅｂｅｒｔ）Ｂａｒｒ）感染同一水稻品种（Ｏｒｙｚａｓａｔｉｖａ Ｌ．ｃｖ．Ｓｈｅｎｘｉａｎｇｇｅｎｇ Ｎｏ．４）的植株产生明显不同的致病和抗病反应,由此建立了有效的感染系统。应用差异显示技术获得两个在侵染早期具有诱导表达特征的ｃＤＮＡ克隆,其中一个同时在致病和抗病反应中进行早期诱导表达,但在抗病反应中的诱导相对早于其在     

Molecular Cloning of Early-expressed cDNAs from Rice in Response to Infection by Rice Blast Fungus Magnaporthe grisea

Compatible and incompatible reactions in rice plants (Oryza sativa L. cv. Shenxianggen No.4) were resulted from inoculation with two different virulent races of rice blast fungus (Magnaporthe grisea (Hebert) Barr), and thus an effective infecting system was established between rice plants and the rice blast pathogen. Two cDNA clones that showed induced and temporal patterns in expression in the very early stage in response to infection of the fungus were obtained from the plants by use of differential display. Of the two cDNA clones, Fastresp-a was induced to express in both compatible and incompatible interactions although it was expressed earlier in the former reaction. The second one, Fastresp-b, was only expressed in incompatible interaction. Southern blot analysis of the rice genomic DNA indicated that both of the two clones were from genome of the plant. No significant homology to the two genes was found from the rice gene database. This suggested that they were novel genes in rice and may play important roles in rice resistant response to infection of rice blast fungus.     

Legumes tolerance to rhizobia is not always observed and not always deserved

Rhizobia display dual lifestyle. These bacteria are soil inhabitants but can also elicit the formation of a special niche on the root of legume plants, the nodules. In such organs, rhizobia can promote the growth of their host by providing them nitrogen they captured from atmosphere. All along the infection process, the plant innate immunity has to be controlled to maintain compatible interaction. However, nodulation does not always result in profit for the plant as compatible interactions include both nitrogen‐fixing and non‐fixing associations. In recent years, our knowledge on the mechanisms involved in the control of plant innate immunity during rhizobia‐legume interactions has greatly improved notably by the identification of bacterial and plant genes activating or suppressing the plant defences. Surprisingly, results also demonstrated that in some cases, plant defence reactions result in abortion of the nodulation process despite that the rhizobial strain has all the genetic potential to establish mutualism. In such situation, experimental evolution approaches highlighted possible rapid switches of incompatible rhizobia either to mutualistic or parasitic behaviour. Here, we review this recent literature.