Stage-specific gene expression during sexual development in Phytophthora infestans

Eight genes that are upregulated during sexual development in the heterothallic oomycete, Phytophthora infestans, were identified by suppression subtractive hybridization. Two genes showed very low but detectable expression in vegetative hyphae and became induced about 40- to >100-fold early in mating, before gametangial initials appeared. The remaining six loci were not induced until later in mating, coincident with the formation of gametangia and oospores, with induction levels ranging from 60- to >100-fold. Five genes were single copy, and three were members of families. Sequence analysis revealed that the predicted products of three of the genes had similarity to proteins that influence RNA stability, namely a ribonuclease activator, the pumilio family of RNA-binding proteins and RNase H. The products of two other mating-induced genes resembled two types of Phytophthora proteins previously shown to elicit plant defence responses. Each mating-induced gene was also expressed in a self-fertile strain, which was shown to be a heterokaryon. However, quantitative and qualitative differences existed in their expression in normal matings and in the self-fertile heterokaryon. Besides the mating-induced genes, two extrachromosomal RNA elements were identified.     

Secondary Sporangium Formation in Phytophthora Palmivora (Butl.) Butl

Very few sporangia of Phytophthora palmivora germinated directlyand produced secondary sporangia in distilled water and in solutionsof amino acids and carbohydrates at 30 °C. Although 1.0per cent (w / v) peptone and yeast-extract stimulated a highpercentage of germination by formation of germ tubes, less than1.0 per cent of the germinated sporangia produced secondarysporangia. Secondary sporangium formation was induced by transferringgerminated primary sporangia from a nutrient medium of sufficientlyhigh concentration to either distilled water or dilute solutionsof organic and inorganic compounds immediately after emergenceof the germ tubes. The percentage of germinated sporangia formingsecondary sporangia was influenced by both the nature and concentrationof the medium into which they were transferred. The secondarysporangia were significantly smaller than the primary sporangia. Phytophthora palmivora, germination, sporangium, Theobroma cacao L., cocoa     

Salmonella typhimurium responses to a bactericidal protein from human neutrophils

Bactericidal/permeability-increasing protein [BPI] is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram-negative bacteria via the lipid A component of lipopolysaccharide. To obtain information about the responses of Salmonella typhimurium to cell-surface damage by BPI, two-dimensional gel electrophoresis and N-terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment. The majority of the affected proteins are involved in central metabolic processes. Upon addition of BPI, the β-subunit of the F₁ portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11-fold. Three of the latter were identified as lipoamide dehydrogenase, enoyl-acyl carrier protein reductase, and the heat-shock protein HtpG. Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes. A conserved direct-repeat motif is present in the regulatory regions of several BPI-inducible genes, including the bipA gene. Only one of the BPI-responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A. We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S. typhimurium even though they bind with high affinity to the same cell-surface component.     

Pseudomonas putida 06909 genes expressed during colonization on mycelial surfaces and phenotypic characterization of mutants

AIMS: The main focus of this study was to gain an overall view of Pseudomonas putida 06909 genes involved in the Pseudomonas-Phytophthora interaction as a biological control mechanism, and to understand the roles of these genes. METHODS AND RESULTS: Sixteen Ps. putida genes with increased expression on Phytophthora mycelial surfaces were identified using in vivo expression technology (IVET) screening. Sequence analysis of these Phytophthora mycelium-induced (pmi) genes revealed that many of them display similarity to genes known or predicted to be involved in carbohydrate catabolism, energy metabolism, amino acid/nucleotide metabolism, and membrane transport processes. Disruption of three pmi genes encoding succinate semialdehyde dehydrogenase, a dicarboxylic acid transporter, and glyceraldehyde-3-phosphate dehydrogenase showed significant phenotypic differences involved in the colonization processes, including motility, biofilm formation on abiotic surfaces, colony morphology, and competitive colonization of fungal mycelia. All three of these pmi genes were induced by glycogen and other substances, such as organic acids and amino acids utilized by Ps. putida. CONCLUSIONS: The IVET screening and mutant characterization can be used to identify bacterial genes that are induced on the mycelial surface and provide insight into the possible mechanisms of mycelial colonization by this bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: The IVET screening through a bacterial genome library might be a huge task. However, because the genes involved in direct interaction with Phytophthora and in bacterial adaptation can be identified, the IVET system will be a valuable tool in studying biocontrol bacteria at the molecular and ecological levels.     

Examination of some morphologically unusual cultures of Phytophthora species using a mitochondrial DNA miniprep technique and a standardised sporangium caducity assessment

Using the mitochondrial DNA miniprep technique, the identity of sixteen morphologically unusual cultures allocated to Phytophthora nicotianae, Phytophthora mexicana or Phytophthora porri was determined by comparison with a library of mtDNA band patterns obtained from reference cultures. Seven cultures were identified as Phytophthora nicotianae (including those assigned to Phytophthora mexicana and Phytophthora porri), six as strains of Phytophthora palmivora with small, ovoid, weakly caducous sporangia, and one as Phytophthora citrophthora. Some cultures of P. nicotianae had a low percentage of caducous sporangia. Percentage sporangium caducity, but not sporangium L : B ratio, is considered a useful taxonomic criterion for separating species morphologically similar to Phytophthora nicotianae. One culture from tobacco in New Zealand had a highly unusual morphology and a unique DNA band pattern, but was not identifiable. One culture from Acacia mearnsii in South Africa had a unique DNA band pattern which was identical to that of an isolate from Annona squamosa from Australia previously identified as Phytophthora palmivora, the precise identity of which is still unclear. The identity of most isolates from diseased durian was found to be Phytophthora palmivora, confirming its role as the main pathogen, but P. nicotianae was also identified from this host. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of Pseudomonas syringae pv. tomato genes induced during infection of Arabidopsis thaliana

Phytopathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. Many of these genes should be induced when the bacteria come in contact with plant tissue. We used a modified in vivo expression technology (IVET) approach to identify genes from the plant pathogen Pseudomonas syringae pv. tomato that are induced upon infection of Arabidopsis thaliana and isolated over 500 in planta-expressed (ipx) promoter fusions. Sequence analysis of 79 fusions revealed several known and potential virulence genes, including hrp/hrc, avr and coronatine biosynthetic genes. In addition, we identified metabolic genes presumably important for adaptation to growth in plant tissue, as well as several genes with unknown function that may encode novel virulence factors. Many ipx fusions, including several corresponding to novel genes, are dependent on HrpL, an alternative RNA polymerase sigma factor that regulates the expression of virulence genes. Expression analysis indicated that several ipx fusions are strongly induced upon inoculation into plant tissue. Disruption of one ipx gene, conserved effector locus (CEL) orf1, encoding a putative lytic murein transglycosylase, resulted in decreased virulence of P. syringae. Our results demonstrate that this screen can be used successfully to isolate genes that are induced in planta, including many novel genes potentially involved in pathogenesis.     

A putative DEAD-box RNA-helicase is required for normal zoospore development in the late blight pathogen Phytophthora infestans

The asexual multinucleated sporangia of Phytophthora infestans can germinate directly through a germ tube or indirectly by releasing zoospores. The molecular mechanisms controlling sporangial cytokinesis or sporangial cleavage, and zoospore release are largely unknown. Sporangial cleavage is initiated by a cold shock that eventually compartmentalizes single nuclei within each zoospore. Comparison of EST representation in different cDNA libraries revealed a putative ATP-dependent DEAD-box RNA-helicase gene in P. infestans, Pi-RNH1, which has a 140-fold increased expression level in young zoospores compared to uncleaved sporangia. RNA interference was employed to determine the role of Pi-RNH1 in zoospore development. Silencing efficiencies of up to 99% were achieved in some transiently-silenced lines. These Pi-RNH1-silenced lines produced large aberrant zoospores that had undergone partial cleavage and often had multiple flagella on their surface. Transmission electron microscopy revealed that cytoplasmic vesicles fused in the silenced lines, resulting in the formation of large vesicles. The Pi-RNH1-silenced zoospores were also sensitive to osmotic pressure and often ruptured upon release from the sporangia. These findings indicate that Pi-RNH1 has a major function in zoospore development and its potential role in cytokinesis is discussed.     

Hypercyst mutants in Rhodospirillum centenum identify regulatory loci involved in cyst cell differentiation

Rhodospirillum centenum is a purple photosynthetic bacterium that forms resting cyst cells when starved for nutrients. In this study, we demonstrate that chalcone synthase gene (chsA) expression is developmentally regulated, with expression of chsA increasing up to 86-fold upon induction of the cyst developmental cycle. Screening for mini-Tn5-induced mutants that exhibit elevated chsA::lacZ expression has led to the isolation of a set of R. centenum mutants that display increased chsA gene expression concomitant with constitutive induction of the cyst developmental cycle. These "hypercyst" mutants have lost the ability to regulate cyst cell formation in response to nutrient availability. Sequence analysis indicates that the mini-Tn5-disrupted genes code for a variety of factors, including metabolic enzymes and a large set of potential regulatory factors, including four gene products with homology to histidine sensor kinases and three with homology to response regulators. Several of the disrupted genes also have sequence similarity to che-like signal transduction components.