Abstract: The mtDNA haplotypes of the plant pathogen Phytophthora infestans present in dried potato and tomato leaves from herbarium specimens collected during the Irish potato famine and later in the 19th and early 20th century were identified. A 100 bp fragment of ribosomal DNA (rDNA) specific for P. infestans was amplified from 90% of the specimens (n=186), confirming infection by P. infestans. Primers were designed that distinguish the extant mtDNA haplotypes. 86% percent of the herbarium specimens from historic epidemics were infected with the Ia mtDNA haplotype. Two mid-20th century potato leaves from Ecuador (1967) and Bolivia (1944) were infected with the Ib mtDNA haplotype of the pathogen. Both the Ia and IIb haplotypes were found in specimens collected in Nicaragua in the 1950s. The data suggest that the Ia haplotype of P. infestans was responsible for the historic epidemics during the 19th century in the UK, Europe, and the USA. The Ib mtDNA haplotype of the pathogen was dispersed later in the early 20th century from Bolivia and Ecuador. Multiple haplotypes were present outside Mexico in the 1940s–60s, indicating that pathogen diversity was greater than previously believed.
Abstract:Phytophthora blight of pepper is a completely destructive plant disease caused by the oomycete pathogen Phytophthora capsici Leonian. Phytophthora disease is responsible for major losses in pepper production and the pathogen can survive in soil in the absence of the host plant for many years. Currently, there are no early diagnostic reagents available that specifically target P. capsici. Therefore, diagnostic tools that can detect Phytophthora are required. In the present study, we screened for P. capsici-binding peptides using M13 phage display. After five rounds of biopanning, we identified P. capsici-binding peptides from a random peptide library that showed high binding affinity and specificity toward P. capsici in the picomolar range. These peptides can be used to develop novel diagnostic probes or potent inhibitors with diverse polyvalencies.
Abstract: The first known families of tRNA-related short interspersed elements (SINEs) in the oomycetes were identified by exploiting the genomic DNA sequence resources for the potato late blight pathogen, Phytophthora infestans. Fifteen families of tRNA-related SINEs, as well as predicted tRNAs, and other possible RNA polymerase III-transcribed sequences were identified. The size of individual elements ranges from 101 to 392 bp, representing sequences present from low (1) to highly abundant (over 2000) copy number in the P. infestans genome, based on quantitative PCR analysis. Putative short direct repeat sequences (6–14 bp) flanking the elements were also identified for eight of the SINEs. Predicted SINEs were named in a series prefixed infSINE (for infestans-SINE). Two SINEs were apparently present as multimers of tRNA-related units; four copies of a related unit for infSINEr, and two unrelated units for infSINEz. Two SINEs, infSINEh and infSINEi, were typically located within 400 bp of each other. These were also the only two elements identified as being actively transcribed in the mycelial stage of P. infestans by RT-PCR. It is possible that infSINEh and infSINEi represent active retrotransposons in P. infestans. Based on the quantitative PCR estimates of copy number for all of the elements identified, tRNA-related SINEs were estimated to comprise 0.3% of the 250 Mb P. infestans genome. InfSINE-related sequences were found to occur in species throughout the genus Phytophthora. However, seven elements were shown to be exclusive to P. infestans.
Abstract: To explore the molecular mechanisms involved in virulence variations in Phytophthora sojae, the low-virulence isolate PS2 was inoculated successively on a resistant soybean (Glycine max) cultivar. After 14 successive inoculations, a high-virulence progeny, termed PS2-vir, was obtained and demonstrated to exhibit lower oospore production. DNA fingerprinting revealed no large-scale DNA differences in PS2 and PS2-vir. A suppression subtractive hybridization (SSH) approach was developed to investigate differences in gene expression between PS2 and PS2-vir in the early stages of soybean infection. Of the 323 sequences chosen for examination, 74 putative unigenes were identified that exhibit high expression in PS2-vir. These sequences are predicted to encode proteins involved in energy production, protein biosynthesis, cell signaling, cell-wall biogenesis, and transcription regulation. Ten clones were selected for temporal expression analysis using RT-PCR based on the results of the dot-blot screens. The possible genetic mechanisms involved in these phenomena are discussed.
Abstract: A large-scale screen for genes induced during sexual development was performed in the heterothallic oomycete Phytophthora infestans, the potato blight agent. Of 15,644 unigenes on an Affymetrix chip, 87 were induced >10-fold during mating, with 28 induced >100-fold. This was validated in independent matings using RNA blots and RT-PCR. Only 44 genes resembled sequences in GenBank. These encoded regulators such as protein kinases, protein phosphatases, and transcription factors, plus enzymes with metabolic, transport, or cell-cycle activities. Several genes were induced during both mating and asexual sporogenesis, suggesting crosstalk between those pathways. In the homothallic species P. phaseoli, 20% of the 87 genes were expressed at higher levels during conditions conducive to oosporogenesis than non-conducive conditions, while the rest were at similar levels. Many of the latter exhibited higher mRNA concentrations in P. phaseoli than in any non-mating culture of P. infestans, suggesting that part of the sexual pathway is active constitutively in homothallics.
Abstract:PnPMA1, a gene encoding a putative P-type plasma membrane H+-ATPase, has been isolated by differential screening of a Phytophthora nicotianae germinated cyst cDNA library. PnPMA1 is differentially expressed during pathogen asexual development with a more than 10-fold increase in expression in germinated cysts, the stage at which plant infection is initiated, compared to vegetative or sporulating hyphae or motile zoospores. PnPMA1 proteins are encoded by two closely linked genes that have no introns and encode identical proteins having 1068 amino acid residues and a molecular mass of 116.3 kDa. PnPMA1 shows moderate identity (30–50%) to plant and fungal plasma membrane H+-ATPases and weak identity to other P-type cation-transporting ATPases. PnPMA1 contains all the catalytic domains characteristic of H+-ATPases but also has a distinct domain of not, vert, similar 155 amino acids that forms a putative cytoplasmic loop between transmembrane domains 8 and 9, a feature that is not present in PMA1 proteins from other organisms. Polyclonal antibodies raised against the 155 residue domain were shown by immunogold labelling to react with a protein in the plasma membrane of P. nicotianae germinated cysts but not with the plasma membrane of motile zoospores. Genetic complementation experiments demonstrated that the P. nicotianae PnPMA1 is functional in yeast, Saccharomyces cerevisiae.
Abstract: 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.
Abstract: Real-time quantitative reverse transcription-PCR (qRT-PCR) has become one of the most commonly used methods for RNA quantification in recent years. To obtain reliable results with biological significance, it is important that qRT-PCR data are normalized with a proper internal control. In this study, 18 housekeeping genes were selected and evaluated for their potential as a suitable internal control for study of gene expression in the oomycete plant pathogen Phytophthora parasitica. Analysis of qRT-PCR data using the geNorm software indicated that, although commonly used as internal controls, β-actin (ACT) and translation elongation factor 1α (eEF1A) might not be the best choice due to variable expression across different life stages of P. parasitica. Instead, other genes would serve as better controls, including ubiquitin-conjugating enzyme (Ubc), WS21, and β-tubulin (Tub-b) for asexual stage, Ubc and Tub-b for sexual reproduction, while Ubc and WS21 for the stage of pathogenesis, because of their constant expression levels in each given subset of RNA samples. Although normalization with more than one gene would generate more reliable results, use of a single stably expressed gene as an internal control would suffice for accurate data normalization in some experiments.
Abstract: The molar fraction of 3-hydroxyvalerate (3HV) in poly(3-hydroxybutyrate-3-hydroxyvalerate) [P(3HB-3HV)] was modulated through the co-amplification of the phbC gene encoding PHB synthase and the zwf gene encoding glucose-6-phosphate dehydrogenase generating NADPH from the metabolism of fructose as a cofactor in Ralstonia eutropha. The biosynthesis of poly-3-hydroxybutyrate (PHB) and P(3HB-3HV) increased appreciably after the co-amplification of the phbC and zwf genes due to the accelerated polymerization reaction and enhanced NADPH supplementation. In particular, the 3HV molar fraction in P(3HB-3HV) was effectively modulated. pH-stat fed-batch cultivation of the R. eutropha transformant co-amplifying the phbC and zwf genes was also carried out to increase the biosynthesis of P(3HB-3HV) and modulate the 3HV molar fraction. The content and molar fraction of P(3HB-3HV) achieved up to 60.1%and 71.9mol%, respectively, through the concurrent feeding of valerate and fructose.
Abstract:Ralstonia sp. KNl-lOA, which was constructed by inserting the tac promoter upstream of the phenol hydroxylase (PI-I) gene in the chromosomal DNA of the wild-type strain, Ralstonia sp. KNl, is a useful recombinant strain for eliminating trichloroethylene (TCE) from contaminated sites because it exhibits constitutive TCE oxidation activity. During TCE degradation by Ralstonia sp. KNl-lOA, noxious chlorinated compounds, such as dichloroacetic acid, trichloroacetic acid,2,2,24richloroethanol, and chloral, were not detected, and more than 95% of chlorine in TCE was released as chloride ions. Among the possible TCE transformation products, only carbon monoxide was detected, and its conversion percentage was 7 mol%. The addition of formate, which Ralstonia sp. KNl-1OA could use as an exogenous electron donor, did not enhance the TCE degradation performance, suggesting that NADH depletion did not limit the degradation. The phenol degradation activity of Ralstonia sp. KNl-1OA that previously degraded TCE was not markedly lower than that of cells not exposed to TCE, suggesting that Ralstonia sp. KNl-1OA was not susceptible to product toxicity associated with TCE degradation. Furthermore, to clarify the mechanisms underlying TCE degradation by PH from Ralstonia sp. KNl, this enzyme was compared with another enzyme, a hybrid aromatic ring dioxygenase exhibiting a high TCE degradation activity in Escherichia coli and Pseudomonas sp. The initial TCE degradation rate of Ralstonia sp. KNl (pKTPlOO), which produced PH, was l/50 lower than that of Rabtonia sp. KNl (pKTF200), which produced the hybrid aromatic ring dioxygenase. However, because of its lower product toxicity, the strain producing PH could degrade 2.3 times more TCE than that generated by the strain producing the hybrid aromatic ring dioxygenase.
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