Abstract: A total of 18 paralogs of xyloglucan-specific endoglucanases (EGLs) from the glycosyl hydrolase family 12 were identified and characterized in Phytophthora sojae and Phytophthora ramorum. These genes encode predicted extracellular enzymes, with sizes ranging from 189 to 435 amino acid residues, that would be capable of hydrolyzing the xyloglucan component of the host cell wall. In two cases, four and six functional copies of these genes were found in tight succession within a region of 5 and 18 kb, respectively. The overall gene copy number and relative organization appeared well conserved between P. sojae and P. ramorum, with apparent synteny in this region of their respective genomes. Phylogenetic analyses of Phytophthora endoglucanases of family 12 and other known members of EGL 12, revealed a close relatedness with a fairly conserved gene sub-family containing, among others, sequences from the fungi Emericella desertorum and Aspergillus aculeatus. This is the first report of family 12 EGLs present in plant pathogenic eukaryotes.
Abstract: Isozyme analysis and cytochrome oxidase sequences were used to examine whether differentiation of P. fragariae var. fragariae and P. fragariae var. rubi at the variety level is justified. In isozyme studies six strains of both P. fragariae varieties were analyzed with malate dehydrogenase (MDH), glucose phosphate isomerase (GPI), aconitase (ACO), isocitrate dehydrogenase (IDH) and phosphogluconate dehydrogenase (PGD), comprising altogether seven putative loci. Five unique alleles (Mdh-1A, Mdh-2B, GpiA, AcoB and Idh-1B) were found in strains of P. fragariae var. fragariae, whereas five unique alleles (Mdh-1B, Mdh-2A, GpiB, AcoA and Idh-1A) were present in strains of P. fragariae var. rubi. It was inferred from these data that there is no gene flow between the two P. fragariae varieties. Cytochrome oxidase I (Cox I) sequences showed consistent differences at 15 positions between strains of Fragaria and Rubus respectively. Based on isozyme data, cytochrome oxidase I sequences, and previously published differences in restyriction enzyme patterns of mitochondrial DNA, sequences of nuclear and mitochondrial genes, AFLP patterns and pathogenicity, it was concluded that both specific pathogenic varieties of P. fragariae are reproductively isolated and constitute a distinct species. Consequently strains isolated from Rubus idaeus are assigned to Phytophthora rubi comb. nov.
Abstract: In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop ‘maca’ (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74–94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 μM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.
Abstract: The Phytophthora Functional Genomics Database (PFGD; http://www.pfgd.org), developed by the National Center for Genome Resources in collaboration with The Ohio State University-Ohio Agricultural Research and Development Center (OSU-OARDC), is a publicly accessible information resource for Phytophthora,–plant interaction research. PFGD contains transcript, genomic, gene expression and functional assay data for Phytophthora infestans, which causes late blight of potato, and Phytophthora sojae, which affects soybeans. Automated analyses are performed on all sequence data, including consensus sequences derived from clustered and assembled expressed sequence tags. The PFGD search filter interface allows intuitive navigation of transcript and genomic data organized by library and derived queries using modifiers, annotation keywords or sequence names. BLAST services are provided for libraries built from the transcript and genomic sequences. Transcript data visualization tools include Quality Screening, Multiple Sequence Alignment and Features and Annotations viewers. A genomic browser that supports comparative analysis via novel dynamic functional annotation comparisons is also provided. PFGD is integrated with the Solanaceae Genomics Database (SolGD; http://www.solgd.org) to help provide insight into the mechanisms of infection and resistance, specifically as they relate to the genus Phytophthora pathogens and their plant hosts.
Abstract: Three putative exo-1,3-β-glucanase genes (Piexo1, Piexo2, Piexo3), one endo-1,3-β-glucanase (Piendo1) and one endo-1,3;1,4-β-glucanase (Piendo2) gene were cloned and characterized from the oomycete Phytophthora infestans. Southern hybridization revealed that Piexo1, Piexo2, Piexo3, and Piendo2 are single copy, and that Piendo1 is encoded by two copies. Furthermore, the analyses showed that for each gene, one or two closely related gene family members were present. The genes contain no introns. Nucleotide sequence analysis of the promoter regions (200 nt upstream of ATG start codon) showed that the regions have 56–81% similarity to a 16-nt core sequence hypothesized to be the initiation of transcription point in oomycetes. The predicted molecular weights (32–83 kDa), iso-electric points (4.2–6.7) and amino acid sequences of the five proteins are diverse. All the genes are expressed in in vitro grown mycelia and sporangia, as well as during infection of potatoes. Further, Piendo1 and Piendo2 are also expressed in germinating cysts, and Piendo2 in zoospores.
Abstract: Reverse transcription followed by quantitative polymerase chain reaction analysis, or qRT-PCR, is an extremely sensitive, cost-effective method for quantifying gene transcripts from plant cells. The availability of nonspecific double-stranded DNA (dsDNA) binding fluorophors, such as SYBR Green, and 384-well-plate real-time PCR machines that can measure fluorescence at the end of each PCR cycle make it possible to perform qRT-PCR on hundreds of genes or treatments in parallel. This has facilitated the comparative analysis of all members of large gene families, such as transcription factor genes (Czechowski et al., 2004Go). Given the relatively low cost of PCR reagents, and the precision, sensitivity, flexibility, and scalability of qRT-PCR, it is little wonder that thousands of research labs around the world have embraced it as the method of choice for measuring transcript levels. However, despite its popularity, we continue to see systematic errors in the application of methods for qRT-PCR analysis, which can compromise the interpretation of results. The letter to the editor by Gutierrez et al. in this issue highlights one of many common sources of error, namely, the inappropriate choice of reference genes for normalizing transcript levels of test genes prior to comparative analysis of different biological samples. The following are 11 golden rules of qRT-PCR that, when observed, should ensure reproducible and accurate measurements of transcript abundance in plant and other cells. These rules are for relative quantification of RNA using two-step RT-PCR (where the product of a single RT reaction is used as template in multiple PCR reactions), SYBR Green to detect gene-specific PCR products, and reference genes for normalizing transcript levels of test genes before comparing samples. Further details can be found elsewhere (Czechowski et al., 2004Go, 2005Go). Most of these rules also apply to relative quantification methods that employ sequence-specific fluorescent probes, such as TaqMan probes, and to absolute quantification methods.
Abstract: A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.
Abstract: Suppression subtractive hybridization (SSH) is an effective approach to identify the genes that vary in expression levels during different biological processes. It is often used in higher eukaryotes to study the molecular regulation in complex pathogenic progress, such as tumorigenesis and other chronic multigene-associated diseases. Because microbes have relatively smaller genomes compared with eukaryotes, aside from the analysis at the mRNA level, SSH as well as its modifications have been further employed to isolate specific chromosomal locus, study genomic diversity related with exceptional bacterial secondary metabolisms or genes with special microbial function. This review introduces the SSH and its associated methods and focus on their applications to detect specific functional genes or DNA markers in microorganisms.
Abstract: A subtracted cDNA library for Davidia involucrata was constructed using suppression subtractive hybridization (SSH). mRNA isolated from young leaves was used as a “driver,” and mRNAs isolated from young bracts were used as “testers.” The differentially expressed cDNA fragments in bracts were identified by differential screening. Of the 16 clones selected randomly from the screened library, 8 were known genes found in GenBank, and 2 had no similar sequences. Northern blot analysis revealed that the expression level of P1A5 cDNAs selected randomly was dominantly expressed in bracts. This indicates that SSH can be used to clone differentially expressed cDNAs inD. involucrata bracts.
Abstract: Glucose-6-phosphate isomerase (GPI) plays a key role in both glycolysis and gluconeogenesis. Isoforms of GPI are common, and therefore, its isozyme pattern is widely used to characterize isolates of Phytophthora infestans. Despite the importance of GPI in P. infestans studies, the gene encoding this enzyme has not yet been characterized. Furthermore, it has been suggested that P. infestans contains multiple copies of the gene but this hypothesis remains to be demonstrated. We have cloned and characterized GPI in various isolates of P. infestans as well as in several species of the genus Phytophthora. The gene contains 1671 bp and encodes a protein with a predicted molecular weight of 60.8 kDa. Multiple different alleles were identified and Southern analysis indicated certain P. infestans isolates carry several copies of the gene. Phylogenetic analysis revealed that P. infestans GPI is most closely related to sequences from Toxoplasma gondii, Arabidopsis thaliana, and Clarkia lewisii.
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