Abstract: Automated and manual annotation of the ATP binding cassette (ABC) superfamily in the Phytophthora ramorum and P. sojae genomes has identified 135 and 136 members, respectively, indicating that this family is comparable in size to the Arabidopsis thaliana and rice genomes, and significantly larger than that of two fungal pathogens, Fusarium graminearum and Magnaporthe grisea. The high level of synteny between these oomycete genomes extends to the ABC superfamily, where 108 orthologues were identified by phylogenetic analysis. The largest subfamilies include those most often associated with multidrug resistance. The P. ramorum genome contains 22 multidrug resistance-associated protein (MRP) genes and 49 pleiotropic drug resistance (PDR) genes, while P. sojae contains 20 MRP and 49 PDR genes. Tandem duplication events in the last common ancestor appear to account for much of the expansion of these subfamilies. Recent duplication events in the PDR and ABCG families in both the P. ramorum and the P. sojae genomes indicate that selective expansion of ABC transporters may still be occurring. In other kingdoms, subfamilies define both domain arrangements and proteins having a common phylogenetic origin, but this is not the case for several subfamilies in oomycetes. At least one ABCG type transporter is derived from a PDR transporter, while transporters in the ABCB-half family cluster with transporters from bacterial, plant, and metazoan genomes. Additional examples of transporters that appear to be derived from horizontal transfer events from bacterial genomes include components of transporters associated with iron uptake and DNA repair.
Abstract: All published QTLs and genes for Phytophthora resistance were projected onto the bins of a high-density reference map of potato. Further, a transcriptome map containing around 700 cDNA-AFLP (TDF) markers was anchored to this map. We have analysed cDNA markers which are co-located with these resistance QTLs by cloning, sequencing and by performing homology searches in public sequence databases. Several interesting homologies were detected with typical resistance and stress response genes. On the other hand, we have screened all known QTL locations on the 12 potato chromosomes via linked SSR markers for the presence of a selectable QTL for Phytophthora resistance in four genetic backgrounds. Progenies descended from different Solanum wild species as resistance sources. Leaf and tuber resistances were analysed. In all case studies, several selectable QTLs were detected which descended from either parent. Tuber and leaf QTLs varied from progeny to progeny and between leaves and tubers. In none of the progenies were resistance levels of leaf and tuber blight correlated.
Abstract: To explore the molecular mechanisms involved in asexual spore development in Phytophthora sojae, the zoospores of strain PS26 were treated with ultraviolet (UV) irradiation. After selection, a mutant progeny, termed PS26-U03, was obtained and demonstrated to exhibit no oospore production. A suppression subtractive hybridization (SSH) approach was developed to investigate differences in gene expression between PS26 and PS26-U03 during asexual sporogenesis. Of the 126 sequences chosen for examination, 39 putative unigenes were identified that exhibit high expression in PS26. These sequences are predicted to encode proteins involved in metabolism, cell cycle, protein biosynthesis, cell signalling, cell defence, and transcription regulation. Seven clones were selected for temporal expression analysis using RT-PCR based on the results of the dot-blot screens. Three of the selected genes, developmental protein DG1037 (UB88), glycoside hydrolase (UB149) and a hypothetical protein (UB145), were expressed only in PS26, whereas the transcripts of phosphatidylinositol-4-phosphate 5-kinase (UB36), FAD-dependent pyridine nucleotide-disulphide oxidoreductase (UB226) and sugar transporter (UB256) were expressed at very low levels in PS26-U03 but at high levels in PS26.
Abstract: Sporulation in Phytophthora infestans is associated with a major remodeling of the transcriptome. To better understand promoter structure and how sporulation-specific expression is determined in this organism, the Pks1 gene was analyzed. Pks1 encodes a protein kinase that is induced at an intermediate stage of sporulation, prior to sporangium maturation. Major and minor transcription start sites mapped throughout the promoter, which contains many T-rich stretches and Inr-like elements. Within the T-rich region are several motifs which bound nuclear proteins in EMSA. Tests of modified promoters in transformants implicated a CCGTTG located 110-nt upstream of the transcription start point as a major regulator of sporulation-specific transcription. The motif also bound a sporulation-specific nuclear protein complex. A bioinformatics analysis indicated that the motif is highly over-represented within co-expressed promoters, in which it predominantly resides 100–300-nt upstream of transcription start sites. Other sequences, such as a CATTTGTT motif, also bound nuclear proteins but did not play an essential role in spore-specific expression.
Abstract: A potato gene, StLRPK1 (Solanum tuberosum L. leucine-rich-repeat receptor-like protein kinases 1), encoding a protein belonging to leucine-rich repeat receptor-like kinases (LRR-RLKs) was identified. It encodes 796 amino acids with 88% of identity to SRF3 of Arabidopsis thaliana and contains a signal peptide, five LRR motifs, a transmembrane domain, two proline-rich regions and a serine/threonine protein kinase domain. The transcripts were present at high levels in flowers and young leaves, while low in other tested organs. The mRNA of StLRPK1 was inducible in potato leaves by Phytophthora infestans, a pathogen causing late blight disease, and showed different profiles after treatment with salicylic acid, methyl jasmonate, ethylene, abscissic acid, wounding, 40°C, 4°C and a salinity stress. The results suggest that StLRPK1 may participate in the responses against environmental stresses and disease resistance in potato.
Abstract: We present a strategy to recover high molecular weight genomic DNA from large numbers of isolates of Phytophthora. Included are steps for generating mycelial mass in 24-well reuseable deep well plates, efficient lyophilization and disruption of the mycelium and genomic DNA extraction with 96-well glass fiber filter plates. The resulting DNA is consistently high molecular weight and is suitable for applications that require high quality DNA such as AFLP analysis and TILLING. A single operator easily can manage mycelium preparation and/or DNA extraction from 384 isolates in a single day and this approach might be useful for other fungi or fungi-like organisms that can be grown in liquid media.
Abstract: Twenty-one homologs of family 5 endo-(1–4)-b-glucanase genes (EGLs) were identified and characterized in the oomycete plant pathogens Phytophthora infestans, P. sojae, and P. ramorum, providing the first comprehensive analysis of this family in Phytophthora. Phylogenetic analysis revealed that these genes constitute a unique eukaryotic group, with closest similarity to bacterial endoglucanases. Many of the identified EGL copies were clustered in a few genomic regions, and contained from zero to three introns. Using reverse transcription PCR to study in vitro and in planta gene expression levels of P. sojae, we detected partially processed RNA transcripts retaining one or more of their introns. In some cases, the positions of intron/exon splicing sites were also found to be variable. The relative proportions of these transcripts remain apparently unchanged under various growing conditions, but differ among orthogolous copies of the three Phytophthora species. The alternate processing of introns in this group of EGLs generates both coding and non-coding RNA isoforms. This is the first report on Phytophthora family 5 endoglucanases, and the first record for alternative intron processing of oomycete transcripts.
Abstract: A correct interpretation of isozyme patterns can be seriously hampered by the lack of supporting genetic data. The availability of the complete genome sequence of Phytophthora ramorum, enabled us to correlate isozyme profiles with the gene models predicted for these enzymes. Thirty-nine P. ramorum strains were characterised employing isozyme analysis on malate dehydrogenase (MDH), NADP-dependent malic enzyme (MDHP), 6-phosphogluconate dehydrogenase (PGD), glucosephosphate isomerase (GPI) and lactate dehydrogenase (LDH) comprising nine putative loci. One isozyme band was enzymatically stained for PGD whereas multiple bands were detected for GPI, MDH, MDHP and LDH. All putative loci were monomorphic except for Ldh-2. Genome mining revealed that the assembled genome sequences of P. ramorum and P. sojae each contain one Gpi and one Pgd gene model. For MDH, two gene models were identified, encoding a cytosolic and mitochondrial type, respectively. Also for MDHP P. ramorum has two gene models that are both duplicated in P. sojae. Both species contain six Ldh gene models, including pseudogenes. The Ldh gene models are clustered and located in regions that show a high level of conserved synteny. This study demonstrates that insight into the gene models encoding isozymes helps to interpret isozyme profiles in Phytophthora.
Abstract: Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 and a compatible infection by Phytophthora megakarya were studied. Ten genes were selected to represent genes involved in defense (TcCaf-1, TcGlu1,3, TcChiB, TcCou-1, and TcPer-1), gene regulation (TcWRKY-1 and TcORFX-1), cell wall development (TcCou-1, TcPer-1, and TcGlu-1), or energy production (TcLhca-1 and TcrbcS). Leaf development was separated into unexpanded (UE), young red (YR), immature green (IG), and mature green (MG). Our data indicates that the constitutive defense mechanisms used by cacao leaves differ between different developmental stages. TcWRKY-1 and TcChiB were highly expressed in MG leaves, and TcPer-1, TcGlu-1, and TcCou-1 were highly expressed in YR leaves. TcGlu1,3 was highly expressed in UE and YR leaves, TcCaf-1 was highly expressed in UE leaves, and TcLhca-1 and TcrbcS were highly expressed in IG and MG leaves. NEP1 encodes the necrosis inducing protein Nep1 produced by Fusarium oxysporum and has orthologs in Phytophthora species. Nep1 caused cellular necrosis on MG leaves and young pods within 24 h of application. Necrosis was observed on YR leaves 10 days after treatment. Expression of TcWRKY-1, TcORFX-1, TcPer-1, and TcGlu-1 was enhanced and TcLhca-1 and TcrbcS were repressed in MG leaves after Nep1 treatment. Expression of TcWRKY-1 and TcORFX-1 was enhanced in YR leaves after Nep1 treatment. Infection of MG leaf disks by P. megakarya zoospores enhanced expression of TcGlu-1, TcWRKY-1, and TcPer-1 and repressed expression of TcChiB, TcLhca-1 and TcrbcS. Five of the six genes that were responsive to Nep1 were responsive to infection by P. megakarya. Susceptibility of T. cacao to P. megakarya includes altered plant gene expression and phytotoxic molecules like Nep1 may contribute to susceptibility.
Abstract: Sequences of the internal transcribed spacer (ITS) region 1 were used to examine the phylogenetic relationships among races of 19 isolates of Phytophthora vignae f. sp. adzukicola and between this forma specialis and three isolates of the closely related P. vignae f. sp. vignae. The ITS 1 sequences were highly conserved (> 98.7% similarity) among representatives of both formae speciales groups. The results of this study indicate that P. vignae is a monophyletic group.
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