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The heat shock transcription factor PsHSF1 of Phytophthora sojae is required for oxidative stress tolerance and detoxifying the plant oxidative burst
Sheng, Yuting ; Wang, Yonglin ; Meijer, H.J.G. ; Yang, Xinyu ; Hua, C. ; Ye, Wenwu ; Tao, Kai ; Liu, Xiaoyun ; Govers, F. ; Wang, Yuanchao - \ 2015
Environmental Microbiology 17 (2015)4. - ISSN 1462-2912 - p. 1351 - 1364.
signal-transduction - in-vivo - pathogen - infestans - expression - sequence - defense - laccase - binding - yeast
In the interaction between plant and microbial pathogens, reactive oxygen species (ROS) rapidly accumulate upon pathogen recognition at the infection site and play a central role in plant defence. However, the mechanisms that plant pathogens use to counteract ROS are still poorly understood especially in oomycetes, filamentous organisms that evolved independently from fungi. ROS detoxification depends on transcription factors (TFs) that are highly conserved in fungi but much less conserved in oomycetes. In this study, we identified the TF PsHSF1 that acts as a modulator of the oxidative stress response in the soybean stem and root rot pathogen Phytophthora sojae. We found that PsHSF1 is critical for pathogenicity in P.¿sojae by detoxifying the plant oxidative burst. ROS produced in plant defence can be detoxified by extracellular peroxidases and laccases which might be regulated by PsHSF1. Our study extends the understanding of ROS detoxification mechanism mediated by a heat shock TF in oomycetes.
Small Homologous Blocks in Phytophthora Genomes Do Not Point to an Ancient Whole-Genome Duplication
Hooff, J.J.E. van; Snel, B. ; Seidl, M.F. - \ 2014
Genome Biology and Evolution 6 (2014)5. - ISSN 1759-6653 - p. 1079 - 1085.
pathogen phytophthora - maximum-likelihood - evolution - genes - consequences - mechanisms - adaptation - repertoire - sequences - infestans
Genomes of the plant-pathogenic genus Phytophthora are characterized by small duplicated blocks consisting of two consecutive genes (2HOM blocks) and by an elevated abundance of similarly aged gene duplicates. Both properties, in particular the presence of 2HOM blocks, have been attributed to a whole-genome duplication (WGD) at the last common ancestor of Phytophthora. However, large intraspecies synteny-compelling evidence for a WGD-has not been detected. Here, we revisited the WGD hypothesis by deducing the age of 2HOM blocks. Two independent timing methods reveal that the majority of 2HOM blocks arose after divergence of the Phytophthora lineages. In addition, a large proportion of the 2HOM block copies colocalize on the same scaffold. Therefore, the presence of 2HOM blocks does not support a WGD at the last common ancestor of Phytophthora. Thus, genome evolution of Phytophthora is likely driven by alternative mechanisms, such as bursts of transposon activity.
Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica
Jiang, R.H.Y. ; Bruijn, I. de; Haas, B.J. ; Belmonte, R. ; Löbach, L. ; Christie, J. ; Ackerveken, G. van den; Bottin, A. ; Bulone, V. ; Díaz-Moreno, S.M. ; Dumas, B. ; Fan, L. ; Gaulin, E. ; Govers, F. ; Grenville-Briggs, L.J. ; Horner, N.R. ; Levin, J.Z. ; Mammella, M. ; Meijer, H.J.G. ; Morris, P. ; Nusbaum, C. ; Oome, S. ; Phillips, A.J. ; Rooyen, D. van; Rzeszutek, E. ; Saraiva, M. ; Secombes, C.J. ; Seidl, M.F. ; Snel, B. ; Stassen, J.H.M. ; Sykes, S. ; Tripathy, S. ; Berg, H. ; Vega-Arreguin, J.C. ; Wawra, S. ; Young, S.K. ; Zeng, Q. ; Dieguez-Uribeondo, J. ; Russ, C. ; Tyler, B.M. ; West, P. van - \ 2013
Plos Genetics 9 (2013)6. - ISSN 1553-7404 - 20 p.
expressed sequence tags - anthocidaris-crassispina eggs - fully automated process - phytophthora-sojae - aphanomyces-euteiches - plant-pathogens - infestans - cells - evolution - reveals
Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler's, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.
Chemotaxis and oospore formation in Phytophthora sojae are controlled by G-protein-coupled receptors with a phosphatidylinositol phosphate kinase domain
Yang, X. ; Zhao, W. ; Hua, C. ; Zheng, X. ; Jing, M. ; Li, D. ; Govers, F. ; Meijer, H.J.G. ; Wang, Y. - \ 2013
Molecular Microbiology 88 (2013)2. - ISSN 0950-382X - p. 382 - 394.
sexual reproduction - gene-expression - mating hormone - plasma-membrane - alpha-subunit - infestans - pathogen - oomycete - isoflavones - identification
G-protein-coupled receptors (GPCRs) are key cellular components that mediate extracellular signals into intracellular responses. Genome mining revealed that Phytophthora spp. have over 60 GPCR genes among which a prominent class of 12 encoding novel proteins with an N-terminal GPCR domain fused to a C-terminal phosphatidylinositol phosphate kinase (PIPK) domain. This study focuses on two GPCR-PIPKs (GKs) in Phytophthora sojae. PsGK4 and PsGK5 are differentially expressed during the life cycle with the highest expression in cysts and during cyst germination, and at late infection stages. In P.¿sojae transformants that constitutively express RFP-tagged PsGK4 and PsGK5, the fusion proteins in hyphae reside in small, rapidly moving vesicular-like structures. Functional analysis using gene silencing showed that PsGK4-silenced transformants displayed higher levels of encystment and a reduced cyst germination rate when compared with the recipient strain. Moreover, GK4 deficiency (or reduction) resulted in severe defects in zoospore chemotaxis towards isoflavones and soybean roots. In contrast, PsGK5-silenced transformants exhibited no obvious defects in asexual development but oospore production was severely impaired. Both, PsGK4- and PsGK5-silenced transformants showed reduced pathogenicity. These results point to involvement of GKs in zoospore behaviour, chemotaxis and oospore development, and suggest that PsGK4 and PsGK5 each head independent signalling pathways.
Genome-wide identification of Phytophthora sojae SNARE genes and functional characterization of the conserved SNARE PsYKT6
Zhao, W. ; Dong, S. ; Ye, W. ; Hua, C. ; Meijer, H.J.G. ; Dou, X. ; Govers, F. ; Wang, Y. - \ 2011
Fungal Genetics and Biology 48 (2011)3. - ISSN 1087-1845 - p. 241 - 251.
pathogen phytophthora - systematic analysis - root-rot - fusion - proteins - infestans - complex - localization - eukaryotes - mechanisms
Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.
Clonal expansion of the Belgian Phytophthora ramorum populations based on new microsatellite markers
Vercauteren, A. ; Dobbelaere, I. de; Grünwald, N. ; Bonants, P.J.M. ; Bockstaele, E. van; Maes, M. ; Heungens, K. - \ 2010
Molecular Ecology 19 (2010)1. - ISSN 0962-1083 - p. 92 - 107.
sudden oak death - fungicide resistance - genotypic diversity - 1st report - metalaxyl resistance - practical experience - ornamental plants - mating-type - infestans - pathogen
Co-existence of both mating types A1 and A2 within the EU1 lineage of Phytophthora ramorum has only been observed in Belgium, which begs the question whether sexual reproduction is occurring. A collection of 411 Belgian P. ramorum isolates was established during a 7-year survey. Our main objectives were genetic characterization of this population to test for sexual reproduction, determination of population structure, evolution and spread, and evaluation of the effectiveness and impact of control measures. Novel, polymorphic simple sequence repeat (SSR) markers were developed after screening 149 candidate loci. Eighty isolates of P. ramorum, broadly representing the Belgian population, were analyzed using four previously described and three newly identified polymorphic microsatellite loci as well as amplified fragment length polymorphisms. SSR analysis was most informative and was used to screen the entire Belgian population. Thirty multilocus genotypes were identified, but 68% of the isolates belonged to the main genotype EU1MG1. Although accumulated mutation events were detected, the overall level of genetic diversity within the Belgian isolates of P. ramorum appears to be limited, indicating a relatively recent clonal expansion. Based on our SSR analysis there is no evidence of sexual recombination in the Belgian population of P. ramorum. Metalaxyl use decreased the genetic diversity of P. ramorum until 2005, when the majority of the isolates had become resistant. Most genotypes were site-specific and despite systematic removal of symptomatic and neighbouring plants, some genotypes were detected over a period of several years at a single site, sometimes discontinuously, indicating (latent) survival of the pathogen at those sites
Letter to the Editor : Standardizing the nomenclature for clonal lineages of the sudden oak death pathogen, Phytophthora ramorum
Grünwald, N.J. ; Goss, E.M. ; Ivors, K. ; Garbelotto, M. ; Martin, F.N. ; Prospero, S. ; Hansen, E. ; Bonants, P.J.M. ; Hamelin, R.C. ; Chastagner, M. ; Werres, S. ; Rizzo, D.M. ; Abad, G. ; Beales, P. ; Bilodeau, G.J. ; Blomquist, C.L. ; Brasier, C. ; Brière, S.C. ; Chandelier, A. ; Davidson, J.M. ; Denman, S. ; Elliott, M. ; Frankel, S.J. ; Goheen, E.M. ; Gruyter, H. de; Heungens, K. ; James, D. ; Kanaskie, A. ; McWilliams, M.G. ; Man in't Veld, W. ; Moralejo, E. ; Osterbauer, N.K. ; Palm, M.E. ; Parke, J.L. ; Perez Sierra, A.M. ; Shamoun, S.F. ; Shishkoff, N. ; Tooley, P.W. ; Vettraino, A.M. ; Webber, J. ; Widmer, T.L. - \ 2009
Phytopathology 99 (2009)7. - ISSN 0031-949X - p. 792 - 795.
in-vitro - north-american - european populations - genotypic diversity - dna polymorphisms - central mexico - toluca valley - united-states - infestans - california
Phytophthora ramorum, the causal agent of sudden oak death and ramorum blight, is known to exist as three distinct clonal lineages which can only be distinguished by performing molecular marker-based analyses. However, in the recent literature there exists no consensus on naming of these lineages. Here we propose a system for naming clonal lineages of P. ramorum based on a consensus established by the P. ramorum research community. Clonal lineages are named with a two letter identifier for the continent on which they were first found (e.g., NA = North America; EU = Europe) followed by a number indicating order of appearance. Clonal lineages known to date are designated NA1 (mating type: A2; distribution: North America; environment: forest and nurseries), NA2 (A2; North America; nurseries), and EU1 (predominantly A1, rarely A2; Europe and North America; nurseries and gardens). It is expected that novel lineages or new variants within the existing three clonal lineages could in time emerge.
A Phytophthora sojae G protein alpha subunit is involved in chemotaxis to soybean isoflavones
Hua, C. ; Wang, Y. ; Zheng, X. ; Dou, D. ; Zhang, Z. ; Govers, F. - \ 2008
Eukaryotic Cell 7 (2008)12. - ISSN 1535-9778 - p. 2133 - 2140.
transcription factor - zoospore encystment - hyphal growth - saccharomyces-cerevisiae - signal-transduction - cyst germination - botrytis-cinerea - beta-subunit - infestans - oomycete
For the soybean pathogen Phytophthora sojae, chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named PsGPA1, is a single-copy gene in P. sojae and encodes a G-protein subunit that shares 96% identity in amino acid sequence with that of Phytophthora infestans. To elucidate the function, expression of PsGPA1 was silenced by introducing antisense constructs into P. sojae. PsGPA1 silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the PsGPA1-silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene, PsCAM1, and two calcium- and calmodulin-dependent protein kinase genes, PsCMK3 and PsCMK4, were increased in the mutant zoospores, suggesting that PsGPA1 negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis
Biologically active Phytophthora mating hormone prepared by catalytic asymmetric total synthesis
Harutyunyan, S.R. ; Zhao, Z. ; Hartog, T. den; Bouwmeester, K. ; Minnaard, A.J. ; Feringa, B.L. ; Govers, F. - \ 2008
Proceedings of the National Academy of Sciences of the United States of America 105 (2008)25. - ISSN 0027-8424 - p. 8507 - 8512.
enantioselective conjugate addition - grignard-reagents - chromosomal heteromorphism - infestans - oomycete - locus - genomics - alpha-1 - enones
A Phytophthora mating hormone with an array of 1,5-stereogenic centers has been synthesized by using our recently developed methodology of catalytic enantioselective conjugate addition of Grignard reagents. We applied this methodology in a diastereo- and enantioselective iterative route and obtained two of the 16 possible stereoisomers of Phytophthora hormone ¿1. These synthetic stereoisomers induced the formation of sexual spores (oospores) in A2 mating type strains of three heterothallic Phytophthora species, P. infestans, P. capsici, and P. nicotianae but not in A1 mating type strains. The response was concentration-dependent, and the oospores were viable. These results demonstrate that the biological activity of the synthetic hormone resembles that of the natural hormone ¿1. Mating hormones are essential components in the sexual life cycle of a variety of organisms. For plant pathogens like Phytophthora, sexual reproduction is important as a source of genetic variation. Moreover, the thick-walled oospores are the most durable propagules that can survive harsh environmental conditions. Sexual reproduction can thus greatly affect disease epidemics. The availability of synthetic compounds mimicking the activity of Phytophthora mating hormone will be instrumental for further unravelling sexual reproduction in this important group of plant pathogens.
Targeted gene mutation in Phytophthora spp.
Lamour, K.H. ; Finley, L. ; Hurtado-Gonzales, O. ; Gobena, D. ; Tierney, M. ; Meijer, H.J.G. - \ 2006
Molecular Plant-Microbe Interactions 19 (2006)12. - ISSN 0894-0282 - p. 1359 - 1367.
phospholipase-d activity - parasitica var.-nicotianae - plant functional genomics - fluorescent protein gfp - induced point mutations - zoospore motility - reverse genetics - infestans - expression - oomycete
The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosis-inducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet heen found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was ohserved. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation
Phytophthora genomics: the plant destroyers' genome decoded
Govers, F. ; Gijzen, M. - \ 2006
Molecular Plant-Microbe Interactions 19 (2006)12. - ISSN 0894-0282 - p. 1295 - 1301.
pathogen phytophthora - oomycete pathogen - saprolegnia-parasitica - microsatellite markers - expressed sequences - downy mildew - infestans - sojae - avirulence - ramorum
The year 2004 was an exciting one for the Phytophthora research community. The United States Department of Energy Joint Genome Institute (JGI) completed the draft genome sequence of two Phytophthora species, Phytophthora sojae and Phytophthora ramorum. In August of that year over 50 people gathered at JGI in Walnut Creek, California, for an annotation jamboree and searched for the secrets and surprises that the two genomes have in petto. This culminated in a paper in Science in September of this year describing the highlights of the sequencing project and emphasizing the power of having the genome sequences of two closely related organisms. This MPMI Focus issue on Phytophthora genomics contains a number of more specialized manuscripts centered on gene annotation and genome organization, and complemented with manuscripts that rely on genomics resources
Nonneutral GC3 and retroelement codon mimicry in Phytophthora
Jiang, R.H.Y. ; Govers, F. - \ 2006
Journal of Molecular Evolution 63 (2006)4. - ISSN 0022-2844 - p. 458 - 472.
dna-base composition - transposable elements - genome evolution - unicellular organisms - expressed sequences - usage biases - g+c content - gene - infestans - fungi
Phytophthora is a genus entirely comprised of destructive plant pathogens. It belongs to the Stramenopila, a unique branch of eukaryotes, phylogenetically distinct from plants, animals, or fungi. Phytophthora genes show a strong preference for usage of codons ending with G or C (high GC3). The presence of high GC3 in genes can be utilized to differentiate coding regions from noncoding regions in the genome. We found that both selective pressure and mutation bias drive codon bias in Phytophthora. Indicative for selection pressure is the higher GC3 value of highly expressed genes in different Phytophthora species. Lineage specific GC increase of noncoding regions is reminiscent of whole-genome mutation bias, whereas the elevated Phytophthora GC3 is primarily a result of translation efficiency-driven selection. Heterogeneous retrotransposons exist in Phytophthora genomes and many of them vary in their GC content. Interestingly, the most widespread groups of retroelements in Phytophthora show high GC3 and a codon bias that is similar to host genes. Apparently, selection pressure has been exerted on the retroelement's codon usage, and such mimicry of host codon bias might be beneficial for the propagation of retrotransposons
Novel phosphatidylinositol phosphate kinases with a G-protein coupled receptor signature are shared by Dictyostelium and Phytophthora
Bakthavatsalam, D. ; Meijer, H.J.G. ; Noegel, A.A. ; Govers, F. - \ 2006
Trends in Microbiology 14 (2006)9. - ISSN 0966-842X - p. 378 - 382.
infestans - plants
G-protein coupled receptors (GPCR) and phosphatidylinositol phosphate kinases (PIPK) are important key switches in signal transduction pathways. A novel class of proteins was identified in the genomes of two unrelated organisms that harbor both a GPCR and a PIPK domain. Dictyostelium discoideum contains one GPCR¿PIPK, which is crucial in cell-density sensing, and the genomes of Phytophthora sojae and Phytophthora ramorum each encode twelve GPCR¿PIPKs. Intriguingly, these are currently the only species that have these two domains combined in one protein. Here, the structural and regulatory characteristics of GPCR¿PIPKs are presented and discussed. It is hypothesized that, upon activation, GPCR¿PIPKs are able to trigger heterotrimeric G-protein signaling and phosphoinositide second-messenger synthesis.
Amplification generates modular diversity at an avirulence locus in the pathogen Phytophthora
Jiang, R.H.Y. ; Weide, R. ; Vondervoort, P.J.I. van de; Govers, F. - \ 2006
Genome Research 16 (2006)7. - ISSN 1088-9051 - p. 827 - 840.
gene amplification - cladosporium-fulvum - effector proteins - cdna-aflp - infestans - resistance - potato - arabidopsis - virulence - disease
The destructive late blight pathogen Phytophthora infestans is notorious for its rapid adaptation to circumvent detection mediated by plant resistance (R) genes. We performed comparative genomic hybridization on microarrays (array-CGH) in a near genome-wide survey to identify genome rearrangements related to changes in virulence. Six loci with copy number variation were found, one of which involves an amplification colocalizing with a previously identified locus that confers avirulence in combination with either R gene R3b, R10, or R11. Besides array-CGH, we used three independent approaches to find candidate genes at the Avr3b¿Avr10¿Avr11 locus: positional cloning, cDNA-AFLP analysis, and Affymetrix array expression profiling. This resulted in one candidate, pi3.4, that encodes a protein of 1956 amino acids with regulatory domains characteristic for transcription factors. Amplification is restricted to the 3' end of the full-length gene but the amplified copies still contain the hallmarks of a regulatory protein. Sequence comparison showed that the amplification may generate modular diversity and assist in the assembly of novel full-length genes via unequal crossing-over. Analyses of P. infestans field isolates revealed that the pi3.4 amplification correlates with avirulence; isolates virulent on R3b, R10, and R11 plants lack the amplified gene cluster. The ancestral state of 3.4 in the Phytophthora lineage is a full-length, single-copy gene. In P. infestans, however, pi3.4 is a dynamic gene that is amplified and has moved to other locations. Modular diversity could be a novel mechanism for pathogens to quickly adapt to changes in the environment
Ancient origin of elicitin gene clusters in Phytophthora genomes
Jiang, R.H.Y. ; Tyler, B.M. ; Whisson, S.C. ; Hardham, A.R. ; Govers, F. - \ 2006
Molecular Biology and Evolution 23 (2006)2. - ISSN 0737-4038 - p. 338 - 351.
plant pathogen phytophthora - lipid-transfer proteins - sterol carrier protein - amino-acid sites - phylogenetic analysis - expressed sequences - effector proteins - ipio gene - infestans - sojae
The genus Phytophthora belongs to the oomycetes in the eukaryotic stramenopile lineage and is comprised of over 65 species that are all destructive plant pathogens on a wide range of dicotyledons. Phytophthora produces elicitins (ELIs), a group of extracellular elicitor proteins that cause a hypersensitive response in tobacco. Database mining revealed several new classes of elicitin-like (ELL) sequences with diverse elicitin domains in Phytophthora infestans, Phytophthora sojae, Phytophthora brassicae, and Phytophthora ramorum. ELIs and ELLs were shown to be unique to Phytophthora and Pythium species. They are ubiquitous among Phytophthora species and belong to one of the most highly conserved and complex protein families in the Phytophthora genus. Phylogeny construction with elicitin domains derived from 156 ELIs and ELLs showed that most of the diversified family members existed prior to divergence of Phytophthora species from a common ancestor. Analysis to discriminate diversifying and purifying selection showed that all 17 ELI and ELL clades are under purifying selection. Within highly similar ELI groups there was no evidence for positively selected amino acids suggesting that purifying selection contributes to the continued existence of this diverse protein family. Characteristic cysteine spacing patterns were found for each phylogenetic clade. Except for the canonical clade ELI-1, ELIs and ELLs possess C-terminal domains of variable length, many of which have a high threonine, serine, or proline content suggesting an association with the cell wall. In addition, some ELIs and ELLs have a predicted glycosylphosphatidylinositol site suggesting anchoring of the C-terminal domain to the cell membrane. The eli and ell genes belonging to different clades are clustered in the genomes. Overall, eli and ell genes are expressed at different levels and in different life cycle stages but those sharing the same phylogenetic clade appear to have similar expression patterns
A transmembrane phospholipase D in Phytophthora; a novel PLD subfamily
Meijer, H.J.G. ; Latijnhouwers, M. ; Ligterink, W. ; Govers, F. - \ 2005
Gene 350 (2005)2. - ISSN 0378-1119 - p. 173 - 182.
phosphatidic-acid - infestans - yeast - motif - phosphoinositides - activation - sequences - beta
Phospholipase D (PLD) is a ubiquitous enzyme in eukaryotes that participates in various cellular processes. Its catalytic domain is characterized by two HKD motifs in the C-terminal part. Until now, two subfamilies were recognized based on their N-terminal domain structure. The first has a PX domain in combination with a PH domain and is designated as PXPH-PLD. Members of the second subfamily, named C2-PLD, have a C2 domain and have, so far, only been found in plants. Here we describe a novel PLD subfamily that we identified in Phytophthora, a genus belonging to the class oomycetes and comprising many important plant pathogens. We cloned Pipld1 from Phytophthora infestans and retrieved full-length sequences of its homologues from Phytophthora sojae and Phytophthora ramorum genome databases. Their promoters contain two putative regulatory elements, one of which is highly conserved in all three genes. The three Phytophthora pld1 genes encode nearly identical proteins of around 1807 amino acids, with the two characteristic HKD motifs in the C-terminal part. Homology of the predicted proteins with known PLDs however is restricted to the two catalytic HKD motifs and adjacent domains. In the N-terminal part Phytophthora PLD1 has a PX-like domain, but it lacks a PH domain. Instead the N-terminal region contains five putative membrane spanning domains suggesting that Phytophthora PLD1 is a transmembrane protein. Since Phytophthora PLD1 cannot be categorized in one of the two existing subfamilies we propose to create a novel subfamily named PXTM-PLD
A combination of baiting and different PCR formats, including measurement of real-time quantitative fluorescence, for the detection of Phytophthora fragariae in strawberry plants
Bonants, P.J.M. ; Gent-Pelzer, M.P.E. van; Hooftman, R. ; Cooke, D.E.L. ; Guy, D.C. ; Duncan, J.M. - \ 2004
European Journal of Plant Pathology 110 (2004)7. - ISSN 0929-1873 - p. 689 - 702.
polymerase-chain-reaction - molecular beacons - dna - hybridization - infestans - identification - amplification - rna - quantification - organization
Phytophthora fragariae, the cause of strawberry red stele disease, is a quarantine pathogen in Europe. Detecting low levels of infection requires sensitive and specific methods. In the past, Dutch and English inspection services have used bait plants to test strawberry propagation stocks destined for export. Increasingly though, PCR is being incorporated into these testing procedures in an effort to increase sensitivity and speed. Various combinations of baiting and PCR assays were compared with existing testing procedures. Water and root samples from the bait test were screened by nested PCR and the PCR amplicon was detected by several methods, including fluorescent labelled probes (TaqMan and Molecular Beacon). PCR amplification was monitored in real-time and semi-quantitative detection was possible. Because PCR reactions are sensitive to inhibitors present in extracted DNA samples, an internal control containing the primer sequences specific for P. fragariae was developed to avoid false negatives
Phylogenetic Analysis of Phytophthora Species Based on Mitochondrial and Nuclear DNA Sequences
Kroon, L.P.N.M. ; Bakker, F.T. ; Bosch, G.B.M. van den; Bonants, P.J.M. ; Flier, W.G. - \ 2004
Fungal Genetics and Biology 41 (2004)8. - ISSN 1087-1845 - p. 766 - 782.
mating-type - central mexico - ribosomal dna - toluca valley - sp nov. - infestans - gene - recombination - origin - populations
A molecular phylogenetic analysis of the genus Phytophthora was performed, 113 isolates from 48 Phytophthora species were included in this analysis. Phylogenetic analyses were performed on regions of mitochondrial (cytochrome c oxidase subunit 1; NADH dehydrogenase subunit 1) and nuclear gene sequences (translation elongation factor 1a; ß-tubulin) and comparisons made to test for incongruence between the mitochondrial and nuclear data sets. The genus Phytophthora was confirmed to be monophyletic. In addition, results confirm that the classical taxonomic grouping as described by [Waterhouse (1963)] does not reflect true phylogenetic relations. Phytophthora species were redistributed into 8 clades, providing a more accurate representation of phylogenetic relationships within the genus Phytophthora. The evolution and transition of morphological, pathogenic, and reproductive traits was inferred from the cladogram generated in this study. Mating system was inferred to be a homoplasious trait, with at least eight independent transitions from homothallism to heterothallism observed.
A Rapid Diagnostic Test to Distinguish Between American and European Populations of Phytophthora ramorum
Kroon, L.P.N.M. ; Verstappen, E.C.P. ; Kox, L.F.F. ; Flier, W.G. ; Bonants, P.J.M. - \ 2004
Phytopathology 94 (2004)6. - ISSN 0031-949X - p. 613 - 620.
mating-type - central mexico - toluca valley - leaf-blight - sp nov. - infestans - california - 1st - differentiation - pathogen
A new devastating disease in the United States, commonly known as Sudden Oak Death, is caused by Phytophthora ramorum. This pathogen, which previously was described attacking species of Rhododendron and Viburnum in Germany and the Netherlands, has established itself in forests on the central coast of California and is killing scores of native oak trees (Lithocarpus densiflora, Quercus agrifolia, Q. kelloggii, and Q. parvula var. shrevei). The phytosanitary authorities in the European Union consider non-European isolates of P. ramorum as a threat to forest trees in Europe. To date, almost all European isolates are mating type A I while those from California and Oregon are type A2. The occurrence of both mating types in the same region Could lead to a population capable of sexual recombination, which Could generate a new Source of diversity. To prevent contact between these two populations, a rapid, reliable, and discriminating diagnostic test was developed to easily distinguish the two populations. Based on a DNA sequence difference in the mitoctiondrial Cytochrome c oxidase subunit 1 (Cox1) gene, we developed a single-nucleotide polymorphism (SNP) protocol to distinguish between isolates of P ramorum originating in Europe and those originating in the United States, A total of 83 isolates of P. ramorum from Europe and 5 1 isolates from the United States were screened and all isolates could be consistently and correctly allocated to either the European or the U.S. populations using the SNP protocol.
High affinity recognition of a Phytophthora protein by Arabidopsis via an RGD motif
Senchou, V. ; Weide, R.L. ; Carrasco, A. ; Bouyssou, H. ; Pont-Lezica, R. ; Govers, F. ; Canut, H. - \ 2004
Cellular and Molecular Life Sciences 61 (2004)4. - ISSN 1420-682X - p. 502 - 509.
plant defense responses - plasma-membrane - cell-wall - extracellular-matrix - ipio gene - thaliana - integrins - sequence - receptors - infestans
The RGD tripeptide sequence, a cell adhesion motif present in several extracellular matrix proteins of mammalians, is involved in numerous plant processes. In plant-pathogen interactions, the RGD motif is believed to reduce plant defence responses by disrupting adhesions between the cell wall and plasma membrane. Photoaffinity cross-linking of [I-125]-azido-RGD heptapeptide in the presence of purified plasma membrane vesicles of Arabidopsis thaliana led to label incorporation into a single protein with an apparent molecular mass of 80 kDa. Incorporation could be prevented by excess RGD peptides, but also by the IPI-O protein, an RGD-containing protein secreted by the oomycete plant pathogen Phytophthora infestans. Hydrophobic cluster analysis revealed that the RGD motif of IPI-O (positions 53-56) is readily accessible for interactions. Single amino acid mutations in the RGD motif in IPI-O (of Asp(56) into Glu or Ala) resulted in the loss of protection of the 80-kDa protein from labelling. Thus, the interaction between the two proteins is mediated through RGD recognition and the 80-kDa RGD-binding protein has the characteristics of a receptor for IPI-O. The IPI-O protein also disrupted cell wall-plasma membrane adhesions in plasmolysed A. thaliana cells, whereas IPI-O proteins mutated in the RGD motif (D56A and D56E) did not.