The repertoire of transfer RNA genes is tuned to codon usage bias in the genomes of Phytophthora sojae and Phytophthora ramorum

In all, 238 and 155 transfer (t)RNA genes were predicted from the genomes of Phytophthora sojae and P. ramorum, respectively. After omitting pseudogenes and undetermined types of tRNA genes, there remained 208 P. sojae tRNA genes and 140 P. ramorum tRNA genes. There were 45 types of tRNA genes, with distinct anticodons, in each species. Fourteen common anticodon types of tRNAs are missing altogether from the genome in the two species; however, these appear to be compensated by wobbling of other tRNA anticodons in a manner which is tied to the codon bias in Phytophthora genes. The most abundant tRNA class was arginine in both P. sojae and P. ramorum. A codon usage table was generated for these two organisms from a total of 9,803,525 codons in P. sojae and 7,496,598 codons in P. ramorum. The most abundant codon type detected from the codon usage tables was GAG (encoding glutamic acid), whereas the most numerous tRNA gene had a methionine anticodon (CAT). The correlation between the frequencies of tRNA genes and the codon frequencies in protein-coding genes was very low (0.12 in P. sojae and 0.19 in P. ramorum); however, the correlation between amino acid tRNA gene frequency and the corresponding amino acid codon frequency in P. sojae and P. ramorum was substantially higher (0.53 in P. sojae and 0.77 in P. ramorum). The codon usage frequencies of P. sojae and P ramorum were very strongly correlated (0.99), as were tRNA gene frequencies (0.77). Approximately 60% of orthologous tRNA gene pairs in P sojae and P. ramorum are located in regions that have conserved synteny in the two species.     

Evolution of the cutinase gene family: evidence for lateral gene transfer of a candidate Phytophthora virulence factor

Lateral gene transfer (LGT) can facilitate the acquisition of new functions in recipient lineages, which may enable them to colonize new environments. Several recent publications have shown that gene transfer between prokaryotes and eukaryotes occurs with appreciable frequency. Here we present a study of interdomain gene transfer of cutinases -- well documented virulence factors in fungi -- between eukaryotic plant pathogens Phytophthora species and prokaryotic bacterial lineages. Two putative cutinase genes were cloned from Phytophthora brassicae and Northern blotting experiments showed that these genes are expressed early during the infection of the host Arabidopsis thaliana and induced during cyst germination of the pathogen. Analysis of the gene organisation of this gene family in Phytophthora ramorum and P. sojae showed three and ten copies in tight succession within a region of 5 and 25 kb, respectively, probably indicating a recent expansion in Phytophthora lineages by gene duplications. Bioinformatic analyses identified orthologues only in three genera of Actinobacteria, and in two distantly related eukaryotic groups: oomycetes and fungi. Together with phylogenetic analyses this limited distribution of the gene in the tree of life strongly support a scenario where cutinase genes originated after the origin of land plants in a microbial lineage living in proximity of plants and subsequently were transferred between distantly related plant-degrading microbes. More precisely, a cutinase gene was likely acquired by an ancestor of P. brassicae, P. sojae, P. infestans and P. ramorum, possibly from an actinobacterial source, suggesting that gene transfer might be an important mechanism in the evolution of their virulence. These findings could indeed provide an interesting model system to study acquisition of virulence factors in these important plant pathogens.     

Comparative analysis of Phytophthora genes encoding secreted proteins reveals conserved synteny and lineage-specific gene duplications and deletions

Comparative analysis of two Phytophthora genomes revealed overall colinearity in four genomic regions consisting of a 1.5-Mb sequence of Phytophthora sojae and a 0.9-Mb sequence of P. ramorum. In these regions with conserved synteny, the gene order is largely similar; however, genome rearrangements also have occurred. Deletions and duplications often were found in association with genes encoding secreted proteins, including effectors that are important for interaction with host plants. Among secreted protein genes, different evolutionary patterns were found. Elicitin genes that code for a complex family of highly conserved Phytophthora-specific elicitors show conservation in gene number and order, and often are clustered. In contrast, the race-specific elicitor gene Avrlb-1 appeared to be missing from the region with conserved synteny, as were its five homologs that are scattered over the four genomic regions. Some gene families encoding secreted proteins were found to be expanded in one species compared with the other. This could be the result of either repeated gene duplications in one species or specific deletions in the other. These different evolutionary patterns may shed light on the functions of these secreted proteins in the biology and pathology of the two Phytophthora spp.     

Recognition of Phytophthora infestans Avr4 by potato R4 is triggered by C-terminal domains comprising W motifs

Oomycete RXLR-dEER effector proteins are rapidly evolving proteins with the selective pressure targeted predominantly at their C-terminal ends. The majority of RXLR-dEER proteins have recognizable motifs of 21–30 amino acids in the C-terminal domain that are named after conserved amino acid residues at fixed positions within the respective motifs. In this article, it is reported that the Phytophthora infestans RXLR-dEER protein Avr4 contains three W motifs and one Y motif in its C-terminal domain. Agroinfection assays using constructs encoding modified forms of PiAvr4 have shown that the region containing the W2 motif, in combination with either the W1 or W3 motif, triggers a necrotic response in potato plants carrying the resistance gene R4 . By mining the superfamily of avirulence homologues (Avh) deduced from three sequenced Phytophthora genomes, several Avh proteins were identified as homologues of PiAvr4: six in P. infestans , one in P. ramorum and seven in P. sojae . One very close homologue of PiAvr4 was cloned from the sibling species, P. mirabilis. This species is not pathogenic on potato but, similar to PiAvr4, PmirAvh4 triggered a necrotic response on potato clones carrying R4 , but not on clones lacking R4 . Genes encoding RXLR-dEER effectors are often located in regions showing genome rearrangements. Alignment of the genomic region harbouring PiAvr4 with syntenic regions in P. sojae and P. ramorum revealed that PiAvr4 is located on a 100-kb indel block and is surrounded by transposable elements.     

Extensive variation in nuclear mitochondrial DNA content between the genomes of Phytophthora sojae and Phytophthora ramorum

Fragments of mitochondrial DNA (mtDNA) transferred to the nuclear genome are called nuclear mitochondrial DNAs (NUMTs). We report here a comparison of NUMT content between genomes from two species of the same genus. Analysis of the genomes of Phytophthora sojae and P. ramorum revealed large differences in the NUMT content of the two genomes: 16.27 x 10(-3) and 2.28 x 10(-3)% of each genome, respectively. Substantial differences also exist between the two species in the sizes of the NUMTs found in each genome, with ranges of 20 to 405 bp for P. sojae and 19 to 137 bp for P. ramorum. Furthermore, in P. sojae, fragments from the mitochondrial genes rns, rnl, coxl, and nad (various subunits) are found most frequently, whereas P. ramorum NUMTs most often originate from the cox3, rpsl4, nad4, and nad5 genes. The large differences in the presumptive mtDNA insertions suggest that the insertions occurred subsequent to the divergence of the two species, and this is supported by sequence comparisons among the NUMTs and the mtDNA sequences of the two species. P. sojae mtDNA sequences inserted in the nuclear genome appear to have been altered as a result of insertions, deletions, inversions, and translocations and provide insights into active mechanisms of sequence divergence in this plant pathogen. No clear examples were found of NUMTs forming functional nuclear genes or of NUMTs inserted into exons or introns of any nuclear gene.     

Genomewide analysis of phospholipid signaling genes in Phytophthora spp.: novelties and a missing link

Phospholipids are cellular membrane components in eukaryotic cells that execute many important roles in signaling. Genes encoding enzymes required for phospholipid signaling and metabolism have been characterized in several organisms, but only a few have been described for oomycetes. In this study, the genome sequences of Phytophthora sojae and P. ramorum were explored to construct a comprehensive genomewide inventory of genes involved in the most universal phospholipid signaling pathways. Several genes and gene families were annotated, including those encoding phosphatidylinositol synthase (PIS), phosphatidylinositol (phosphate) kinase (PI[P]K), diacylglycerol kinase (DAG), and phospholipase D (PLD). The most obvious missing link is a gene encoding phospholipase C (PLC). In all eukaryotic genomes sequenced to date, PLC genes are annotated based on certain conserved features; however, these genes seem to be absent in Phytophthora spp. Analysis of the structural and regulatory domains and domain organization of the predicted isoforms of PIS, PIK, PIPK, DAG, and PLD revealed many novel features compared with characterized representatives in other eukaryotes. Examples are transmembrane proteins with a C-terminal catalytic PLD domain, secreted PLD-like proteins, and PIPKs that have an N-terminal G-protein-coupled receptor-transmembrane signature. Compared with other sequenced eukaryotes, the genus Phytophthora clearly has several exceptional features in its phospholipid-modifying enzymes.     

Expressed sequence tags from phytophthora sojae reveal genes specific to development and infection

Six unique expressed sequence tag (EST) libraries were generated from four developmental stages of Phytophthora sojae P6497. RNA was extracted from mycelia, swimming zoospores, germinating cysts, and soybean (Glycine max (L.) Merr.) cv. Harosoy tissues heavily infected with P. sojae. Three libraries were created from mycelia growing on defined medium, complex medium, and nutrient-limited medium. The 26,943 high-quality sequences obtained clustered into 7,863 unigenes composed of 2,845 contigs and 5,018 singletons. The total number of P. sojae unigenes matching sequences in the genome assembly was 7,412 (94%). Of these unigenes, 7,088 (90%) matched gene models predicted from the P. sojae sequence assembly, but only 2,047 (26%) matched P. ramorum gene models. Analysis of EST frequency from different growth conditions and morphological stages revealed genes that were specific to or highly represented in particular growth conditions and life stages. Additionally, our results indicate that, during infection, the pathogen derives most of its carbon and energy via glycolysis of sugars in the plant. Sequences identified with putative roles in pathogenesis included avirulence homologs possessing the RxLR motif, elicitins, and hydrolytic enzymes. This large collection of P. sojae ESTs will serve as a valuable public genomic resource.     

Targeted gene mutation in Phytophthora spp

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 necrosisinducing 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 been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.     

Cross-species global proteomics reveals conserved and unique processes in Phytophthora sojae and Phytophthora ramorum

Phytophthora ramorum and Phytophthora sojae are destructive plant pathogens. P. sojae has a narrow host range, whereas P. ramorum has a wide host range. A global proteomics comparison of the vegetative (mycelium) and infective (germinating cyst) life stages of P. sojae and P. ramorum was conducted to identify candidate proteins involved in host range, early infection, and vegetative growth. Sixty-two candidates for early infection, 26 candidates for vegetative growth, and numerous proteins that may be involved in defining host specificity were identified. In addition, common life stage proteomic trends between the organisms were observed. In mycelia, proteins involved in transport and metabolism of amino acids, carbohydrates, and other small molecules were up-regulated. In the germinating cysts, up-regulated proteins associated with lipid transport and metabolism, cytoskeleton, and protein synthesis were observed. It appears that the germinating cyst catabolizes lipid reserves through the beta-oxidation pathway to drive the extensive protein synthesis necessary to produce the germ tube and initiate infection. Once inside the host, the pathogen switches to vegetative growth in which energy is derived from glycolysis and utilized for synthesis of amino acids and other molecules that assist survival in the plant tissue.     

Expressed peptide tags: an additional layer of data for genome annotation

While genome sequencing is becoming ever more routine, genome annotation remains a challenging process. Identification of the coding sequences within the genomic milieu presents a tremendous challenge, especially for eukaryotes with their complex gene architectures. Here, we present a method to assist the annotation process through the use of proteomic data and bioinformatics. Mass spectra of digested protein preparations of the organism of interest were acquired and searched against a protein database created by a six-frame translation of the genome. The identified peptides were mapped back to the genome, compared to the current annotation, and then categorized as supporting or extending the current genome annotation. We named the classified peptides Expressed Peptide Tags (EPTs). The well-annotated bacterium Rhodopseudomonas palustris was used as a control for the method and showed a high degree of correlation between EPT mapping and the current annotation, with 86% of the EPTs confirming existing gene calls and less than 1% of the EPTs expanding on the current annotation. The eukaryotic plant pathogens Phytophthora ramorum and Phytophthora sojae, whose genomes have been recently sequenced and are much less well-annotated, were also subjected to this method. A series of algorithmic steps were taken to increase the confidence of EPT identification for these organisms, including generation of smaller subdatabases to be searched against, and definition of EPT criteria that accommodates the more complex eukaryotic gene architecture. As expected, the analysis of the Phytophthora species showed less correlation between EPT mapping and their current annotation. While approximately 76% of Phytophthora EPTs supported the current annotation, a portion of them (7.7% and 12.9% for P. ramorum and P. sojae, respectively) suggested modification to current gene calls or identified novel genes that were missed by the current genome annotation of these organisms.     

Computational and comparative analyses of 150 full-length cDNA sequences from the oomycete plant pathogen Phytophthora infestans

Phytophthora infestans is a devastating phytopathogenic oomycete that causes late blight on tomato and potato. Recent genome sequencing efforts of P. infestans and other Phytophthora species are generating vast amounts of sequence data providing opportunities to unlock the complex nature of pathogenesis. However, accurate annotation of Phytophthora genomes will be a significant challenge. Most of the information about gene structure in these species was gathered from a handful of genes resulting in significant limitations for development of ab initio gene-calling programs. In this study, we collected a total of 150 bioinformatically determined near full-length cDNA (FLcDNA) sequences of P. infestans that were predicted to contain full open reading frame sequences. We performed detailed computational analyses of these FLcDNA sequences to obtain a snapshot of P. infestans gene structure, gauge the degree of sequence conservation between P. infestans genes and those of Phytophthora sojae and Phytophthora ramorum, and identify patterns of gene conservation between P. infestans and various eukaryotes, particularly fungi, for which genome-wide translated protein sequences are available. These analyses helped us to define the structural characteristics of P. infestans genes using a validated data set. We also determined the degree of sequence conservation within the genus Phytophthora and identified a set of fast evolving genes. Finally, we identified a set of genes that are shared between Phytophthora and fungal phytopathogens but absent in animal fungal pathogens. These results confirm that plant pathogenic oomycetes and fungi share virulence components, and suggest that eukaryotic microbial pathogens that share similar lifestyles also share a similar set of genes independently of their phylogenetic relatedness.     

A Phytophthora infestans cystatin-like protein targets a novel tomato papain-like apoplastic protease

There is emerging evidence that the proteolytic machinery of plants plays important roles in defense against pathogens. The oomycete pathogen Phytophthora infestans, the agent of the devastating late blight disease of tomato (Lycopersicon esculentum) and potato (Solanum tuberosum), has evolved an arsenal of protease inhibitors to overcome the action of host proteases. Previously, we described a family of 14 Kazal-like extracellular serine protease inhibitors from P. infestans. Among these, EPI1 and EPI10 bind and inhibit the pathogenesis-related (PR) P69B subtilisin-like serine protease of tomato. Here, we describe EPIC1 to EPIC4, a new family of P. infestans secreted proteins with similarity to cystatin-like protease inhibitor domains. Among these, the epiC1 and epiC2 genes lacked orthologs in Phytophthora sojae and Phytophthora ramorum, were relatively fast-evolving within P. infestans, and were up-regulated during infection of tomato, suggesting a role during P. infestans-host interactions. Biochemical functional analyses revealed that EPIC2B interacts with and inhibits a novel papain-like extracellular cysteine protease, termed Phytophthora Inhibited Protease 1 (PIP1). Characterization of PIP1 revealed that it is a PR protein closely related to Rcr3, a tomato apoplastic cysteine protease that functions in fungal resistance. Altogether, this and earlier studies suggest that interplay between host proteases of diverse catalytic families and pathogen inhibitors is a general defense-counterdefense process in plant-pathogen interactions.     

Strawberry Tree Blight in Spain, a New Disease Caused by various Phytophthora Species

During surveys for Phytophthora ramorum in garden centres in Majorca, Spain, 31 isolates of Phytophthora were recovered from potted strawberry trees ( Arbutus unedo ) showing leaf and twig blights. Many isolates of Phytophthora syringae and Phytophthora citrophthora as well as single isolates of P. ramorum, Phytophthora tropicalis and Phytophthora nicotianae were identified on morphological features and on the sequences of the internal transcribed spacer regions from ribosomal DNA genes. Phytophthora syringae was collected most frequently in late autumn and winter, whereas P. citrophthora was dominant during late summer and autumn. In vitro pathogenicity of P. syringae and P. citrophthora was compared with that of P. ramorum by inoculating intact detached leaves of A. unedo with zoospores and twigs with mycelial plugs. In addition, in vitro sporangial production was examined on inoculated excised leaves and on agar plugs at 12, 15 and 20°C. Phytophthora citrophthora produced the largest lesions both on leaves and on twigs at all temperatures. Phytophthora ramorum formed lesions comparable in size to those of P. syringae , but it significantly produced more sporangia on excised leaves and agar plugs. In a log inoculation assay, P. syringae caused large lesions in the inner bark, whereas those of P. ramorum were moderate. Strawberry tree blight has not yet been observed in natural ecosystems in the western Mediterranean areas. Possible biological and environmental limitations hindering disease spread in the wild are discussed.     

Ancient origin of elicitin gene clusters in Phytophthora genomes

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.     

Rapid identification of Phytophthora ramorum using PCR-SSCP analysis of ribosomal DNA ITS-1

AIMS: The primary objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis can be used for rapid identification of Phytophthora ramorum, an important quarantine plant pathogen worldwide, and to further assess the potential of the SSCP technique as a taxonomic tool for the genus Phytophthora. METHODS AND RESULTS: SSCP of ribosomal DNA internal transcribed spacer 1 was characterized for 12 isolates of P. ramorum, using a recently reported protocol. The SSCP patterns of this species then were compared with those of 18 closely related Phytophthora species. Phytophthora ramorum had a unique pattern and was easily distinguished from genetically, morphologically and ecologically close relatives. CONCLUSION: An immediate benefit of this study is provision of a highly effective and efficient identification tool for P. ramorum in the quarantine process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study also provides additional evidence demonstrating that the SSCP is an ideal DNA marker for species differentiation within the genus Phytophthora.     

Adaptive evolution has targeted the C-terminal domain of the RXLR effectors of plant pathogenic oomycetes

Oomycete plant pathogens deliver effector proteins inside host cells to modulate plant defense circuitry and to enable parasitic colonization. These effectors are defined by a conserved motif, termed RXLR (for Arg, any amino acid, Leu, Arg), that is located downstream of the signal peptide and that has been implicated in host translocation. Because the phenotypes of RXLR effectors extend to plant cells, their genes are expected to be the direct target of the evolutionary forces that drive the antagonistic interplay between pathogen and host. We used the draft genome sequences of three oomycete plant pathogens, Phytophthora sojae, Phytophthora ramorum, and Hyaloperonospora parasitica, to generate genome-wide catalogs of RXLR effector genes and determine the extent to which these genes are under positive selection. These analyses revealed that the RXLR sequence is overrepresented and positionally constrained in the secretome of Phytophthora relative to other eukaryotes. The three examined plant pathogenic oomycetes carry complex and diverse sets of RXLR effector genes that have undergone relatively rapid birth and death evolution. We obtained robust evidence of positive selection in more than two-thirds of the examined paralog families of RXLR effectors. Positive selection has acted for the most part on the C-terminal region, consistent with the view that RXLR effectors are modular, with the N terminus involved in secretion and host translocation and the C-terminal domain dedicated to modulating host defenses inside plant cells.     

Identification of 18 genes encoding necrosis-inducing proteins from the plant pathogen Phytophthora capsici (Pythiaceae: Oomycetes)

Phytophthora capsici is an aggressive plant pathogen that affects solanaceous and cucurbitaceous hosts. Necrosis-inducing Phytophthora proteins (NPPs) are a group of secreted toxins found particularly in oomycetes. Several NPPs from Phytophthora species trigger plant cell death and activate host defense gene expression. We isolated 18 P. capsici NPP genes, of which 12 were active during hypha growth from a Phytophthora stain isolated from pepper (Capsicum annuum) plants in China. The 18 predicted proteins had a sequence homology of 46.26%. The 18 Pcnpp sequences had a conserved GHRHDWE motif and fell into two groups. Eleven sequences in group 1 had two conserved cysteine residues, whereas the other seven sequences in group 2 lacked these two cysteine residues. A phylogenetic tree was constructed on the basis of the alignment of the predicted protein sequences of 52 selected NPP genes from oomycetes, fungi and bacteria from Genbank. The tree did not rigorously follow the taxonomic classification of the species; all the NPPs from oomycetes formed their own clusters, while fungal sequences were grouped into two separate clades, indicating that based on NPPs, we can separate oomycetes from fungi and bacteria, and that expansion of the NPP family was a feature of Phytophthora evolution.