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.     

The plant pathogen Phytophthora andina emerged via hybridization of an unknown Phytophthora species and the Irish potato famine pathogen, P. infestans

Emerging plant pathogens have largely been a consequence of the movement of pathogens to new geographic regions. Another documented mechanism for the emergence of plant pathogens is hybridization between individuals of different species or subspecies, which may allow rapid evolution and adaptation to new hosts or environments. Hybrid plant pathogens have traditionally been difficult to detect or confirm, but the increasing ease of cloning and sequencing PCR products now makes the identification of species that consistently have genes or alleles with phylogenetically divergent origins relatively straightforward. We investigated the genetic origin of Phytophthora andina, an increasingly common pathogen of Andean crops Solanum betaceum, S. muricatum, S. quitoense, and several wild Solanum spp. It has been hypothesized that P. andina is a hybrid between the potato late blight pathogen P. infestans and another Phytophthora species. We tested this hypothesis by cloning four nuclear loci to obtain haplotypes and using these loci to infer the phylogenetic relationships of P. andina to P. infestans and other related species. Sequencing of cloned PCR products in every case revealed two distinct haplotypes for each locus in P. andina, such that each isolate had one allele derived from a P. infestans parent and a second divergent allele derived from an unknown species that is closely related but distinct from P. infestans, P. mirabilis, and P. ipomoeae. To the best of our knowledge, the unknown parent has not yet been collected. We also observed sequence polymorphism among P. andina isolates at three of the four loci, many of which segregate between previously described P. andina clonal lineages. These results provide strong support that P. andina emerged via hybridization between P. infestans and another unknown Phytophthora species also belonging to Phytophthora clade 1c.     

The malarial host-targeting signal is conserved in the Irish potato famine pathogen

Animal and plant eukaryotic pathogens, such as the human malaria parasite Plasmodium falciparum and the potato late blight agent Phytophthora infestans, are widely divergent eukaryotic microbes. Yet they both produce secretory virulence and pathogenic proteins that alter host cell functions. In P. falciparum, export of parasite proteins to the host erythrocyte is mediated by leader sequences shown to contain a host-targeting (HT) motif centered on an RxLx (E, D, or Q) core: this motif appears to signify a major pathogenic export pathway with hundreds of putative effectors. Here we show that a secretory protein of P. infestans, which is perceived by plant disease resistance proteins and induces hypersensitive plant cell death, contains a leader sequence that is equivalent to the Plasmodium HT-leader in its ability to export fusion of green fluorescent protein (GFP) from the P. falciparum parasite to the host erythrocyte. This export is dependent on an RxLR sequence conserved in P. infestans leaders, as well as in leaders of all ten secretory oomycete proteins shown to function inside plant cells. The RxLR motif is also detected in hundreds of secretory proteins of P. infestans, Phytophthora sojae, and Phytophthora ramorum and has high value in predicting host-targeted leaders. A consensus motif further reveals E/D residues enriched within approximately 25 amino acids downstream of the RxLR, which are also needed for export. Together the data suggest that in these plant pathogenic oomycetes, a consensus HT motif may reside in an extended sequence of approximately 25-30 amino acids, rather than in a short linear sequence. Evidence is presented that although the consensus is much shorter in P. falciparum, information sufficient for vacuolar export is contained in a region of approximately 30 amino acids, which includes sequences flanking the HT core. Finally, positional conservation between Phytophthora RxLR and P. falciparum RxLx (E, D, Q) is consistent with the idea that the context of their presentation is constrained. These studies provide the first evidence to our knowledge that eukaryotic microbes share equivalent pathogenic HT signals and thus conserved mechanisms to access host cells across plant and animal kingdoms that may present unique targets for prophylaxis across divergent pathogens.     

Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato

Comparative genomics provides a tool to utilize the exponentially increasing sequence information from model plants to clone agronomically important genes from less studied crop species. Plant disease resistance (R) loci frequently lack synteny between related species of cereals and crucifers but appear to be positionally well conserved in the Solanaceae. In this report, we adopted a local RGA approach using genomic information from the model Solanaceous plant tomato to isolate R3a, a potato gene that confers race-specific resistance to the late blight pathogen Phytophthora infestans. R3a is a member of the R3 complex locus on chromosome 11. Comparative analyses of the R3 complex locus with the corresponding I2 complex locus in tomato suggest that this is an ancient locus involved in plant innate immunity against oomycete and fungal pathogens. However, the R3 complex locus has evolved after divergence from tomato and the locus has experienced a significant expansion in potato without disruption of the flanking colinearity. This expansion has resulted in an increase in the number of R genes and in functional diversification, which has probably been driven by the co-evolutionary history between P. infestans and its host potato. Constitutive expression was observed for the R3a gene, as well as some of its paralogues whose functions remain unknown.     

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.     

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.     

A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences

Phytophthora species are devastating plant pathogens in both agricultural and natural environments. Due to their significant economic and environmental impact, there has been increasing interest in Phytophthora genetics and genomics, culminating in the recent release of three complete genome sequences (P. ramorum, P. sojae, and P. infestans). In this study, genome and other large sequence databases were used to identify over 225 potential genetic markers for phylogenetic analyses. Here, we present a genus-wide phylogeny for 82 Phytophthora species using seven of the most informative loci (approximately 8700 nucleotide sites). Our results support the division of the genus into 10 well-supported clades. The relationships among these clades were rigorously evaluated using a number of phylogenetic methods. This is the most comprehensive study of Phytophthora relationships to date, and many newly discovered species have been included. A more resolved phylogeny of Phytophthora species will allow for better interpretations of the overall evolutionary history of the genus.     

A multi-locus phylogeny for Phytophthora utilizing markers derived from complete genome sequences.

Phytophthora species are devastating plant pathogens in both agricultural and natural environments. Due to their significant economic and environmental impact, there has been increasing interest in Phytophthora genetics and genomics, culminating in the recent release of three complete genome sequences (P. ramorum, P. sojae, and P. infestans). In this study, genome and other large sequence databases were used to identify over 225 potential genetic markers for phylogenetic analyses. Here, we present a genus-wide phylogeny for 82 Phytophthora species using seven of the most informative loci (approximately 8700 nucleotide sites). Our results support the division of the genus into 10 well-supported clades. The relationships among these clades were rigorously evaluated using a number of phylogenetic methods. This is the most comprehensive study of Phytophthora relationships to date, and many newly discovered species have been included. A more resolved phylogeny of Phytophthora species will allow for better interpretations of the overall evolutionary history of the genus.     

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.     

Transcriptome of Aphanomyces euteiches: new oomycete putative pathogenicity factors and metabolic pathways

Aphanomyces euteiches is an oomycete pathogen that causes seedling blight and root rot of legumes, such as alfalfa and pea. The genus Aphanomyces is phylogenically distinct from well-studied oomycetes such as Phytophthora sp., and contains species pathogenic on plants and aquatic animals. To provide the first foray into gene diversity of A. euteiches, two cDNA libraries were constructed using mRNA extracted from mycelium grown in an artificial liquid medium or in contact to plant roots. A unigene set of 7,977 sequences was obtained from 18,864 high-quality expressed sequenced tags (ESTs) and characterized for potential functions. Comparisons with oomycete proteomes revealed major differences between the gene content of A. euteiches and those of Phytophthora species, leading to the identification of biosynthetic pathways absent in Phytophthora, of new putative pathogenicity genes and of expansion of gene families encoding extracellular proteins, notably different classes of proteases. Among the genes specific of A. euteiches are members of a new family of extracellular proteins putatively involved in adhesion, containing up to four protein domains similar to fungal cellulose binding domains. Comparison of A. euteiches sequences with proteomes of fully sequenced eukaryotic pathogens, including fungi, apicomplexa and trypanosomatids, allowed the identification of A. euteiches genes with close orthologs in these microorganisms but absent in other oomycetes sequenced so far, notably transporters and non-ribosomal peptide synthetases, and suggests the presence of a defense mechanism against oxidative stress which was initially characterized in the pathogenic trypanosomatids.     

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.     

Chromosomal heteromorphism and an apparent translocation detected using a BAC contig spanning the mating type locus of Phytophthora infestans

Genetic and physical irregularities associated with the mating type locus of the oomycete, Phytophthora infestans, were revealed by analyzing a contig spanning the locus that was constructed using a bacterial artificial chromosome library. Contigs from both homologs of an A1 strain A/a genotype at mating type locus) had chromosome-specific differences, flanked by regions of similarity. Such heteromorphism was detected within multiple isolates. The mating type locus was narrowed to a 60-70kb interval by genetic mapping of candidate genes, identified using a cDNA library. During these analyses, an unusual isolate of P. infestans was identified in which the mating type determinant had apparently translocated from its location in typical strains. Comparative mapping of the cDNAs between P. infestans and P. parasitica revealed partial synteny between the species however; substantial rearrangements existed and no cDNA was tightly linked to mating type in P. parasitica. These findings add to previous observations of unusual genetic behavior involving mating type in Phytophthora.     

Strategies of attack and defense in plant-oomycete interactions, accentuated for Phytophthora parasitica Dastur (syn. P. Nicotianae Breda de Haan)

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to their particular physiological characteristics, no efficient treatments against diseases caused by these microorganisms are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. Available data are scarce, and genomic approaches were mainly developed for the two species, Phytophthora infestans and Phytophthora sojae. However, these two species are exceptions from, rather than representative species for, the genus. P. infestans is a foliar pathogen, and P. sojae infects a narrow range of host plants, while the majority of Phytophthora species are quite unselective, root-infecting pathogens. To represent this majority, Phytophthora parasitica emerges as a model for the genus, and genomic resources for analyzing its interaction with plants are developing. The aim of this review is to assemble current knowledge on cytological and molecular processes that are underlying plant-pathogen interactions involving Phytophthora species and in particular P. parasitica, and to place them into the context of a hypothetical scheme of co-evolution between the pathogen and the host.     

PCR amplification of species-specific DNA sequences can distinguish among Phytophthora species.

We used PCR to differentiate species in the genus Phytophthora, which contains a group of devastating plant pathogenic fungi. We focused on Phytophthora parasitica, a species that can infect solanaceous plants such as tomato, and on Phytophthora citrophthora, which is primarily a citrus pathogen. Oligonucleotide primers were derived from sequences of a 1,300-bp P. parasitica-specific DNA segment and of an 800-bp P. citrophthora-specific segment. Under optimal conditions, the primers developed for P. parasitica specifically amplified a 1,000-bp sequence of DNA from isolates of P. parasitica. Primers for P. citrophthora similarly and specifically amplified a 650-bp sequence of DNA from isolates of P. citrophthora. Detectable amplification of these specific DNA sequences required picogram quantities of chromosomal DNA. Neither pair of primers amplified these sequences with DNAs from other species of Phytophthora or from the related genus Pythium. DNAs from P. parasitica and P. citrophthora growing in infected tomato stem tissue were amplified as distinctly as DNAs from axenic cultures of each fungal species. This is the first report on PCR-driven amplification with Phytophthora species-specific primers.     

Characterization of the glucose-6-phosphate isomerase gene in Phytophthora infestans reveals the presence of multiple alleles

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 1671bp and encodes a protein with a predicted molecular weight of 60.8kDa. 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.     

Initial assessment of gene diversity for the oomycete pathogen Phytophthora infestans based on expressed sequences

A total of 1000 expressed sequence tags (ESTs) corresponding to 760 unique sequence sets were identified using random sequencing of clones from a cDNA library constructed from mycelial RNA of Phytophthora infestans. A number of software programs, represented by a relational database and an analysis pipeline, were developed for the automated analysis and storage of the EST sequence data. A set of 419 nonredundant sequences, which correspond to a total of 632 ESTs (63.2%), were identified as showing significant matches to sequences deposited in public databases. A putative cellular identity and role was assigned to all 419 sequences. All major functional categories were represented by at least several ESTs. Four novel cDNAs containing sequences related to elicitins, a family of structurally related proteins that induce the hypersensitive response and condition avirulence of P. infestans on Nicotiana plants, were among the most notable genes identified. Two of these elicitin-like cDNAs were among the most abundant cDNAs examined. The set also contained several ESTs with high sequence similarity to unique plant genes.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

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 microM. 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.     

Mitochondrial and Nuclear DNA Restriction Enzyme Analysis of the Closely Related Phytophthora Species P. infestans, P. mirabilis, and P. phaseoli

To determine relatedness of the phytopathogenic fungi Phytophthora infestans , P. mirabilis , and P. phaseoli restriction fragment patterns of mitochondrial DNAs of several isolates and hybridization patterns of nuclear DNAs after Southern hybridization with a specific homologous probe were analyzed.
All but two isolates of P. infestans and P. mirabilis show very similar restriction fragment patterns differing only in the length of one fragment due to small insertion/deletion(s). Two isolates of P. mirabilis have one additional site for Scr FI. On the contrary at least six sites differ in P. phaseoli when compared to the other two species. The mitochondrial genome of P. phaseoli is considerably smaller (approx. 6 kbp) than those of P. infestans and P. mirabilis .
A cloned 430 bp multicopy DNA sequence, derived from P. infestans , hybridized specifically with P. infestans, P. mirabilis , and P. phaseoli out of 61 species of Peronosporales ( Phytophthora, Halophytophthora, Pythium, Albugo, Bremia, Peronospora, Plasmopara ) tested and therefore has potential as a diagnostic probe. Restriction patterns revealed by this probe are invariant intraspecifi-cally but differ between the three species.
We consider P. mirabilis a forma specialis of P. infestans because of the very high similarly in its mitochondrial DNA restriction patterns.