Multiple Horizontal Gene Transfer Events and Domain Fusions Have Created Novel Regulatory and Metabolic Networks in the Oomycete Genome

Complex enzymes with multiple catalytic activities are hypothesized to have evolved from more primitive precursors. Global analysis of the Phytophthora sojae genome using conservative criteria for evaluation of complex proteins identified 273 novel multifunctional proteins that were also conserved in P. ramorum. Each of these proteins contains combinations of protein motifs that are not present in bacterial, plant, animal, or fungal genomes. A subset of these proteins were also identified in the two diatom genomes, but the majority of these proteins have formed after the split between diatoms and oomycetes. Documentation of multiple cases of domain fusions that are common to both oomycetes and diatom genomes lends additional support for the hypothesis that oomycetes and diatoms are monophyletic. Bifunctional proteins that catalyze two steps in a metabolic pathway can be used to infer the interaction of orthologous proteins that exist as separate entities in other genomes. We postulated that the novel multifunctional proteins of oomycetes could function as potential Rosetta Stones to identify interacting proteins of conserved metabolic and regulatory networks in other eukaryotic genomes. However ortholog analysis of each domain within our set of 273 multifunctional proteins against 39 sequenced bacterial and eukaryotic genomes, identified only 18 candidate Rosetta Stone proteins. Thus the majority of multifunctional proteins are not Rosetta Stones, but they may nonetheless be useful in identifying novel metabolic and regulatory networks in oomycetes. Phylogenetic analysis of all the enzymes in three pathways with one or more novel multifunctional proteins was conducted to determine the probable origins of individual enzymes. These analyses revealed multiple examples of horizontal transfer from both bacterial genomes and the photosynthetic endosymbiont in the ancestral genome of Stramenopiles. The complexity of the phylogenetic origins of these metabolic pathways and the paucity of Rosetta Stones relative to the total number of multifunctional proteins suggests that the proteome of oomycetes has few features in common with other Kingdoms

Bioinformatic Inference of Specific and General Transcription Factor Binding Sites in the Plant Pathogen Phytophthora infestans

Plant infection by oomycete pathogens is a complex process. It requires precise expression of a plethora of genes in the pathogen that contribute to a successful interaction with the host. Whereas much effort has been made to uncover the molecular systems underlying this infection process, mechanisms of transcriptional regulation of the genes involved remain largely unknown. We performed the first systematic de-novo DNA motif discovery analysis in Phytophthora. To this end, we utilized the genome sequence of the late blight pathogen Phytophthora infestans and two related Phytophthora species (P. ramorum and P. sojae), as well as genome-wide in planta gene expression data to systematically predict 19 conserved DNA motifs. This catalog describes common eukaryotic promoter elements whose functionality is supported by the presence of orthologs of known general transcription factors. Together with strong functional enrichment of the common promoter elements towards effector genes involved in pathogenicity, we obtained a new and expanded picture of the promoter structure in P. infestans. More intriguingly, we identified specific DNA motifs that are either highly abundant or whose presence is significantly correlated with gene expression levels during infection. Several of these motifs are observed upstream of genes encoding transporters, RXLR effectors, but also transcriptional regulators. Motifs that are observed upstream of known pathogenicity-related genes are potentially important binding sites for transcription factors. Our analyses add substantial knowledge to the as of yet virtually unexplored question regarding general and specific gene regulation in this important class of pathogens. We propose hypotheses on the effects of cis-regulatory motifs on the gene regulation of pathogenicity-related genes and pinpoint motifs that are prime targets for further experimental validation

A High-Quality Grapevine Downy Mildew Genome Assembly Reveals Rapidly Evolving and Lineage-Specific Putative Host Adaptation Genes

Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94 Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5 kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant–pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in gene-sparse, repeat-rich regions and evolving rapidly, as indicated by pairwise dN/dS values. We also used short reads to assemble the genome of Plasmopara muralis, a closely related species infecting grape ivy (Parthenocissus tricuspidata). The lineage-specific proteins identified by comparative genomics analysis included a large proportion of RxLR cytoplasmic effectors and, more generally, genes with high dN/dS values. We identified 270 candidate genes under positive selection, including several genes encoding transporters and components of the RNA machinery potentially involved in host specialization. Finally, the Pl. viticola genome assembly generated here will allow the development of robust population genomics approaches for investigating the mechanisms involved in adaptation to biotic and abiotic selective pressures in this species

Identification of the First Oomycete Mating-type Locus Sequence in the Grapevine Downy Mildew Pathogen, Plasmopara viticola

Mating types are self-incompatibility systems that promote outcrossing in plants, fungi, and oomycetes. Mating-type genes have been widely studied in plants and fungi but have yet to be identified in oomycetes, eukaryotic organisms closely related to brown algae that cause many destructive animal and plant diseases. We identified the mating-type locus of Plasmopara viticola, the oomycete responsible for grapevine downy mildew, one of the most damaging grapevine diseases worldwide. Using a genome-wide association approach, we identified a 570-kb repeat-rich non-recombining region controlling mating types, with two highly divergent alleles. We showed that one mating type was homozygous, whereas the other was heterozygous at this locus. The mating-type locus encompassed 40 genes, including one encoding a putative hormone receptor. Functional studies will, however, be required to validate the function of these genes and find the actual determinants of mating type. Our findings have fundamental implications for our understanding of the evolution of mating types, as they reveal a unique determinism involving an asymmetry of heterozygosity, as in sex chromosomes and unlike other mating-type systems. This identification of the mating-type locus in such an economically important crop pathogen also has applied implications, as outcrossing facilitates rapid evolution and resistance to harsh environmental conditions

Distinctive expansion of potential virulence genes in the genome of the oomycete fish pathogen Saprolegnia parasitica.

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

Effector Genomics Accelerates Discovery and Functional Profiling of Potato Disease Resistance and Phytophthora Infestans Avirulence Genes

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties

Runaway repeats force expansion of the Phytophthora infestans genome

Sequencing of the potato late blight pathogen Phytophthora infestans provides insight into the structure and evolution of its genome