Karyotype variation, spontaneous genome rearrangements affecting chemical insensitivity, and expression level polymorphisms in the plant pathogen Phytophthora infestans revealed using its first chromosome-scale assembly.

Natural isolates of the potato and tomato pathogen Phytophthora infestans exhibit substantial variation in virulence, chemical sensitivity, ploidy, and other traits. A chromosome-scale assembly was developed to expand genomic resources for this oomyceteous microbe, and used to explore the basis of variation. Using PacBio and Illumina data, a long-range linking library, and an optical map, an assembly was created and coalesced into 15 pseudochromosomes spanning 219 Mb using SNP-based genetic linkage data. De novo gene prediction combined with transcript evidence identified 19,981 protein-coding genes, plus about eight thousand tRNA genes. The chromosomes were comprised of a mosaic of gene-rich and gene-sparse regions plus very long centromeres. Genes exhibited a biased distribution across chromosomes, especially members of families encoding RXLR and CRN effectors which clustered on certain chromosomes. Strikingly, half of F1 progeny of diploid parents were polyploid or aneuploid. Substantial expression level polymorphisms between strains were identified, much of which could be attributed to differences in chromosome dosage, transposable element insertions, and adjacency to repetitive DNA. QTL analysis identified a locus on the right arm of chromosome 3 governing sensitivity to the crop protection chemical metalaxyl. Strains heterozygous for resistance often experienced megabase-sized deletions of that part of the chromosome when cultured on metalaxyl, increasing resistance due to loss of the sensitive allele. This study sheds light on diverse phenomena affecting variation in P. infestans and relatives, helps explain the prevalence of polyploidy in natural populations, and provides a new foundation for biologic and genetic investigations.

A Variant of LbCas12a and Elevated Incubation Temperatures Enhance the Rate of Gene Editing in the Oomycete Phytophthora infestans.

CRISPR-Cas editing systems have proved to be powerful tools for functional genomics research, but their effectiveness in many non-model species remains limited. In the potato and tomato pathogen Phytophthora infestans, an editing system was previously developed that expresses the Lachnospiracae bacterium Cas12a endonuclease (LbCas12a) and guide RNA from a DNA vector. However, the method works at low efficiency. Based on a hypothesis that editing is constrained by a mismatch between the optimal temperatures for P. infestans growth and endonuclease catalysis, we tested two strategies that increased the frequency of editing of two target genes by about 10-fold. First, we found that editing was boosted by a mutation in LbCas12a (D156R) that had been reported to expand its catalytic activity over a broader temperature range. Second, we observed that editing was enhanced by transiently incubating transformed tissue at a higher temperature. These modifications should make CRISPR-Cas12a more useful for interrogating gene and protein function in P. infestans and its relatives, especially species that grow optimally at lower temperatures. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Metabolic Diversity and Novelties in the Oomycetes.

The eukaryotic microbes called oomycetes include many important saprophytes and pathogens, with the latter exhibiting necrotrophy, biotrophy, or obligate biotrophy. Understanding oomycete metabolism is fundamental to understanding these lifestyles. Genome mining and biochemical studies have shown that oomycetes, which belong to the kingdom Stramenopila, secrete suites of carbohydrate- and protein-degrading enzymes adapted to their environmental niches and produce unusual lipids and energy storage compounds. Despite having limited secondary metabolism, many oomycetes make chemicals for communicating within their species or with their hosts. Horizontal and endosymbiotic gene transfer events have diversified oomycete metabolism, resulting in biochemical pathways that often depart from standard textbook descriptions by amalgamating enzymes from multiple sources. Gene fusions and duplications have further shaped the composition and expression of the enzymes. Current research is helping us learn how oomycetes interact with host and environment, understand eukaryotic diversity and evolution, and identify targets for drugs and crop protection chemicals.

Spray-induced gene silencing for disease control is dependent on the efficiency of pathogen RNA uptake.

Recent discoveries show that fungi can take up environmental RNA, which can then silence fungal genes through environmental RNA interference. This discovery prompted the development of Spray-Induced Gene Silencing (SIGS) for plant disease management. In this study, we aimed to determine the efficacy of SIGS across a variety of eukaryotic microbes. We first examined the efficiency of RNA uptake in multiple pathogenic and non-pathogenic fungi, and an oomycete pathogen. We observed efficient double-stranded RNA (dsRNA) uptake in the fungal plant pathogens Botrytis cinerea, Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger and Verticillium dahliae, but no uptake in Colletotrichum gloeosporioides, and weak uptake in a beneficial fungus, Trichoderma virens. For the oomycete plant pathogen, Phytophthora infestans, RNA uptake was limited and varied across different cell types and developmental stages. Topical application of dsRNA targeting virulence-related genes in pathogens with high RNA uptake efficiency significantly inhibited plant disease symptoms, whereas the application of dsRNA in pathogens with low RNA uptake efficiency did not suppress infection. Our results have revealed that dsRNA uptake efficiencies vary across eukaryotic microbe species and cell types. The success of SIGS for plant disease management can largely be determined by the pathogen's RNA uptake efficiency.

Niche-specific metabolic adaptation in biotrophic and necrotrophic oomycetes is manifested in differential use of nutrients, variation in gene content, and enzyme evolution.

The use of host nutrients to support pathogen growth is central to disease. We addressed the relationship between metabolism and trophic behavior by comparing metabolic gene expression during potato tuber colonization by two oomycetes, the hemibiotroph Phytophthora infestans and the necrotroph Pythium ultimum. Genes for several pathways including amino acid, nucleotide, and cofactor biosynthesis were expressed more by Ph. infestans during its biotrophic stage compared to Py. ultimum. In contrast, Py. ultimum had higher expression of genes for metabolizing compounds that are normally sequestered within plant cells but released to the pathogen upon plant cell lysis, such as starch and triacylglycerides. The transcription pattern of metabolic genes in Ph. infestans during late infection became more like that of Py. ultimum, consistent with the former's transition to necrotrophy. Interspecific variation in metabolic gene content was limited but included the presence of γ-amylase only in Py. ultimum. The pathogens were also found to employ strikingly distinct strategies for using nitrate. Measurements of mRNA, 15N labeling studies, enzyme assays, and immunoblotting indicated that the assimilation pathway in Ph. infestans was nitrate-insensitive but induced during amino acid and ammonium starvation. In contrast, the pathway was nitrate-induced but not amino acid-repressed in Py. ultimum. The lack of amino acid repression in Py. ultimum appears due to the absence of a transcription factor common to fungi and Phytophthora that acts as a nitrogen metabolite repressor. Evidence for functional diversification in nitrate reductase protein was also observed. Its temperature optimum was adapted to each organism's growth range, and its Km was much lower in Py. ultimum. In summary, we observed divergence in patterns of gene expression, gene content, and enzyme function which contribute to the fitness of each species in its niche.

The Genome of Peronospora belbahrii Reveals High Heterozygosity, a Low Number of Canonical Effectors, and TC-Rich Promoters.

Along with Plasmopara destructor, Peronosopora belbahrii has arguably been the economically most important newly emerging downy mildew pathogen of the past two decades. Originating from Africa, it has started devastating basil production throughout the world, most likely due to the distribution of infested seed material. Here, we present the genome of this pathogen and results from comparisons of its genomic features to other oomycetes. The assembly of the nuclear genome was around 35.4 Mbp in length, with an N50 scaffold length of around 248 kbp and an L50 scaffold count of 46. The circular mitochondrial genome consisted of around 40.1 kbp. From the repeat-masked genome, 9,049 protein-coding genes were predicted, out of which 335 were predicted to have extracellular functions, representing the smallest secretome so far found in peronosporalean oomycetes. About 16% of the genome consists of repetitive sequences, and, based on simple sequence repeat regions, we provide a set of microsatellites that could be used for population genetic studies of P. belbahrii. P. belbahrii has undergone a high degree of convergent evolution with other obligate parasitic pathogen groups, reflecting its obligate biotrophic lifestyle. Features of its secretome, signaling networks, and promoters are presented, and some patterns are hypothesized to reflect the high degree of host specificity in Peronospora species. In addition, we suggest the presence of additional virulence factors apart from classical effector classes that are promising candidates for future functional studies.

Phytophthora infestans Sporangia Produced in Artificial Media and Plant Lesions Have Subtly Divergent Transcription Profiles but Equivalent Infection Potential and Aggressiveness.

Sporangia of the potato late blight agent Phytophthora infestans are often used in studies of pathogen biology and plant responses to infection. Investigations of spore biology can be challenging in oomycetes because their sporangia are physiologically active and change in response to environmental factors and aging. Whether sporangia from artificial media and plant lesions are functionally equivalent has been a topic of debate. To address these issues, we compared the transcriptomes and infection ability of sporangia from rye-sucrose media, potato and tomato leaflets, and potato tubers. Small differences were observed between the mRNA profiles of sporangia from all sources, including variation in genes encoding metabolic enzymes, cell-wall-degrading enzymes, and ABC transporters. Small differences in sporangia age also resulted in variation in the transcriptome. Taking care to use sporangia of similar maturity, we observed that those sourced from media or plant lesions had similar rates of zoospore release and cyst germination. There were also no differences in infection rates or aggressiveness on leaflets, based on single-spore inoculation assays. Such results are discordant with those of a recent publication in this journal. Nevertheless, we conclude that sporangia from plant and media cultures are functionally similar and emphasize the importance of using "best practices" in experiments with sporangia to obtain reliable results.

Defining Transgene Insertion Sites and Off-Target Effects of Homology-Based Gene Silencing Informs the Application of Functional Genomics Tools in Phytophthora infestans.

DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora spp. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spread to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5' ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for 12 different gene targets indicated that neighbors within 500 nt were often cosilenced, regardless of whether hairpin or sense constructs were employed and the direction of transcription of the target. However, this cis spreading of silencing did not occur in all transformants obtained with the same plasmid. Genome-wide studies indicated that unlinked genes with partial complementarity with the silencing-inducing transgene were not usually down-regulated. We learned that hairpin or sense transgenes were not cosilenced with the target in all transformants, which informs how screens for silencing should be performed. We conclude that transformation and gene silencing can be reliable tools for functional genomics in Phytophthora spp. but must be used carefully, especially by testing for the spread of silencing to genes flanking the target.

A MADS-box transcription factor regulates a central step in sporulation of the oomycete Phytophthora infestans.

MADS-box transcription factors play significant roles in eukaryotes, but have not yet been characterized in oomycetes. Here, we describe a MADS-box protein from Phytophthora infestans, which causes late blight of potato. P. infestans and most other oomycetes express a single MADS-box gene. PiMADS is not transcribed during vegetative growth, but is induced early during asexual sporulation. Its mRNA levels oscillate in response to light, which suppresses sporulation. The protein was not detected in nonsporulating mycelia, but was found in sporulating mycelia and spores. Both mRNA and protein levels decline upon spore germination. A similar expression pattern as well as nuclear localization was observed when the protein was expressed with a fluorescent tag from the native promoter. Gene silencing triggered by a construct expressing 478 nt of MADS sequences indicated that PiMADS is required for sporulation but not hyphal growth or plant colonization. A comparison of wild type to a silenced strain by RNA-seq indicated that PiMADS regulates about 3000 sporulation-associated genes, and acts before other genes previously shown to regulate sporulation. Analysis of the silenced strain also indicated that the native gene was not transcribed while the transgene was still expressed, which contradicts current models for homology-dependent silencing in oomycetes.

Phosphagen kinase function in flagellated spores of the oomycete Phytophthora infestans integrates transcriptional regulation, metabolic dynamics and protein retargeting.

Flagellated spores play important roles in the infection of plants and animals by many eukaryotic microbes. The oomycete Phytophthora infestans, which causes potato blight, expresses two phosphagen kinases (PKs). These enzymes store energy in taurocyamine, and are hypothesized to resolve spatial and temporal imbalances between rates of ATP creation and use in zoospores. A dimeric PK is found at low levels in vegetative mycelia, but high levels in ungerminated sporangia and zoospores. In contrast, a monomeric PK protein is at similar levels in all tissues, although is transcribed primarily in mycelia. Subcellular localization studies indicate that the monomeric PK is mitochondrial. In contrast, the dimeric PK is cytoplasmic in mycelia and sporangia but is retargeted to flagellar axonemes during zoosporogenesis. This supports a model in which PKs shuttle energy from mitochondria to and through flagella. Metabolite analysis indicates that deployment of the flagellar PK is coordinated with a large increase in taurocyamine, synthesized by sporulation-induced enzymes that were lost during the evolution of zoospore-lacking oomycetes. Thus, PK function is enabled by coordination of the transcriptional, metabolic and protein targeting machinery during the life cycle. Since plants lack PKs, the enzymes may be useful targets for inhibitors of oomycete plant pathogens.

Novo&Stitch: accurate reconciliation of genome assemblies via optical maps.

Motivation: De novo genome assembly is a challenging computational problem due to the high repetitive content of eukaryotic genomes and the imperfections of sequencing technologies (i.e. sequencing errors, uneven sequencing coverage and chimeric reads). Several assembly tools are currently available, each of which has strengths and weaknesses in dealing with the trade-off between maximizing contiguity and minimizing assembly errors (e.g. mis-joins). To obtain the best possible assembly, it is common practice to generate multiple assemblies from several assemblers and/or parameter settings and try to identify the highest quality assembly. Unfortunately, often there is no assembly that both maximizes contiguity and minimizes assembly errors, so one has to compromise one for the other. Results: The concept of assembly reconciliation has been proposed as a way to obtain a higher quality assembly by merging or reconciling all the available assemblies. While several reconciliation methods have been introduced in the literature, we have shown in one of our recent papers that none of them can consistently produce assemblies that are better than the assemblies provided in input. Here we introduce Novo&Stitch, a novel method that takes advantage of optical maps to accurately carry out assembly reconciliation (assuming that the assembled contigs are sufficiently long to be reliably aligned to the optical maps, e.g. 50 Kbp or longer). Experimental results demonstrate that Novo&Stitch can double the contiguity (N50) of the input assemblies without introducing mis-joins or reducing genome completeness. Availability and implementation: Novo&Stitch can be obtained from https://github.com/ucrbioinfo/Novo_Stitch.

Gene Expression and Silencing Studies in Phytophthora infestans Reveal Infection-Specific Nutrient Transporters and a Role for the Nitrate Reductase Pathway in Plant Pathogenesis.

To help learn how phytopathogens feed from their hosts, genes for nutrient transporters from the hemibiotrophic potato and tomato pest Phytophthora infestans were annotated. This identified 453 genes from 19 families. Comparisons with a necrotrophic oomycete, Pythium ultimum var. ultimum, and a hemibiotrophic fungus, Magnaporthe oryzae, revealed diversity in the size of some families although a similar fraction of genes encoded transporters. RNA-seq of infected potato tubers, tomato leaves, and several artificial media revealed that 56 and 207 transporters from P. infestans were significantly up- or down-regulated, respectively, during early infection timepoints of leaves or tubers versus media. About 17 were up-regulated >4-fold in both leaves and tubers compared to media and expressed primarily in the biotrophic stage. The transcription pattern of many genes was host-organ specific. For example, the mRNA level of a nitrate transporter (NRT) was about 100-fold higher during mid-infection in leaves, which are nitrate-rich, than in tubers and three types of artificial media, which are nitrate-poor. The NRT gene is physically linked with genes encoding nitrate reductase (NR) and nitrite reductase (NiR), which mobilize nitrate into ammonium and amino acids. All three genes were coregulated. For example, the three genes were expressed primarily at mid-stage infection timepoints in both potato and tomato leaves, but showed little expression in potato tubers. Transformants down-regulated for all three genes were generated by DNA-directed RNAi, with silencing spreading from the NR target to the flanking NRT and NiR genes. The silenced strains were nonpathogenic on leaves but colonized tubers. We propose that the nitrate assimilation genes play roles both in obtaining nitrogen for amino acid biosynthesis and protecting P. infestans from natural or fertilization-induced nitrate and nitrite toxicity.

How Does Phytophthora infestans Evade Control Efforts? Modern Insight Into the Late Blight Disease.

The infamous oomycete Phytophthora infestans has been a persistent threat to potato and tomato production worldwide, causing the diseases known as late blight. This pathogen has proved to be remarkably adept at overcoming control strategies including host-based resistance and fungicides. This review describes the features of P. infestans that make it such a daunting challenge to agriculture. These include a stealthy lifestyle that helps P. infestans evade plant defenses, effectors that suppress host defenses and promote susceptibility, profuse sporulation with a short latent period that enables rapid dissemination, and a genome structure that promotes the adaptive evolution of P. infestans by fostering genetic diversity. Nevertheless, there is reason to be optimistic that accumulated knowledge about the biology of P. infestans and its hosts will lead to improved management of late blight.

Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria.

BACKGROUND: An important feature of eukaryotic evolution is metabolic compartmentalization, in which certain pathways are restricted to the cytosol or specific organelles. Glycolysis in eukaryotes is described as a cytosolic process. The universality of this canon has been challenged by recent genome data that suggest that some glycolytic enzymes made by stramenopiles bear mitochondrial targeting peptides. RESULTS: Mining of oomycete, diatom, and brown algal genomes indicates that stramenopiles encode two forms of enzymes for the second half of glycolysis, one with and the other without mitochondrial targeting peptides. The predicted mitochondrial targeting was confirmed by using fluorescent tags to localize phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase in Phytophthora infestans, the oomycete that causes potato blight. A genome-wide search for other enzymes with atypical mitochondrial locations identified phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which form a pathway for generating serine from the glycolytic intermediate 3-phosphoglycerate. Fluorescent tags confirmed the delivery of these serine biosynthetic enzymes to P. infestans mitochondria. A cytosolic form of this serine biosynthetic pathway, which occurs in most eukaryotes, is missing from oomycetes and most other stramenopiles. The glycolysis and serine metabolism pathways of oomycetes appear to be mosaics of enzymes with different ancestries. While some of the noncanonical oomycete mitochondrial enzymes have the closest affinity in phylogenetic analyses with proteins from other stramenopiles, others cluster with bacterial, plant, or animal proteins. The genes encoding the mitochondrial phosphoglycerate kinase and serine-forming enzymes are physically linked on oomycete chromosomes, which suggests a shared origin. CONCLUSIONS: Stramenopile metabolism appears to have been shaped through the acquisition of genes by descent and lateral or endosymbiotic gene transfer, along with the targeting of the proteins to locations that are novel compared to other eukaryotes. Colocalization of the glycolytic and serine biosynthesis enzymes in mitochondria is apparently necessary since they share a common intermediate. The results indicate that descriptions of metabolism in textbooks do not cover the full diversity of eukaryotic biology.

RNA-seq of life stages of the oomycete Phytophthora infestans reveals dynamic changes in metabolic, signal transduction, and pathogenesis genes and a major role for calcium signaling in development.

BACKGROUND: The oomycete Phytophthora infestans causes the devastating late blight diseases of potato and tomato. P. infestans uses spores for dissemination and infection, like many other filamentous eukaryotic plant pathogens. The expression of a subset of its genes during spore formation and germination were studied previously, but comprehensive genome-wide data have not been available. RESULTS: RNA-seq was used to profile hyphae, sporangia, sporangia undergoing zoosporogenesis, motile zoospores, and germinated cysts of P. infestans. Parallel studies of two isolates generated robust expression calls for 16,000 of 17,797 predicted genes, with about 250 transcribed in one isolate but not the other. The largest changes occurred in the transition from hyphae to sporangia, when >4200 genes were up-regulated. More than 1350 of these were induced >100-fold, accounting for 26% of total mRNA. Genes encoding calcium-binding proteins, cation channels, signaling proteins, and flagellar proteins were over-represented in genes up-regulated in sporangia. Proteins associated with pathogenicity were transcribed in waves with subclasses induced during zoosporogenesis, in zoospores, or in germinated cysts. Genes involved in most metabolic pathways were down-regulated upon sporulation and reactivated during cyst germination, although there were exceptions such as DNA replication, where transcripts peaked in zoospores. Inhibitor studies indicated that the transcription of two-thirds of genes induced during zoosporogenesis relied on calcium signaling. A sporulation-induced protein kinase was shown to bind a constitutive Gß-like protein, which contributed to fitness based on knock-down analysis. CONCLUSIONS: Spore formation and germination involves the staged expression of a large subset of the transcriptome, commensurate with the importance of spores in the life cycle. A comparison of the RNA-seq results with the older microarray data indicated that information is now available for about twice the number of genes than before. Analyses based on function revealed dynamic changes in genes involved in pathogenicity, metabolism, and signaling, with diversity in expression observed within members of multigene families and between isolates. The effects of calcium signaling, a spore-induced protein kinase, and an interacting Gß-like protein were also demonstrated experimentally. The results reveal aspects of oomycete biology that underly their success as pathogens and potential targets for crop protection chemicals.

Lifestyle, gene gain and loss, and transcriptional remodeling cause divergence in the transcriptomes of Phytophthora infestans and Pythium ultimum during potato tuber colonization.

BACKGROUND: How pathogen genomes evolve to support distinct lifestyles is not well-understood. The oomycete Phytophthora infestans, the potato blight agent, is a largely biotrophic pathogen that feeds from living host cells, which become necrotic only late in infection. The related oomycete Pythium ultimum grows saprophytically in soil and as a necrotroph in plants, causing massive tissue destruction. To learn what distinguishes their lifestyles, we compared their gene contents and expression patterns in media and a shared host, potato tuber. RESULTS: Genes related to pathogenesis varied in temporal expression pattern, mRNA level, and family size between the species. A family's aggregate expression during infection was not proportional to size due to transcriptional remodeling and pseudogenization. Ph. infestans had more stage-specific genes, while Py. ultimum tended towards more constitutive expression. Ph. infestans expressed more genes encoding secreted cell wall-degrading enzymes, but other categories such as secreted proteases and ABC transporters had higher transcript levels in Py. ultimum. Species-specific genes were identified including new Pythium genes, perforins, which may disrupt plant membranes. Genome-wide ortholog analyses identified substantial diversified expression, which correlated with sequence divergence. Pseudogenization was associated with gene family expansion, especially in gene clusters. CONCLUSION: This first large-scale analysis of transcriptional divergence within oomycetes revealed major shifts in genome composition and expression, including subfunctionalization within gene families. Biotrophy and necrotrophy seem determined by species-specific genes and the varied expression of shared pathogenicity factors, which may be useful targets for crop protection.

SNP-Based Differentiation of Phytophthora infestans Clonal Lineages Using Locked Nucleic Acid Probes and High-Resolution Melt Analysis.

Phytophthora infestans, the cause of the devastating late blight disease of potato and tomato, exhibits a clonal reproductive lifestyle in North America. Phenotypes such as fungicide sensitivity and host preference are conserved among individuals within clonal lineages, while substantial phenotypic differences can exist between lineages. Whole P. infestans genomes were aligned and single nucleotide polymorphisms (SNPs) identified as targets for the development of clonal-lineage-specific molecular diagnostic tools. Informative SNPs were used to develop high-resolution melt (HRM) assays and locked nucleic acid (LNA) probes to differentiate lineage US-23, the predominant lineage in the Eastern United States for the past several years, from three other U.S. lineages. Three different primer pairs targeting one to three SNPs were capable of separating lineage US-23 from lineages US-8, US-11, and US-24 using HRM analysis. A fourth HRM primer pair targeted a highly variable genomic region containing nine polymorphisms within 63 bp. These primers separated US-23, US-11, and US-8 plus US-24 into three separate groups following HRM analysis but did not separate US-8 from US-24. Additionally, two LNA probes were designed to target a portion of the P. infestans genome containing two SNPs diagnostic for US-23. A single multiplex quantitative polymerase chain reaction assay containing both differentially labeled LNA probes differentiated individuals belonging to lineage US-23 from those belonging to US-8, US-11, and US-24.

Genome-wide prediction and functional validation of promoter motifs regulating gene expression in spore and infection stages of Phytophthora infestans.

Most eukaryotic pathogens have complex life cycles in which gene expression networks orchestrate the formation of cells specialized for dissemination or host colonization. In the oomycete Phytophthora infestans, the potato late blight pathogen, major shifts in mRNA profiles during developmental transitions were identified using microarrays. We used those data with search algorithms to discover about 100 motifs that are over-represented in promoters of genes up-regulated in hyphae, sporangia, sporangia undergoing zoosporogenesis, swimming zoospores, or germinated cysts forming appressoria (infection structures). Most of the putative stage-specific transcription factor binding sites (TFBSs) thus identified had features typical of TFBSs such as position or orientation bias, palindromy, and conservation in related species. Each of six motifs tested in P. infestans transformants using the GUS reporter gene conferred the expected stage-specific expression pattern, and several were shown to bind nuclear proteins in gel-shift assays. Motifs linked to the appressoria-forming stage, including a functionally validated TFBS, were over-represented in promoters of genes encoding effectors and other pathogenesis-related proteins. To understand how promoter and genome architecture influence expression, we also mapped transcription patterns to the P. infestans genome assembly. Adjacent genes were not typically induced in the same stage, including genes transcribed in opposite directions from small intergenic regions, but co-regulated gene pairs occurred more than expected by random chance. These data help illuminate the processes regulating development and pathogenesis, and will enable future attempts to purify the cognate transcription factors.

Metalaxyl Resistance in Phytophthora infestans: Assessing Role of RPA190 Gene and Diversity Within Clonal Lineages.

Prior work has shown that the inheritance of resistance to metalaxyl, an oomycete-specific fungicide, is complex and may involve multiple genes. Recent research indicated that a single nucleotide polymorphism (SNP) in the gene encoding RPA190, the largest subunit of RNA polymerase I, confers resistance to metalaxyl (or mefenoxam) in some isolates of the potato late blight pathogen Phytophthora infestans. Using both DNA sequencing and high resolution melt assays for distinguishing RPA190 alleles, we show here that the SNP is absent from certain resistant isolates of P. infestans from North America, Europe, and Mexico. The SNP is present in some members of the US-23 and US-24 clonal lineages, but these tend to be fairly sensitive to the fungicide based on artificial media and field test data. Diversity in the level of sensitivity, RPA190 genotype, and RPA190 copy number was observed in these lineages but were uncorrelated. Controlled laboratory crosses demonstrated that RPA190 did not cosegregate with metalaxyl resistance from a Mexican and British isolate. We conclude that while metalaxyl may be used to control many contemporary strains of P. infestans, an assay based on RPA190 will not be sufficient to diagnose the sensitivity levels of isolates.