Genome-Wide Association Study Identifies Single Nucleotide Polymorphism Markers Associated with Mycelial Growth (at 15, 20, and 25°C), Mefenoxam Resistance, and Mating Type in Phytophthora infestans.

Phenotypic diversity among individuals defines the potential for evolutionary selection in a species. Phytophthora infestans epidemics are generally thought to be favored by moderate to low temperatures, but temperatures in many locations worldwide are expected to rise as a result of global climate change. Thus, we investigated variation among individuals of P. infestans for relative growth at different temperatures. Isolates of P. infestans came from three collections: (i) individual genotypes recently dominant in the United States, (ii) recently collected individuals from Central Mexico, and (iii) progeny of a recent sexual recombination event in the northeastern United States. In general, these isolates had optimal mycelial growth rates at 15 or 20°C. However, two individuals from Central Mexico grew better at higher temperatures than did most others and two individuals grew relatively less at higher temperatures than did most others. The isolates were also assessed for mefenoxam sensitivity and mating type. Each collection contained individuals of diverse sensitivities to mefenoxam and individuals of the A1 and A2 mating type. We then searched for genomic regions associated with phenotypic diversity using genotyping-by-sequencing. We found one single nucleotide polymorphism (SNP) associated with variability in mycelial growth at 20°C, two associated with variability in mycelial growth at 25°C, two associated with sensitivity to mefenoxam, and one associated with mating type. Interestingly, the SNPs associated with mefenoxam sensitivity were found in a gene-sparse region, whereas the SNPs associated with growth at the two temperatures and mating type were found both at more gene-dense regions.

Loop-mediated isothermal amplification for detection of the tomato and potato late blight pathogen, Phytophthora infestans.

AIMS: To design and validate a colorimetric loop-mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA. METHODS AND RESULTS: Two sets of loop-mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans. ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross-reacted with the closely related species Phytophthora nicotianae. Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross-react with P. nicotianae. The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina, P. ipomoeae, P. mirabilis and P. phaseoli. Cross-reactions occurred with P. andina and P. mirabilis, but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. CONCLUSIONS: Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of a LAMP assay for the detection of P. infestans, the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.

Genomic Analyses of Dominant U.S. Clonal Lineages of Phytophthora infestans Reveals a Shared Common Ancestry for Clonal Lineages US11 and US18 and a Lack of Recently Shared Ancestry Among All Other U.S. Lineages.

Populations of the potato and tomato late-blight pathogen Phytophthora infestans are well known for emerging as novel clonal lineages. These successions of dominant clones have historically been named US1 through US24, in order of appearance, since their first characterization using molecular markers. Hypothetically, these lineages can emerge through divergence from other U.S. lineages, recombination among lineages, or as novel, independent lineages originating outside the United States. We tested for the presence of phylogenetic relationships among U.S. lineages using a population of 31 whole-genome sequences, including dominant U.S. clonal lineages as well as available samples from global populations. We analyzed ancestry of the whole mitochondrial genome and samples of nuclear loci, including supercontigs 1.1 and 1.5 as well as several previously characterized coding regions. We found support for a shared ancestry among lineages US11 and US18 from the mitochondrial genome as well as from one nuclear haplotype on each supercontig analyzed. The other nuclear haplotype from each sample assorted independently, indicating an independent ancestry. We found no support for emergence of any other of the U.S. lineages from a common ancestor shared with the other U.S. lineages. Each of the U.S. clonal lineages fit a model where populations of new clonal lineages emerge via migration from a source population that is sexual in nature and potentially located in central Mexico or elsewhere. This work provides novel insights into patterns of emergence of clonal lineages in plant pathogen genomes.

Five Reasons to Consider Phytophthora infestans a Reemerging Pathogen.

Phytophthora infestans has been a named pathogen for well over 150 years and yet it continues to "emerge", with thousands of articles published each year on it and the late blight disease that it causes. This review explores five attributes of this oomycete pathogen that maintain this constant attention. First, the historical tragedy associated with this disease (Irish potato famine) causes many people to be fascinated with the pathogen. Current technology now enables investigators to answer some questions of historical significance. Second, the devastation caused by the pathogen continues to appear in surprising new locations or with surprising new intensity. Third, populations of P. infestans worldwide are in flux, with changes that have major implications to disease management. Fourth, the genomics revolution has enabled investigators to make tremendous progress in terms of understanding the molecular biology (especially the pathogenicity) of P. infestans. Fifth, there remain many compelling unanswered questions.

Accessibility of ribosomal genes to trimethyl psoralen in nuclei of Physarum polycephalum.

We have probed the accessibility of the genes for rRNA in Physarum polycephalum by using the photoreactive DNA cross-linking agent 4,5',8-trimethyl psoralen. Nuclei isolated from actively growing Physarum were treated with trimethyl psoralen and irradiated with 360-nm light in order to form cross-links. The palindromic, extrachromosomal rDNA then was isolated, and the positions of cross-links were determined by electron microscopy of the DNA under totally denaturing conditions. The results indicate that the frequency of cross-linking, after correction for base sequence bias of the reaction, is up to sixfold higher in the transcribed regions than in the central or the terminal spacer regions. There is no detectable heterogeneity among the different rDNA molecules or between the halves of a single molecule. Cross-linked molecules invariably occur in a linear as opposed to a cruciform structure. The preferential cross-linking of the transcribed region is nearly eliminated in spherules, a dormant transcriptionally inactive form in the Physarum life cycle.

Genetic and physical variability at the mating type locus of the oomycete, Phytophthora infestans.

Mating type in the oomyceteous fungus, Phytophthora infestans, is determined by a single locus. In a previous study of a few isolates, the locus segregated in a manner genetically consistent with its linkage to a system of balanced lethal loci. To determine the prevalence of this phenomenon within P. infestans, genetic analyses were performed using isolates representative of the diversity within the species that had been selected by DNA fingerprinting using probes linked to mating type. Non-Mendelian segregation of the mating type locus was observed in crosses performed with each isolate. An unusual group of isolates was identified in which the mating type determinants had been rearranged within the genome; these strains also produced an aberrantly large number of self-fertile progeny. Curiously, in all isolates, markers linked to the mating type locus appeared prone to duplication, transposition, deletion, or other rearrangement. This was not observed for loci unlinked to mating type. Data from the crosses and analyses of marker variation were used to erect models to explain the bases of mating type determination and of the unusual segregation of the chromosomal region containing the mating type locus.

Genetic mapping and non-Mendelian segregation of mating type loci in the oomycete, Phytophthora infestans.

DNA markers linked to the determinants of mating type in the oomycete, Phytophthora infestans, were identified and used to address the genetic basis of heterothallism in the normally diploid fungus. Thirteen loci linked to the A1 and A2 mating types were initially identified by bulked segregant analysis using random amplified polymorphic DNA markers (RAPDs) and subsequently scored in three crosses polymorphisms (SSCP), cleaved amplified polymorphisms (CAPS), or allele-specific polymerase chain reaction markers (AS-PCR). All DNA markers mapped to a single region, consistent with a single locus determining both mating types. Long-range restriction mapping also demonstrated the linkage of the markers to one region and delimited the mating type locus to a 100-kb region. The interval containing the mating type locus displayed non-Mendelian segregation as only two of the four expected genotypes were detected in progeny. This is consistent with a system of balance lethal loci near the mating type locus. A model for mating type determination is presented in which the balanced lethals exclude form progeny those with potentially conflicting combinations of mating type alleles, such as those simultaneously expressing A1 and A2 functions.

Highly abundant and stage-specific mRNAs in the obligate pathogen Bremia lactucae.

Germinating spores of the obligate pathogen Bremia lactucae (lettuce downy mildew) contain several unusually abundant species of mRNA. Thirty-nine cDNA clones corresponding to prevalent transcripts were isolated from a library synthesized using poly(A)+ RNA from germinating spores; these clones represented only five distinct classes. Each corresponding mRNA accounted for from 0.4 to 9 percent by mass of poly(A)+ RNA from germinating spores and together represented greater than 20 percent of the mRNA. The expression of the corresponding genes, and a gene encoding Hsp70, was analyzed in spores during germination and during growth in planta. The Hsp70 mRNA and mRNA from one abundant cDNA clone (ham34) were expressed constitutively. Two clones (ham9 and ham12) hybridized only to mRNA from spores and germinating spores. Two clones (ham37 and ham27) showed hybridization specific to germinating spores. Quantification of the number of genes homologous to each cDNA clone indicated that four clones corresponded to one or two copies per haploid genome, and one hybridized to an approximately 11-member family of genes. A sequence of the gene corresponding to ham34 was obtained to investigate its function and to identify sequences conferring high levels of gene expression for use in constructing vectors for the transformation of B. lactucae.

Transformation of the oomycete pathogen, Phytophthora infestans.

A stable transformation procedure has been developed for Phytophthora infestans, an oomycete fungus that causes the late blight diseases of potato and tomato. This is the first description of reliable methods for transformation in an oomycete pathogen. Drug-resistant transformants were obtained by using vectors that contained bacterial genes for resistance to hygromycin B or G418 fused to promoters and terminators from the Hsp70 and Ham34 genes of the oomycete, Bremia lactucae. Using polyethylene glycol and CaCl2, vector DNA was introduced into protoplasts as a complex with cationic liposomes or with carrier DNA only. Transformants were obtained at similar frequencies with each combination of promoter and selectable marker and were confirmed by DNA and RNA hybridization and phosphotransferase assays. Transformation occurred through the integration of single or tandemly repeated copies of the plasmids into genomic DNA, conferring mitotically stable drug-resistant phenotypes. The sizes of the marker gene mRNAs in each transformant and the results of transcript mapping studies were consistent with the function of the B. lactucae regulatory sequences in P. infestans. A hygromycin-resistant transformant was tested and found to maintain pathogenicity, indicating that the gene transfer procedure will be useful for the molecular analysis of genes relevant to disease.

Structure and expression of a gene encoding heat-shock protein Hsp70 from the Oomycete fungus Bremia lactucae.

A gene encoding a protein homologous to a 70-kDa heat-shock protein (Hsp70) was isolated from Bremia lactucae and its structure and pattern of expression were determined. This is the first report on the structure of a protein-coding gene from an Oomycete fungus. The cloned gene is a member of a small multigene family. The level of hsp70 mRNA in germlings increases from a low constitutive level in response to heat or cold treatment. A high level of the mRNA is also detected in spores. The hsp70 gene is expressed as a primary transcript of 2241 nucleotides (nt) and contains a continuous open reading frame of 2025 nt. Near the C terminus of the coding sequence is an unusual region that contains repeated enhancer-like sequences. This insert has not been described in other hsp70 genes and is not an intron. Upstream from the 5' terminus of the mRNA are multiple CCAAT motifs, a sequence similar to a consensus heat-shock regulatory element, and an A + T-rich putative 'TATA' box. A canonical polyadenylation recognition sequence is present downstream from the coding sequence. The deduced amino acid sequence is equally similar to yeast and maize Hsp70, providing further evidence of the dissimilarity between Oomycetes and true fungi. The cloning of this gene is part of our strategy to develop a transformation system for B. lactucae.

Expression and antisense inhibition of transgenes in Phytophthora infestans is modulated by choice of promoter and position effects.

Procedures were identified for manipulating the expression of genes in the oomycete fungus, Phytophthora infestans. The activities of five putative promoter sequences, derived from the 5' regions of oomycete genes, were measured in transient assays performed in protoplasts and in stable transformants. The sequences tested were from the ham34 and hsp70 genes of Bremia lactucae, the actin-encoding genes of P. infestans and P. megasperma, and a polyubiquitin-encoding gene of P. infestans. Experiments using the GUS reporter gene (encoding beta-glucuronidase) demonstrated that each 5' fragment had promoter activity, but that their activities varied over a greater than tenfold range. Major variation was revealed in the level of transgene expression in individual transformants containing the same promoter::GUS or promoter::lacZ fusion. The level of expression was not simply related to the number of genes present, suggesting that position effects were also influencing expression. Fusions between the ham34 promoter, and full-length and partial GUS genes in the antisense orientation blocked the expression of GUS in protoplasts and in stable transformants.

Enhanced Polymerase Chain Reaction Methods for Detecting and Quantifying Phytophthora infestans in Plants.

ABSTRACT Sensitive and specific primer sets for polymerase chain reaction (PCR) for Phytophthora infestans, the oomycete that causes late blight of potato and tomato, were developed based on families of highly repeated DNA. The performance of these primers was compared to those developed previously for the internal transcribed spacer (ITS) of ribosomal DNA. The detection limit using the new primers is 10 fg of P. infestans DNA, or 0.02 nuclei. This is about 100 times more sensitive than ITS-directed primers. Nested polymerase chain reaction (PCR) allows the measurement of down to 0.1 fg of DNA using the new primers. To enhance the reliability of diagnostic assays, an internal positive control was developed using an amplification mimic. The mimic also served as a competitor for quantitative PCR, which was used to assess the growth of P. infestans in resistant and susceptible tomato. A key dimension of this study was that two laboratories independently checked the specificity and sensitivity of each primer set; differences were noted that should be considered when PCR is adopted for diagnostic applications in any system.

Multiple Loci Determining Insensitivity to Phenylamide Fungicides in Phytophthora infestans.

ABSTRACT The diversity of mechanisms causing insensitivity to phenylamide fungicides in Phytophthora infestans was addressed by comparative genetic analyses of isolates from North America, Europe, and Mexico. Both semidominant major loci (MEX loci) and genes of minor effect were previously shown to determine insensitivity based on studies of isolates from Europe and Mexico. In this investigation, genetic analyses of three highly insensitive isolates from the United States and Canada revealed a similar pattern involving major and minor loci. However, MEX alleles in two Canadian isolates conferred higher levels of insensitivity than those examined previously, particularly in a heterozygous state. This suggested that not all MEX alleles in P. infestans were functionally equivalent. The chromosomal locations of the major insensitivity loci were also shown to vary in different isolates based on linkage analyses performed with the aid of DNA markers. The major determinant of insensitivity in the North American, Dutch, and Mexican isolates mapped to the same locus, which was named MEX1. In a British isolate, a different locus, dubbed MEX2, was implicated that mapped to the same linkage group as MEX1 but to a distinct site.

Genetic Analysis of Metalaxyl Insensitivity Loci in Phytophthora infestans Using Linked DNA Markers.

ABSTRACT Previous studies indicated that incompletely dominant loci determine insensitivity by oomycetes to phenylamide fungicides such as metalaxyl. To compare the bases of insensitivity in different strains of the late blight pathogen, Phytophthora infestans, crosses were performed between sensitive isolates and isolates from Mexico, the Netherlands, and the United Kingdom that displayed varying levels of insensitivity. Segregation analyses indicated that metalaxyl insensitivity was determined primarily by one locus in each isolate, and that two of the isolates were heterozygous and the other homozygous for the insensitive allele. Metalaxyl insensitivity was also affected by the segregation of additional loci of minor effect. DNA markers linked to insensitivity were obtained by bulked segregant analysis using random amplified polymorphic DNA (RAPD) markers and the Dutch and Mexican crosses. By studying the linkage relationships between these markers and the insensitivity in each cross by RAPD or restriction fragment length polymorphism analysis, it appeared that the same chromosomal locus conferred insensitivity in the Mexican and Dutch isolates. However, a gene at a different chromosomal position was responsible for insensitivity in the British isolate.

Chromosomal heteromorphism linked to the mating type locus of the oomycete Phytophthora infestans.

The mating type locus of the oomycete, Phytophthora infestans, is embedded in a region of DNA that displays distorted and non-Mendelian segregation. By using DNA probes linked to the mating type locus to genetically and physically characterize that region, a large zone of chromosomal heteromorphism was detected. Locus S1 was shown to represent a tandemly repeated array of DNA that was typically present in a hemizygous state in A1 isolates while being absent from A2 isolates. The analysis of the parents and progeny of seven crosses indicated that the tandem array was linked in cis to the A1-determining allele of the mating type locus. A worldwide survey of genotypically diverse field isolates of P. infestans indicated that S1 was present in each of 48 isolates of the A1 mating type that were tested, but was absent in 46 of 47 A2 strains. Physical analysis of S1 indicated that the tandemly repeated DNA sequence spanned about 300 kb and had evolved from a 1.35-kb monomer. Internal deletions occurred within S1 during sexual propagation. This and other mutations apparently contributed to a high degree of polymorphism within the S1 array.

The genetics and biology of Phytophthora infestans: modern approaches to a historical challenge.

The oomyceteous fungus Phytophthora infestans, which causes the late blight diseases of potato and tomato, has a history that is closely associated with that of mycology and plant pathology. Nevertheless, P. infestans and other oomycetes remain poorly understood relative to fungi in other groups. A resurgence in the worldwide impact of late blight has recently increased interest in the species. Fortunately, over the past decade improved tools for laboratory analysis have been developed which provide an opportunity to advance our understanding of this important pathogen. Since oomycetes do not have a close taxonomic affinity with well-characterized organisms such as ascomycetes and basidiomycetes, it is likely that studies of P. infestans will yield novel biological findings. This review provides an update on the status of research into the fundamental aspects of the biology, genetics, and pathology of P. infestans and describes prospects for future advances.

Intermolecular ligation mediates efficient cotransformation in Phytophthora infestans.

The processing of DNA molecules during transformation was characterized in the oomycete Phytophthora infestans. Linear and circular forms of non-replicating transformation vectors supported similar rates of stable transformation. Remarkably, digestion of plasmids within the selectable marker genes neomycin phosphotransferase (npt) or hygromycin phosphotransferase (hpt) had little effect on the recovery of drug-resistant transformants, and the cleaved sites were shown to be reconstituted in the transformants. An assay for the transient expression of beta-glucuronidase (GUS) in protoplasts treated with partial or disrupted GUS genes demonstrated that active genes could be reconstituted through intramolecular and/or intermolecular ligation between compatible ends, while incompatible ends were inefficiently joined. Stable transformation studies also demonstrated that complementing portions of incomplete npt or hpt genes joined through homologous recombination. Based on the indication of efficient ligation between DNA molecules during transformation, an efficient procedure for cotransformation was developed. The frequency of cotransformation between vectors expressing selected genes (npt or hpt) and nonselected sequences (GUS, beta-galactosidase, or streptomycin phosphotransferase) approached unity when the plasmids were linearized with the same restriction enzyme before transformation. In contrast, cotransformation between circular plasmids or those cut with different enzymes occurred infrequently (10%). Hybridization analysis of DNA from cotransformants demonstrated that linearized plasmids became colocalized within genomic DNA, while circular plasmids typically inserted at unliked sites.

Maturation of asparagine-linked oligosaccharides in Dictyostelium discoideum analyzed with modification-specific probes.

Lysosomal enzymes in Dictyostelium discoideum contain high mannose oligosaccharides that contain mannose 6-phosphate and several unusual structures. The synthesis and distribution of these post-translational modifications were studied using probes for different carbohydrate groups. These probes include lectin-like antibodies directed to two distinct sulfated and one nonsulfated N-linked determinants, the lectin Con A, and the mammalian 215-kDa phosphomannosyl receptor. Only Con A binds to newly synthesized alpha-mannosidase present in the rough endoplasmic reticulum. The other modifications are acquired at different rates and are first detected on protein in light density Golgi-like membranes. Mutations which prevent protein transport to Golgi membranes block synthesis of these moieties, but inhibitors which prevent later transport steps have no effect. The majority of modified proteins are in lysosomes but significant amounts are delivered to nonlysosomal destinations. Different lysosomal proteins contain unequal amounts of each modification.

Mating-type loci segregate aberrantly in Phytophthora infestans but normally in Phytophthora parasitica: implications for models of mating-type determination.

In the oomycete, Phytophthora infestans, mating type is determined by a locus that segregates in a non-Mendelian manner consistent with its linkage to a system of balanced lethals. The significance of this unusual phenomenon was addressed by studying the segregation patterns of DNA markers linked to mating type in the related species, P. parasitica. This was done using loci identified by either RAPD analysis of P. parasitica crosses or by cross-hybridization with RFLP markers linked to mating type in P. infestans. The resulting data revealed that, unlike P. infestans, mating type in P. parasitica was regulated by a locus displaying Mendelian segregation. An improved model for mating-type determination in Phytophthora is presented.

A linear RNA replicon from the oomycete Phytophthora infestans.

An unusual RNA element was discovered in an isolate of the oomyceteous fungus Phytophthora infestans. The RNA exists predominantly as single-stranded molecules of about 625 nucleotides with complementary strands present at a ratio of approximately 130:1. Gel mobility and PCR assays indicated that the element was linear. The RNA appeared to be an autonomous element, since P. infestans DNA did not contain cross-hybridizing sequences. Standard methods for virus purification yielded no evidence for encapsidation of the RNA, or for other virus particles in the isolate bearing the replicon. The replicon contained polyU and polyA tracts at its 5' and 3' termini, respectively, with a central region that had a GC content of 47%, and lacked obvious ORFs. Two-thirds of the replicon co-purified with nuclei, at about 200 copies per nucleus, while one-third resided in a cytoplasmic but non-mitochondrial location. Maternal inheritance was observed in sexual crosses, with a few exceptions. The replicon was not widely distributed throughout the species and had little effect on growth or pathogenicity. The data suggest that the RNA is best characterized as a novel linear RNA plasmid.