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.

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.

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.

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.

The hAT -like DNA transposon DodoPi resides in a cluster of retro- and DNA transposons in the stramenopile Phytophthora infestans.

A family of transposable elements belonging to the hAT group of DNA transposons is described from an oomycete, the plant pathogen Phytophthora infestans. The family, named DodoPi, was identified by studying a hotspot for retro- and DNA transposon insertions adjacent to the mating type locus. The DodoPi family comprises a small number of full-length copies, each of which is 2.7 kb long and predicted to encode a transposase-like protein consisting of 617 amino acids, and several truncated copies. Both types contain 12-bp terminal inverted repeats and are flanked by 8-bp target site duplications. Despite the detection of a DodoPi transcript and of many polymorphisms between isolates, conclusive evidence of recent transposition was not obtained. A phylogenetic analysis indicated that DodoPi was novel, with only modest similarity to some elements from plants and fungi. Relatives were detected in only some members of the genus. This is the first DNA transposon identified in the stramenopile group of eukaryotes.

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.

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.

Construction of a bacterial artificial chromosome library of Phytophthora infestans and transformation of clones into P. infestans.

A bacterial artificial chromosome (BAC) library of Phytophthora infestans was constructed in a derivative of pBELOBACII that had been modified by adding a npt selectable marker gene for transforming P. infestans. A total library of 8 genome equivalents was generated and 16,128 clones with inserts averaging 75 kb (4.9 genome equivalents) were individually picked and stored as an arrayed library in microtiter plates. This coverage was confirmed by screening the library for 11 DNA loci by colony hybridization and by polymerase chain reaction of DNA pools. Transformation of P. infestans with BAC clones containing inserts of 93 to 135 kb was demonstrated. The efficiency of transformation with most BACs was noticeably higher than that with smaller plasmids. Detailed analyses of transformants obtained with a 102-kb BAC indicated that entire inserts were present in about one-quarter of the transformants.

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.

Families of repeated DNA in the oomycete Phytophthora infestans and their distribution within the genus.

The abundance, genomic organization, species distribution, and structure of 33 distinct families of repetitive DNA in Phytophthora are described. The families were identified by screening a library of Phytophthora infestans DNA for repetitive sequences. These was subsequently characterized within 26 species distributed within each of the six taxonomic groups traditionally defined within the genus. Some repeat elements were specific to P. infestans and its close relative, Phytophthora mirabilis, while other repeated sequences were present in most species. The distribution of the DNA families did not conform to the traditional taxonomic groups used for the genus. Characterization of the repeated sequences in P. infestans indicated that they included both dispersed and tandemly repeated elements, with copy numbers ranging from 70 to 8400 per haploid genome. In total, these repeats were estimated to represent 51% of the nuclear genome of P. infestans. Reverse transcriptase motifs were detected in seven of the repeat families that were widely distributed throughout the genus.

The ipiO gene of Phytophthora infestans is highly expressed in invading hyphae during infection.

The expression of the in planta-induced gene ipiO of the potato late blight pathogen Phytophthora infestans was analyzed during various developmental stages of its life cycle. ipiO mRNA was detected in zoospores, cysts, germinating cysts, and young mycelia, but not in sporangia or in old mycelia grown in vitro. ipiO is not only expressed in stages prior to infection but also during colonization of potato and tomato leaves. In disease lesions, ipiO mRNA was detected in the water-soaked area and the healthy-looking plant tissue surrounding it. In contrast, ipiO mRNA was not found in necrotized tissue or in sporulating areas of a lesion. To determine more precisely the location and time of ipiO gene expression in planta, cytological assays were performed using a P. infestans transformant expressing a transcriptional fusion between the ipiO1 promoter and the beta-glucuronidase (GUS) reporter gene. GUS staining was found specifically in the subapical and vacuolated area of tips of invading hyphae. The histochemical GUS assays demonstrate that ipiO is expressed during biotrophic stages of the disease cycle.

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.

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.

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.

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.

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.

Inactivation of transgenes in Phytophthora infestans is not associated with their deletion, methylation, or mutation.

The mitotic and meiotic stabilities of transgenes were evaluated in the oomycete, Phytophthora infestans. Genes encoding beta-glucuronidase (GUS), neomycin phosphotransferase (NPT) and hygromycin phosphotransferase (HPT), fused to one of six promoters from P. infestans or other oomycetes, were usually stably expressed during continued asexual culture and transmitted to progeny. However, the activity of these genes became undetectable in many strains during asexual or sexual propagation. Over 33 months of growth, transgene expression stopped each month in 1-3% of the transformants. Silencing of the genes was not associated with their deletion, mutation, or hypermethylation. The conformation of the integrated sequences was similar in strains destined to continue or terminate expression of the transgenes. Expression of the genes was not associated with a loss of fitness during growth in vitro and in planta, which might otherwise have selected for silencing events.

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.

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.

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.