Sequence Variation and Genomic Amplification of a Family of Gypsy-like Elements in the Oomycete Genus Phytophthora

Howard S. Judelson

Department of Plant Pathology, University of California, Riverside


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
A family of sequences resembling Gypsy retroelements was identified and shown to be widely distributed throughout the genus Phytophthora, a member of the algallike oomycete fungi. Polymerase chain reaction (PCR) using specific and degenerate primers detected the family in 29 of 37 species tested. DNA hybridization also failed to detect the sequences in the eight species that were negative in PCR. The element appears to have been a major force in the shaping of Phytophthora genomes because its abundance varied drastically from about 10 to more than 10,000 copies per genome within the species containing the element. Family members diverged from each other by single-base changes, insertions, and deletions, with a mean nucleotide divergence of 16.7%. By constructing phylogenies of the elements, lineages were identified that predated speciation events within Phytophthora and subfamilies that had diverged more recently. The element was studied in detail in Phytophthora infestans, in which about 30 copies are dispersed throughout the genome. Phylogenetic comparisons of the reverse transcriptases placed the family within the Ty3/Gypsy group of long terminal repeat (LTR) retrotransposons, with the closest affinities to elements from plants. However, each of 12 family members sequenced contained defects that would render their protein products inactive, including frameshift mutations within reverse transcriptase domains and truncations that appeared to eliminate gag, protease, and terminal repeat sequences.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Phytophthora is a major genus of plant pathogens within the group of diploid, funguslike organisms known as oomycetes. There are at least 50 species within the genus (Erwin and Ribeiro 1996Citation ), of which the most notorious is Phytophthora infestans, which caused the Irish potato famine (potato late blight disease). Classical taxonomic schemes cluster oomycetes with the true fungi (ascomycetes and basidiomycetes) because of their common filamentous growth styles. However, contemporary assessments using molecular and biochemical criteria indicate that oomycetes are more properly classified with diatoms and brown algae within the Kingdom Chromista, not the Kingdom Fungi (Gunderson et al. 1987Citation ; Baldauf et al. 2000Citation ).

The molecular biology of oomycetes began to be investigated only recently, but an interesting finding has been that their genomes are larger than those of most lower eukaryotes and contain substantial repetitive DNA. Most studies have involved Phytophthora, in which genomes vary in size from 6.2 x 107 to 2.4 x 108 bp and contain 50% or more repetitive DNA (Tooley and Therrien 1987Citation ; Mao and Tyler 1991Citation ). A survey in P. infestans indicated that some repeated DNA contains reverse transcriptase motifs, suggesting that this repeated DNA evolved from retroelements (Judelson and Randall 1998Citation ).

To identify factors that have shaped the evolution and genetics of Phytophthora, we are characterizing the nature of its transposable elements. It is also anticipated that this will lead to the development of new molecular tools and an understanding of the phenotypic instability of isolates, in which sectoring for growth rate, morphology, virulence, and other traits are reported (Stamps 1953Citation ; Buddenhagen 1958Citation ; Erwin et al. 1963Citation ). Transposable elements are known to be a major source of spontaneous variation and mutation in many eukaryotes (Bennetzen 2000Citation ). In addition, transposable elements may alter chromosome structure by expanding the size of genomes through amplification and by providing regions of homology for illegitimate recombination (Sanmiguel et al. 1998Citation ). This may be a driving force in speciation (Loennig and Saedler 1997Citation ; Hurst and Schilthuizen 1998Citation ). Indeed, comparing the phylogenies of transposable elements and their hosts is useful for analyzing the evolutionary characteristics of both (Kossida et al. 2000Citation ).

This study describes a family of repeated DNA within Phytophthora that resemble the Ty3/Gypsy group of retrotransposons, which were detected in 29 of 37 species tested. The family appears to have been a major force in the evolution of Phytophthora chromosomes, representing a miniscule part of the genome in some taxa and as much as 25% in others. Distinct evolutionary lineages of the retroelementlike sequence were identified, showing significant intraspecific and interspecific variation. Some relationships between lineages matched phylogenies of species within the genus, whereas others represented either basal lineages predating speciation within Phytophthora or horizontal transfer events. Most elements appeared to be defective.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Polymerase Chain Reaction of Retroelements
Primers RetroA and RetroB (5'-CGATTGGTAATAGACGACACGATCAC-3' and 5'-CTTCGGGGCGAGCGAGATAC-3', respectively) were based on a P. cinnamomi sequence (Judelson and Messenger-Routh 1996Citation ) and were used to amplify the region between reverse transcriptase domain 6 and the N-terminus of the RNAse H domain (Xiong and Eickbush 1990Citation ). These primers were used in 25-µl reactions that involved 35 cycles of denaturation (92°C, 30 s), annealing (50°C, 30 s), and extension (72°C, 1 min), followed by incubation for 2 min at 72°C. Reactions contained 10–100 ng of template DNA, 1.5 mM MgCl2, 1 unit Taq polymerase, 10 mM Tris (pH 8.3), 50 mM KCl, 0.2 mM deoxynucleotide triphosphates, and 0.1% gelatin. Reaction products were separated on 1%–1.3% agarose in 90 mM Tris-borate and 2 mM ethylenediaminetetraacetic acid (EDTA) and were visualized using ethidium bromide. Some PCR used degenerate primers (5'-RCCAMGATCGGCGCYTC-3' and 5'-ATACCCGTNRTVGGGTGT-3'), which amplify the region just C-terminal to reverse transcriptase domain 7 and the N-terminus of the RNAse H domain. Genomic DNA was prepared as described (Judelson and Tooley 2000Citation ) from strains provided by Dr. Paul Tooley (USDA-ARS, Fort Detrick, Md.) or Dr. Michael Coffey and Dr. Helga Förster (University of California-Riverside, Calif.). These individuals also provided some DNA samples. Internal transcribed spacer (ITS) sequencing was performed as described (Förster, Cummings, and Coffey 2000Citation ).

DNA Hybridization
Gel blots of genomic DNA, and replicas of colonies from a bacterial artificial chromosome (BAC) library (Randall and Judelson 1999Citation ), were prepared on nylon membranes as described (Judelson and Fabritius 2000Citation ). Dot-blots were made by placing genomic DNA (100 µg/µl) or appropriate standards in a microtiter dish in 10 x SSPE (SSPE is 0.18 M NaCl, 10 mM NaH2PO4, 1 mM EDTA) plus 0.1 µg/ml SybrGold (Molecular Probes, Eugene, Ore.). These were spotted to replicate membranes using a slotted, surfactant-treated 96-pin tool designed to deliver 0.5 µl (V&P Scientific, San Diego, Calif.). To correct for errors in initial DNA quantitations, bound DNA was quantitated under long-wave UV light using a Fluor-S camera and QuantityOne software for Macintosh (Biorad, Richmond, Calif.). Membranes were prepared for hybridization by soaking each membrane for 10 min in 0.4 M NaOH followed by 3 x SSPE, hybridized as described (Judelson and Fabritius 2000Citation ), and washed at 50°C in 1 x SSPE, 0.3% sodium dodecyl sulfate, 0.1% sodium pyrophosphate. Signals were detected by phosphorimager analysis and quantified using QuantityOne software (Biorad).

Sequencing of PCR Products and BAC Clones
Retroelement fragments obtained by PCR were cloned into pGEMT-EZ (Promega, Madison, Wis.) and sequenced using T7 and SP6 primers. A region of genomic DNA cross-hybridizing with the retroelement was isolated from a BAC clone and subcloned as HindIII and XbaI-BglII fragments in pBS-KS2+ (Stratagene, La Jolla, Calif.), which were sequenced in both directions by primer walking. Sequences were assembled using Seqman for Macintosh (DNASTAR, Madison, Wis.). Sequences were deposited in GenBank under accession numbers AF490269AF490339.

Alignments and Phylogenetic Analysis
Preliminary studies were performed using the Alignment program of Vector NTI (Informax, Bethesda, Md.). Data were then reformatted and analyzed using programs from the PHYLIP 3.57c package for Macintosh (distributed by J. Felsenstein, Department of Genetics, University of Washington, Seattle). For DNA-based phylogenies, a consensus tree was developed from 1,000 bootstrapped data sets using DNADIST followed by NEIGHBOR (Neighbor-Joining option) and CONSENSE. Protein phylogenies were developed from 1,000 bootstrapped data sets analyzed using the PROTDIST program (PAM matrix) followed by CONSENSE.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Detection of Retroelementlike Sequences
A repetitive DNA fragment from P. cinnamomi, originally isolated as a target for a disease diagnosis procedure (Judelson and Messenger-Routh 1996Citation ), was sequenced and found to have a reverse transcriptase motif. The strongest similarities were against plant members of the Ty3/Gypsy family of retroelements. In anticipation that such a clone might prove useful for studying retroelements in Phytophthora, several PCR primers based on the P. cinnamomi sequence were designed and tested against members of the genus as listed in table 1 . The table also shows the classification of those species in the Waterhouse system (Waterhouse 1963Citation ), which is based on morphological criteria such as spore structure, and in a scheme based on ITS regions of ribosomal DNA (Cooke et al. 2000Citation ).


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Table 1 Isolates of Phytophthora Used in This Study and Results of Polymerase Chain Reaction

 
The primers RetroA and RetroB proved most effective at amplifying fragments from most species of Phytophthora. These primers amplified bands of the expected size, about 400 bp, from 28 species (table 1 ). These primers flanked the interval between reverse transcriptase domain 6 and the N-terminus of the RNAse H domain of canonical retroelement polyproteins (Xiong and Eickbush 1990Citation ); primers based on more N-terminal reverse transcriptase domains were not as successful. The amplicons obtained using RetroA and RetroB were sequenced, which confirmed that each contained the reverse transcriptase-RNAse H region. In several species, amplicons of other sizes were also obtained; however, they had no sequence similarity to retroelements, and most contained the RetroB primer at both termini.

Alternatives were tested for amplifying the region from the nine species recalcitrant to PCR with RetroA and RetroB. This included using RetroA, RetroB, and other specific primers at reduced annealing temperatures, or degenerate primers. Success was realized only for P. lateralis, using degenerate primers. That the DNA from the eight remaining refractory species was suitable for amplification was confirmed by PCR using random amplified polymorphic DNA and ITS primers.

Relationships Between Family Members
The evolution of the retroelementlike family within Phytophthora was examined by comparing 75 clones. These generally included two to three elements for each of the 29 species. The sequences were aligned and a phylogenetic tree was developed (fig. 1 ).



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Fig. 1.—Phylogram of retroelementlike sequences from Phytophthora. Amplicons as shown in figure 1 were cloned, sequenced, and an unrooted phylogram was constructed on the basis of Neighbor-Joining analysis. Confidence of groupings was estimated using 1,000 bootstrap replicates; numbers near each branch indicate the percentage of replicates supporting each branch. Labels on the right indicate the species, isolate, Waterhouse group (Waterhouse 1963Citation ), and ITS clade (Cooke et al. 2000Citation ). Where multiple clones were analyzed from an isolate, sequences are differentiated by letter suffixes (A, B, etc.). The bar at the bottom represents 5% sequence divergence

 
Two major types of relationships between family members were revealed by this analysis. In a minority of instances, retroelementlike sequences clustered most closely with those from the same species, indicating sequence divergence after speciation. This was the case for the clones from P. cinnamomi isolates 283 and 6379, for example. More commonly, distinct retroelement lineages appeared to have evolved before speciation. For example, the P. drechsleri 1795A sequence most closely resembled the P. vignae 3018 sequence. However, such conclusions are tentative because this study did not sequence all retroelements from each genome.

Whereas retroelementlike sequences generally did not cluster with those from the same species, the phylogram revealed strong relationships between related species. This was most obvious for sequences from P. infestans, P. mirabilis, and P. phaseoli, which are shown at the bottom of figure 1 . These species reside in Group IV of the Waterhouse scheme, and analyses of ITS and other loci confirmed their close association (Moeller, De Cock, and Prell 1993Citation ; Tooley, Carras, and Falkenstein 1996Citation ). To help explore the relationship between these three species, 23 retroelementlike sequences were analyzed. All 23 intermingled within the phylogram, reflecting the evolution of lineages predating the radiation of the three species from their common ancestor. That distinct sublineages were already present in such an ancestor is suggested by the low bootstrap values obtained for many nodes. These sequences did not have affinity with the retroelementlike sequences from another Group IV species, P. colocasiae, and other Group IV members such as P. ilicis and P. hibernalis were negative in PCR. This confirms ITS-based data that contend that the P. infestans–like group (ITS clade 1c) is poorly related to such other Group IV taxa, which lie in ITS clades 2 and 3 (Cooke et al. 2000Citation ).

Other clusters were also evident, such as between members of Waterhouse Groups II and III (including ITS clades 2 and 5) and between Groups V and VI (ITS clades 7a and 8a). Exceptions to such patterns were also noted. For example, the P. erythroseptica 340 sequence (Group VI) clustered with Group V and VI sequences, whereas P. erythroseptica 6180 resided in a clade largely from Groups II and III. This latter clade includes two additional Group VI sequences (P. vignae 7825 and P. cambivora 6412), suggesting that it may represent a retroelement lineage that predates radiation of the Group II-III and Group V-VI clades, or which underwent horizontal transfer. That such observations were not an artifact of isolate misidentification was shown by ITS analyses.

Nature of Differences Between Family Members
Several molecular phenomena distinguish the different retroelementlike lineages. A combination of base changes, insertions, and deletions were observed. These changes, and particularly the insertions and deletions, support many conclusions drawn from the phylogram. For example, a cluster of five sequences from P. infestans, P. mirabilis, and P. phaseoli, presumed to represent a lineage predating radiation of those species, was supported by the observation that they each contained the same novel deletions and insertions (not shown). Excluding gaps, the mean nucleotide divergence was 16.7%.

The potential translation products of the sequences were determined and compared with that of functional reverse transcriptase polyproteins. Nearly all contained frameshifts or stop mutations. This indicated that most were obtained from defective elements.

Copy Number of Element
Extraordinary divergence in the abundance of the retroelement sequence in different species of Phytophthora was revealed using dot-blot assays (fig. 2 ) and blots of gels of genomic DNA (not shown). For the dot-blots, genomic DNA and standards for copy number were spotted on a membrane that was hybridized at low stringency with a 32P-labeled "average" retroelement probe. This probe and the copy number standard were made by pooling DNA from 24 sequences representing each subclade in figure 1 .



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Fig. 2.—Quantitation of retroelementlike sequences in different species. Similar amounts of genomic DNA from each species were spotted on a membrane, stained with SybrGold, and then hybridized at low stringency with the 24-species pooled probe. The number of copies of the sequence was derived from the ratio of the hybridization signal ("R" in the insert, measured using a phosphorimager) to the amount of genomic DNA bound to the membrane before hybridization ("S" in the insert, quantitated using a charge-coupled device camera). Standard curves were used to calibrate the amount of genomic DNA on the membrane and the relationship between bound radioactivity and copy number. Values are expressed in copies per average-sized genome of 108 base pairs. Spots from one of two replicate filters are shown; bars represent the range of values from the replicates

 
From the dot-blots, it was calculated that species that were positive for the retroelement sequence in PCR contain from a low of 10 copies per 108 base pairs (an average-sized genome) to 11,000 copies, as in the cases of P. palmivora and P. iranica, respectively (fig. 2 ). No hybridization was observed against species that were negative in PCR, such as P. humicola. This suggested that the PCR failures were not attributable to simple base differences in primer-binding sites. Instead, either the family was absent or had diverged to a nonhybridizing form. It was calculated that the latter required >30% divergence from each of the 24 sequences used to make the hybridization probe or 46% divergence from any single sequence.

The values shown in figure 2 represent only estimates of copies per genome for two reasons. Firstly, the calculations invoke a 108-bp genome, which was selected on the basis of the Phytophthora species measured to date which include P. infestans at 2.4 x 108 bp (Tooley and Therrien 1987Citation ), P. porri at 1.2 x 108 bp (F. Mauch, personal communication), and P. sojae at 6.2 x 107 bp (Mao and Tyler 1991Citation ). Secondly, not all of the 24 sequences in the probe may have hybridized to all potential targets at maximum efficiency. Nevertheless, copy values from the dot-blot approach were similar to values from alternative methods. For example, whereas the dot-blots indicated that P. infestans contained 32 copies per genome (correcting for its known genome size), a value of 25 copies was calculated from the fraction of colonies from a BAC library that hybridized to the probe. Secondly, the value calculated for P. cinnamomi from dot-blots hybridized with the 24-sequence probe was only 24% less than that obtained using a P. cinnamomi–derived probe (not shown).

The enormous interspecific variation in copy number was confirmed by analyzing blots of electrophoresed genomic DNA (not shown). Using the 24-sequence probe, for example, massive hybridization was observed against P. cinnamomi, P. cambivora, and P. iranica which were calculated from dot-blots to have >6,000 copies each. In contrast, very low hybridization was recorded against P. infestans, P. megasperma, and P. porri, which were calculated to have <=35 copies. This intensity difference was not an artifact of some bias caused by using the 24-sequence probe. For example, a probe based on P. infestans sequences showed much greater hybridization against DNA from P. iranica than against DNA from P. infestans. These blots also confirmed the absence of cross-hybridizing sequences in the species negative in PCR including P. humicola, P. ilicis, P. macrochlamydospora, P. quininea, and P. richardiae.

Distribution of Family in P. infestans
The retroelementlike family appeared to be dispersed throughout the genome of P. infestans, based on the patterns of hybridization observed in blots of genomic DNA restricted with different enzymes plus analyses of BAC clones isolated by hybridization with the 24-sequence probe. In genomic blots, for example, the pattern observed was consistent with a dispersed pattern or a localized array of highly variable sequences (fig. 3 , left). The dispersed pattern seemed more likely, however, because only single bands were observed in restriction digests of the BACs (fig. 3 , right).



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Fig. 3.—Genomic organization of retroelementlike family in P. infestans. Genomic DNA of isolate 1391 digested with the indicated enzymes (left panel), and DNA from eight independent BAC clones digested with HindIII (right panel), was electrophoresed, blotted, and hybridized at low stringency with a probe made from a pool of three P. infestans, three P. mirabilis, and three P. phaseoli amplicons. Molecular weight standards were derived from a DNA ladder (Invitrogen)

 
The BACs were also used to demonstrate that sequences amplified with the primers RetroA and RetroB were representative of all cross-hybridizing elements. To do this, 12 BACs were selected that represented independent chromosomal loci on the basis of whether they had nonoverlapping patterns in restriction digests. When used as templates in PCR using RetroA and RetroB, each yielded the expected products. Because the BACs were obtained by hybridization, the primers appeared to have provided a relatively unbiased sampling of retroelementlike sequences in the previous experiments (i.e., fig. 1 ).

Detailed Analysis of Individual Family Members in P. infestans
The majority of elements in P. infestans appeared defective, based on sequencing portions of the retroelementlike loci from the 12 BAC clones. Eleven loci were quickly shown to have frameshift or nonsense mutations. The remaining clone first appeared to contain an intact retroelement on the basis of the presence of >1 kb of an intact reading frame and was therefore analyzed in detail. However, subsequent data showed that it also contained frameshift and nonsense mutations and was truncated (fig. 4 ).



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Fig. 4.—Structure of retroelement-containing locus in P. infestans. Portrayed are the results of sequencing approximately 10 kb of DNA from a BAC clone, corresponding to the BAC in lane 8 of figure 3 . The location of the region amplified by primers RetroA and RetroB is indicated as a dark bar above the restriction map. Below the map are shown two truncated retrolementlike sequences containing motifs for reverse transcriptase (RT), RNAse H (RH), and integrase (INT) plus frameshift (F) and nonsense (T) mutations interrupting the reading frame. A region corresponding to a putative truncated expressed sequence tag is shown above the map (EST). The solid bar portrays the part of the EST showing homology to the genomic sequence, and the shaded portion represents the portion of the EST that is absent. Restriction enzyme sites are Bg, BglII; E, EcoRI; H, HindIII; P, PstI; S, SacI; V, EcoRV; X, XbaI; and Xh, XhoI

 
Unexpectedly, the twelfth clone contained two retroelementlike sequences in an inverted repeat, each containing defective open reading frames. Regions encoding reverse transcriptase, RNAse H, and integrase were present, but the element lacked gag, protease, or other transposable element–like sequences. The left- and right-facing elements appeared truncated just 5' of reverse transcriptase domains 4 and 2, respectively (Xiong and Eickbush 1990Citation ). No terminal repeats were detected within the 10-kb interval of DNA that was sequenced, as was expected because the 5' ends of each element were absent. The two elements were diverged, having 52% and 64% amino acid identity and similarity. They had 51% GC, which can be compared to 49% and 58% for total genomic DNA and expressed sequences in P. infestans (unpublished data). When compared with the sequences in figure 1 , they were most similar to the P. infestans-P. mirabilis clade at the base of that figure.

To test for activity of the retroelementlike family, both the 24-sequence pooled probe and the one derived from six P. infestans elements were hybridized to blots of DNA of 20 genetically and geographically diverse isolates of P. infestans. Patterns in HindIII and PvuII digests were monomorphic, consistent with a lack of recent transposition (not shown). Nevertheless, a weak 5-kb transcript was detected in RNA blots, and relatives were present in an expressed sequence tag database (EST) of P. infestans (not shown). Also, possible historical transposition activity was revealed by observing that the 5' end of the right-facing element bordered a truncated version of a sequence in the EST database (94% identity). The data were consistent with a deletion of the 3' two-thirds of a gene that predated the retroelement at the locus, as shown in figure 4 .

The P. infestans elements were compared with those from other organisms by generating a phylogram incorporating reverse transcriptase–containing viruses and retrotransposons (fig. 5 ). The two P. infestans retroelementlike sequences, labeled as PIRETA and PIRETB, had the strongest affinity with the Ty3/Gypsy group and especially those of plant origin defined as class B plant elements by Marin and Llorens (2000)Citation . The sequence closest to PIRETA and PIRETB fell into the Tma subfamily of Ty3/Gypsy elements from Arabidopsis (Marin and Llorens 2000Citation ). It is consequently proposed that PIRETA and PIRETB had evolved from a long terminal repeat (LTR) retroelement, even though LTRs were absent from the copies that were sequenced.



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Fig. 5.—Phylogenetic tree showing relationship between reverse transcriptase proteins from the P. infestans sequences (PIRETA and PIRETB) and other LTR and non-LTR elements. Neighbor-Joining was performed as described in Materials and Methods, after correcting for frameshift and nonsense mutations. The tree was generated on the basis of the portion of the reverse transcriptase–RNAseH-integrase region in the P. infestans sequences from the BAC shown in figure 3 ; the total alignment length was 1,055 amino acids. Similar results were obtained when the analysis was limited to the reverse transcriptase region. Sequences used and their GenBank accession numbers included Arabidopsis, AF128395 (Marin and Llorens 2000Citation ); Rice, AB014738 (RIRE3) (Kumekawa et al. 1999Citation ); Gypsy-like polyprotein from tomato, T17459 (Parniske and Jones 1999Citation ); Skippy, S60179 (Anaya and Roncero 1995Citation ); Cft-1, AAF21678 (Mchale et al. 1992Citation ); MarY1, BAA78625 (Murata and Yamada 2000Citation ); Maggy, AAA33420 (Farman et al. 1996Citation ); REAL, BAA89272 (Kaneko, Tanaka, and Tsuge 2000Citation ); Mdg3, T13798 (Dzhumagaliev et al. 1986Citation ); Mag, S08405 (Michaille et al. 1990Citation ); Gypsy, AAB50148 (Tchurikov et al. 1989Citation ); Ty3, S69842 (Hansen, Chalker, and Sandmeyer 1988Citation ); Cre1, A34728 (Gabriel et al. 1990Citation ); Jockey, P21328 (Priimagi, Mizrokhi, and Ilyin 1988Citation ); DIRS-1, C24785 (Cappello, Handelsman, and Lodish 1985Citation ); CAMV, M90543 (Chenault and Melcher 1993Citation ); FIV, S23820 (Olmsted et al. 1989Citation ); Ty1, B22671 (Vandenbol et al. 1995Citation ); Tnt1, P10978 (Grandbastien, Spielmann, and Caboche 1989Citation ); and copia, P04146 (Mount and Rubin 1985Citation ). Percentage boostrap values, based on 1,000 replicates, are shown when above 40%

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
This study has addressed several fundamental issues in phylogenetic biology, namely, the evolution and transmission of transposable elements, the development of genomes, and the evolution of species within a genus. In oomycetes such as Phytophthora, as in most other organisms, transposable elements constitute a significant fraction of repeated DNA within the genome. The widespread distribution of the retroelementlike sequence within Phytophthora suggests that it is an ancient component of the genome. Clues to its origin may be provided by its closest affinity to LTR retrotransposons of plants. Phytophthora and many other oomycetes are plant pathogens; consequently, horizontal transfer from a plant host (or vice versa) is an attractive model. Insertion of the element into a common ancestor of plants and oomycetes is an alternative, but it is difficult to assess because of unknowns about the evolutionary history of oomycetes. ITS-based phylogenies place oomycetes, along with brown algae and diatoms, in a clade lacking affinity with plants, true fungi, animals, slime molds, and other well-characterized groups (Gunderson et al. 1987Citation ). A protein-based phylogeny suggested a relationship between oomycetes and plants, although the data were not conclusive (Baldauf et al. 2000Citation ). These hypotheses for the origin of the element could be tested by searching for related sequences in other oomycete genera, diatoms, and brown algae.

The groupings of the retroelement sequences analyzed in this study cannot be used to unambiguously infer species phylogenies within Phytophthora because of the propensity of such elements for horizontal transfer and their multicopy nature. Moreover, for each species, only two or three retroelement sequences were analyzed; therefore, a full sampling of their diversity remains to be performed. Nevertheless, the retroelement data are generally congruent with most conclusions of the ITS-based phylogenies (Cooke et al. 2000Citation ; Förster, Cummings, and Coffey 2000Citation ). This includes their disagreement with some of the Waterhouse classifications, although in fairness it should be noted that Waterhouse recognized that her scheme might not always reflect true phylogenies (Waterhouse 1963Citation ).

The spread of the element within the Phytophthora groups may have involved both vertical and horizontal transmission. Vertical distribution explains the similar phylogenies of many elements and host species, as illustrated by the cluster of P. infestans-P. mirabilis-P. phaseoli sequences. In contrast, those sequences from Group VI species that were close to Groups II and III may reflect horizontal transfer. Many Phytophthora species share the same environmental niche, facilitating horizontal transfer. In fact, there are examples of new species formed by natural interspecific hybridization (Man In't Veld et al. 1998Citation ; Brasier, Cooke, and Duncan 1999Citation ).

Interspecific variation in the copy number of the element was striking. At one extreme, PCR and hybridization assays identified species lacking the element. This was less surprising for P. insolita, P. macrochlamydospora, and P. richardiae than for P. humicola and P. ilicis because Cooke et al. (2000)Citation placed the first three in clades 9 and 10, far from other Phytophthora taxa (clades 1–8). Although there are contradictory data on this placement (Förster, Cummings, and Coffey 2000Citation ), the ancestral retroelement may have inserted in the progenitor of the main Phytophthora clade, bypassing the P. insolita cluster. The absence of the element in P. humicola and P. ilicis, by contrast, is harder to reconcile because both are well rooted with element-containing species in both the Waterhouse and ITS phylogenies. The element could have been eliminated, before substantial amplification had occurred, by the loss of a chromosome arm, ectopic excision, or recombinational editing (Wichman et al. 1993Citation ).

At the other extreme, species such as P. iranica and P. cactorum contained about 10,000 copies of the retroelementlike sequence. If the entire element were in the 5-kb range, this would likely account for >25% of their genomes. Cooke et al. (2000)Citation clustered these species in clades 1a and 1b; the much lower abundance in clade 1c species like P. infestans (20–30 copies) helps fix the stage at which a burst of amplification likely occurred. The hypothesis that amplification was blocked in species like P. infestans is consistent with the truncations, frameshift mutations, and other defects found by this study. In species such as P. iranica, the Gypsy-like retroelement family possibly includes both full-length and truncated copies, as seen in organisms in other kingdoms (Marin and Llorens 2000Citation ).

This study has presented the first comprehensive analysis of a family of transposable element–like sequences distributed throughout Phytophthora. Besides the Gypsy relative revealed by this study, a copia-like sequence was previously reported from P. infestans (Tooley and Garfinkel 1996Citation ). Additional types of retroelements likely reside within Phytophthora. Twelve distinct families of LTR retrotransposons exist in C. elegans, for example (Bowen and McDonald 1999Citation ). Whether all transposable elements in Phytophthora are retroelements also remains to be determined. In yeast, retrotransposons are the only class of transposable elements (Sandmeyer 1998Citation ), whereas plants contain elements replicating through either RNA or DNA intermediates (Bennetzen 2000Citation ). Considering the taxonomic placement of oomycetes, it will be interesting to learn which paradigm best fits their genomes.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The author is grateful to Mark Springer for helpful discussions; Syngenta Corp. for its collaboration in establishing an EST database; Audrey Ah Fong for commenting on the manuscript; Dr. Michael Coffey, Dr. Helga Förster, and Dr. Paul Tooley for generously providing experimental materials; and the United States Department of Agriculture for providing support under NRI award 98-35303-6770.


    Footnotes
 
Thomas Eickbush, Reviewing Editor

Keywords: Gypsy Phytophthora oomycete speciation evolution repeated DNA Back

Address for correspondence and reprints: Department of Plant Pathology, University of California, Riverside, California 92521. howard.judelson{at}ucr.edu Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

    Anaya N., M. I. G. Roncero, 1995 Skippy, a retrotransposon from the fungal plant pathogen Fusarium oxysporum Mol. Gen. Genet 249:637-647[ISI][Medline]

    Baldauf S. L., A. J. Roger, I. Wenk-Siefert, W. F. Doolittle, 2000 A Kingdom-level phylogeny of eukaryotes based on combined protein data Science 290:972-977[Abstract/Free Full Text]

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Accepted for publication April 11, 2002.