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Department of Plant Pathology and Microbiology, The Program for the Biology of Filamentous Fungi, Texas A&M University, College Station, Texas 77843-2132
Larry D. Dunkle 1
Crop Production and Pest Control Research, U.S. Department of Agriculture-Agricultural Research Service, Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907-2054
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Two fungal pathogens, Cercospora zeae-maydis Groups I and II, cause gray leaf spot of maize. During the sequencing of a cosmid library from C. zeae-maydis Group I, we discovered a sequence with high similarity to Maggy, a transposable element from Magnaporthe grisea. The element from C. zeae-maydis, named Malazy, contained 194-base-pair terminal repeats and sequences with high similarity to reverse transcriptase and integrase, components of the POL gene in the gypsy-like retrotransposons in fungi. Sequences with similarity to other POL gene components, protease and ribonuclease, were not detected in Malazy. A single copy of the element was detected by PCR and Southern analyses in all six North American isolates of C. zeae-maydis Group I but was not detected in the four isolates of C. zeae-maydis Group II from three continents or in phylo-genetically related species. Fragments of the core domains of reverse transcriptase and integrase contained a high frequency of stop codons that were conserved in all six isolates of Group I. Additional C:G to T:A transitions in occasional isolates usually were silent mutations, while two resulted in isolate-specific stop codons. The absence of Malazy from related species suggests that it was acquired after the divergence of C. zeae-maydis Groups I and II. The high frequency of stop codons and the presence of a single copy of the element suggest that it was inactivated soon after it was acquired. Because the element is inactive and because reading frames for other genes were not found in sequences flanking the element, Malazy does not appear to be the cause of differences leading to speciation or genetic diversity between C. zeae-maydis Groups I and II.
Key words: genetic variability, gray leaf spot of maize, gypsy, retrotransposon
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Ward et al 1999). Analyses of amplified fragment length polymorphism (AFLP) profiles indicated that the pathogen population in the United States consists of two genetically distinct groups (Wang et al 1998). These two pathogens, designated C. zeae-maydis Group I and Group II, are conspecific based on commonly applied taxonomic criteria but differ substantially in growth rates and their ability to produce the phytotoxin, cercosporin, in culture (Dunkle and Levy 2000, Wang et al 1998). Phylogenetic analyses employing nucleotide sequences of the internal transcribed spacer (ITS) and 5.8S rDNA region indicated that the two maize pathogens, in fact, are distinct species (Goodwin et al 2001b), but little information is available to explain the mechanism by which the two pathogens diverged.
One mechanism directing phenotypic change via genomic rearrangement and rapid alteration of chromosomal structure in filamentous fungi is through the effects of transposable elements (Davière et al 2001, Kempken and Kück 1998, Wei et al 1996). In addition significantly increased rates of spontaneous mutation can be induced by transpositions (Kempken and Kück 1998, Skimmer et al 1993, Talbot et al 1993). Transposable elements have been detected in several plant pathogenic fungi, including Magnaporthe grisea, Cladosporium fulvum, Colletotrichum gloeosporioides and Fusarium oxysporum (Kempken and Kück 1998). Mycosphaerella graminicola, a close relative of C. zeae-maydis, contains a transposable element that is active during both sexual and asexual reproduction (Goodwin et al 2001a), but the phenotypic consequences of this transposable element were not demonstrated.
During the sequencing of a genomic library from C. zeae-maydis (Shim and Dunkle 2002), we obtained a sequence contig with high similarity to Maggy, a retrotransposon in the rice blast fungus, M. grisea (Farman et al 1996). Sequence analysis revealed that the element has characteristics of gypsy-like retrotransposons, such as a long terminal repeat (LTR) and components of the POL gene encoding enzymatic functions that are essential for retrotransposition in other transposable elements (Kempken and Kück 1998). Upon analysis via the translated BLAST algorithm (BLASTX), the highest degree of amino acid identity was located in the reverse-transcriptase domain. We named this putative transposable element Malazy (Maggy-like element in Cercospora zeae-maydis). The objectives of the study were to describe the genetic characteristics of the element and determine its distribution in geographically diverse isolates of C. zeae-maydis and in phylogenetic relatives. We present evidence that Malazy is present in Group I isolates of C. zeae-maydis but not in Group II isolates or in closely related species. Sequence data revealed an abundance of stop codons throughout the POL gene, suggesting that Malazy is degenerate and therefore inactive in the genome of C. zeae-maydis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Single-spore isolates were recovered from conidia produced from individual gray leaf spot lesions collected from diverse regions of the United States and Mexico over a period of four years and maintained as described by Wang et al (1998). Mycelium for DNA extraction was prepared as described and stored at –80 C. Restriction fragment-length polymorphism (RFLP) analyses of the ITS-rDNA sequence and growth rates were used to designate each isolate as Group I or Group II (Dunkle and Levy 2000, Wang et al 1998).
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).
PCR amplification of the Malazy POL gene components.— –
A 1.9 kb region of the Malazy POL gene was amplified by PCR with primers pPOLF2 (5'-TACGCTCTAAAGGCATAACG-3') and pPOLR2 (5'-GCTTGCACTTTAGCTGCG-3') (FIG. 1). PCR amplification was performed in a 50-µL reaction mixture with Taq DNA polymerase (Promega, Madison, Wisconsin) in a DNA thermal cycler 480 (Perkin Elmer Cetus, Norwalk, Connecticut). The reactions were carried out for 30 cycles of 45 s of denaturation at 94 C, 45 s of annealing at 58 C, and 90 s of extension at 72 C.
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). The conditions and temperature program for PCR were 30 cycles of 45 s of denaturation at 94 C, 45 s of annealing at 56 C, and 30 s of extension at 72 C. After electrophoresis in a 1.0% agarose gel, the PCR products were purified with a GeneClean Spin Kit (BIO 101, Carlsbad, California) and, as necessary, cloned with the TA Cloning Kit (Invitrogen, Carlsbad, California).
Southern analyses.— –
Close relatives of C. zeae-maydis (Goodwin et al 2001b), including members of the teleomorph genus Mycosphaerella, also were analyzed by Southern blots to test for the presence of the transposable element in those species. Genomic DNA samples from C. kalmiae, C. kikuchii, C. sojina, C. sorghi var. maydis, Mycosphaerella fijiensis (anamorph Paracercospora fijiensis), M. graminicola (anamorph Septoria tritici), M. musicola (anamorph Pseudocer-cospora musicola) and Septoria lysimachiae, as well as the more distantly related species, Aspergillus nidulans and M. grisea, were extracted and digested with EcoRI before Southern hybridization at a lower stringency of 60 C with the 1.9 kb POL amplicon as a probe. The Malazy sequence lacks an EcoRI site.
Genomic DNA samples from six C. zeae-maydis Group I isolates and three Group II isolates (TABLE I) were digested with EcoRV prior to Southern hybridization. The 527 bp RT amplicon (FIG. 1) was used as a probe, and the hybridization was performed at normal stringency (65 C).
DNA sequencing and sequence analysis.— –
We used the EZ:: TN 
TET-1
Insertion Kit (Epicentre, Madison, Wisconsin) to generate random transposon insertions in a cosmid containing Malazy and used the Forward and Reverse Sequencing Primers from the kit to perform high-throughput sequencing and contig assembly at the Agricultural Genome Center, Purdue University. Sequencing of PCR-amplified POL DNA fragments (1.9 kb) from six isolates of C. zeae-maydis Group I was performed at the DNA Sequencing and Synthesis Facility, Iowa State University. Similarity searches were done via the BLAST algorithm (Altschul et al 1990) against genes in GenBank. Multiple sequence alignments were done via Clustal W (Thompson et al 1994). The nucleotide sequence of Malazy, including the putative ORF2 (POL gene), was submitted to GenBank (accession No. AY170475
[GenBank]
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). In common with LTRs found in other retrotransposons (Farman et al 1996, Goyon et al 1996, Kempken and Kück 1998, Zhu and Oudemans 2000), 8-bp inverted repeats (TATAAGAG/CTCTTATA) were identified at the 5' and 3' termini of the LTRs. Translation of the Malazy nucleotide sequence revealed two conserved domains that show significant alignments with the core domains of reverse transcriptase (RT) and integrase (INT) (FIG. 1).
The putative translation products of Malazy contained an unusually high frequency of stop codons in the reading frame, 71 stop codons in 1201 amino acids (5.9%) (data not shown). This high frequency of stop codons hindered our efforts to identify ORF1 or conserved domains of other genes (protease and ribonuclease) commonly found in ORF2 of fungal transposable elements. However, when we used the BLOSUM80 scoring matrix of BLASTX instead of the default BLOSUM62 to analyze the putative ORF1, we discovered a sequence with high similarity to the Gag-Pol precursor in Oryza sativa. Within the 2.3 kb sequence spanning the region between the INT gene and the LTR (FIG. 1), no ORFs or homologs were identified in database searches.
Detection of Malazy in C. zeae-maydis isolates by polymerase chain reaction.— –
Malazy was identified initially in the OH strain of C. zeae-maydis. We subsequently analyzed five other isolates of C. zeae-maydis Group I isolates from widely distributed areas of North America and four Group II isolates from three continents (TABLE I). PCR with primers, pPOLF2 and pPOLR2, designed to amplify a 1.9 kb region of the POL gene in Malazy (FIG. 1) produced an amplicon of the expected size only in Group I isolates of C. zeae-maydis (FIG. 2).
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) was digested with EcoRV for Southern analysis. Results confirmed that Malazy was present in these widely distributed Group I isolates but absent from Group II isolates. The lack of polymorphism for the Malazy loci in different C. zeae-maydis Group I isolates (FIG. 4) suggests that Malazy is present at the same insertion points within the genomes of all Group I isolates.
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). When the sequence alignment of the RT domain (500 bp) of six Group I isolates was analyzed, nucleotide differences were observed at 20 sites and all such point mutations were C:G to T:A transitions (FIG. 5A). Of the eight stop codons in this domain, seven were conserved in all six isolates. The eighth stop codon resulted from a C to T transition at nucleotide 490 and occurred in two isolates (OH and KS). Similar results were found in the INT sequence alignment (FIG. 5B). Among the six isolates, nucleotide differences were observed at 21 sites in 500 bp of the INT domain. Of the 12 stop codons recognized, 11 were conserved. The one unconserved stop codon resulted from two C:G to T:A transitions at nucleotides 401and 402 and was found in only one isolate (KS).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Kempken and Kück 1998, Wei et al 1996). In addition, their distribution in host-specific strains of some pathogenic fungi provide clues to evolutionary history and phylogenetic relationships among fungi. For example, Maggy, a gypsy-like LTR-retrotransposon was identified in a number of M. grisea isolates from different hosts, including rice, in which it is present in high copy numbers (Farman et al 1996). The fact that Maggy was located at identical sites in the genome of geographically dispersed strains suggested that the insertion of the element in the M. grisea genome occurred in a progenitor of the rice-specific form during fungal evolution (Farman et al 1996). In contrast, MRPAN, a Maggy-like sequence from a common millet isolate of M. grisea, was found in the genome as a single copy and in a highly degenerate form (Nakayashiki et al 1999). The authors suggested that MRPAN was inactivated before amplification of the element. Our data similarly showed that Malazy is present as a single copy in the genome of C. zeae-maydis Group I isolates but absent from the genomes of C. zeae-maydis Group II and closely related fungal species. These observations suggest that the element is species specific and was introduced into the genome of C. zeae-maydis Group I relatively recently but after divergence from the ancestors of Group II. Southern analysis (FIG. 4) showing the lack of polymorphism among the Group I isolates suggests that Malazy probably was inactivated rapidly before it could be amplified.
In fungi the two well-known mechanisms by which fungi defend themselves against intragenic parasites or foreign sequences (e.g., transposable elements or mycoviruses) are RIP (repeat-induced point mutations) and MIP (methylation induced premeiotically) (Goyon and Faugeron 1989, Graïa et al 2001, Selker 1997, Selker and Garrett 1988). RIP inactivates repetitive sequences characteristically via C:G to T:A transitions. Although sequence analysis of Malazy revealed a high frequency of mutations and conceptual translations of putative ORFs resulted in numerous stop codons, we could not determine whether the stop codons are the direct result of nucleotide transitions because an active progenitor of Malazy has not been discovered.
We examined the highly conserved POL gene sequence of six C. zeae-maydis isolates from North America to determine the mutation pattern and whether such nucleotide differences were responsible for stop codons during translation. From two 500 bp amplicons sequenced and aligned from the Malazy POL gene (RT and INT domains) of six Group I isolates, we identified 41 sites (4.1%) where a nucleotide difference was present (FIG. 5). This mutation frequency is surprising given that the ITS sequences of four Group I isolates were identical (Wang et al 1998). With the exception of three transitions resulting in two, isolate-specific stop codons, all transitions were silent mutations, resulting in no change in the amino acid sequence. Of the 20 stop codons observed in these DNA sequences, 18 were conserved in all six isolates.
Our results suggest that Malazy is a degenerate, inactive transposable element that was acquired by the fungus after the two Cercospora pathogens of maize diverged. Sequence analysis of DNA (ca. 1.3 kb at the 5' end and 80 bp at the 3' end) flanking the Malazy insertion site revealed no significant similarity to genes in the GenBank (data not shown). Although we cannot eliminate the possibility that the insertion site is in a regulatory cis-acting element or trans-acting factor in the fungal genome, our sequence analysis suggests that Malazy is not directly involved in gene disruption or inactivation in C. zeae-maydis. Therefore, Malazy is not considered to be a cause of phenotypic and genetic differences between Group I and Group II isolates nor a contributing factor in species divergence. Nevertheless, the unique occurrence of Malazy only in Group I isolates provides additional evidence to corroborate the conclusion (Goodwin et al 2001b) that these two maize pathogens are distinct taxonomic species.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author: E-mail: dunkle{at}purdue.edu
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LITERATURE CITED |
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(pPOLF2) and 
(pPOLR2). The primers pPOLF2 and pMRTR1, which is indicated with 
, were used to amplify the 527 bp RT probe. The fragments used for sequence alignment shown (FIG. 5
. Malazy lacks an EcoRI restriction site. LTR, long terminal repeats; GAG, gene for structural proteins; PR, protease; RT, reverse transcriptase; RH, RNaseH; INT, integrase; ORF, open reading frame.
