Pyret, a Ty3/Gypsy retrotransposon in Magnaporthe grisea contains an extra domain between the nucleocapsid and protease domains

Abstract

A novel Ty3/Gypsy retrotransposon, named Pyret, was identified in the plant pathogenic fungus Magnaporthe grisea (anamorph Pyricularia oryzae). Pyret-related elements were distributed in a wide range of Pyricularia isolates from various gramineous plants. The Pyret element is 7250 bp in length with a 475 bp LTR and one conceptual ORF. The ORF contains seven nonsense mutations in the reading frame, indicating that the Pyret clone is lightly degenerate. Comparative domain analysis among retroelements revealed that Pyret exhibits an extra domain (WCCH domain) beyond the basic components of LTR retrotransposons. The WCCH domain consists of approximately 300 amino acids and is located downstream of the nucleocapsid domain. The WCCH domain is so named because it contains two repeats of a characteristic amino acid sequence, W-X(2)-C-X(4)-C-X(2)-H-X(3)-K. A WCCH motif-like sequence is found in the precoat protein of some geminiviruses, viral RNA-dependent RNA polymerase and also in an Arabidopsis protein of unknown function. Interestingly, detailed sequence analysis of the gag protein revealed that Pyret, as well as some other chromodomain-containing LTR retrotransposons, displays significant sequence homology with members of the gammaretroviruses (MLV-related retroviruses) in the capsid and nucleocapsid domains. This suggests that chromodomain-containing LTR retrotransposons and gammaretroviruses may share a common ancestor with the gag protein.

Figure 1

Partial structure of the cosmid clone S8-C-6. Arrows represent long terminal repeats. The shaded box indicates an MGR586-like element inserted in Pyret. The nucleotide sequence of Pyret has been deposited in the DDBJ/EMBL/Genbank nucleotide databases under accession no. AB062507. B, BamHI; E, EcoRV; K, KpnI; S, SacI; X, XhoI.

Figure 2

Amino acid alignment of CA-like domains of chromodomain-containing LTR retrotransposons. The bottom block includes authentic CA domains from several gammaretrovirses. Coloring was generated by Clustal X using default values. Accession numbers for the elements in protein databases are as follows: Cgret, AAG24791; Skippy, AAA88790; CfT-1, CAA77890; MarY1, BAA78624; REAL, BAA89271; MAGGY, AAA33419; RETRO, AAD13304; Sushi, AAC33525; KIAA1051, AB028974; Moloney murine leukemia virus (MoMLV), AAB59942; feline leukemia virus (FeLV), AAA43054; baboon endogenous retrovirus (BaEV), BAA00923. Nucleotide accession numbers for the elements which were conceptually translated from their DNA sequences are as follows: Del1, X13886; RIRE3, AB014738; Legolas, AC006570; Gimli, AL049655; human endogenous retrovirus (HERV-E), M10976.

Figure 3

Comparison of overall structure of Pyret with five chromodomain-containing LTR retrotransposons and MoMLV. Boxes indicate ORFs, including putative frames, and vertical lines correspond to stop codons in them. The positions of domains in the gag and pol proteins are indicated. MA, matorix protein; CA, capsid protein; NC, nucleocapsid protein; RT, reverse transcriptase; INT, integrase; CHR, chromodomain; env, envelop protein; MHR, major homology region; ZF, zinc finger motif; W, WCCH motif.

Figure 4

Alignment of putative WCCH motifs from four fungal retrotransposons. The bottom block includes WCCH motif-like sequences in various proteins available in the databases. The WCCH motif occurs twice in Pyret (Pyret-1 and Pyret-2) and three times in Cgret (Cgret-1, Cgret-2 and Cgret-3). The numbers after the hyphen are given according to the order of occurrence in the WCCH domain. Coloring was generated by Clustal X using default values. Accession numbers for the proteins in the databases are as follows: Arabidopsis thaliana putative protein (A.thaliana), CAA16935; tomato leaf curl geminivirus AV2 protein (ToLCV), AAA92806; Lelystad virus orf1A,1B protein (Lelystad), CAA01837.

Figure 5

A phylogenic tree of fungal Ty3/Gypsy LTR retrotransposons based on an alignment of the sum of the amino acids in the reverse transcriptase, RNase H and integrase domains. The tree is constructed by the neighbor-joining method (16) and rooted using insect LTR retrotransposons Gypsy and Tom as outgroups. The number indicates the frequency with which a given branch appeared in 1000 bootstrap replications. Accession numbers for the elements in databases are as follows: Tom, Z24451; gypsy, M12927; Ty3, M34549; Tf1, M38526; CfT-1, Z11866; Skippy, L34658; Cgret, F264028; Pyret, AB062507; marY1, AB028236; Sushi, AF030881; MAGGY, L35053; REAL, AB025309; grasshopper, M77661; MGRL3, AF314096.

Figure 6

DNA gel blot analysis of the distribution of Pyret-related elements in Pyricularia isolates. Genomic DNA was digested with EcoRI and probed with a Pyret probe (see Materials and Methods). Isolates used and their host plants were as follows: lane 1, UG-77-7-1-1 (Eleusine indica); lane 2, G10-1 (E. coracana); lane 3, NI986 (Eragrostis lehmanniana); lane 4, Br58 (Avena sativa); lane 5, 0903-4 (Oryza sativa); lane 6, Br127.11 (Triticum aestivum); lane 7, Br130.1 (T.aestivum); lane 8, NI919 (Leersia oryzoides); lane 9, NI979 (Pennisetum clandestinum); lane 10, HYZiM101-1-1-1 (Zingiber mioga); lane 11, NI907 (Digitaria sanguinalis); lane 12, Br29 (Digitaria horizontaris).