Transposable elements in phytopathogenic Verticillium spp.: insights into genome evolution and inter- and intra-specific diversification

Abstract

Background: Verticillium dahliae (Vd) and Verticillium albo-atrum (Va) are cosmopolitan soil fungi causing very disruptive vascular diseases on a wide range of crop plants. To date, no sexual stage has been identified in either microorganism suggesting that somatic mutation is a major force in generating genetic diversity. Whole genome comparative analysis of the recently sequenced strains VdLs.17 and VaMs.102 revealed that non-random insertions of transposable elements (TEs) have contributed to the generation of four lineage-specific (LS) regions in VdLs.17.

Results: We present here a detailed analysis of Class I retrotransposons and Class II "cut-and-paste" DNA elements detected in the sequenced Verticillium genomes. We report also of their distribution in other Vd and Va isolates from various geographic origins. In VdLs.17, we identified and characterized 56 complete retrotransposons of the Gypsy-, Copia- and LINE-like types, as well as 34 full-length elements of the "cut-and-paste" superfamilies Tc1/mariner, Activator and Mutator. While Copia and Tc1/mariner were present in multiple identical copies, Activator and Mutator sequences were highly divergent. Most elements comprised complete ORFs, had matching ESTs and showed active transcription in response to stress treatment. Noticeably, we found evidences of repeat-induced point mutation (RIP) only in some of the Gypsy retroelements. While Copia-, Gypsy- and Tc1/mariner-like transposons were prominent, a large variation in presence of the other types of mobile elements was detected in the other Verticillium spp. strains surveyed. In particular, neither complete nor defective "cut-and-paste" TEs were found in VaMs.102.

Conclusions: Copia-, Gypsy- and Tc1/mariner-like transposons are the most wide-spread TEs in the phytopathogens V. dahliae and V. albo-atrum. In VdLs.17, we identified several retroelements and "cut-and-paste" transposons still potentially active. Some of these elements have undergone diversification and subsequent selective amplification after introgression into the fungal genome. Others, such as the ripped Copias, have been potentially acquired by horizontal transfer. The observed biased TE insertion in gene-rich regions within an individual genome (VdLs.17) and the "patchy" distribution among different strains point to the mobile elements as major generators of Verticillium intra- and inter-specific genomic variation.

Figure 1

Structure and organization of Class I and II elements identified in the genome of Verticillium dahliae (strain VdLs.17).A) Class I LTR retrotransposons. Schematic representations of the structure of the Ty1/Copia-like and Ty3/Gypsy-like LTR elements, as well as a LINE-like non-LTR retrotransposon . The conserved domains identified in the putative protein sequences encoded by the GAG and POL genes are indicated below the open reading frames (ORFs). VdLTRE1 (upper schematic) contains direct long terminal repeats (LTR), and a fused GAG and POL ORF, while VdLTRE5 GAG and POL ORFs are separated by a UGA stop codon. The highly similar sequences of VdLTRE2, VdLTRE3, and VdLTRE4 (VdLTRE2/3/4) have overlapping GAG and POL ORFs. VdLINE sequence contains linked, but separate, GAG and POL ORFs. Domain abbreviations: INT, integrase (pfam00665); RVT2, reverse transcriptase 2 (pfam07727); RnaseH (pfam0075.16), GAG, group specific antigen; ZK, zinc knuckle (pfam00098); PR, Retropepsin protease (CD00303); RVT_1, reverse transcriptase 1; CR, chromatin organization modifier (CD00024); EEP, endonuclease-exonuclease-phosphatase; B) Class II transposable elements of the superfamilies Tc1/mariner, Activator and Mutator. Each TE type possesses terminal inverted repeats (TIRs) of different length flanking a transposase gene (shown is the corresponding ORF). The Tc1/mariner transposases are characterized by the endonuclease superfamily motif DDE_1 (pfam03184) and the presence of the additional N-terminal DNA-binding domains helix-turn-helix_pipsqueak (here indicated as psq, pfam05225), and helix-turn-helix_Tnp_Tc5 (here indicated as Tc5, pfam3221). The relative position of the hAT dimerization (pfam05699) and the MULE (pfam10551) domains characterizing the Activator and Mutator transposases are also shown. Flanking the TIRs are the nucleotide sequences of the direct target site duplications (TSDs) generated in the fungal genome by the TE insertion.

Figure 2

Phylogenetic analysis of VdLs.17 Class I and II elements. A) Ty1/Copia LTR retrotransposons, analysis of the reverse transcriptases (amino acid sequences); B) Ty3/Gypsy LTR retrotransposons, analysis of the reverse transcriptases (amino acid sequences); C) Tc1/mariner-like DAHLAIE 1, analysis of the full-length elements (TIR-comprising nucleotide sequences). Each element is identified by its chromosome location (supercontig and nucleotide position); D) Tc1/mariner-like DAHLAIE 1 to 3, analysis of complete transposases (amino acid sequences); E) Activator-like VdHAT elements, analysis of hAT transposase domains (amino acid sequences); F) Mutator-like VdMULE elements, analysis of MULE transposase domains (amino acid sequences). The trees indicate the GenBank accession number of the amino acid sequences analyzed, along with the abbreviation of the scientific names of organisms of origin. Abbreviations are as follow: Aa, Alternaria alternata (A) , Aspergillus awamori (D) ; Ac, Ajellomyces capsulatus; Af, Aspergillus flavus (A) , Aspergillus fumigatus (D) ; Ad/And, Aspergillus nidulans (A) , Ag/Ang, Aspergillus niger (D) ; At, Aspergillus terreus; Bf, Botryotinia fuckeliana; Cn, Cryptococcus neoformans; Cgl/Cg, Chaetomium globosum; Cf, Cladospororium fulvum; Cgs, Colletotrichum gloeosporioides; Dm, Drosophila melanogaster; Ds, Drosophila simulans; Fo/Fox, Fusarium oxysporum; Fom, Fusarium oxysporum melonis; Ma, Metharhizium anisopliae; Mo, Magnaporthe oryzae; Mt, Medicago truncatula; Pc, Penicillium crysogenum; Pm, Penicillium marneffei; Pn, Phaesphaeria nodorum; Pt, Pyrenophora tritici-repentis; Sc, Saccharomyces cerevisiae; Ss, Sclerotinia sclerotiorum; Sm, Sordaria macrospora; Ts, Talaromyces stipitatus. Scale bar corresponds to 0.1 substitutions per amino acid or nucleotide.

Figure 3

Distribution of Class I and II TEs within the VdLs.17 genome. The ChromoMap genome browser available online at the BROAD Institute website was used to visualize the TE sequences against a synteny map of the VdLs.17 and VaMs.102 sequenced genomes. The biased chromosome localization of the retroelements ( A) and the DNA elements ( B) in the “lineage-specific” regions of Vd.Ls17 are outlined in black.

Figure 4

Genetic context of Class I and II transposable elements in VdLs.17. The sequence segments include 90 kb from supercontig 4 nucleotide positions 162,000-222,000 (top), and 100 kb from supercontig 8 nucleotide positions 1006000 – 1106000 (bottom), of chromosome 3. The arrow indicates the position of the putative cyanide hydratase fusion protein (VDAG_04885) containing both nitrilase and MULE transposase functional domains (pfam 00795 and 10551, respectively). *Predicted gene names were assigned by the Broad Institute (www.broadinstitute.org/annotation/genome/verticillium_dahliae).