V. dahliae and V. albo-atrum are the parental genomes for two paralogue transcription

3.4.3. V. dahliae and V. albo-atrum are the parental genomes for two paralogue transcription factor isogene pairs of V. longisporum

We extended our analysis by examining two orthologues of transcriptional regulatory genes VTA1 and VTA2, which are expressed in the cDNA library of V. longisporum and identified from the yeast complementation assays. They belong to the zinc finger family and are conserved within filamentous ascomycetes (Figure S4B, S5B). The VTA1 gene encodes a protein containing the Zn(II)2Cys6 conserved domain. This domain is similar to the conserved domain of the AflR transcription factor in Aspergilli required for the biosynthesis of the cancerogenic aflatoxin (Yu et al., 1996). The VTA2 gene containing a C2H2 conserved motif is an orthologue of the CON7 gene in the rice blast fungus Magnaporthe grisea. The CON7 protein controls approximately 100 genes and is essential for appressorium formation and growth in planta (Odenbach et al., 2007).

V. longisporum carries two isogenes for VTA1 without introns. They correspond to single genes in V. dahliae or V. albo-atrum. The two 1.275-1.278 kb VTA1 sequences display a 95 to 99% identity to each other and to the corresponding genes of the corresponding putative parents with a significant different signature: a three-nucleotide sequence in the longer VTA1 sequence (VTA1-1) of V. longisporum is only present in V.

dahliae. The V. albo-atrum as well as the shorter VTA1 sequence (VTA1-2) of V.

longisporum carry a small deletion of this sequence (Figures 41, S4A). Comparison of the Southern hybridization pattern revealed two loci for the VTA1 genes in both rDNA types of V. longisporum. One locus corresponds to V. dahliae. The other gene which carries the deletion of the corresponding V. albo-atrum gene shows a different pattern. This could be due to a point mutation in the V. albo-atrum genome acquired during speciation after hybridization or could reflect another V. albo-atrum strain carrying this point mutation as parent (Figure 40C). The data corroborating V. longisporum as an interspecies hybrid between V. dahliae and V. albo-atrum (or a close relative) support that the different rDNA types of V. longisporum might share a common original hybridization event.

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Figure 41. Two VTA1 isogenes of V. longisporum (VTA1-1, VTA1-2) in comparison to the corresponding single genes of V. dahliae and V. albo.atrum. VTA1-1 shares with the V. dahliae orthologue the same signature consisting of a three-nucleotide insertion in the coding region, whereas VTA1-2 and the V. albo-atrum orthologue VTA1 lacks this insertion.

The origin of V. longisporum as interspecies hybrid from a V. dahliae and a V. albo-atrum parent was further confirmed when we compared the VTA2 genes of all three species. VTA2 genes are broadly conserved within filamentous fungi and the Verticillium isogenes are related to fungal plant pathogens such as Fusarium species and Magnaporthe grisea (Figure S5C). Comparison of the expressed sequence tags to the genomic sequences amplified by PCR revealed a complex gene structure for VTA2, which includes four introns and five exons. The V. dahliae and V. albo-atrum VTA2 orthologues vary slightly in size with 1.701 kb and 1.754-1.759 kb, respectively (Figures S5A and S5D). The first exon of V. dahliae VTA2 has an insertion of nine nucleotides (ACGCTCACC) when compared with V. albo-atrum VTA2. In addition, the third intron (I3) of the V. dahliae gene (217 bp) is 68 nucleotides shorter than V. albo-atrum I3 (285 bp). In contrast to the single parental genes, both types of V. longisporum rDNA types carry again two paralogues which carry two distinct gene signatures which classify them either as V. dahliae or V. albo-atrum genes (Figures 42, S5A).

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Figure 42. Comparative scheme of VTA2 homologues from all three Verticillium species.

VTA2-1 and VTA2-2 isogenes of V. longisporum are derivatives from V. dahliae and V. albo-atrum, respectively. Characteristic signatures include a nine-nucleotide insertion (ACGCTCACC) in the first exon (E1) and the length of the third intron (I3). VTA2-1 and the V. dahliae orthologue carries the nine-nucleotide insertion in the first exon and a shortened third intron (217 bp), whereas VTA2-2 corresponds to the V. albo-atrum orthologue lacking the insertion in the first exon and carries and extended third intron (282 bp).

We compared the two VTA2 isogenes of the eight European and the three American V. longisporum isolates (Table 2) to further analyze a nascent species during geographic distribution. The two V. longisporum VTA2 isogenes VTA2-1 and VTA2-2 varied in length slightly between 1.701 and 1.755 kb. VTA2-1 is identical in all 11 V. longisporum isolates and differs only slightly from the corresponding V. dahliae gene (99% identity). It carries the characteristic ACTCTCACC insertion in the first exon and the shortened third intron of 217 bp as V. dahliae VTA2. The other VTA2-2 isogene is also identical in all 11 V.

longisporum isolates and resembles VTA2 of V. albo-atrum (97% identity). It carries the characteristic first exon of V. albo-atrum lacking the nine-nucleotide insertion as well as the longer third intron of 282 bp. Southern hybridization confirmed that the two V.

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longisporum VTA2 isogenes are either a derivative of V. dahliae or of V. albo-atrum (Figure 40D).

The VTA2 gene analyses corroborated that the two isogenes VTA2-1 and VTA2-2 are completely conserved within all 11 V. longisporum isolates derived from Europe and America and have not (yet) been changed during geographical separation. In contrast, these isolates represent two different rDNA types of V. longisporum. Sequencing of the corresponding VTA2 genes from seven V. dahliae isolates from different geographical regions also reveals an absolute conservation of this gene (100% identity). In contrast, V.

albo-atrum isolates display minor changes in VTA2 genes depending on hosts. The VTA2 genes from the Va-1 isolate deriving from potato in Wisconsin and from the Va-2 isolate from alfalfa from the United Kingdom exhibit a slight reduction of 98% identity to each other (Figure S5D) but still carry the same signatures for the first exon and the third intron.

This variation in small nucleotide polymorphisms in V. albo-atrum might be the reason for the differences in the hybrid V. longisporum where the VTA2-2 gene from V. albo-atrum carries more SNPs than VTA2-1 from V. dahliae (Figure S5A).

In total, our data show that single genes in the V. dahliae or V. albo-atrum genome are present as isogene pairs in the hybrid V. longisporum. Both rDNA types of V.

longisporum carry identical isogene pairs suggesting a single hybridization event and subsequent homogenization of rDNA where either parental rDNA could be lost.

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