Characterization, distribution and activity of transposable elements in Crinipellis perniciosa, casual agent of the witches’ broom disease in cocoa (Theobroma cacao)

In this work, Class 1 (RNA) and Class 2 (DNA) transposable elements were characterized in the genome of Crinipellis perniciosa, the causal agent of the witches’ broom disease of cocoa. C. perniciosa presents a high genetic variability and transposable elements have been reported as the main agents responsible for the high plasticity and adaptability presented by phytopathogenic fungi. Once the genome of this fungus has been sequenced, preliminary searches in the database of the witches’ broom genome project revealed the presence of a DNA sequence with similarity to the reverse transcriptase of gypsy/Ty3-like retrotransposons. This sequence was amplified in different C. perniciosa isolates that belong to the C-, S-, and L-biotypes and to different geographical areas. The sequencing of the amplified fragments revealed the occurrence of several G:C to A:T transitions, indicating the existence of a possible RIP-like silencing mechanism. Southern analyses showed a high copy number of the reverse transcriptase gene with different profiles among the C-, S- and L- biotypes, indicating that the elements that contain these sequences may have an important role in C. perniciosa genetic variability. The DNA fragment containing the reverse transcriptase gene sequence was used to isolate different recombinant phages from a C. perniciosa genomic library. Gypsy/Ty3-like retrotransposons, named CpSaci1 and CpSaci2, were characterized from a new sequence present in the sequencing project database and from one recombinant phage. CpSaci1 contains 6.856 bp and long direct terminal repeats (LTRs) of 423 bp that do not present regions usually found in LTRs. Two open reading frame (ORFs) were identified and they code proteins with similarity to GAG and to a POL polyprotein showing protease, reverse transcriptase, RNase H, and integrase domains. Partially characterized DNA sequence of CpSaci2 possesses about xi82% of identity to CpSaci1. The copies CpSaci1 and CpSaci2 present punctual base insertions and stop codons in the structural region indicating that they are inactive copies. However, RT-PCR analyses revealed the presence of CpSaci- like transcripts expressed in C. perniciosa, presenting a higher expression under nutritional stress conditions. CpSaci is present in high copy number in the genome of the C-, S- and L-biotypes. The C-biotype isolates from Bahia state, Brazil, present two hybridization profiles and some DNA fragments similar to those of the isolates from the Amazon region. Once CpSaci presents a high copy number and activity, it is probably related to the generation of genetic variability in C. perniciosa. Besides the CpSaci retrotransposon, a Class II transposable element was also characterized. Two copies, named Boto1 and Boto2, were obtained from the genome sequencing database and from a recombinant phage obtained from a C. perniciosa genomic library, respectively. Boto1 is flanked by a 3 bp duplication (TAA) and possesses two ORFs, one that codes a transposase and another that codes a protein with low similarity to a plant transcription regulator. The structural regions that code the transposases of Boto1 and Boto2 are interrupted by a 53 bp intron with high A+T content. The aminoacid sequence of Boto1 and Boto2 transposases are highly similar to plant PIF-like transposases. These transposases are related to those of bacterial insertion sequences of the group IS5 and they are members of a new superfamily of Class II transposons named PIF/IS5. Once this superfamily is rarely found in fungi and phylogenetic analyses indicate that Boto1 and Boto2 transposases present an independent evolutionary branch, it seems that these sequences were horizontally transferred to C. perniciosa genome. Although Boto-like transcripts were amplified in C. perniciosa normally cultivated in culture medium, they were not activated by nutritional stress. This is the first report of elements of the PIF/IS5 superfamily in a phytopathogenic fungus.