Repeat Induced Point-Mutation (RIP) Analysis

All of the ten transposable elements associated with the EAS cluster appeared to be degenerate relics. The Tahi and Rua sequences associated with the LTM cluster were also observed to be degenerate and the AT-richness of these elements was proposed to be evidence of RIP (Young et al., 2005), which results in C:G to T:A transitions. RIP is a fungal mechanism first discovered and best characterised in Neuropora crassa, which disables repeated genomic sequences (Cambareri et al., (1989), reviewed in Selker, (2002)). The genome is scanned pre-meiosis for repeats, which are then mutated by converting cytosine residues to thymines, resulting in guanine to adenine transitions on the complementary strand. Cytosine and histone methylation is known to be important (Freitag et al., 2002; Selker et al., 2002) but the exact mechanism for conversion of the C to T mutations is not known. While RIP only occurs during sexual reproduction, evidence of RIP can be ascertained from previously RIPed sequences based on the sequence context of SNPs between copies (Margolin et al., 1998). This analysis was performed on each of the transposons characterised in this chapter.

5.5.1 Epichloë Transposons Have Been Subject to RIP

In N. crassa, RIP occurs on cytosines preferentially at the CpA context (Grayburn and Selker, 1989). The ratio of TpA (resulting from CpA conversion) to ApT (a dinucleotide with the same composition) and CpA + TpG to ApC + GpT is thus indicative of whether or not RIP has occurred for a given sequence (Margolin et al., 1998). These ratios were determined for a representative of each class of transposons identified in this study and also for the entire EAS cluster sequence with transposons removed (Table 5.5). (Galagan et al., 2003) suggest that a ratio of TpA/ApT of greater than 2.0 or CpA + TpG/ApC + GpT less than 0.7 is indicative of RIP mutation in N. crassa. Each of the elements but Rima satisfy at least one of these criteria while the EAS cluster sequence without transposons does not. The

presumed to have occurred in the sexual progenitor strain. In N. crassa RIP only occurs on repeat sequences of greater than ~400 bp, it would thus be unexpected for the MITEs Toru and Iwa to have undergone this process. However, it would seem from this analysis that in E. festucae RIP may function on smaller sequences than is possible in N. crassa.

5.5.2 De-RIP Wha and Ono-nui and E. festucae RIP Sequence

Preference

Different ascomycetes in which RIP has been observed, either experimentally or by sequence analysis, appear to have C to T conversion at different sequence contexts in terms of the base 3ʹ to the cytosine (Grayburn and Selker, 1989; Idnurm and Howlett, 2003; Clutterbuck, 2004). None of the transposon families identified contained copies in the E. festucae E2368 genome that were not degenerated. Two of the larger elements, Wha and Ono-nui, for which RIP can confidently be asserted to have taken place, were thus “deRIPed”, similar to the analysis done by Attard et al., (2005) with the Leptosphaeria maculans Pholy retrotransposon. Transposon sequences were aligned and wherever at least one of the sequences contained a C rather than T, or G rather than A, this base was inserted into the consensus sequence, resulting in a sequence that was “deRIPed” and likely to be more similar to the progenitor sequence.

Table5.5. RIP indices for transposons associated with the EAS cluster

element size stop

codonsa

CpA* TpG* TpA* TpA/ApT CpA +TpG/

ApC+GpT Toru-1a 141 9.2 0.67 0.80 2.05 2.02 0.56 Wha 2553 11.2 0.39 0.43 2.97 1.58 0.67 Rima 291 11.2 0.98 0.65 2.12 1.63 1.0 Ono-nui 5435 10.5 0.27 0.30 2.91 1.64 0.47 Whitu 8939 10.1 0.07 0.08 2.92 1.50 0.15 Waru 3075 10.5 0.37 0.32 2.91 1.50 0.55 Iwa 234 11.1 0.34 0.48 3.09 2.11 0.38 Tekau 4038 10.8 0.07 0.10 3.57 1.83 0.11 EAS cluster 36612 4.7 1.10 1.13 0.91 0.83 1.21

*ratio of observed to expected

Table 5.6. RIP sequence context preference summed for both strandsa

element CpA -> TpA CpG -> TpG CpC -> TpC CpT -> TpT non C ->T SNP

Wha 23 12 12 22 17

Ono-nui 96 40 21 87 20

combined 119 52 33 109 37

a_{Derived from comparison of Wha sequence from E. festuae E2368 contig 1978 and Ono-nui sequence from contig}

12 with “deRIPed” sequences

Table 5.7. Ascomycete RIP sequence context preference

Species 3ʹ base preference†

E. festucae A>T>>G>C

N. crassa A>>T>G>C

A. nidulans G>A>>C≈T

Leptosphaeria maculans G≈A

F. oxysporum A>G

M. grisea T>A>>C>G

†_{Preferences for base 3ʹ of mutated cytosine from fungi other than E. festucae were taken from (Clutterbuck, 2004)}

The deRIP consensus sequence for both Wha and Ono-nui contained fewer stop codons than degenerate copies; 307 reduced to 276 and 572 to 545 respectively. DeRIPing improved the BLASTX match for both elements to the top match (E values 1e-13 reduced to 3e-34 and 4e-26 to 2e-28 respectively) and included a direct match to the F. oxysporum Hop transposase (E value 2e-08) for the deRIP Wha, which was not observed in the list of matches for the degenerate Wha sequence. Although the BLAST matches were still weaker than might be expected for a non-

of protein alignment and reduction in stop codons suggested that the deRIP sequences more accurately represented the original sequences for both elements.

Each sequence was compared with a representative degenerate copy and the base 3ʹ of the mutated cytosine was determined on each strand (Table 5.6). Total C to T transitions for both elements showed 119 CpA to TpA, 109 CpT to TpT, 52 CpG to TpG and 33 CpC to TpC transitions (Table 5.6). This was a similar RIP site preference to N. crassa (Table 5.7), however, preference for CpG may be underestimated as mutation of the cytosine in either strand changes the context of the other.