ANÁLISIS CONCEPTUAL DEL CONCEPTO INCLUSIÓN

The E. festucae E2368 genome was recently sequenced in the laboratory of Professor Chris Schardl at the University of Kentucky. To determine whether the eas genes identified in this study were present, BLASTN analysis of the genome sequence was performed. This revealed that each of the eas genes identified in this study, along with lpsA and dmaW, were present (Table 3.6) with almost identical nucleotide sequences in N. lolii Lp19 and E. festucae E2368. The EAS cluster sequence in the genome assembly was not as connected as the cloned N. lolii cluster, being found on four different contigs (Table 3.6).

Two genes, easC and easD, were present in the C. purpurea and A. fumigatus EAS clusters but not identified in this study. TBLASTN analysis was thus undertaken, using the C. purpurea predicted EasC and EasD protein sequences, to determine whether these genes were present in the E. festucae genome sequence. Both easC and easD were identified (Fig. 3.17), completing the suite of eas genes in epichloë that are present in the C. purpureaEAS cluster.

Table 3.6.N. loliiEAS genes in the E. festucae E2368 genome

*easF is incomplete on two contigs, total gene length 1103 bp. †_{The dmaW sequence from}

Neotyphodium sp. Lp1 (AY259837) was used to query the E. festucae genome as the N. lolii

gene was not sequenced. a_{Total Neotyphodium sp. Lp1 gene length 1485.} ‡_Accession

AF368420

Gene Identity (identical

bp/total bp)

E. festucae contig (length) Position

lpsB 4125/4130 1241 (10033) 4708 – 8837 easA 1143/1143 3372 (1391) 163 – 1305 easE 1942/1951 1241 (10033) 1488 – 3438 easF 1008/1018* 54/54* 1241 (10033) 2216 (1771) 1 – 1018 1718 – 1771 easG 1058/1059 2216 (1771) 616 – 1674 easH 556/558 2048 (2125) 1297 – 1854 dmaW† _1483/1487a _{1139 (14909) 3020 – 4506} lpsA‡ _{10890/10922 1183 (12626) 842 – 11763}

Figure 3.17. Physical map and GC% of E. festucae E2368 Contig 1139. Predicted genes are shown as arrows indicating the direction of transcription. Introns are shown as gaps. The end of a Tahi retrotransposon relic is shown as a dark brown bar.

Both easC and easD were found on the 14909-bp contig 1139, along with dmaW and cloA (Fig. 3.17). This contig was very AT-rich at each end, from 1.7 kb upstream of dmaW at the left end and from 1.24 kb upstream of easD at the right end. The AT- rich sequence at the right end of the cluster had 87 % identity with a Tahi LTR retrotransposon relic from the N. lolii LTM cluster. The left end sequence did not match transposon sequences from the database but did have several repeats in the E. festucae genome sequence and thus was likely to represent a novel transposable element. (This and other transposons from the EAS cluster are further analysed in Chapter 5.)

Genome sequence analysis, combined with N. lolii data, thus indicates that the EAS cluster is found in three mini-clusters separated by AT-rich sequence in E. festucae: cluster one contains easA, easE, easF, easG, easH and lpsB, cluster two contains dmaW, cloA, easC and easD, and cluster three contains just lpsA. The N. lolii cluster may be even more disjointed, as dmaW does not appear to be linked to cloA (Fig. 3.14).

3.8.1 Bioinformatics Analysis of E. festucaeeasC, easD, and

Full-Length easH and cloA

3.8.1.1 The easH Gene and Predicted Dioxygenase EasH

The easH gene was truncated at one end of the EAS cluster sequence isolated from N. lolii. This sequence was used to identify the full-length gene on the 2125-bp contig 2048 (Fig. 3.18). easH is the only predicted gene found on this contig, with the other end of the contig being highly AT-rich (Fig. 3.18). The predicted full- length easH gene is 921 bp in length and contains no introns. The C. purpureaeasH sequence is not deposited in GenBank and so percentage identity could not be determined and the easH gene found in A. fumigatus is a pseudogene and is unlikely to encode a protein product.

The predicted EasH protein is 307 aa long, a predicted unmodified molecular mass of 33.6 kD. Analysis with the CDD revealed a conserved phytanoyl-CoA dioxygenase (PhyH) domain (pfam05721.5). The top functionally-characterised protein match was to Fum3p from Gibberella moniliformis (30% identity), a dioxygenase involved in the fumonisin pathway (Ding et al., 2004). EasH is thus likely to catalyse a hydroxylation step subsequent to the steps shared with A. fumigatus, possibly the conversion of agroclavine to elymoclavine or lysergyl peptide lactam to ergovaline (Schardl et al., 2006).

Figure 3.18. Physical map and GC% of E. festucae E2368 Contig 2048. The easH gene is shown as a blue arrow indicating the direction of transcription.

3.8.1.2 The cloA Gene and P450 Monooxygenase CloA

The 3ʹ end of the cloA gene was identified downstream of dmaW in N. coenophialum (section 3.6) and the full-length gene was identified in EAS cluster 2, on contig 1139. The E. festucaecloA gene is 2140 bp in length and is predicted to contain eight introns (Fig. 3.17), substantially more than other eas genes.

The predicted CloA protein is 527 aa long, a predicted unmodified molecular mass of 60 kD. CloA is likely to be an orthologue of the C. purpurea protein, to which it is 63% identical. CloA is a P450 monooxygenase required for the conversion of elymoclavine to lysergic acid (Haarmann et al., 2006).

3.8.1.3 The easC Gene and Predicted Catalase-Like Protein EasC

The easC gene is 1491 bp and manual annotation predicts it to contain a rare GC- AG intron of 63 bp (Fig 3.17). The predicted protein is 476 aa long and a predicted unmodified molecular mass of 54 kD. Analysis with the CDD revealed a catalase domain (cd00328.2) covering the length of the protein. BLASTP results reflected this, showing EasC to be similar to many predicted and characterised catalases. The top functionally characterised match (38% identical), for which a structure has also been solved, was to Catalase A (Cta1p) from Saccharomyces cerevisiae (Mate et al., 1999). EasC is 73% and 62% identical respectively with the C. purpurea and A. fumigatus orthologues. A role for a catalase in ergot alkaloid synthesis is not immediately obvious.

3.8.1.4 The easD Gene and Predicted Oxidoreductase EasD

The easD gene is 850 bp in length and is predicted to contain one intron of 64 bp (Fig 3.17). The predicted EasD protein is 262 aa long and a predicted unmodified molecular mass of 28 kD. Analysis with the CDD predicted a short-chain dehydrogenase domain (COG4221, COG1028). These enzymes are NAD(P)H dependant and reduce a wide range of substrates (Jornvall et al., 1995). BLASTP hits were largely to hypothetical oxidoreductases. The top hit to a functionally characterised gene was to ABA4 (29% identical) from the Botryotinia fuckeliana

67% identical respectively to the C. purpurea and A. fumigatus orthologues respectively. There are several oxidation/reduction reactions in the ergot alkaloid pathway that EasD could potentially catalyse.