Conclusions and future work

4.1 The DOTH biosynthesis gene clusters in P. arachidicola

Although it is not known if the three regions containing DOTH biosynthetic genes are clustered in P. arachidicola, they are each similar to mini-clusters found in the fragmented DOTH cluster in D. septosporum.

Southern blots using DIG-labelled P. arachidicola probes suggested that there is only one copy of Pa-pksA, Pa-dotA, Pa-vbsA and Pa-cypA genes in the whole genome. Based on Southern blot results that showed the size of fragments that contained the DOTH genes in P. arachidicola, EcoRI 5-11 kb and 11-20 kb size-fractionated genomic libraries were made and screened. Four positive clones were found, two of which overlapped to make three sequences of 11.5 kb, 5.7 kb and 9.3 kb were obtained. Sequence analysis identified 11 putative DOTH biosynthesis genes separated into three mini-clusters, similar to the DOTH biosynthesis gene cluster in D. septosporum. Most of the predicted DOTH biosynthesis genes in P. arachidicola had high amino acid identity to homologous DOTH genes in D. septosporum, and were therefore predicted to have the same functions. Homologous genes had about 73% to 96% amino acid identity between the species, and most of the Pa-DOTH genes had similar gene order and direction of transcription to Ds-DOTH genes. No regulatory genes were identified in either P. arachidicola or D. septosporum, but conserved aflR binding sites were identified in the upstream and coding region of several DOTH genes in two species. The DOTH genes in P. arachidicola may have a similar co-expression pattern to DOTH genes in D. septosporum.

There are three different points between the DOTH biosynthesis gene clusters in P. arachidicola and D. septosporum. Firstly Pa-epoA may not functional due to a premature stop codon in the coding region. Secondly the Pa-moxA and Ds-moxA had different transcription orientations. Thirdly a MFS transporter, Pa-mfs, was found in P.

arachidicola that is not seen in D. septosporum. This was additional to the Pa-dotC MFS gene upstream of Pa-mfs in mini-cluster 3. The amino acid identity between Pa-dotC and Pa-mfs was quite low, only 12.9%, while Pa-mfs had 54% amino acid identity to the corresponding region of a MFS transporter in A. fumigatus. The DOTH biosynthesis genes in P. arachidicola had different gene organization and direction of transcription to homologous AF or ST biosynthesis genes in Aspergillus species. Several tandem and inverted repeat sequences were identified in DOTH gene cluster intergenic regions in P. arachidicola, but the distribution of those repeats appears to be random, suggesting that the fragmentation of the DOTH biosynthesis gene cluster may not due to retrotransposon activity or recombination between repeat sequences. The DOTH biosynthesis gene cluster in P. arachidicola was predicted to be ancestral to the AF/ST biosynthesis clusters. Understanding the DOTH biosynthesis gene cluster in P. arachidicola could provide insights into the evolution of AF/ST biosynthesis cluster

Studies showed that DOTH is not required for D. septosporum to cause Dothistroma needle blight of pines (Schwelm, 2007). The growth of other pine-needle inhabitants were inhibited by DOTH in vitro, so DOTH may provide an advantage to D. septosporum in growth competition with other fungi. It may play a role in competition of D. septosporum with other fungi in its ecological niche (Schwelm et al., in press). So the DOTH produced by P. arachidicola may have a similar role.

4.2 Future works

4.2.1 Identification of other DOTH biosynthesis genes in P. arachidicola

The three mini-clusters identified in P. arachidicola so far are not complete. It is of interest to know the regions flanking the DOTH biosynthesis genes. First, according to Southern blot results and sequence analysis of the three mini-clusters, 7-13 kb and 13-20 kb size-fractionated ScaI genomic libraries should contain positive clones that cover more regions of mini-clusters 1 and 2, so further efforts could be made to obtain and sequence these. Second, a genome walking strategy could be used to identify

flanking sequences adjacent to known regions. This PCR based method involves gene-specific primer (GSP) and a tailing step with a 5’-RACE abridged anchor primer (AAP) (Leoni et al., 2008). Third, because there is high amino acid identity of DOTH genes between P. arachidicola and D. septosporum, new degenerate primers can be designed according to D. septosporum gene sequences and used for PCR to identify fragments containing DOTH genes in P. arachidicola.

4.2.2 Identification of DOTH gene functions in P. arachidicola

The Pa-vbsA and Pa-dotA genes from P. arachidicola were recently shown to be involved in DOTH biosynthesis by complementation experiments in this lab (unpublished data). The Pa-vbsA and Pa-dotA genes were able to rescue D. septosporum Ds-vbsA and Ds-dotA mutant strains and allowed them to reproduce DOTH. A Ds-pksA knockout strain is also available that cannot produce DOTH. It would be interesting to see if Pa-pksA is able to rescue the Ds-pksA mutant strain. D. septosporum knockout strains were obtained by transformation of the Ds-gene containing vector to D. septosporum wild type strain using a protoplast mediated method. Because there are some difficulties in making P. arachidicola protoplast, D. septosporum knockout strains were used for complementation experiments. An alternative transformation system such as Agrobacterium mediated transformation would be a possibility for making P. arachidicola knockout strains.

Another gene of interest was Pa-mfs which did not have a homologue in D. septosporum. If a transformation system could be developed, a Pa-mfs knockout mutant could be obtained by gene replacement. The DOTH biosynthesis and toxin secretion can be monitored by ELISA assay. It is also of interest to compare the effect of Pa-dotC or Pa-mfs mutant strain for DOTH biosynthesis and toxin secretion. This may help to understand the relation of these two MFS transporters.

Based on similarities between P. arachidicola and D. septosporum gene clusters seen so far, it is expected that further analysis will reveal a fragmented gene cluster in P. 103 

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arachidicola, and that the genes will have similar functions to their homologues. There are plans in our laboratory to sequence the genome of D. septosporum. This is expected to reveal other DOTH genes and their locations. From this, further studies of the P. arachidicola DOTH gene cluster can be made that will lead to a more complete understanding of the evolutionary origins of these gene clusters.

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