Characterization of three DNA transposons in the Dutch elm disease fungi and evidence of repeat-induced point (RIP) mutations

Abstract

Transposable elements (TEs) are fundamental components of eukaryotic genomes and can contribute in various ways to genome plasticity and evolution. We describe here the first three DNA transposons in the Dutch elm disease (DED) pathogens Ophiostoma ulmi and O. novo-ulmi, named OPHIO1, OPHIO2 and OPHIO3. We demonstrate that OPHIO transposons, which show high homology to Fot1/pogo TEs within the Tc1/mariner superfamily, have different distribution patterns and specificity in the DED fungi and that interspecific hybrids could act as genetic bridges for transmission of TEs between closely related fungal species. OPHIO3 was found to have undergone repeat-induced point mutations (RIP). We have also developed a complementary method to Margolin’s ratios based on the computation of cumulative transition scores (CTS) in order to visualize rapidly RIP signatures on individual DNA strands of OPHIO transposons and TEs found in other ascomycete fungi.

Introduction

Transposable elements are repetitive and ubiquitous genomic DNA sequences present in most eukaryotic organisms (Craig, 2002). They account for 15% of the genome in fruit flies, 45% in humans and more than 80% in barley and Lilium species (Feschotte et al., 2002, Goyon et al., 1996). In the fungal phylum Ascomycota, TEs have been estimated to represent about 14% of the genome (Goyon et al., 1996). The principal characteristic of TEs is their ability to move (or transpose) from one chromosomal location to another and to replicate themselves. This characteristic is what distinguishes the autonomous TEs from the non-autonomous ones, depending on their ability to synthesize proteins that allow them to transpose. Mobility is the essence of their evolutionary success and is certainly at the origin of implications for the dynamics between TEs and host genomes (Bowen and Jordan, 2002). TEs are generally believed to be a major driving force of genome evolution and plasticity by generating chromosomal rearrangements and breakage, and promoting ectopic recombination, as well as variability in gene expression (Kidwell and Lisch, 2002). For example, transposon MGR386 was found to decrease virulence in the plant pathogenic fungus Magnaporthe oryzae (formerly Magnaporthe grisea) (Farman et al., 1996), whereas Pot3 had the opposite effect (Kang et al., 2001).

Transposable elements can be divided into two major classes (class I =  retrotransposons, class II = DNA transposons) according to their structural organization and transposition mechanism. As of 2005, 52 Class I and 58 Class II TE families have been described in 22 species or subspecies in the phylum Ascomycota. Of the currently nine recognized superfamilies, only three have been identified in fungi: Tc1/mariner, hAT and Mutator, with an over representation of the pogo/Fot1 group within the Tc1/mariner superfamily (Chalvet et al., 2003, Daboussi and Capy, 2003, Kempken, 1999, Zhang et al., 2001). DNA transposons move from a chromosomal site to another through a DNA intermediate. They demonstrate specific structures, such as terminal inverted repeats (TIRs) that flank an open reading frame (ORF) encoding a transposase enzyme required for mobility and a target site duplication (TSD) recognized during insertion (Richardson et al., 2006).

Following a collaborative initiative of four research teams, the Canadian Ophiostoma Genome Project was launched in 2001 with the aim of understanding functional and structural genomic variations in three ascomycete fungi belonging to the genus Ophiostoma (Sordariomycetes, Ophiostomatales, Ophiostomataceae): the saprobe O. piceae (Dogra and Breuil, 2004) and the pathogens O. ulmi and O. novo-ulmi (Bernier et al., 2004). The latter two species were responsible for two devastating pandemics of Dutch elm disease (DED) which have resulted in heavy losses in elm populations. The first pandemic, caused by O. ulmi, started in northwestern Europe in 1910 and lasted until the late 1960s (Brasier et al., 2004). The second, much more destructive, pandemic is thought to have begun in the 1940s and is due to the highly pathogenic O. novo-ulmi. The latter is divided into two subspecies, according to their initial repartition area: O. novo-ulmi subsp. americana (formerly known as the North American [NAN] race) and O. novo-ulmi subsp. novo-ulmi (formerly called the Eurasian [EAN] race) (Brasier and Kirk, 2001). These species and subspecies differ either by their phenotype (morphological or biochemical variations) or their genotype (functional or structural variations) (Brasier et al., 2004, Pipe et al., 1995). However, although speciation between O. ulmi and O. novo-ulmi was demonstrated (Brasier et al., 1993), rare interspecific hybrids with reproductive capacities can be found in natural populations (Brasier et al., 1998). O. ulmi and O. novo-ulmi display extensive chromosome polymorphisms (Dewar and Bernier, 1993, Dewar and Bernier, 1995, Dewar et al., 1997). Pathogenicity of these species is believed to be under additive polygenic control (Brasier, 1986) but the molecular bases of this important trait remain unresolved.

The DED fungi are vectored principally by elm bark beetles (Insecta, Coleoptera, Scolytidae). Upon emerging from breeding galleries in which they may have acquired Ophiostoma spores, young adults fly to the crown of healthy elms where they will feed in twig crotches. In doing so, the beetles inoculate the pathogen into xylem vessels where it will multiply and induce the blockage of the water-conducting tissue within the tree. Soon after infection, susceptible elm species will display characteristic wilting, curling and yellowing of leaves, and usually die (Lanier and Peacock, 1981).

In order to develop a global understanding of the genome of the DED fungi and to supplement ongoing studies on differential gene expression, we explored whether strains of O. ulmi and O. novo-ulmi harbored DNA transposons in their nuclear genome. We describe here the first three transposable elements found in the Ophiostoma genome, termed OPHIO1, OPHIO2 and OPHIO3. These TEs show various characteristics of class II transposons, including sequences coding for a transposase, TIRs and TSD. Comparative analysis of Southern profiles allowed us to observe that the three OPHIO transposons differed in their distribution patterns and specificities in the DED fungi. In addition, it would seem that interspecific hybrids may have acted as genetic carriers for TEs between O. ulmi and O. novo-ulmi. On the other hand, only OPHIO3 exhibited mutation patterns typical of repeat-induced point (RIP) mutation. RIP is a process that efficiently detects and mutates duplicated sequences during the sexual cycle. The mechanism identifies duplications and introduces C:G to T:A mutations into both copies of the duplicated sequences (Galagan and Selker, 2004, Selker, 2002). The occurrence of RIP silencing is usually deduced from the computation of Margolin’s ratios (Margolin et al., 1998). We developed a complementary method, called “cumulative transition score” (CTS), to visualize C:G to T:A transitions in OPHIO3 and transposase sequences from various ascomycete fungi.