Identification of mariner-like elements from the root-knot nematode Meloidogyne spp.

Abstract

The Meloidogyne species are agriculturally important pests widespread in the world. These polyphagous endoparasitic nematodes possess an astonishing ability to bypass the plant resistance genes in few generations. However, the genes and mechanisms involved in this molecular determinism are not yet known. Except cytogenetic and cytotaxonomic studies, few data are available concerning their genome. There is therefore an important need of molecular tools for genetic investigation of their virulence character and other aspects of host–pathogen interactions. In that respect, the presence of mariner-like-elements (MLEs) was assessed in these endoparasitic nematodes by a polymerase chain reaction (PCR) assay using degenerate primers designed from two conserved regions of the mariner transposase open reading frame (ORF). Four Meloidogyne species of the five tested revealed the presence of MLEs in their genome. Southern blot analysis indicated that sequences hybridizing to the mariner transposase-like PCR clones occur at a moderate to low copy number in the different Meloidogyne spp. genomes. The phylogenetic analysis show that the Meloidogyne MLEs may form new subfamilies of mariner. Moreover, five PCR clones were shown to possess a continuous ORF suggesting the presence of putative transposase-like coding regions.

Introduction

Transposable elements are widespread in all major phylogenetic groups. Because of their ability to move from one location to another, they are valuable tools for genetic analyses as mutagenic agents for the mapping and isolation of genes. In the nematode Caenorhabditis elegans, the Tc1 element has proved a valuable tool for reverse genetics [1]. Due to the intense interest in C. elegans as an experimental organism for developmental genetic studies and the availability of sophisticated genetics, most information about transposons is known in this species. However, except this model, few elements are known in other nematode species. Some of them are present in nematodes of medical interest, such as TAS in Ascaris lumbricoides [2] or mle-1 in Trichostrongylus colubriformis [3]. But so far, there is no data concerning plant parasitic nematodes.

Endoparasitic nematodes, such as Meloidogyne, Globodera or Heterodera species, are pests of great agronomic importance, infecting thousands of plant species [4]. Some of them including Meloidogyne javanica, Meloidogyne incognita, Meloidogyne arenaria, reproduce exclusively by mitotic parthenogenesis. Nevertheless, in their clonal progeny, virulent individuals can appear on plants carrying genetic resistance. However, due to lack of sexual reproduction, the inheritance of avirulence and virulence cannot be directly tested but must await development of new strategies to study genes involved in this mechanism. In this connection, transposable elements could be used in reverse genetics as molecular tools for gene tagging, since such a strategy has been used to isolate a range of genes in a large number of species [5], [6], [7].

The mariner element was first found in Drosophila mauritiana [8]. Afterwards, it has been identified in an exceedingly wide variety of genomes ranging from fungi to plants or humans [9], [10], [11], [12], [13], [14]. Mariner-like-elements (MLEs) are members of the mariner/Tc1 transposon superfamily. Members of this superfamily contain a single open reading frame (ORF) encoding a transposase, possess inverted terminal repeats, and transpose via a DNA intermediate expressed in both the germline and the soma [for review see [15]]. MLEs are a family of about 1250 bp transposons with 20–40 bp ITRs, encoding a transposase of about 345 amino acids. However, functional elements constitute only a small subset of the mariner elements that have been sequenced. To date, the only known MLEs naturally active are represented by Mos1 and closely related mauritiana MLEs [16]. Mos1 is an autonomous copy of mariner isolated from D. mauritiana [17]. All other MLEs that have been examined are nonfunctional either because of missense mutations in the transposase ORF or because of small deletions or point mutations that frameshift the ORF. Most of the MLEs have been detected via PCR experiments by using primers designed on conserved regions of the mariner transposase gene, representing a third of the element [10], [18]. Several MLEs have been isolated from nematodes and cestodes, two types have been found in the genome of the nematode C. elegans and a related sequence has been isolated from the planarian worm Dugesia tigrina [11], [19], [20].

In this study, data on the presence of MLEs in the plant parasitic nematode Meloidogyne is reported. These mariner elements, identified for the first time in phytoparasitic nematodes, may form new distinct groups as compared with the mariner subfamilies known until now. Among them, five PCR fragments show a continuous ORF, without stop codons, frameshifts or deletions. These data strongly suggest that some Meloidogyne species may possess mariner transposase-like coding regions.