Adler Ray Dillman and Dr. Byron J. Adams Microbiology and Molecular Biology
Background
The phylum Nematoda is a large and diverse group, making-up an estimated 80% of Metazoans [1]. Of specific agricultural importance are the plant-parasitic nematodes, which are responsible for the loss of an estimated 12.3% of the world’s annual crop production [2]. We looked at the most destructive members of the family Heteroderidae, the cyst (Heterodera and Globodera spp.) and root-knot (Meloidogyne spp.) nematodes.
The cyst and root-knot nematodes are able to extensively modify host cell gene expression and morphology through enzymatic secretions [3]. These modifications result in the development of special food cells that accommodate the sedentary parasitic lifestyle of these nematodes.
Several enzymes have already been identified and characterized from these gland secretions including pectinases and cellulases [4]. In each of these and several subsequent studies we dealt with the question of how nematodes acquired these genes. Several mechanisms have been hypothesized including; ancestral heritage and modification, mutation and adaptation, and horizontal gene transfer (HGT) [5]. HGT is a debated topic and while generally accepted as a method of evolution in prokaryotes it still lacks evidence in eukaryotes, with many early claims of HGT in eukaryotes being later refuted [6]. We utilized TreeSAAP, [5] a novel computer program to help us explore putative HGT events with several genes that have already been proposed as good HGT candidates [7]. Looking at selection in these genes should help us further differentiate between convergent evolution and HGT events as hypothesized in these genes.
Materials and Methods
Using the contiguous ID’s provided by Scholl et al., [7] we acquired the rough EST’s from nematode.net for the twelve genes previously identified as being good candidates for HGT. After blasting these nematode EST’s in a series of BlastX queries in GenBank, and aligning and editing these sequences using ClustalX [8] and MacClade [9] (See Figure 1) we were left with only three sequence alignments to analyze with PAUP [10], ModelTest, [11] and TreeSAAP [5] because the other nine sequences presented major alignment ambiguities or because the nucleotide sequences were too dissimilar to obtain significant results using TreeSAAP. The Goal in using these programs was to detect selection in these genes and find evidence that supports rejecting the null hypothesis of HGT. (Figure 1. An example MacClade alignment, representative of the sequence divergence we found.)
The BlastX queries showed some similarity to prokaryotic sequences. However, they also had significant similarity to eukaryotic sequences including many fungi. The alignment doesn’t seem to be in favor of HGT as the sequences show extensive variation. Initial characterizations of these enzymes lead to speculations of HGT, but we believe that as more genes are sequenced, there will be an increase in homologous eukaryotic sequences. Our TreeSAAP analysis revealed that all three genes had several amino acid (AA) properties that were under positive destabilizing selection and several other properties under negative stabilizing selection. In all three genes there were several amino acid properties under negative stabilizing selection including power to be at the N-terminal, coil tendencies and average number of surrounding residues. Also, in all three genes both the power to be at the N terminal and refractive index properties are under positive destabilizing selection at certain sites in the sequence.
Following the HGT hypothesis, one would predict that several aspects of the protein gene product would need to be conserved (under negative stabilizing selection) in order to preserve function, while other properties of the AA would need to be altered (under positive destabilizing selection) in order to be used parasitically. However, these genes seem to show a surprising amount of variation if indeed they were laterally acquired. We find it interesting that all three genes have several properties in common that are under selection. These findings persuade us not to reject the null hypothesis in this case.
Future research will include further investigation of the properties that are under selection as well as the actual location of these with respect to the functional domains of the proteins. At the time of this study there were still relatively few nematode pectinase, endoglucanase, etc., genes that have been made publicly accessible through GenBank and this research will be greatly advance as more and more of these genes are sequenced and analyzed in a phylogenetic context and made available in GenBank.
References
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- Posada, D. and Crandall. K.A., 1998. Modeltest: testing the model of DNA substitution. Bioinformatics 14(9): 817-818