Submitted to: Molecular and Biochemical Parasitology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 2/23/2010
Publication Date: 4/1/2010
Citation: Wubben, M., Callahan, F.E., Scheffler, B.E. 2010. Transcript Analysis of Parasitic Females of the Sedentary Semi-Endoparasitic Nematode Rotylenchulus reniformis. Molecular and Biochemical Parasitology. 172:31-40. Interpretive Summary: Limiting the damage caused by plant-parasitic nematodes (PPNs) to economically important plant species is an intense area of research from both the traditional plant breeding and molecular biology perspectives. In many instances, natural host resistance to PPNs has not been identified or has yet to be incorporated into elite crop varieties. The reniform nematode, Rotylenchulus reniformis, is a serious pest of many crops, particularly cotton, where annual yield losses in the United States can approach $230 million. The targeted silencing of PPN genes by RNA-interference (RNAi) has shown tremendous promise as a transgenic alternative to natural resistance for cyst and root-knot nematodes. In order to develop RNAi-based strategies for controlling the reniform nematode, genes must be identified that are essential to the nematode’s ability to parasitize plants or are required for normal growth, development, and reproduction. In this study, we identified 556 reniform nematode genes from a total of 2,517 expressed sequence tags (ESTs) which were derived from the parasitic life-stage of the nematode. Within this collection of 556 genes, four were identified that likely play a role in the formation of the nematode feeding site. Eight genes were found that likely perform crucial functions in normal growth and development based on the fact that RNAi-based silencing of highly similar genes in the model nematode Caenorhabditis elegans results in lethality or abnormal development. Eighteen genes were identified that appeared to be present only in PPN species, suggesting that these genes may have evolved specifically for plant parasitism. This study represents the first EST analysis of the reniform nematode and provides the required gene sequence data necessary for investigating the potential for RNAi in controlling this damaging plant pathogen.
Technical Abstract: The reniform nematode, Rotylenchulus reniformis Linford & Oliveira, is a sedentary semi-endoparasitic roundworm that infects the roots of many economically important plant species. Engineered resistance to plant-parasitic nematodes (PPNs) via RNA-interference (RNAi) has shown promise in providing host resistance where natural resistance is lacking or insufficient. A prerequisite for investigating the efficacy of RNAi-mediated resistance to PPNs is a knowledge of the genes expressed by the parasite that are directly necessary for parasitism or for conserved cellular processes. Here, we describe the first expressed sequence tag (EST) dataset generated for the PPN R. reniformis. A cDNA library was constructed from the sedentary parasitic female life-stage, and 2,880 ESTs were sequenced and clustered to give 556 putative unigenes. BLASTx analysis revealed that 41% of all unigenes showed similarity to at least one known protein. Four unigenes represented ca. 36% of the EST dataset and included predicted peptides similar to VITillogenin structural proteins, a C-type lectin, and transthyretin-like family members. R. reniformis genes homologous to known parasitism genes were also identified and included ß-1,4-endoglucanase, fatty acid and retinol binding proteins, and two esophageal gland cell-specific genes from Heterodera glycines of unknown function. In addition to parasitism genes, a unigene was identified that showed a high level of similarity to an enzyme involved in thiamin biosynthesis, a metabolic pathway thought to exist solely in prokaryotes, fungi, and plants. Unigenes were also discovered that showed high levels of similarity to Caenorhabditis elegans orthologs known to produce lethal RNAi-mediated phenotypes. tBLASTx analysis of the unigenes identified eighteen sequences with homologs derived exclusively from PPN species. The work described here offers a glimpse into the transcriptome of a sedentary semi-endoparasitic nematode which i) provides the functional genomic data necessary for investigating engineered resistance against R. reniformis and ii) hints at the existance of a thiamin biosynthesis pathway in an animal.