1a. Objectives (from AD-416):
Develop new sources of soybean resistance against root-knot and reniform infection using biotechnology.
1b. Approach (from AD-416):
Esophageal gland cells of reniform nematode will be isolated from the vermiform and sedentary life-stages. The transcriptional profile of these gland cells will be determined via Next-Generation-Sequencing (NGS). Candidate parasitism genes will be identified from the NGS data by their homology with known parasitism genes from other plant-parasitic nematode species and by the presence/absence of specific protein motifs and domains. Candidate reniform nematode parasitism genes will be further characterized in an effort to determine whether they are potential targets for gene silencing using RNA-interference. If gland cells cannot be collected from each life-stage, then entire nematodes from the problematic life-stage will be sequenced and the common housekeeping genes filtered by comparison to transcripts found in the gland cells.
3. Progress Report:
Thirty-one reniform nematode parasitism gene candidates were identified from a collection of reniform nematode gene sequences based on the following characteristics: (1) presence of a signal peptide in the predicted protein; (2) lack of a transmembrane domain within the predicted protein; and (3) lack of homologous sequences within the Caenorhabditis spp. genomes. The expression level of all 31 genes within the life-stages of reniform nematode (egg, second-stage juvenile (J2), J3, J4, vermiform adult, and parasitic female adult) was determined. Eleven of the 31 genes showed specific and high levels of expression during the parasitic life-stage of the nematode; therefore, these 11 genes were selected for further analysis. Full-length complementary deoxyribonucleic acid (cDNA) and genomic DNA sequences for each candidate gene were collected and analyzed. In addition, biological material was collected from a mixture of juvenile and vermiform adult life-stages and processed for esophageal gland cell sequencing, which was unsuccessful on the first attempt. Quarterly reports were submitted by each collaborator to the lead Principle Investigator. The objectives of this project relate directly to the objectives of the in-house project including the development of cotton germplasm resistant to reniform nematodes through the development of a reniform nematode functional genomics platform that can be used to characterize genes potentially involved in reniform nematode parasitism.