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.
Preparations were made for in situ hybridization analysis of (11) reniform nematode candidate parasitism genes. Emphasis was placed on one gene in particular, named Rr-cle-1, that was similar to known parasitism genes from other plant-parasitic species. Rr-cle-1 transcript was identified exclusively within the dorsal esophageal gland cells of reniform nematode sedentary females by in situ hybridization. Two regions of the Rr-cle-1 open reading frame were targeted for ribonucleic acid interference (RNAi)-mediated silencing. Preliminary experiments to produce transgenic soybean hairy root lines expressing Rr-cle-1 hairpinRNAi constructs were successful and these lines are currently being propagated so that nematode infection assays can be performed. Experiments were also initiated to perform in situ hybridization studies on two additional parasitism gene candidates. We also continued to work with collaborators in refining methods for the isolation of total ribonucleic acid (RNA) from reniform nematode esophageal gland cells. This project was monitored through email, phone conversations, and one meeting that was sponsored by the United Soybean Board that was held at the University of Georgia on March 12-13, 2013. Quarterly reports were submitted by each collaborator to the lead Principle Investigator.