We have previously shown the ability to isolate intact cyst nematode gland cells and generate transcriptome sequences via both 454 and Illumina-based technologies. These sequences indicated that we were indeed generating gland-cell derived sequences, based on the rediscovery of many of our previously reported SCN effectors. For the last six months of our first year on this grant, we have been optimizing the quality of the libraries generated for sequencing. We recently have been trying a Clontech SMARTer Ultra Low RNA kit, to improve cDNA library quality, as this kit is optimized for cDNA synthesis from low starting input, which our pooled gland cell approach requires. It is also optimized to be used with lower quality RNA as starting material, which our ethanol-fixed gland cell approach requires. Using this kit, we have generated several independent libraries from cyst nematodes, and these libraries are of good yield and have an appropriate size range for proceeding with sequencing. Two of these libraries have been finalized and are currently being sequenced via Illumina MiSeq technology at the Iowa State University Sequencing Facility.
During the first year of this project we also have made progress in characterizing the interesting effector 4EO2. This is a novel effector from SCN and we have been using the Arabidopsis-sugar beet cyst nematode (BCN) pathosystem for its characterization which has proven very useful in past. We discovered that this effector specifically interacts with vacuolar protease RD21A and relocalizes it to cytoplasm. 4EO2 overexpression alters transgenic plants’ response to multiple pathogens including nematodes suggesting its role in defense modulation. The overexpression of 4EO2 also modifies biochemical components of the cell wall in transgenic plant lines. We conducted yeast 2-hybrid analyses using RD21A as bait to identify its targets. The preliminary analysis of its interactors suggests that RD21A interacts with multiple proteins involved in cell wall biosynthesis and modification. As the development of nematode feeding site (syncytium) involves massive cellular reprogramming and cell wall modification, our discovery of interaction between the nematode effector with RD21A could be of great significance. We will continue to build on these results for the subsequent funding cycle.
Year 2: During the second year, we will complete the bioinformatics analysis of the transcriptome data and identify potentially novel effectors. We will begin conducting insitu hybridization to confirm their gland specific expression. We are also anticipating publishing the characterization work completed during the first year (4EO2) and begin work on new effectors.
Year 3: During the third year, we will complete the list of newly identified effectors from our gland mining efforts and we will develop a manuscript to announce our results to the research community. We are also anticipating break through results from the characterization efforts that we would have started during the second year.
During the cycle of 3 years we are anticipating to complete enough research projects to generate multiple manuscripts detailing our findings. The ISA will be kept abreast with our progress by submitting progress reports in timely manner.