We have analyzed the gene expression of newly hatched aphids (Biotype1) feeding on resistant (Rag1; avirulent aphids) and susceptible (SD: virulent aphids) plants. Overall, there were a total of 3340 genes that were differentially expressed (p<0.05) in virulent and avirulent insects. In virulent aphids, a total of 1552 and 1788 genes were upregulated and downregulated respectively as compared to avirulent insects. A large proportion (~41%) of these genes has no match in the public database and thus encodes for proteins whose functions are not known. Among the rest, majority of them encode for proteins that perform basic cellular functions such as transport, binding, enzymatic catalysis etc. in insects. The antibiotic mode of resistance in Rag1 plants was manifested by upregulation of Cytochrome P450 (CYP450) genes of avirulent insects. Out of 24 CYP450 genes identified from soybean aphid transcriptome, 18 were highly upregulated in insects feeding on Rag1 plants. Currently we are investigating the gene expression of Biotype2 and Biotype3 of soybean aphid on resistant and susceptible plants. The differential gene expression among different biotypes of this insect can elucidate the mechanism of biotype development at the genetic level in soybean aphid.

To understand the soybean-soybean aphid interactions at molecular level, we have analyzed and compared the gene expression of soybean aphid (Biotype1) feeding on susceptible and resistant plants. Overall, there were a total of 1,759 genes that were differentially expressed (p<0.01) in soybean aphid feeding upon resistant and susceptible plants. On resistant plants, a total of 797 and 962 insect genes were upregulated and downregulated, respectively, as compared to insects feeding on susceptible soybean. Our analysis clearly indicates that in soybean aphid feeding on Rag1-soybean plants, there is a selective regulation of genes that encode for detoxification enzymes. Our data shows that soybean aphid does have genes for all major detoxification enzymes to overcome natural and synthetic toxins, but only a few detoxification genes are differentially expressed in response to toxins encountered in Rag1-soybean. Specific genes, called cytochrome P450s, are well known for the role in the development of resistance against plant allelochemicals and insecticides. Quantitative PCR has confirmed that 3 specific P450s that seem to be upregulated when feeding on resistant plants. Currently, we are sequencing RNA from biotype 2 on resistant and susceptible plants to compare gene expression to our data on biotype 1.

We have confirmed differential gene expression of ten P450s in biotype 1 aphids from Rag1 and susceptible leaves. All ten P450s analyzed were upregulated in biotype 1 aphids on Rag1 plants. Using enzyme activity assays, however, we did not see any increase in P450 activity or concentration. This indicates that despite increased P450 gene expression, additional P450 enzymes are not being manufactured to combat the allelochemical response from Rag1 plants which leads to the lethal phenotype of avirulent aphids. We have finished assembly of RNA-Seq results with biotype 2 on Rag1 and susceptible and are comparing differential gene expression for consistency with biotype 1.

Sequencing from Illumina is currently being assembled and compared to the soybean genome. Data are also being analyzed for differential gene expression among lines. To increase coverage and robustness of data we have sent samples to be sequenced on the SOLiD system. Overall, RNA from PI243540 (Rag2) was extracted from soybean leaves 6hrs and 12hrs from infested and uninfested leaves. This data will be compared to Illumina sequencing to determine differentially expressed soybean genes.

Additional markers and SNPs are being validated with aphids collected on Rag1 and susceptible plants. Preliminary results indicate at least 3 SNPs with significant genetic differentiation, suggesting possible candidates for diagnostic markers. Additional testing is ongoing to validate SNPs in wider collections sampled last year. Statistical analysis of biotype 1, 2 and 3 frequencies revealed no significant differences across geographic locations, but significantly lower frequencies of biotype 2 and 3. However, most frequencies were well above assumptions provided in host-plant durability models, suggesting that longevity of single-gene Rag resistance varieties is limited and further work to develop multi-genic resistance is needed.

A manuscript detailing resistance in this variety will be submitted in May. We have screened our 3 aphid biotype colonies to determine virulence and this variety exhibits resistance to biotype 1 and biotype 2. We will compare gene expression changes with candidate genes determined in Objective 1 with aphids on this variety to evaluate shared virulence mechanisms.