2012 Annual Report
1a.Objectives (from AD-416):
Identify candidate proteins and genes that are responsible for aphid resistance of Rag2 gene.
1b.Approach (from AD-416):
Proteomics and transcriptomics research on soybean aphid infested and control tissue samples from BC4 near siogenic lines (NILs) with and without the Rag2 gene will be perfomed to identify the proteins and transcriptomes that may express differentially between NILs. Samples will be collected after 0, 2, 4, 8, 24, 48, and 72 hours after infestation with aphids. The proteins and transcriptomes that are unique to the resistant lines will be used as the candidates for further research.
Data coming from the second sequencing of the libraries were analyzed. A gene was considered expressed when reads matching this gene were detected for 2 biological replicates of the same time point. About 46 000 genes were found expressed in both Rag2 (resistant) and rag2 (susceptible) lines. More than 80% of these genes were found expressed in the 5 time points of the kinetic for both the resistant and the susceptible lines. Statistical analysis (EdgeR p-Value<0.05, Cuttdiff p-Value<0.05, fold change ratio >2 or <0.5) identified a total of 2,361 genes regulated between Rag2 and rag2 lines. The number of genes found regulated are 124, 633, 1204, 233, 929 for t0, t4, t8, t24, and t48, respectively. About 70% of these genes were found upregulated in the resistant line in comparison to the susceptible one. Three genes present in the Rag2 locus were found upregulated in the resistant line in at least one time point. However, Glyma13g26000.1 encoding a putative resistant gene (nucleotide-binding site leucine-rich repeat protein) was not found regulated. Williams82 soybeans were bombarded using gold particles coated with the different Virus Induced Gene Silencing (VIGS) constructs in order to produce the viral particles which were planned to be used to infect Rag2 soybean to silence the seven genes present in the rag2 locus. However, the viral infection symptoms were too weak to use these plants for subsequent infections. Rag2 soybeans may have to be bombarded directly using the viral particle.
Life time summary: The soybean aphid, a plant sap sucking insect, has become an important soybean pest in the USA and infestation of soybean by this insect can lead to significant yield losses. The Rag2 gene of soybean, providing resistance to soybean aphid biotypes I (Illinois) and II (Ohio), was identified by researchers in Ohio. A proteomic analysis is currently being performed on near isogenic lines (NILs) with the Rag2 gene for resistance or rag2 for susceptibility to the soybean aphid to unravel mechanisms of aphid resistance. Soybeans were grown in an environment controlled greenhouse to the V1 (first trifolitate) stage and infested with 20 adult soybean aphids of biotype II. Leaves were collected in four replicates at 0, 2, 4, 8, 24, 36, 48, 72 and 96 hours after infestation. The biological material was produced at the Ohio State University and then shipped on dry ice to researchers at the University of Missouri-Columbia for proteomic analysis. Proteins and ribonucleic acid (RNA) from 4 biological replicates of Rag2 and rag2 NILs 48 hours after aphid infestation were extracted using the Trizol method, enabling us to compare transcriptomic and proteomic data coming from identical biological material. Proteomic analysis was performed the first time. Proteins (50 µg) were separated by 1D gel electrophoresis and stained by colloidal Coomassie blue. Each line of the gel was divided into 12 bands and digested by Trypsin. Tryptic peptides were analyzed on Time of Flight-Mass spectrometer using a 43 mm chip. Fourteen hundred and eighty proteins were detected in at least 3 biological replicates of Rag2 or rag2 NILs. Statistical analysis identified 112 proteins differentially regulated between NILs 48 hours after aphid infestation. The cutoffs of fold change ratio used were 1.8 for the upregulated proteins (p) and 0.55 for the downregulated proteins. The p-value cutoff used was p<0.05. Proteins and RNA were extracted from other time points including 0, 2, 4, 8, 24, 36, 72 and 96 hours after infestation. Trypsin digestion was performed on proteins extracted from infested leaves 24 hours after aphid infestation. Since December 2010, proteins from the first biological replicate of Rag2 and rag2 lines 0, 8, 24, 36, 48, 72 and 96 hours after aphid infestation were separated by 1D gel electrophoresis, cut into 12 bands and digested with trypsin. Tryptic peptides were analyzed on a Linear Trap Quadripole -Orbitrap. Preliminary results show the identification of about 2000 proteins per sample. Protein from Rag2 and rag2 lines (replicate.
1)2 and 4 hours were digested with trypsin and are ready to be analyzed by mass spectrometry. A high throughput RNA sequencing (RNA-seq) approach was used to examine Messenger RNA expression in Rag2 and rag2 soybean leaves 0, 4, 8, 24 and 48 hours after aphid infestation. About 20 million reads were obtained for each sample. Data is currently being analyzed in order to identify genes differentially regulated in aphid -resistant and aphid-sensitive soybean lines. More sequencing was done using latest Illumina technology in 2011. The new sequencing data gave in depth transcriptomic analysis and enabled us to identify more genes differentially regulated between the two soybean lines that differ in their resistance to aphids. The correlation between the Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and RNA-seq data was not satisfactory enough using the first pipeline. A second pipeline to analyze RNA-seq data was developed using Cufflinks and cutdiff. The overlap of genes found differentially regulated using both pipelines correlates satisfactorily with qRT-PCR data. These two pipelines will always be used in parallel in the future. VIGS approach is used to silence the 7 Rag2 genes in the resistant lines and to study the phenotype of the transformed plants towards aphid infestation. Sequence analysis of the 7 genes was performed to design a specific fragment for each gene that can be used for VIGS experiment. Primers were designed on these specific fragments and all seven genes were amplified from the resistant line. PCR products were then cloned, positive colony PCR were obtained for all the genes using specific primers.
This project enhances/expands on objective 2B (Characterize and map aphid resistance genes in three soybean plant introductions) of the parent project.