Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Research scientists, especially aphidologists, world-wide. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Numerous individuals were received training at each of several levels: Visiting scientist: Wei Wang (visiting from Tsinhua University, Beijing) Post-doctoral research associates: Feng Cui, Changzhong Sheng, Minglin Wang, Chandrasekar Raman. Graduate students (master’s level): Matthew Heerman, Matthew Aksamit, Jarrod Bechard Graduate students (doctoral level): Chris Miller, James Balthazor How have the results been disseminated to communities of interest? Our results have been published in peer review journals. What do you plan to do during the next reporting period to accomplish the goals? To follow the plan given in my project proposal for the current 5 year period
Impacts
What was accomplished under these goals?
We have studied several proteins of saliva from the pea aphid (Acyrthosiphon pisum) and in addition conducted transcriptomic studies on salivary gland RNA from both the pea aphid and from the Russian Wheat Aphid (Diuraphis noxia). Protein studies Armet. This is a calcium binding protein that occurs widely in vertebrates and invertebrates alike. It is a protein whose mRNA or transcript is enriched in salivary glands compared to the insect body as a whole. We have expressed the protein and studied its properties and obtained antibodies to be able to detect the protein in various sources. We found that the protein is transferred to plants during aphid feeding, using immunoblotting. Transcript knockdown, using dsRNA injection, demonstrates that the presence of Armet is needed for efficient feeding of the pea aphid on a host plant. We believe that Armet may be a good target for genetically engineered resistance through expression of dsArmet-RNA in the crop-plant in question. Glutathione peroxidase. This is the enzyme with the greatest enrichment of its transcript in salivary glands compared to the aphid body as a whole. We have expressed the enzyme and studied its enzymatic properties. In addition, we have knocked down its transcript using dsRNA injection. The enzyme studies not only verified that the gene/transcript in question does encode an active enzyme, but also that the enzyme can act on either hydrogen peroxide or on oxidized lipid as substrate. This suggests the possibility that the enzyme is used in the sieve element to repair damage to the cell membrane, thus disguising the presence of and penetration by the aphid stylet into the cell. Curiously, however, knockdown of the transcript by dsRNA injection does not lead to decreased life span of the pea aphid. Transcriptomics Pea aphid. We have spent a good deal of our time study the salivary-gland transcriptome of the pea aphid and the Russian Wheat Aphid. In the case of the pea aphid, we collaborated with James Carolan and Tim Wilkinson of University College Dublin to combine our transcriptomic results with their proteomic results and thus produce a proposed salivary gland secretome for the pea aphid (Carolan et al. 2011), the most comprehensive secretome proposed for any aphid species. We assume that further transcripts/proteins will be added to this catalog from continued studies in our laboratory and from other laboratories. Our salivary gland transcriptomics studies contributed to gene identification in the pea aphid genome and I was therefore one of the (many) authors of the paper on the determination of and annotation of the pea aphid genome. Russian Wheat Aphid. We investigated the genes and transcripts of proteins of saliva in the Russian Wheat Aphid in two ways. First, in collaboration with our colleagues (including a former post-doc of mine) at the Chinese Academy of Science, we investigated the role of methylation of several genes encoding proteins of saliva. The results clearly indicated differences in the methylation of these genes in two biotypes, RWA1 and RWA2, and thus provide an insight into the evolution of these biotypes. We also investigated sequence polymorphisms in 17 transcripts encoding putative proteins of saliva. We found that RWA1 and RWA2 typically shared the most abundant variant (allele) in each biotype, but that minor biotypes were typically distinct for each species. The results open the possibility that functional differences (in host specificity for instance) in the two biotypes is due to the range of alleles in each biotype rather than in the major biotype (which, again, is typically shared between the two biotypes).
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Liu X, Khajuria C, Li J, Trick HN, Huang L, Gill BS, Reeck GR, Antony G, White FF, Chen MS (2013) Wheat Mds-1 encodes a heat-shock protein and governs susceptibility towards the Hessian fly gall midge. Nature Commun. 2013;4:2070. doi: 10.1038/ncomms3070
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Zehedina Khatun, Md Nurunnabi, Gerald R Reeck, Kwang Jae Cho, Yong-Kyu Lee (2013). Oral delivery of taurocholic acid linked heparin-docetaxel conjugates for cancer therapy. Journal of Controlled Release 05/2013; DOI:10.1016/j.jconrel.2013.04.024
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Md Nurunnabi, Zehedina Khatun, Gerald R Reeck, Dong Yun Lee, Yong-Kyu Lee (2013). Near infra-red photoluminescent graphene nanoparticles greatly expand their use in noninvasive biomedical imaging. Chemical Communications 04/2013; DOI:10.1039/c3cc42334d.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Our output is research. Our focus in this project is on the mechanisms by which aphids feed on plants, including important crop plants such as wheat. We study the genetic model among aphids, the pea aphid, and also an aphid that can cause a good deal of damage to wheat crops, namely, the Russian Wheat Aphid. Our specific outputs include: the enumeration and sequences (at the mRNA and protein levels) of components of aphid saliva; methods for transcript knockdown in aphids; identification of components of saliva that overcome plant defense systems; mentoring of students (undergrad and grad) and post-doctoral research associates. PARTICIPANTS: Gerald Reeck, PI; Matthew Heerman, graduate student; Matthew Aksamit, graduate student; Chandrasekar Raman, postdoctoral student; Owain Edwards (CSIRO, Australia), colaborator TARGET AUDIENCES: Scientists in the same and similar fields. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We are contributing, through this project, in a sort of revolution in the understanding of the proteins and enzymes of aphid saliva and in evaluating the essentiality of the roles of individual proteins and enzymes. We have now demonstrated, using transcript knockdown, that two proteins of aphid saliva, Protein C002 and the protein Armet play crucial roles. These are in fact the first two proteins of the saliva of any aphid species that have been shown to be essential for feeding on a host plant. We are continuing to explore the roles of individual aphid proteins and enzymes and are expanding our scope to include the components of saliva in two subpopulations (biotypes) of the Russian Wheat Aphid. In this case, we hope to gain insight, through analysis of the proteins and enzymes of their saliva, into the differing abilities of the two biotypes to feed on various cultivars of wheat and barley. The change that we are generating will continue to be reflected in peer-reviewed research publications.
Publications
- Gong, L., Cui, F., Sheng, C., Lin, Z., Reeck, G., Xu, L., and Kang, L. (2012). Polymorphism and Methylation of Four Genes Expressed in Salivary Glands of Russian Wheat Aphid (Homoptera: Aphididae) Journal Economic Entomology. 105: 232-241.
- Cui, F., Smith, C.M., Reese, J., Edwards, O., and Reeck, G. (2012). Polymorphisms in salivary-gland transcripts of Russian wheat aphid biotypes 1 and 2. Insect Science 19: 429-440.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Our output in this project is research, focusing on proteins of aphid saliva, and approaching these proteins through their transcripts. The project is mainly on the pea aphid (Acyrthosiphon pisum) and the Russian Wheat Aphid (Diuraphis noxia). Our specific outputs include: sequences of transcripts from the salivary glands of these species; prediction of those transcripts that encode secreted proteins of saliva and therefore of the proteome of saliva in these two species. Another output is effect of transcript knockdown by injection of dsRNA into aphid. A final output is the mentoring of students (both undergraduate and graduate) and of post-doctoral research associates. PARTICIPANTS: Gerald Reeck, PI; Matthew Heerman, graduate student; Owain Edwards (CSIRO, Australia), colaborator TARGET AUDIENCES: Scientists in the same and similar fields. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Through this project we are creating a change in knowledge of the proteins and enzymes of aphid saliva. During the current project year we have made a major advance that, when completed in the next project year, will make a major contribution to understanding the differences in proteins of saliva of different biotypes of the same species, namely the Russian Wheat Aphid. Based on the work of the previous project year, we have, in the current year, successfully dissected hundreds of salivary glands of three Russian Wheat Aphid biotypes: biotype 1, biotype 2, and the Hungarian biotype. RNA purified from these preparations was purified and subjected to "deep sequencing" or RNASeq at a collaborating laboratory. The sequencing results are being analyzed now in our Bioinformatics Center at KSU. The comparison of the transcript sequences from the biotypes should provide a wealth of information about differences, at the molecular genetic level, among the three biotypes. We have also worked out methods, during this year, for injection of Russian Wheat Aphid with dsRNA and in the next year will be conducting transcript knockdown experiments on selected genes encoding proteins of saliva. The change that we are generating will continue to be reflected in peer-reviewed research publications.
Publications
- Carolan, J., Caragea, D., Reardon, K., Dittmer, N., Pappan, K., Cui, F., Castaneto, M., Poulain, J., Dossat, C., Tagu, D., Reeck, J., Reeck, G., Wilkinson, T., Edwards, O. (2011) Predicted effector molecules in the salivary secretome of the pea aphid (Acyrthosiphon pisum): a dual transcriptomic/proteomic approach. J Proteome Res. 10: 1505-18.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The outputs of this project are research activities. In particular we are researching the aphid salivary gland and the proteins and enzymes synthesized in it. Our work currently deals with three aphid species: the pea aphid (Acyrthosiphon pisum), the Russian Wheat Aphid (Diuraphis noxia), and the greenbug (Schizaphis graminum). Our outputs include: nucleotide sequences of transcripts encoding aphid saliva proteins and enzymes; properties of recombinantly expressed aphid saliva proteins; physiological effects of decreasing the concentrations of transcripts of individual aphid proteins; the localization of individual aphid proteins among the secretory cells of the aphid salivary gland. Some of the participants in the project are students (both undergraduate and graduate students) and post-doctoral research associates, so another major output is the mentoring of young scientists. PARTICIPANTS: Gerald R. Reeck, PI; Changzhong Sheng, Research Associate; Matthew Heerman, Graduate Student; Xiaoxi Zhu, Graduate Student; Kent Downing, Undergraduate; Owain Edwards (CSIRO, Australia), International Consultant TARGET AUDIENCES: Scientists in the same and similar fields. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The project has resulted in a change in knowledge. Two such changes in the reporting year are properties of pea aphid glutathione peroxidase and improvement of dissection methods for Russian Wheat Aphid salivary glands. Pea aphid recombinantly expressed glutathione peroxidase is most efficient as an enzyme when the oxidizing agent in the reaction is phospholipid hydroperoxide. This sort of compound arises as a result of membrane wounds. The result suggests that glutathione peroxidase in aphid saliva could function to repair wounds to plant cell membranes. We have also found a restricted distribution of the enzyme itself in the pea aphid salivary gland, consistent with its being a secreted protein, i.e, a component of aphid saliva. Secondly, we have worked out improved methods for dissection of salivary glands form the small, but very important, Russian Wheat Aphid. We are in the process of dissecting hundreds of salivary glands from three biotypes of the aphid, in preparation for sequencing of transcripts present in those glands. These changes will be reflected in the coming months and years by publication as peer-reviewed journal articles.
Publications
- Cui, F., Lin, Z., Wang , H., Liu, L., Chang, H., Reeck, G., Qiao, C., Raymond, M., and Kang, L. (2010). Two single mutations commonly cause qualitative change of nonspecific carboxylesterases in insects. J. Insect Biochem. Mol. Biol. (in press)
- International Aphid Genomics Consortium (including GR Reeck as one of the many members/authors). Genome sequence of the pea aphid Acyrthosiphon pisum (2010) PLoS Biology 8: e1000313. doi:10.1371/journal.pbio.1000313
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: This is a laboratory research project and the outputs are therefore research activities. In particular we are researching the aphid salivary gland and the proteins and enzymes synthesized in it. Our work currently deals with three aphid species: the pea aphid (Acyrthosiphon pisum), the Russian Wheat Aphid (Diuraphis noxia), and the greenbug (Schizaphis graminum). Our outputs include: nucleotide sequences of transcripts encoding aphid saliva proteins and enzymes; properties of recombinantly expressed aphid saliva proteins; physiological effects of decreasing the concentrations of transcripts of individual aphid proteins; the localization of individual aphid proteins among the secretory cells of the aphid salivary gland. Some of the participants in the project are students (both undergraduate and graduate students) and post-doctoral research associates, so another major output is the mentoring of young scientists. PARTICIPANTS: Gerald R. Reeck, Yoonseong Park, Feng Cui, Sheng ChangZhong, Matthew Heerman, Doina Caragea, Owain Edwards (CSIRO, Australia) TARGET AUDIENCES: Other Research Investigators and scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The project has resulted in a change in knowledge. One of the major changes has been in our investigation of a major enzyme of pea aphid saliva, glutathione peroxidase. We identified this as being encoded by the most abundant EST present in pea aphid salivary gland cDNA libraries. We have expressed the protein in E coli and verified that the expressed enzyme has a glutathione peroxidase enzymatic activity. We are now in the process of examining the enzyme's substrate specificity. Another major change in knowledge has been the identification of the location of another saliva protein, Armet, in the secretory cells of the salivary gland. This protein appears to be expressed preferentially in a subset of those cells, but a different subset than the cells which express Protein C002, a protein previously studied in our laboratory in the same way. This change in knowledge has led to a change in condition: namely, it has revealed that different proteins of aphid saliva are produced in different subsets of secretory cells of the salivary gland. These changes will be reflected in the coming months and years by publication as peer-reviewed journal articles.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Among the members of the salivary gland secretome, we have focused attention on a protein that we call Protein C002. We have established that this protein is produced by the secretory cells of salivary glands, that the protein is transferred to plants in aphid-feeding and that it is crucial to the ability of the pea aphid to feed on a host plant, the fava bean. This, then, is the first aphid protein so demonstrated to be essential to the interaction of aphid and plant. PARTICIPANTS: Gerald R. Reeck - Principle Investigator; Yoonseong Park; Feng Cui - molecular biology experiments; Sheng ChangZhong - molecular biology experiments; Matthew Heerman - molecular biology experiments; Doina Caragea - bioinformatic analysis of ESTs and genomic data; Owain Edwards (CSIRO, Australia) - experimental design and consultation TARGET AUDIENCES: Other research investigators and scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
For the two months that this report covers, we have had two emphases. One is the feeding of double-stranded RNA to aphids, in artificial diets, to effect transcript knockdown. These efforts continue and our current work is designed to understand the effect of control RNA, when fed to insects. At high levels, non-aphid-related double stranded RNA has some ability to kill aphids and we are varying the size and concentration of such RNA to understand the effect better. Also, we are working with a new gene/transcript/protein of interest, namely that encoding pea aphid Armet. The protein has been expressed in E. coli and has been found to bind calcium with high affinity. This is basic research, and the outcomes will therefore be publications, and the impacts are impacts on future research both in my laboratory and more broadly.
Publications
- No publications reported this period
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