Progress 08/15/05 to 08/14/08
Outputs
OUTPUTS: The long term goal of the proposed project is to find a suppressor to prevent posttranscriptional gene silencing (PTGS) of transgenes without interfering with plant growth and development. Objective 1. Comparison of the effects of viral suppressors of gene silencing on small RNA populations, mRNA targets of small RNAs, and development in transgenic and wild-type (WT) sugarcane plants. We have quantified and analyzed mRNA levels in 5 SrMV HC-Pro and 6 SCYLV P0 transgenic lines for 5 endogenous sugarcane genes (Unigene numbers: Sof.1592, Sof.2682, Sof.3770, Sof.4677, and CA227452) predicted to be regulated by miR393, miR166, miR167, miR160, and miR159 respectively. We found no evidence for major alterations of endogenous gene expression in these PTGS suppressor transgenic lines. We have also examined the production of six miRNAs (miR156, miR160, miR165/166, miR171, miR393, and miR394) in high and low expressors of P0sc and HC-Pro using qPCR. Three of these miRNAs (miR166, miR171, and miR394) were not detectable in young leaves from either P0sc or SrMV HC-Pro lines regardless of the expression levels of the suppressor in these plants. Three other miRNAs (miR156, miR160, and miR393) were produced in the P0sc and SrMV HC-Pro lines tested, but no discernable differences were found between low and high expressors of either P0sc or SrMV HC-Pro. Objective 2. Confirmation of the putative interactors of ScYLV P0 (P0sc). We have further characterized the function of P0sc. Using the Nicotiana benthamiana system, we have determined that P0sc has additional activities not associated with P0ca or P0bw: it triggers cell death in infiltrated cells and suppresses systemic PTGS. To determine which parts of the P0sc protein are required for the different activities, a series of deletion/mutation constructs were assembled and tested in leaf infiltration experiments with N. benthamiana. SCYLV P0, like the previously characterized P0 proteins, contains a possible F-box-like domain. Deletion of this region abolished suppression of local and systemic PTGS, and induction of cell death. All three activities were also lost when the first 15 N-terminal amino acid residues were deleted. Deletion of 15 C-terminal amino acid residues indicates this region of the protein is required to suppress systemic PTGS and for induction of cell death, but is not required to suppress local PTGS. Small RNA blot hybridizations indicated that full-length P0 suppresses the accumulation of two size classes of GFP siRNAs in local leaves, when induced by sGFP. Strikingly, the P0-212-256, P0-226-256, and P0-241-256 constructs, which suppress local PTGS but not systemic PTGS, suppress accumulation of the smaller size class, but not the larger. Objective 3. Development of a transient expression system in sugarcane using Agrobacterium infiltration. We have completed work on this objective, finding that this method is not useful in sugarcane. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In 2006, Hawaii sugarcane plantations were able to harvest 1.6 million tons of sugarcane from 42,100 acres and produce 213,000 tons of raw sugar. Sugar prices during 2006 were ~ $351 per ton of raw sugar which translated to a total value of $74.8 million. Using sugarcane plants to produce high value proteins could significantly increase the economic competitiveness of the Hawaii sugar industry. For example, recombinant GM-CSF for human therapeutic use is produced in yeast and sold under the name Leukine by Berlex (www.berlex.com). World sales of Leukine totaled $77 million in 2003. US Medicare payments for 50 ug doses of Sargramostin (generic drug name) are set at $24.47. At that price, the 2003 world sales of Leukine represent ~ 157 g GM-CSF. Because of the biosecurity and other advantages that sugarcane provides, there are several significant efforts underway in other countries to develop sugarcane as a biofactory system. In Australia, the Bureau of Sugar Experiment Stations and the University of Queensland have developed transgenic sugarcane that produces the thermoplastic polyhydroxybutyrate, and are also working on other high value products. Research by Dr. Mirkov is being developed by ECOR Corp. through its subsidiary proCANE LLC to produce pharmaceutical-grade proteins for human therapeutic use. Other efforts to produce high value proteins in sugarcane are said to be underway in Brazil, South Africa, and elsewhere.
Publications
- Tichaona Mangwende, T., Wang, M.-L., Borth, W., Hu, J., Moore, P. H., Mirkov, T. E., Albert, H. H. (2008) The P0 gene of Sugarcane yellow leaf virus encodes an RNA silencing suppressor with novel activities. Submitted to Virology July, 2008.
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Progress 08/15/06 to 08/14/07
Outputs
OUTPUTS: Previously 120 transgenic lines containing helper component-proteinase (HC-Pro) from Sorghum mosaic virus (SrMV) or the P0 protein from Sugarcane yellow leaf virus (ScYLV). These lines have been analyzed for steady-state mRNA levels of suppressor, test transgene, and endogenous genes. mRNA levels for 5 endogenous sugarcane genes predicted to be regulated by miRNAs have been analyzed for 5 SrMV HC-Pro and 6 SCYLV P0 transgenic lines. No evidence was found for major alterations of endogenous gene expression in these PTGS suppressor transgenic lines. Small RNA blot hybridizations determined no significant change in RNA levels for 6 tested miRNA genes. We have now produced 34 additional sugarcane lines incorporating the P25 gene from Potato virus X (PVX), and P38 from Turnip crinkle virus (TCV). The same analysis carried out on the HC-Pro and P0 lines will now be performed on these new lines. To determine the suppressor protein levels in transgenic sugarcane lines requires
specific antibodies against these proteins. We have now produced ScYLV P0 in E. coli; this required the development of new methods to recover soluble protein. In a synergistic project to functionally characterize SCYLV P0 using the Nicotiana benthamiana system we previously determined that expression of ScYLV P0 induces cell death. This feature distinguishes ScYLV P0 from previously characterized P0 proteins from dicot-infecting viruses. A manuscript we submitted earlier this year reporting our research results with P0 was rejected by reviewers, in part because they felt the cell-death we reported could be an artifact. We have now obtained P0 genes from Cucurbit aphid borne yellows virus and Beet western yellows virus. Plant expression vectors have now been produced for these P0 genes; experiments are underway comparing these genes side-by-side with ScYLV P0. We have completed work on this objective, finding that this method is not useful in sugarcane. In N. benthamiana, we (and many
other labs) have excellent results using agrobacterium leaf infiltration to obtain high levels of transient gene expression. In most other plant systems, however, this system works much less efficiently. We found that infiltration of sugarcane leaves requires much greater pressure than N. benthamiana leaves, presumably reflecting differences in leaf anatomy and stomatal conductance. This made it very difficult to introduce adequate volumes of agrobacterium suspension. Transient expression of reporter genes introduced by this method was undetectable. Because sugarcane is not a natural host for agrobacterium, cell infection/transformation rates are expected to be low. This, combined with the physical resistance to introducing the suspension results in an extremely low (perhaps zero) number of infected cells to express any introduced gene expression constructs. This result is not unexpected based on our experience adapting this method to papaya and other "non-model" plant species.
Because the method is so powerful in facile species (like N. benthamiana) we felt it was worth making the effort for sugarcane, even though the probability of success was low.
PARTICIPANTS: Wayne Borth Plant Pathologist University of Hawaii
TARGET AUDIENCES: plant virologists
Impacts
This project is advancing our understanding of the basic science behind RNA silencing. Scientists are becoming increasingly aware of an important role for this phenomena in fields of human health and genetics, genomic research, and pathology. Our own work in sugarcane will advance basic understanding of plant development, plant defense against viruses, and regulation of endogenous genes. RNA silencing has also emerged as an extremely powerful tool for functional genomic studies. As ever increasing amounts genomic sequence data are acquired for many plants, tools that allow the functional study of the genes identified in the sequence data are very important. For some model systems like Arabidopsis, large-scale efforts involving many labs across the globe have produced insertion mutants in a large percentage of known Arabidopsis genes. This provides a very powerful tool for scientists to study the function of these genes, but such large-scale efforts are not feasible for
less widespread non-model (i.e. real crop plants) systems. RNA silencing can provide a means for creating down-regulation of specific genes with much more modest investments
Publications
- Wang, M.-L., T. Mangwende, W. Borth, T. E. Mirkov, J. Hu, P. H. Moore and H. H. Albert (2007). Constitutive expression of viral suppressors of PTGS in sugarcane Plant and Animal Genome XV Conference, San Diego.
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Progress 08/15/05 to 08/14/06
Outputs
Monocot expression vectors have been made for 4 viral suppressors of PTGS and introduced to sugarcane by bombardment; to date 120 stable lines have been recovered. PCR for presence of the transgenes in SrMV HC-Pro and SCYLV P0 lines found 80% of tested lines positive for all transgenes, 88% positive for suppressor and selection genes. RNA has been extracted from selected transgenic lines and used to make cDNA. This cDNA has been analyzed by qPCR to determine relative levels of suppressor, test transgene, and endogenous gene mRNAs. Some potentially interesting lines (high levels of suppressor and test transgene mRNA) have been identified. mRNA levels for 5 endogenous sugarcane genes predicted to be regulated by miRNAs have been analyzed by qPCR for 5 HC-Pro and 6 P0 transgenic lines. No evidence was found for major alterations of endogenous gene expression in these PTGS suppressor transgenic lines. In a synergistic project to functionally characterize SCYLV P0 using
the Nicotiana benthamiana system, 26 deletion and mutation constructs have been made & tested in leaf infiltration assays. This work confirmed the presence of an F box domain required for suppressor function and identified a second domain required for suppressor activity which may constitute a second protein-protein interaction domain. Two grant applications have been submitted to NRI for outside support in 2004 and 2005. Both failed to obtain funding, but 2005 reviewers' comments were positive and encouraged a new submission this year. To date no manuscripts reporting this work have been submitted to peer review.
Impacts
Posttranscriptional gene silencing (PTGS) is being extensively studied in dicot plants, but to a much lesser extent in monocots, which include most of our most important crop plants. Viral suppressors of PTGS have proven to be very useful tools for understanding the plant pathways controlling PTGS, which are now known to be critical for anti-viral defense, for control of transposable genetic elements, and for regulation of many endogenous genes. Advancing the basic understanding of these pathways in monocots provides much needed information to deepen our knowledge of many critical aspects of crop plant biology. One specific application may be the use of viral suppressors of PTGS to control silencing of transgenes; this is very important in crop improvement through biotechnology and in the use of plants as biofactories. This is especially important for sugarcane, which has several important advantages in transgene containment, and so may be an ideal biofactory system.
Using a viral suppressor to control transgene silencing will require the basic knowledge gathered in this project, as it will require detailed knowledge of the PTGS pathways and PTGS suppressors in order to control transgene silencing without simultaneously compromising important aspects of normal plant biology.
Publications
- No publications reported this period
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