Plant BiotechnologyEPA Grant Number: EM832933
Title: Plant Biotechnology
Investigators: Schumacher, Dorin
Institution: The Consortium for Plant Biotechnology Research Inc.
EPA Project Officer: Levinson, Barbara
Project Period: January 1, 2006 through December 31, 2011
Project Amount: $1,224,500
RFA: Targeted Research Grant (2006) Recipients Lists
Research Category: Health Effects , Food Allergy , Targeted Research
The objectives of the work proposed by the Consortium for Plant Biotechnology Research (CPBR) is to foster and facilitate promising research that leads to environmentally practical applications. CPBR will bring together the resources of member organizations to address the problems of protecting human health and safeguarding the natural environment through a program of environmentally beneficial plant related research.
Since the initial EPA funding under this grant, awarded in September 2006, CPBR made six subawards to investigators at member universities and to collaborators at minority institutions. Appendix A lists all of the projects funded in the 2006 and 2008 ERTT competitions. Appendix B contains the required Final Project Summaries of the projects funded. Appendix C contains a list of inventions and their status.
CPBR’s 2008 ERTT competition was the last competition held with this grant. All the projects supported by this grant are now completed. At the beginning of each competition, the principal investigators presented preproposals at the annual CPBR Symposium (held either February or March in Washington, DC). CPBR member companies evaluated the preproposals and discussed posters with the researchers. Those preproposals with high enough industry evaluations were requested to submit proposals. Ad hoc peer review and Scientific Consultant evaluations were used by the Project Recommendation Committee to rank the proposals. EPA funding was awarded to the highest ranked and most environmentally relevant proposals. In addition, investigators funded in the 2006 ERTT and 2008 ERTT competitions presented talks at the Symposium describing the research they did with their EPA funding.
Michael Antal, Jr., University of Hawaii-Manoa Flash-Carbonization TM Catalytic Afterburner Development
Scaleup of laboratory equipment to commercial size is not an easy job. During this 2 year project we overcame many scaleup hurdles by making major modifications of our Demo Reactor and CAB. We also learned how different ignition procedures heavily influence the combustion and carbonization phenomena within the Demo Reactor. Although we did not realize our goal of meeting state and federal emissions regulations, we believe we have learned how to operate our existing equipment to eliminate smoke. A test of our new operating procedure is imminent. Successful elimination of smoke will assure rapid progress towards meeting state and federal emissions regulations.
Bryony Bonning, Iowa State University Plant resistance to insect pests mediated by a protease
Specific Aim 1We tried a variety of different approaches for production of the ScathL-lectin fusion protein (FP) using the yeast expression system but encountered significant technical difficulties. It was only possible to produce small amounts (<10%) of the intact ScathL-GNA or ScathL- garlic lectin Allium sativum II (ASAII) fusion proteins, where the lectin domain was fused to the C-terminus of ScathL, due to instability to self-proteolysis during production. We then made new constructs for FP production with the lectin sequence incorporated between the propeptide and the proteinase domain, in the Nterminal region of the mature protein. The site of insertion was chosen on the basis of structural models for ScathL (based on human cathepsin L), with the aim of avoiding any disruption to the C1a cathepsin proteinase domain. However, the yeast expression products showed GNAimmunoreactivity, but no ScathL immunoreactivity, along with evidence of extensive degradation. There was no evidence from the western blots for the expected intact GNA-ScathL polypeptide, and there was no ScathL activity. The construct appeared to be unstable to posttranslational proteolysis in yeast - possibly due to failure to fold correctly. We then went back to our original constructs with the lectin domain was fused to the C-terminus of ScathL, and used a clone encoding a small protein proteinase inhibitor from silk which is effective at inhibiting yeast proteinases, to block degradation of the fusion protein. However, once again, yields of intact fusion protein were low. The fusion protein was very unstable and hence inappropriate folding is suspected. Finally, we tested the feasibility of chemically linking GNA to ScathL, but this strategy also failed. Although in vitro production of FP using the yeast expression system was not successful for our purposes, the instability of recombinant FP produced in yeast culture may not be applicable to the stability of FP produced in planta.
Specific Aim 2Transgenic Arabidopsis were constructed expressing GNA fused to the C-terminus of ScathL. We screened the F3 generation of these transgenic plants for resistance to aphids. Three transgenic lines of Arabidopsis that express GNA (F4), ScathL/GNA (F3 of line 7-2-5 and line 11-8-2), ScathL (F3 of line 3-2) along with a negative control line transfected with an empty vector, were used for bioassays. The plants were maintained at 24oC with 24 hours of light. A single first instar nymph of M. persicae was transferred carefully on to each test plant using a soft brush. The aphid population on each plant was counted daily until the third generation was produced (10 days). Aphids were examined for behavioral and morphological variations during the course of the bioassay. We observed morphological variation in the transgenic Arabidopsis with plants expressing GNA alone and 40% of the plants in the 11-8-2 (ScathL/GNA) line having smaller, but more leaves. Western blot analysis confirmed strong expression of GNA in the control line, but the results were unclear for the transgenic lines expressing the ScathL/GNA fusion. Bands of the expected size for the fusion protein (49 kD: GNA is 14 kD and ScathL is 35 kD) were seen inconsistently in western blots. Aphid population growth on the ScathL/GNA and GNA transgenic plants was significantly reduced compared to control plants. There was no significant difference between the numbers of aphids on the ScathL/GNA and GNA transgenic plants. A significant difference was observed however between line 11-8-2 expressing ScathL/GNA and 3-2 expressing ScathL alone, and between the test (GNA, ScathL/GNA and ScathL-expressing plants) and control transgenic plants (p<0.05; student’s T-test). These results indicate that (1) GNA expressed in Arabidopsis has a significant, negative impact on aphid population growth, (2) fusion of ScathL to GNA did not significantly increase the insecticidal impact of GNA alone, and (3) based on inconsistent western blot results, the ScathL/GNA fusion may be unstable in the plant. Despite numerous additional attempts, we were unable to get consistent detection of plant expressed GNA-ScathL by western blot analysis.
Dilip Shah, Donald Danforth Plant Science Center Ear rot resistant mycotoxin-free transgenic corn
Transgenic corn lines expressing an antifungal protein MtDef4 have been generated. These lines have been tested in the field at two different locations in North Carolina. Two of the lines exhibit reduced symptoms of the ear rot disease than the control lines.
Bryony Bonning, Iowa State University Plant resistance to aphids mediated by an insect virus
Objective 1. Characterize and optimize infectivity of the infectious clone.We constructed a full-length infectious clone of the RhPV genome, which is the first infectious clone for any member of the emerging Dicistroviridae family. However, this clone included 13 non-viral bases at the 5’ end, 10 non-viral bases at the 3’ end, and lacked the poly(A) tail that is present on the natural viral genome. On the basis that all of these differences from wild type virus are likely deleterious to viral replication, we modified the infectious clone (RhPV6.1) for optimized infectivity.
Objective 2. Determine whether an additional gene can be added to the RhPV genome 2.1 Selection of insertion site. We examined the genomic sequence of RhPV closely to identify the optimal strategy for insertion of additional coding sequence into the genome, without disrupting viral function. The potential insertion sites are shown figure 3. It is well established that the 5’ untranslated region (UTR) and IGR include IRES elements, which are essential for viral function. Hence we focused on bioinformatic analysis of the 3’ UTR as a potential insertion site and came to the following conclusions: 1) There is no clear alignment between the 3’ UTR sequences among dicistroviruses. The 3’ UTR varies greatly in length and is not well-conserved among viruses related to RhPV. 2) Comparison of the 5’ end of the 3’ UTR of RhPV with that of the closely related aphid virus Aphid lethal paralysis virus (ALPV) by Tcoffee and Clustal showed no sequence homology. 3) The use of Mfold for secondary RNA structure predictions showed no conserved secondary structure within the 3’ UTR. We plan to insert the additional ORF into the 5’ end of the 3’ UTR of RhPV62A, which should tolerate insertions and deletions necessary to express foreign sequences.
2.2 IRES for translation of inserted ORF. We plan to use the IGR IRES from ALPV to drive expression of GFP encoded by the inserted ORF. Sequence alignment of the IGR IRES of ALPV and RhPV using Vector NTI and T-coffee indicated that there is sufficient difference between the two sequences to minimize the risk of recombination. There are also structural differences between these two IGR IRES elements (3, 4).
Objective 3. Characterize RhPV assembled in planta We are using northern blots to determine whether RhPV will replicate in planta, as indicated by the presence of negative strand RNA, and to examine RNA stability. Preliminary northern analysis suggested that RhPV does not replicate in oat protoplasts. Having only recently obtained the improved RhPV62A infectious clone, we are just beginning inoculation of protoplasts to determine whether this clone will replicate in planta. We will also use RT-PCR for detection of negative strand RNA in protoplasts transfected with positive strand RhPV62A RNA as an additional approach to address whether RhPV62A will replicate in the plant cellular environment.
Andrew Paterson, University of Georgia A TILLING resource for cotton
Although funds were not actually received until 2009, it was agreed by the PI and the matching sponsor to plant the first year of the study in the 2008 field season. A total of 3,200 plots were evaluated for about 20 phenotypes agreed to by the co-PIs and the matching sponsor, emphasizing measures of the key components of fiber quality (length, strength, fineness and elongation) as well as relevant aspects of plant development (trichomes in particular, which are thought to be the predecessors of fibers and may share some genes in common). A total of 50 bolls per plot were hand-harvested to have sufficient seed for 2009 studies.
The top 5% of genotypes (plots) for each of the key components of fiber quality, and also for an aggregate ‘index’ reflecting the combined fiber quality measures each equally weighted, were planted in replicated studies in the 2009 field season, each replicated 3x in both Georgia and Texas. We have visually confirmed those lines that were identified based on discrete mutations. Phenotyping of these lines has been completed, validating the hypothesis that we were able to select novel variants with improved fiber qualities from the mutant population. Analysis of these data has been completed in a student thesis.
While the study identified an appreciable number of potentially valuable mutant alleles (indeed, heritable improvements were found for each of seven primary fiber quality traits, including strength, length, micronaire, elongation, uniformity, color, and lint percent), it has become clear that the population is not sufficiently pure for TILLING. Specifically, in the 2008 study at the Georgia location, we noted appreciable variation at frequencies too high to be accounted for by newly-induced mutants. We included a morphological mutant (okra leaf) as a border row in the study, and the 2009 growout suggested that outcrossing rates of about 10% had been experienced. While outcrossing is likely to be lower in the Texas location where the population was developed, limited DNA sequence analysis supported the conclusion hat the population was not sufficiently pure for TILLING.
A fringe benefit of the study is that we have shed light on the long-standing hypothesis that fiber development and trichome development are related. We included in the 2009 study about 100 lines that were selected because they contained discrete mutations in trichome development. If trichome development and fiber development were related, we expect that the population of trichome mutants as a whole will also have altered fiber quality. If the trichome mutants have the same average fiber quality as non-mutants, then it suggests that the development of these respective organs is largely independent. The hypothesis that trichome mutants would have altered fiber properties was largely validated. We found 100 lines with perturbations of stem trichomes, and were able to associate these perturbations with statistically significant differences in 5 of the 7 primary fiber traits (strength, micronaire, elongation, color, and lint percent). Only 13 lines were found with perturbations of leaf trichomes, and in this small number of mutants we could only associate variation in leaf trichomes with two fiber quality traits (micronaire, color). This association, particularly regarding stem trichomes, indicates that some of the same genes account for genetic variations in both trichome and fiber development. This is important because trichome development is very well understood, and many relevant genes have been identified, implicating these genes in fiber development.
Steve Strauss, Oregon State University Inducible systems for graft transmission of FT-stimulated flowering
The goal of this project was to explore the use of the FT gene for inducing rapid flowering in poplar to speed research and breeding. Although we did develop the FT gene to be a useful research and biotechnology tool, due to the biology of poplar its value was more limited than we had hoped for. Despite numerous attempts we were unable to see any evidence of graft transmission of the flowering response, a major goal that could have facilitated research and allowed acceleration of breeding without having a transgene in final varieties. There is no evidence that graft transmission, if it occurs, is of sufficient magnitude for floral induction, even when the FT rootstock:non-FT scion ratio was ~10 or 100:1 in mass.
We were, however, able to identify a protocol for heat induction of the FT gene to cause flowering in the greenhouse within 6 weeks, a major advance for research. Unfortunately, the level of floral development is limited; we are unable to induce the formation of fertile pollen or ovules in poplar (though this is successful in some genotypes in Prunus: R. Scorza, pers. comm.). Thus, FT acceleration does allow us to observe floral morphology, but not actually produce seed or pollen that can be used in breeding.
We have used heat induction of the FT gene to initiate flowering, however, we also constructed an ecdysone inducible FT gene and tested it in poplar. It gave only modifications to vegetative growth, indicative of FT effects, but did not appear to induce sufficient FT signal in the right tissues, for flowering.
We have conducted a number of studies to help understand the limits of the FT system, some of which are still underway. We have found that a number of 35S:FT transgenics lost their early flowering phenotype, and explored the use of dormancy treatments to “awaken” the genes. This failed, but we are using quantitative reverse transcription, real-time PCR (qPCR) to confirm our suspicion that the gene was indeed transcriptionally silenced in those lines. We have observed that many insertion events never flower even with heat induction and suspect that there is a required threshold of FT expression needed for flowering. We are using qPCR to compare flowering vs. non-flowering FT transgenics to define what the critical threshold is.
We are collaborating with Cetin Yuccer at Mississippi State University to transform and monitor GFP:FT protein fusion genes into poplar to study FT movement in stems, including through grafts, to help understand why graft induction was not effective, and perhaps how to modify it to enable movement.
Finally, as originally proposed, because the graft method of FT induction did not work, we have spent considerable effort retransforming sterility genes into highly inducible FT genotypes, and also transforming FT genes into poplars that had sterility genes previously inserted. This work required that we reconfigure all of our vectors to have a distinctive second selectable marker. However, we now have five different sterility constructs transformed into FT genotypes and have begun to study their flowering after FT induction. Thus, the original EPA relevant goal of this work, to develop a tool to speed development of containment technologies for genetically trees to mitigate environmental impacts, is well underway. The analysis of these plants is continuing.