Grantee Research Project Results

2007 Progress Report: Plant Biotechnology

EPA Grant Number: EM832933
Title: Plant Biotechnology
Investigators: Schumacher, Dorin , Antal, Michael , Bonning, Bryony C. , Gatehouse, John , Shah, Dilip
Current Investigators: Schumacher, Dorin
Institution: The Consortium for Plant Biotechnology Research, Inc
EPA Project Officer: Packard, Benjamin H
Project Period: January 1, 2006 through December 31, 2011
Project Period Covered by this Report: January 1, 2007 through December 31,2007
Project Amount: $1,224,500
RFA: Targeted Research Grant (2006) Recipients Lists
Research Category: Targeted Research , Human Health

Objective:

Flash-Carbonization^TM Catalytic Afterburner Development

The primary objective of this proposal is the development of a catalytic afterburner that will enable the UH commercial-scale, Flash Carbonization^TM Demo Reactor to satisfy all State and Federal emission requirements.  

Plant resistance to insect pests mediated by a protease

The objective of this project is to test the concept that the insecticidal protease ScathL can be used in combination with a lectin, such as Galanthus nivalis agglutinin (GNA) for production of insect resistant transgenic plants. The objectives are to test the insecticidal efficacy of the protease-lectin fusion protein (FP) by using both in vitro (aim 1) and in vivo (aim 2) techniques. For Specific Aim 1, Determine whether FP is toxic on ingestion by aphids and moth larvae, the objective is to produce and test recombinant FP for insecticidal efficacy against lepidopteran (moth) larvae and aphids. For Specific Aim 2, Construct transgenic plants that express FP, the objective is to produce and test transgenic plants (Arabidopsis) to demonstrate insecticidal efficacy in planta. We will test the hypotheses that (1) recombinant FP will be expressed at a high level in Arabidopsis from the 35S promoter, (2) FP will move into the hemocoel of aphids and moth larvae that feed on transgenic Arabidopsis, (3) delivery of FP from the plant will result in basement membrane damage and rapid insect mortality. We expect that the technology will then be applied to agriculturally relevant crops of interest to the sponsoring company. 

Ear-rot resistant mycotoxin-free transgenic corn

1. Determine in vitro antifungal activity of an antifungal defensin MtDef4 against the field isolates of Fusarium verticillioides and F. proliferatum, causal fungal pathogens of an ear rot disease in corn.  
2. Determine if constitutive expression of MtDef4 in transgenic corn confers resistance to these pathogens in the greenhouse and in the field and results in significant reduction in the levels of a mycotoxin called fumonisin.   

Progress Summary:

Flash-Carbonization^TM Catalytic Afterburner Development

1. We completed many runs using the lab scale FC reactor.  Some of these runs were designed to mimic that performance of the Demo reactor.  Results to date indicate that data taken from the lab scale FC reactor can be used reliably for scaleup.  Other runs were designed to provide data for publications. 
2. Together with Prof. Donald G.M. Anderson at Harvard University, we are developing a FORTRAN computer program to simulate the ignition and burn characteristics of FC reactors.  This work has been ongoing for 5 years and as a result of a recent “breakthrough” we are now able to reliably simulate the performance of the lab scale FC reactor.  More work is needed to simulate the Demo reactor’s performance.  This computer program will be very useful for scaleup and optimization purposes.  Likewise, we have developed a computer program to simulate the performance of the CAB (see Appendix 2). This simulation program is designed to estimate the CAB monolith catalyst bed height required to diminish carbon monoxide from the effluent.  We are going to verify this model by comparing with experimental data. This program will be useful for developing a new CAB and for estimating the effluent composition from CAB.
3. Our sponsor (Carbon Diversion Corp.) provided us a representative sample of waste tire material.  I carried this material to Budapest where Dr. Marianne Blazso and I performed PYGC-MS studies of it using sophisticated instrumentation available in the Hungarian Academy of Sciences laboratories.  The tests revealed that very little sulfur or nitrogen containing compounds were emitted during the pyrolysis of the waste rubber tire.  This is an auspicious result relative to our sponsor’s hope to carbonize waste rubber tires in his FC reactor.  Details of these tests are attached as Appendix 3.
4. Kingsford Products Co. recently licensed the University of Hawaii’s FC patents.  Kingsford engineers will come to Honolulu in the fall for training in my laboratory.

Plant resistance to insect pests mediated by a protease

Specific Aim 1 (Dr. John Gatehouse, University of Durham, UK). Technical difficulties encountered in producing the ScathL-lectin fusion protein (FP) using the yeast expression system (aim 1) have now been resolved. It proved impossible to produce usable amounts of the ScathLGNA 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. Only small amounts of intact fusion protein could be produced. The fusion protein rapidly selfdegraded, and after purification the product inevitably consisted of cleaved fusion protein components, with small amounts (<10%) of residual fusion protein.

To attempt to address this problem, a different strategy was designed, in which the fusion between the proteinase and lectin domains was reversed, so that the lectin was attached to the Nterminus of the proteinase. However, there is a significant technical problem in making a fusion of this type, in that the ScathL coding sequence contains an N-terminal propeptide which needs to be removed to activate the proteinase. Thus, fusing the lectin domain to the N-terminus of the proprotein will not work, as the lectin will be attached to the propeptide, not the mature protein (see Fig. 1). Similarly, attaching the lectin domain to the N-terminus of the mature protein and omitting the propeptide will not work either, because the proteinase will be active once synthesised, and will be lethal to the yeast expression system. The solution arrived at was to incorporate the lectin sequence between the propeptide and the proteinase domain, in the N-terminal region of the mature protein (see Fig. 1). 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. Research on this construct is ongoing.

Fig. 1.  Schematic diagrams showing the original ScathL-ASAII fusion protein construct, and possible ASAII-ScathL fusion protein constructs.

Specific Aim 2 (Dr. Bryony Bonning, Iowa State University). On the basis that expression of FP in yeast would not be representative of expression of FP in planta, transgenic Arabidopsis were constructed expressing GNA fused to the C-terminus of ScathL with the native, insect-derived cDNA sequence, or with the coding sequence optimized according to Arabidopsis stress- or housekeeping gene codon preference (Chiapello, Lisacek et al. 1998; Wright, Yau et al. 2004). Control plants expressed GNA or ScathL alone. Integration of genes into the plant genome was confirmed by PCR and transcription of the transgenes was confirmed by RT-PCR. Plant expressed proteins were detected by western blot in some plants with purified anti-ScathL antiserum. Preliminary bioassays show that one construct (GNA fused to ScathL with housekeeping codon preference) appears to suppress aphid population growth (Fig. 2). Further work is underway for screening and analysis of the proteins expressed by these plants, and their impact on aphid population growth. Screening of the F3 generation of plants for insecticidal activity and physiological impact will provide the focus for research in Year 2.  

Fig. 2 Suppression of Myzus persicae population growth on four transgenic plants expressing GNA/ScathL (11-8 series; housekeeping codon preference). For reference, control, wild type plants typically had a population of 180 aphids by day 12. 

Ear-rot resistant mycotoxin-free transgenic corn

A. In vitro antifungal activity of MtDef4 against field isolates of F. verticillioides and F. proliferatum.  

In nature, ear rot disease is often caused by a mixed infection of F. verticillioides and F. proliferatum.  We therefore determined the in vitro antifungal activity of MtDef4 against several field isolates of these two fungal pathogens.  Two isolates of F. verticillioides and three isolates of F. proliferatum, provided by our collaborator, Dr. Don White of the University of Illinois, were used for this analysis.  The results are shown in Table 1.  The IC₅₀ values for the different isolates range from 0.77 µM to 1.28 µM, indicating that MtDef4 is active against different isolates of both fungal pathogens at micromolar concentrations.  Based on the observed in vitro antifungal activity against different isolates, we believe that MtDef4 expression of 2 ppm or higher in transgenic corn should provide strong resistance to these isolates in the greenhouse or in the field.  The isolates used in this study will also be used to challenge transgenic corn lines expressing MtDef4 protein in the greenhouse in 2007 and in the field in 2008 (Table I).  Both of these pathogens can also cause stem rot of corn, sometimes in conjunction with F.  graminearum.  Ubiquitous expression of MtDef4 may prove to protect the plants against this disease as well.  We have previously published the activity of MtDef4 against F. graminearum (Ramamoorthy et al., 2007).

Table I. Antifungal activity of MtDef4 against Fusarium field isolates

Pathogen	IC50 (µM)	SD
F. verticillioides ISU94445	1.19	1.2
F. verticillioides ISU95082	1.24	0.6
F. proliferatum 19	1.28	0.8
F. proliferatum 310	0.77	0.2
F. proliferatum 37-2	1.17	0.7

IC50 is the Def4 concentration at which 50% of the growth is inhibited and is determined by reading the optical density of the culture 40 or 60 hours after exposure to Def4.  The number is the average of three experiments that contained four replicates. SD is standard deviation. 

B. Generation of transgenic corn lines expressing MtDef4

In year 1, we have generated 29 independent events of transgenic corn containing the MtDef4 gene construct.  We have carried out Agrobacterium tumefaciens-mediated transformation of two maize inbreds, namely H99 and Hi II, using paromomycin and glufosinate resistance genes as selectable markers, respectively.  (Sidorov et al 2005, Frame et al 2002,). The chimeric gene constructs used for transformation are shown below.

The constructs have the same MtDef4 gene expression cassette, consisting of the maize ubiquitin promoter and intron, the tobacco etch virus (TEV) leader, the monocot-codonoptimized MtDef4 coding sequence, and the CaMV 35S terminator.  The constructs differed in the selectable marker gene and its promoter, namely the Npt II gene conferring resistance to paromomycin, driven by Cauliflower mosaic virus (CaMV) 35S promoter and the Basta resistance gene conferring resistance to glufosinate, driven by CaMV 35S promoter .

T₀ plants were screened by PCR for the Def4 gene cassette.  Fifteen events were received from the Iowa State University Plant Transformation Facility in the Hi II background.  For many events, multiple clones were available, thus increasing the overall survival rate.  When multiple clones within one event were positive, they were carried forward and delineated as families within the event. Overall, 80% of the T0 events in the Hi II background were positive.  Thirtynine potential events were received from the Donald Danforth Plant Science Center Plant Tissue Culture Facility. Fifty percent of these events were positive, and many events consisted of multiple positive clones. (Table II)

Table II.  T₀ transgenic maize events

T0	# lines received in agar	# lines survived transplant	Def4 PCR positive	% T0 events positive
Hi II*	15	15	12	80%
H99^	39	34	17	50%

^*Hi II selected by glufosinate        ^{^}H99 selected by paromomycin

Selected transgenic lines have been carried forward to the T1 generation.  For each event, 1620 T1 plants have been subjected to both PCR and ELISA screens (Table III).  Sixteen plants is an appropriate progeny size to ascertain which families are segregating for a single copy insertion based on segregation ratios at P=0.01.  Chi squared (χ²) tests were analyzed for all event families at one degree of freedom to determine which lines carried a single insert.  Lines that appeared to contain multiple inserts were not carried onto the next generation.   Additionally, in the T1 generation, a sandwich ELISA with MtDef4 antibodies was used to eliminate families that did not express the MtDef4 protein.  14 lines are expressing detectable amounts of protein.  Quantitative ELISA will be done in the T2 generation.

Table III.  Independent events tested in the T1 generation 

Maize Inbred	# of lines tested	Lines PCR+ for MtDef4	Lines with single insert1	Detect of Def4 protein2	Percent selected
H99	14	8	8	6	42.9%
Hi II	12	12	9	8	66.7%

[1] Insertions determined by segregation of transgene and c2 test.

^[2]Def4 protein detected by sandwich ELISA

 

Table IV. Segregation data of selected MtDef4 events.

• ns: Not significant at p = 0.05 (χ² = 3.84, 1 df).
• 1. Data expressed as number of positive and negative plants based on PCR detection of the transgene cassette.
• 2. T0 generation is the original transgenic material; T1 is derived from selfing the original transgenic plant; BC0F1 is derived from crossing the transgenic material to the public inbred B73; BC1F1 is derived from subsequent backcrossing to B73.
• nd: Plants are currently undergoing segregation analysis
• na: not applicable, the T0 generation consists of one individual plant         

The plants have all currently reached the T2 seed stage and we are now in the process of growing and testing the T2 plants.  Through the T1 generation, we have seen no negative effects of the MtDef4 transgene expression on the growth and development of corn. The T2 plants are currently being grown. Thus far, multiple families of 3 independent events have been tested for heritability and will be backcrossed to B73.  The plants will be challenged with a mixture of pathogens that cause ear rot disease in late summer of 2007.  The mixture consists of three field isolates of F. verticillioides and three field isolates of F. proliferatum.  This ear rot disease evaluation will be performed in the greenhouse by Dr. Don White at the University of Illinois as described. The cobs will be evaluated for overall disease severity and for fumonisin content.  

Future Activities:

Flash-Carbonization^TM Catalytic Afterburner Development

1. We plan to employ the FTIR to measure the CO content of the effluent of the CAB and compare this data to State and Federal emissions requirements.  These measurements will enable us to determine if we are likely to satisfy emissions requirements.  To accomplish this we must make some changes to our sampling system to protect instruments from pressure surges.  
2. When we achieve favorable emissions results, we will engage a professional emissions test company to measure the emissions from the CAB.  Our interactions with local authorities suggest that this is the only way for us to have a credible emissions measurement.
3. We plan to begin studies of the carbonization of shredded tires and “fluff” (shredded fabric from cars) during the month of September.  The shredded waste will be loaded on top of corn cobs or other biomass in the canister.  After ignition, the biomass will be carbonized, followed by the shredded waste.  In this way the tarry hydrocarbon vapors from the shredded waste will pass through the very hot bed of charcoal prior to entering the afterburner.  We expect that these vapors will crack and burn, or simply be reduced on the hot carbon.  Results described in Appendix 3 (see item 8 above) support this expectation.  We will use existing Gas Chromatographs to analyze the gas for noxious pollutants. 

Plant resistance to insect pests mediated by a protease

In consultation with the sponsoring company, the plan for Year 2 of this project is to screen the F3 generation of transgenic plants that express the ScathL-lectin fusion for insecticidal activity, with particular emphasis on aphids. Given the widespread use of transgenic plants that express toxins derived from Bacillus thuringiensis (Bt) that are highly effective against lepidopteran (moth) larvae, there is an urgent need for the development of aphid resistance technologies. Indeed, hemipteran pests that include aphids and plant hoppers are compromising the success of the Bt technology (Greene, Turnipseed et al. 1999; Greene, Turnipseed et al. 2001).

During Year 2 of the project, we propose to conduct bioassays with the F3 generation of transgenic Arabidopsis constructed in Year 1 that express the lectin-protease fusion protein. We will monitor the mortality of insects maintained on these plants and compare to mortality of insects maintained on control plants. We will examine the insects for the presence of the recombinant proteins in the hemocoel, and for the impact of ScathL on its target site, the basement membrane. 

Ear-rot resistant mycotoxin-free transgenic corn

We will be determining expression of the MtDef4 gene quantitatively in transgenic corn lines.  We will also be testing transgenic corn lines for resistance to the ear rot fungi in the greenhouse as well as in the field.  Particular attention will be paid to the levels of fumonisins in these lines challenged with the pathogen. We anticipate that high level expression of the antifungal MtDef4 protein in transgenic corn will confer strong resistance to ear rot resulting in significantly reduced levels of mycotoxin in the seed.  Should constitutive expression of the MtDe4 gene result in any negative impact on the normal growth and development of transgenic corn lines in the field, we will develop transgenic corn lines where the expression of this gene is restricted to the floral tissue which is normally colonized by the pathogens. 

References:

• Chiapello, H., F. Lisacek, et al. (1998). "Codon usage and gene function are related in sequences of Arabidopsis thaliana." Gene 209(1-2): GC1-GC38.
• Greene, J. K., S. G. Turnipseed, et al. (1999). "Boll Damage by Southern Green Stink Bug (Hemiptera: Pentatomidae) and Tarnished Plant Bug (Hemiptera: Miridae) Caged on Transgenic Bacillus thuringiensis Cotton." Journal of Economic Entomology 92: 941-944.
• Greene, J. K., S. G. Turnipseed, et al. (2001). "Treatment Thresholds for Stink Bugs (Hemiptera: Pentatomidae) in Cotton " Journal of Economic Entomology 94: 403-409.
• Wright, S. I., C. B. K. Yau, et al. (2004). "Effects of gene expression on molecular evolution in Arabidopsis thaliana and Arabidopsis lyrata." Mol. Biol. Evol. 21(9): 1719-1726.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Publications Views

Other project views:	All 18 publications	8 publications in selected types	All 5 journal articles

Publications

Type	Citation	Project	Document Sources
Journal Article	Antal Jr. MJ, Wade SR, Nunoura T. Biocarbon production from Hungarian sunflower shells. Journal of Analytical and Applied Pyrolysis 2007;79(1-2):86-90.	EM832933 (2007) EM832933 (Final)	Abstract: ScienceDirect-Abstract Exit

Supplemental Keywords:

Charcoal, biocarbon, Flash Carbonization process, Insect resistance; Protease; Lectin; Transgenic plant, Antifungal defensin, mycotoxin, corn.

Progress and Final Reports:

Original Abstract

2006

2008

2009

2010

Final Report