INFORMATION SYSTEMS FOR BIOTECHNOLOGY


January 2005
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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LONG DISTANCE POLLEN-MEDIATED GENE FLOW FROM CREEPING BENTGRASS
Lidia S. Watrud

Creeping bentgrass (Agrostis stolonifera L.) is a cosmopolitan, cool season, perennial, highly outcrossing, wind-pollinated species, grown primarily for use on golf courses. The natural, low to the ground, spreading or "creeping" growth pattern of the grass designated by its species epithet "stolonifera" reflects its ability to reproduce asexually by means of stolons. Outside of cultivation, in preferred non-agronomic disturbed or natural habitats such as drainages, riparian areas, wetlands, and wet meadows, A. stolonifera may form dense patches by vegetative spread via new plantlets established by rootings from stolons. Seed dispersal by wind, water, wildlife, and mechanical means may also occur. Under highly managed conditions such as golf courses, creeping bentgrass thrives even with frequent mowings, due at least in part to its ability to spread via stolons.

To further enhance the aesthetic quality of golf greens by reducing grass weeds, Scotts and Monsanto companies have jointly developed and are seeking commercial approval for bentgrass resistant to glyphosate, the active ingredient in Roundup® herbicide (see "Regulatory News," this issue). To that end, under permit from the USDA-APHIS, the companies planted fields totaling approximately 400 acres in central Oregon in 2002. When the genetically modified (GM) creeping bentgrass crop fields flowered for the first time in the early summer of 2003, a unique opportunity was presented to test methods to track gene flow from the GM fields to compatible relatives in surrounding, largely non-agronomic areas.

The experimental design used to measure gene flow by researchers from the U.S. EPA’s Office of Research & Development in Corvallis, OR, encompassed a geographic scale much larger than that typically used under agronomic conditions. In contrast to studies of gene flow that have measured gene flow to distances of hundreds of feet1,2, the design used by US EPA researchers3 attempted to measure gene flow on a much larger landscape scale that included a variety of largely non-agronomic habitats. Key assumptions used in the sampling design were an estimated time of pollen viability of three hours4 and prevailing north and northwest winds of 5–6 mph at the time of day (mid- to late morning) when anthesis was expected to occur.

GM fields were located on an irrigated plain above the Deschutes River canyon within a "control district" designated by the Oregon Department of Agriculture. Central Oregon was chosen as the location for the control district to address concerns of grass seed growers in the Willamette Valley of Oregon located 90 miles to the west, where approximately 70% of US grass seed is produced. The sampling design that US EPA researchers used included both temporarily deployed sentinel plants of A. stolonifera and resident or wild (naturalized or native) plants of A. stolonifera and A. gigantea Roth (redtop) that were expected to be compatible with the GM crop. All of the sentinel plants and resident plants used in the study were located outside the control district. Sentinel plants were placed along highway and riparian rights-of-way in a variety of largely non-agronomic habitats that included sagebrush steppe, natural and man-made drainages, and residential and light industrial areas. Resident plants were found primarily in wetter habitats such as riparian zones and drainages. The sampling grid for the sentinel plants had shorter interval spacings (e.g., one half mile) when located closer to the control district, and progressively larger intervals (one and two miles) farther away from it.

The sentinel plants were placed in position in mid-June of 2003. Approximately six weeks later, after seed set and prior to the harvest of the GM fields, bagged seedheads of sentinel and resident plants were collected for greenhouse and laboratory analyses to determine if hybridization had occurred between the GM crop fields and the sentinel or resident plants. Analyses performed included greenhouse tests for resistance of seedlings germinated from the harvested seeds to Roundup® herbicide, and confirmatory tests for production of the engineered protein (CP4 EPSPS), based on the use of a commercial lateral flow immunological strip test (TraitChekTM). Molecular analyses of DNA isolated from sub-samples of herbicide resistant and TraitChekTM positive seedling progeny by PCR and sequencing additionally confirmed the presence of the engineered CP4 EPSPS gene.

The highest relative frequencies of gene flow from the GM crop to sentinel or resident plants were observed within approximately one mile of the perimeter of the control district, in the direction of prevailing winds. However, pollen-mediated movement of the GM gene was recorded as far as 13 miles away from the perimeter of the control district in sentinel plants, five miles away in resident A. stolonifera, and nine miles away in resident A. gigantea. A total of 625 observations of progeny positive for the GM gene were noted among 32,000 sentinel seedlings that were screened (overall frequency of 2%); a total of 157 observations of positive seedling progeny were noted among 565,000 resident A. stolonifera seedlings analyzed (0.03% ); and 159 positive progeny were noted among 397,000 (0.04% ) A. gigantea resident plant seedling progeny. The much lower frequencies of hybridization observed among the resident plant seedling progeny as compared to the sentinel plant progeny are believed to be due to less floral synchrony (i.e., resident plants tended to flower later than the crop fields in their typically moist habitat locations) and possibly higher pollen loads and competition around resident plants.

Sentinel plants were deployed to repeat the test in 2004; however, most of the 400 acres of crop fields that flowered in 2003 were taken out of production, and less than 10 acres are estimated to have shed pollen in 2004. We anticipate that our 2004 study results will reflect the much smaller source of GM pollen available to fertilize compatible sentinel or resident plants. In addition to the second year study of gene flow, efforts are in place to determine whether establishment and recruitment of hybrids between the GM crop and resident plants have occurred under field conditions.

With suitable technical modifications to detect specific traits of interest, a sampling design that utilizes both sentinel and resident plants may be amenable to measuring gene flow from other types of GM crops. Crops particularly suitable for those sorts of studies are ones that are wind pollinated and which may have compatible crop or non-crop relatives whose flowering periods may overlap with the GM crop of interest.

The information in this document has been funded in its entirety by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory’s Western Ecology Division and approved for publication. Approval does not signify that the contents reflect the views of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

References

1. Wipff JK & Fricker C (2001) Int. Turfgrass Soc. Res. J. 9, 224-242

2. Berlanger FC, Meagher TR, Day PR, Plumley K, and Meyer WA (2003) Crop Sci. 43, 240-246

3. Watrud LS, Henry Lee E, Fairbrother A, Burdick C, Reichman JR, Bollman M, Storm M, King G, & Van de Water PK (2004) PNAS 101, 14533-14538

4. Fei S & Nelson E (2003) Crop Sci. 43, 2177-2181

Lidia S. Watrud
US Environmental Protection Agency, Office of Research & Development
National Health & Environmental Effects Research Laboratory
Western Ecology Division
Corvallis, Oregon
watrud.lidia@epa.gov


GENETIC ACCLIMATION FOR FREEZING TOLERANCE
Kan Wang

A late spring cold wind-current or early fall frost can cause severe damage to crop yield. A number of strategies using recombinant DNA technology and genetic transformation has been utilized to enhance crop freezing tolerance in recent years1,2. These approaches include the overexpression of biosynthetic enzymes for osmoprotectants (such as mannitol, proline, treholose, or glycine betaine), the constitutive expression of stress-induced proteins (such as late embryogenesis abundant [LEA] proteins or heat shock proteins [HSPs]), altering the enzyme activity of antioxidants (such as superoxide dismutase [SOD] or glutathione S-transferase [GST]) that are involved in the detoxification of active oxygen species (AOS) accumulated during the stress environment, and expression of transcriptional factors (such as dehydration-responsive element [DRE] or CRT-binding factors [CBFs]) that bind to water deficit and cold responsive genes. Recently, it was reported that overexpression of a plasma membrane-associated phospholipase Dδ could enhance freezing tolerance in Arabidopsis3. While most work utilizes Arabidopsis as a model system, we carried out experiments on maize, a frost-sensitive crop plant originated from subtropical regions. The freezing tolerance enhancement described here involves the constitutive expression of a protein kinase in an oxidative stress signaling pathway4.

Many plant species increase their tolerance to cold or freezing temperature after they are exposed to a sub-optimal temperature. This process is called cold acclimation. Exposure to acclimation temperature causes many changes, including mild oxidative stress in plants, which can consequently induce chilling tolerance. At the molecular level, extensive alteration in gene expression has been observed during this process. Oxidative stress generates and accumulates active oxygen species such as H2O2 in plants, which triggers the activation of a mitogen-activated protein kinase (MAPK) cascade. The activation of the MAPK pathway induces production of a number of stress responsive proteins, such as heat shock proteins (HSPs), which in turn protect plants from stresses.

The MAPK signal transduction pathway is conserved among different organisms. It is usually activated only upon stress conditions. However, in some cold stress conditions, plants may be severely damaged before they even get a chance to turn on their protective mechanisms. Our hypothesis was then, if a plant was acclimated genetically, namely, its stress-induced pathways or proteins were turned on without first being stressed, could it withstand sudden severe stress such as subzero freezing temperatures?

We introduced a tobacco MAP kinase kinase kinase gene (NPK1) into maize through Agrobacterium-mediated transformation. The cDNA fragment encoding a 268-amino acid catalytic domain of NPK1, which is under a constitutive CaMV 35S promoter, was shown previously to enhance tolerance to freezing, salt, and heat stresses in transgenic tobacco5. Two dozen transgenic NPK1 maize lines were generated. According to the NPK1 gene transcript levels in R1 plants, we categorized maize lines into high, medium, and low expressers. Two events, A4-9 and A4-15, representing the medium and high levels of gene expression, respectively, were used for freezing analysis.

We performed two types of freezing tests for these transgenic plants: graduated freezing and constant freezing. In the graduated freezing test, the temperature of the growth chamber was set at –1oC and continually decreased at the rate of 1ºC per hour until it reached a temperature of –6oC, while in the constant freezing test, the temperature was set at –5ºC. Maize plants were grown under normal growth conditions (25ºC, 14 hr day length) to the three-leaf stage before they were subjected to freezing treatments. Cellular damage of treated seedlings due to freeze-induced membrane lesions was estimated by measuring electrolyte leakage (EL) from the leaves of treated plants. The higher the EL, the more severe the damage to the plant membrane, thus indicating that samples had less tolerance to freeze challenging. Considering the possibility of genetic variation among these transgenic events, we used the null segregants from each transgenic event as the negative control. We observed that leaf EL increased with a decrease in environmental temperature. When the temperature dropped to –4ºC, the EL of negative segregants increased extensively, indicating that severe membrane damage had been caused by freezing stress. The EL of transgenic plants of A4-9 and A4-15, on the other hand, did not increase until the temperature dropped to –5ºC and –6ºC, respectively. This result indicates that these transgenic maize events were able to tolerate up to 2ºC lower freezing temperature than their negative control siblings.

The increased freezing tolerance in transgenic plants of events A4-15 and A4-9 were confirmed in the constant freezing test. The EL of A4-15 transgenic plants did not increase until 5 hours at –5ºC temperature. The EL of A4-15 negative control siblings, however, increased to 57% and 90% after 3 and 4 hours of –5ºC treatment, respectively. This result indicates that transgenic A4-15 plants can survive 1–2 more hours than their negative control siblings at a temperature of –5ºC. A similar difference in freezing tolerance between transgenic plants and their non-transgenic siblings was observed in event A4-9 in which transgenic plants survived for 3 hours at –5ºC, while the negative siblings survived for 1 hour.

To understand the mechanism of freezing tolerance in NPK1 transgenic maize plants, we measured total soluble sugar content (TSC). An increase in TSC was positively correlated with enhanced freezing tolerance in plants. It is believed that soluble sugars function as cryoprotectants and osmolytes that protect cells from freezing damage. In our study, NPK1-expressing transgenic plants had higher TSC both under non-acclimated (25ºC) and cold-acclimated (4ºC, 24 or 48 hrs) conditions compared to their negative non-transgenic siblings in all treatments. Cold acclimations significantly increased TSC levels in all plants. Under normal growth conditions (non-acclimated), event A4-9 contained significantly higher total soluble sugar content compared to its null segregants (P<0.02). It is interesting that the increase of TSC in transgenic plants was not tightly correlated with NPK1 transgene expression level or freezing tolerance performance in our case. It is possible that while the NPK1 transgene induced cold-acclimation-like biochemical processes that elevated TSC, factors other than the NPK1 transgene also affected sugar levels in the maize seedlings.

We also conducted microarray analysis to investigate whether enhanced freezing tolerance in NPK1 transgenic maize was due to the activation of MAPK cascades resulting from oxidative stress. Using a fiber-optic BeadArray™ technology, we compared the expression levels of several stress-induced genes between transgenic and non-transgenic plants with or without cold acclimation. The fiber-optic array uses randomly ordered, self-assembled arrays of beads for parallel analysis of complex biological samples6. Since the miniature fiber optic arrays that interrogate hundreds to over one thousand targets are built into a 96- or 384-array matrix that matches microtiter plates, it allows multiple assays to be carried out rapidly and efficiently. Twenty-eight maize EST sequences based on the protein sequences of putative stress-related Arabidopsis and tobacco orthologues, together with a housekeeping gene (18S rRNA) and the transgene NPK1, were chosen for analysis. The expression of three genes, GST (glutathione S-transferase), HSP17.8 (small heat shock protein), and PR1 (pathogenesis-related), was up-regulated (> 1.5) in NPK1 transgenic plants under either normal or cold-acclimated conditions. Two of these genes, GST and HSP17.8, are involved in oxidative signaling pathways5. While constitutive expression of transgene NPK1 up-regulated the gene expression of GST and HSP17.8, cold acclimation treatment (4ºC, 48 hr) did not additionally increase their transcript levels.

As discerned from array analysis, about 50% of stress-induced genes tested in our study showed no significant increase in NPK1 transgenic maize lines. One explanation is that the transgene NPK1 expression level may be too low to up-regulate these stress related genes. It is also possible that these genes were only transiently induced and our assay condition did not capture their expression at the right moments.

We have generated transgenic maize plants that constitutively express a tobacco MAP kinase kinase kinase gene (NPK1) with enhanced freezing tolerance. In field evaluation of agronomic performance of 22 events, we detected no significant differences in plant heights and leaf numbers between transgenic plants and their non-transgenic segregants, suggesting that expression of transgene NPK1 did not affect maize growth under normal field conditions. Our results demonstrate that maize freezing tolerance level could be enhanced through a genetic acclimation (instead of cold acclimation) process in which stress-induced proteins for plant protection is achieved upon the activation of the oxidative signaling pathway through manipulation of the MAPK cascade.

References

1. Cushman JC, & Bohnert HJ (2000) Genomic approaches to plant stress tolerance. Curr. Opin. Plant Biol. 3: 117-124

2. Iba K (2002) Acclimative response to temperature stress in higher plants: Approaches of gene engineering for temperature tolerance. Annul Rev. Plant Biol. 53: 225-245

3. Li W, Li M, Zhang W, Welti R, & Wang, X (2004) The plasma membrane-bound phospholipase Dδ enhances freezing tolerance in Arabidopsis thaliana. Nat. Biotechnol 22, 427-433

4. Shou H, Bordallo P, Fan J-B, Yeakley JM, Bibikova M, Sheen J, & Wang K (2004) Expression of an active tobacco MAP kinase kinase kinase enhances freezing tolerance in transgenic maize. Proc. Natl. Acad. Sci. (USA) 101, 3298-3303

5. Kovtun, Y, Chiu, WL, Tena, G, & Sheen, J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc. Natl. Acad. Sci. 97, 2940-2945

6. Yeakley JM, Fan J-B, Doucet D, Luo L, Wickham E, Ye Z, Chee MS, & Fu XD. (2002) Profiling alternative splicing on fiber-optic arrays. Nat. Biotechnol 20, 353-358

Kan Wang
Department of Agronomy
Iowa State University
kanwang@iastate.edu


ENGINEERED RIBOSOMAL PROTEIN LIMITS PLANT RESISTANCE TO MYCOTOXINS
Gerhard Adam and Rudolf Mitterbauer

Fusarium head blight (FHB) is a serious disease that limits cereal production in many parts of the world, leading to yield reduction and contamination of grain with mycotoxin deoxynivalenol (DON). Favorable weather conditions and shifts in agricultural practices has resulted in a dramatic reemergence of Fusarium head blight in the U.S. The cumulated economic impact during the period of 1991 to 1997 was calculated as 4.8 billion dollars, with dramatic consequences for farmers1.

FHB outbreaks are infrequent in Europe, but locally high levels of DON have been observed. To protect consumers, EU-wide regulatory measures are in preparation. The European Commission has reevaluated the toxicological hazard of DON (http://europa.eu.int/comm/food/fs/sc/scf/out44_en.pdf) and has collected occurrence data (http://www.europa.eu.int/comm/food/fs/scoop/task3210.pdf) to assess dietary intake. Germany has already enacted legally binding maximum tolerated DON levels for various commodities (Table 1). In comparison, the U.S. "advisory" level for DON is 1 ppm (1000 µg/kg) in final milled products (flour, bran etc.) for human food use.

Table 1: Maximum tolerated DON levels* in Germany

Commodity:

DON (µg/kg):

Cereal grain for human consumption (except durum wheat)

500

Bread, bakery goods

350

Cereal products intended for production of infant food

100

*blending is prohibited.

Production of DON, which is a member of the large group of toxins known as trichothecenes, is a virulence factor of Fusarium graminearum2. Engineering trichothecene tolerance in transgenic crop plants is therefore an obvious strategy and one that is already advanced to field testing stage in the case of a fungal acetyltransferase that converts DON into less toxic 3-Acetyl-DON (Syngenta, US Patent 6.346.655 B1). Recently, a plant glucosyltransferase, which detoxifies DON by formation of DON-3O-glucoside, has been described by our group3. In addition to detoxification, we also work on toxin efflux by ABC transporter proteins (PDR5-like genes; Mitterbauer et al., in preparation) and on toxin target modification.

In the mid 1970s the mode of action of trichothecenes as inhibitors of eukaryotic protein biosynthesis was elucidated. Semidominant Saccharomyces cerevisiae mutants resistant to trichodermin were identified. It was determined that the trichodermin resistance gene encodes ribosomal protein L3 (RPL3). The goal of our work summarized below was to identify mutations in yeast RPL3 conferring resistance to the Fusarium toxin DON and to test whether improved toxin resistance can be engineered in transgenic plants expressing a modified plant RPL3 cDNA. Unfortunately we discovered that a posttranscriptional effect, leading to preferential utilization of endogenous Rpl3 protein, severely limits the efficacy of this approach4.

Methods
All yeast work was performed in DON-sensitive pdr5 mutant strains (pleiotropic drug resistance). A strain expressing RPL3 under control of the GAL1 promoter and containing a deletion of the chromosomal copy, rpl3::LYS2, was constructed and used as a host in a shuttle mutagenesis procedure. A RPL3 plasmid was mutagenized, and plasmids conferring DON resistance in yeast were selected and characterized by sequencing.

For plant transformation, a tomato (Lycopersicon esculentum) cDNA encoding LeRPL3 was identified and placed behind the 35S promoter. An amino acid alteration leading to DON resistance and an epitope tag were introduced into this gene. The resulting construct and appropriate controls were introduced into Nicotiana tabacum (cultivar Samsun NN)4.

Results
We sequenced changes in three trichodermin-resistant yeast strains. The changes identified were G765T and G765C, both leading to the conversion of tryptophan into cysteine (W255C). This amino acid change also conferred resistance to DON and to all other trichothecenes tested thus far. In the systematic screen of DON-resistant mutants, we additionally found the changes S2P, P9L, W255R, and H256Y. In order to detect expression of engineered RPL3, we constructed C terminally epitope-tagged versions of yeast RPL3 gene. The constructs were capable of replacing the wild type RPL3 gene. Epitope-tagged constructs containing the W255C mutation also conferred increased resistance in strains containing the endogenous wild type RPL3 gene. We therefore reasoned that this semi-dominance should allow engineering of plants with improved trichothecene resistance by introduction of a modified RPL3 gene.

For proof of principle, a RPL3 cDNA from tomato was isolated. Alignment of Rpl3 amino acid sequences showed that the change corresponding to W255C in yeast is W258C in tomato. This alteration was introduced into the LeRPL3 cDNA alone and in combination with HA- and c-Myc epitope tags. The constructs were used for Agrobacterium-mediated transformation. At least 10 independent transgenic tobacco plants expressing either wild type untagged tomato cDNA, the W258C allele, or the corresponding constructs with either the HA-tag or c-Myc-tag were regenerated and characterized by PCR and Southern blotting.

Western blot screening of tobacco protein extracts revealed that c-Myc epitope-tagged gene products were undetectable in plants containing a modified transgene (W258C allele of LeRPL3), in contrast to those encoding the wild type LeRPL3 allele. Reverse transcriptase PCR showed that both types of transformants had about equal amounts of transcript from the respective LeRPL3-transgenes. In agreement with the lack of mutant protein, the engineered RPL3 gene did not confer toxin resistance when plants were challenged with concentrations of DON inhibitory for the control. Yet, when sublethal concentrations were applied, the transformants containing the modified RPL3 gene had improved ability to adapt to DON4. The mutant protein was only temporarily detectable in the toxin-stressed plants.

These results led us to reinvestigate the semidominance observed in yeast. Tagged Rpl3 proteins were easily detectable in all strains that contained solely the tagged version of the gene, regardless of the W255C mutation. Likewise, in a strain that contains an endogenous Rpl3 protein, the tagged wild type protein was detected without problem (Fig. 1). In contrast, when the tagged W255C alleles were introduced into this strain, the modified Rpl3 protein was undetectable on medium without toxin. Since plate assays showed that introduced Rpl3 mutant alleles also confer DON resistance in the wild type background, we grew heterozygous strains in the presence of variable amounts of DON in liquid culture. Modified protein was undetectable in the control lacking DON, but increased with increasing concentration of DON in the medium (see Fig. 1). In summary, in yeast as well as tobacco, the mutant Rpl3 protein is utilized in a toxin-dependent manner.

Figure 1. Increasing DON concentrations enforce incorporation of W255C mutated ribosomal protein L3 into yeast ribosomes. Western blot using monoclonal antibody 9E10 (anti-c-Myc) and horseradish peroxidase-conjugated goat anti-mouse IgG.

Left: A strain containing, in addition to the endogenous RPL3 gene, a plasmid-encoded mutant RPL3W255C-Myc gene was grown in SC-Leu in the presence of 0, 60, 120, and 180 ppm DON, respectively. Note the absence of a signal in the medium without toxin.

Right: Serial dilutions of protein extracts of a strain containing in addition to the endogenous chromosomal RPL3 gene a plasmid-encoded wild type RPL3-Myc gene. The tagged wild type protein is easily detectable in medium without toxin.

Discussion
Trichothecenes are suspected fungal virulence factors. A possible reason for the high structural diversity of trichothecenes is that plants have evolved detoxification mechanisms that the pathogen can evade by producing structural variants. Our DON inducible UDP-glucosyltransferase4 can inactivate DON and 15 ADON, but not other trichothecenes. It does not, for instance, provide protection against nivalenol, the predominant F. graminearum toxin in Asia. The identified target alteration RPL3W255C confers resistance to all trichothecenes tested so far, a prerequisite for engineering durable resistance.

In contrast to the clear semidominant toxin resistance phenotype in yeast, transgenic LeRPL3W258C tobacco plants showed no relevant increase in toxin resistance. Plants were as sensitive as controls whenever test conditions were applied that caused an immediate block of protein synthesis. Yet, when plants were pretreated with only partially inhibiting concentrations of DON, transformants containing the modified RPL3 transgene could adapt to otherwise inhibitory DON concentrations.

While this work was ongoing, a Canadian group5 claimed that introduction of a modified RPL3 cDNA of rice, into which the W258C change had also been introduced, leads to DON tolerance of transgenic plants (U.S. patent 6.060.646). The apparent contradiction may be due solely to different expectations. Whether or not the RPL3 transgenic plants show increased resistance to very low concentrations of toxin (5 ppm) in assays that require weeks is most likely irrelevant for the field situation. Toxin levels in individual Fusarium-infected kernels were reported to be in the 250 ppm range. Plant cells close to the toxin-secreting fungus could be confronted locally with much higher levels. It seems reasonable to assume that plant defensive gene expression within the first few hours determines the outcome of pathogen attack.

With the help of epitope-tagged Rpl3, we obtained clear evidence that engineered Rpl3 protein is not accumulating in transgenic plants prior to toxin contact, and that this is a post-transcriptional effect. The same preferential utilization was also observed in yeast. The explanation for the weak phenotype of our transgenic plants and the clear resistance phenotype in yeast could be that ribosome biogenesis in the rapidly dividing microorganism is extremely efficient compared to plant cells.

Additional basic research is needed to understand the discovered post-transcriptional effect. The observed allele-specific interaction in the yeast two-hybrid system between Rpl3 and Rrb1 (a protein required for ribosome biogenesis)4 suggests that differences in ribosome assembly could be responsible for the preferential incorporation of the wild type Rpl3 protein into ribosomes. Our current hypothesis to explain the accumulation of mutant Rpl3 protein in the presence of toxin is that ribosomes blocked by the toxin could be preferentially degraded in vivo, eventually leading to a higher steady state level of modified Rpl3 protein. Improved understanding of basic processes could allow us to fix the problem and to engineer high level RPL3-mediated trichothecene resistance.

Acknowledgements
This work was funded by the Austrian Science Foundation (FWF P-11551-MOB), the Austrian Academy of Sciences, and by the European Commission (project FUCOMYR, QLRT-2000-02044).

References

1. Leonard KJ & Bushnell WR, eds. (2003) Fusarium Head Blight of Wheat and Barley. APS Press: St. Paul, MN, USA. (ISBN 0-89054-302-X)

2. BM für Verbraucherschutz, Ernährung und Landwirts-chaft:Verordnung zur Änderung der Mykotoxin-Höchstmengenverordnung und der Diätverordnung. (Bonn, 12. February 2004) BGBL. 2004 Teil 1 Nr. 5 pp. 151-152. (http://www.mykotoxin.de/Dokumente/bgbl104s0151.pdf)

3. Poppenberger B et al. (2003) Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana. J. Biol. Chem. 278, 47905-47914

4. Mitterbauer R et al. (2004) Toxin-dependent utilization of engineered ribosomal protein L3 limits trichothecene resistance in transgenic plants. J. Plant Biotechnol. 2, 329-340

5. Harris LJ & Gleddie SC. (2001) A modified Rpl3 gene from rice confers tolerance of the Fusarium graminearum mycotoxin deoxynivalenol to transgenic tobacco. Physiol. Mol. Plant Pathol. 58, 173-181

Gerhard Adam and Rudolf Mitterbauer
Institute of Applied Genetics and Cell Biology
BOKU – Univ. of Natural Resources and Applied Life Sciences Vienna, Austria
gerhard.adam@boku.ac.at
(rudolf.mitterbauer@boku.ac.at)



APPROVAL FOR GENETICALLY ENGINEERED BENTGRASS CREEPS THROUGH AGENCY TURFS
Phillip B. C. Jones

Monsanto Company and The Scotts Company want to market creeping bentgrass (Agrostis stolonifera) genetically modified (GM) for tolerance to the glyphosate herbicide, Roundup®. The companies plan to sell the GM plant for use in commercial grass seed production and on golf courses. Among the benefits attributed to GM bentgrass, the companies claim that it will enhance the uniformity, quality, aesthetics, and playability of golf course turf. Before GM bentgrass can be sold within the United States, however, Monsanto and Scotts must obtain approval for commercial use from the Food and Drug Administration, the Environmental Protection Agency, and the Department of Agriculture.

Monsanto and Scotts Make a Hole in One at the FDA
The Federal Food, Drug, and Cosmetic Act authorizes the FDA to monitor food and feed safety. Monsanto and Scotts provided the agency with a summary of a safety and nutrition evaluation to permit a possible feed use of glyphosate-tolerant bentgrass straw and chaff. Based on the companies’ assessments, the FDA understood that glyphosate-tolerant bentgrass forage derived from the new GM plant does not materially differ in composition and safety from creeping bentgrass forage currently on the market. The agency stated that genetically engineered bentgrass does not raise issues requiring pre-market review or approval by the FDA.

Companies Pitch for a Deuce at the EPA
The EPA regulates two aspects of GM bentgrass. Under the authority of the Federal Insecticide, Fungicide, and Rodenticide Act, the EPA requires the registration of herbicides prior to distribution or sale, unless exempt by EPA regulation. In the case of Roundup®-tolerant GM bentgrass, the EPA must approve a new use or different pattern of use for the herbicide. Monsanto and Scotts filed proposed supplemental labels for Roundup® PRO herbicide for uses in seed production of the GM bentgrass and for general weed control in glyphosate-tolerant bentgrass turf planted on golf courses.

The EPA also establishes residue tolerances for herbicides, an activity authorized by the Federal Food, Drug, and Cosmetic Act. The agency must determine residue tolerances when the use of herbicide on a GM plant may result in increased residues in a food or feed crop for which the herbicide is currently registered, or may result in new residues in a crop for which the herbicide is not currently registered. Here, the EPA determined that it is not necessary to revise the existing tolerance for a minimal use of creeping bentgrass straw and chaff as animal feed.

Deregulation: the Line of Play at APHIS
In April 2003, Monsanto and Scotts advanced their commercialization plans by filing a request with the USDA’s Animal Plant and Health Inspection Service for a determination of nonregulated status for GM bentgrass. The companies asserted that APHIS should not regulate the GM bentgrass because the engineered plant does not present a plant pest risk.

In a preliminary risk assessment, the agency reached the following conclusions: there appear to be no major unintended effects resulting from the introduction of the glyphosate-resistance gene into the creeping bentgrass genome; GM bentgrass is not sexually compatible with any federally acknowledged threatened or endangered species, or with any species on the federal noxious weed list; and GM bentgrass does not differ in pest and pathogen susceptibility or resistance from its parent. APHIS seemed to be on the verge of approving the new plant.

Yet the agency, in its experience, considered GM bentgrass to be unique. Unlike deregulated articles it had previously considered, APHIS characterized GM bentgrass as a widespread perennial species that establishes without cultivation in a variety of habitats. The agency noted that creeping bentgrass can form hybrids with at least 12 other U.S. naturalized or native species of bentgrasses and rabbit’s foot grasses. This raised the possibility that GM bentgrass, or its glyphosate-tolerant progeny, would establish in various urbanized and natural areas.

In January 2004, APHIS opened a 60-day period for public input, soliciting comments and information on whether GM bentgrass presents potential risks to the environment, including a plant pest risk. APHIS’ request garnered over 480 remarks. About 339 expressed support for the deregulation petition, while 134 expressed concern or opposed the petition.

University-based weed and turf grass specialists, golf course superintendents, and golf course operators numbered among the strongest supporters; commenters associated with plant societies, environmental and consumer groups, and certain federal, state, and city officials opposed deregulation. The opposers echoed APHIS’ concerns about the aggressiveness of Agrostis and the possible spread of the glyphosate-tolerant transgene with potential loss of glyphosate for control of invasive perennial grasses.

APHIS reported results of the public comment request in September 2004 and announced its decision to prepare an environmental impact statement to examine potential environmental effects associated with a determination of nonregulated status for GM bentgrass. The agency identified issues that it proposed to explore and opened a 30-day period for public comment on this proposed scope of study.

Genetic and Legal Hazards for GM Bentgrass Commercialization
One of the issues that APHIS identified for further examination in the environmental impact statement process was whether deregulation of GM bentgrass might result in its establishment and persistence in situations where it is unintended or unexpected. On the same day that APHIS released its notice about the proposed environmental document, the Proceedings of the National Academy of Sciences electronically published the study of Lidia Watrud et al. As detailed elsewhere in this ISB News Report, Watrud and her colleagues documented that GM bentgrass’ glyphosate-tolerance trait can indeed spread to unintended or unexpected locations.

On October 5, a group led by the International Center for Technology Assessment and the Center for Food Safety responded to the PNAS paper by filing against the USDA with a request for an immediate injunction. The Center asked a Washington, D.C., federal court to bar the USDA from allowing or approving any further field tests of GM bentgrass and to order the agency to terminate all such current permits for field tests. The injunction request is part of a case filed in January 2003 that seeks to block environmental releases of the engineered bentgrass. A copy of the injunction can be found at the Center for Food Safety’s website (http://www.centerforfoodsafety.org/).

APHIS Plays Through
When they filed a request for an injunction, the plaintiffs had requested a hearing within twenty days. However, the case bogged down as the parties obtained extensions of time to reply to each other’s assertions, and the judge allowed Scotts Company to intervene as another defendant with its own assertions. The twenty-day deadline passed without a hearing.

Meanwhile, APHIS continues to review the petition for deregulation. On November 18, the agency reopened the comment period for an additional two weeks and announced a decision to hold a public meeting to encourage further public participation in defining the scope of the environmental impact study. APHIS plans to announce the date and location of the meeting on its website and in the Federal Register.

When APHIS does complete its environmental document, the agency will make it available for public input. After evaluating these comments, APHIS will either approve the petition in whole or in part, or deny the petition. The agency will then publish a notice in the Federal Register announcing the regulatory status of the GM bentgrass.

Glyphosate-tolerant bentgrass has been under development for about six years and has been released for field tests in over 30 states. Nevertheless, GM bentgrass still has a fair way to go before it appears on golf courses.

Selected References

APHIS (2004) Monsanto Co. and The Scotts Co.; Availability of petition for determination of nonregulated status for genetically engineered glyphosate-tolerant creeping bentgrass. Federal Register 69, 315-317, January 5, 2004

APHIS (2004) Environmental impact statement; Petition for deregulation of genetically engineered glyphosate-tolerant creeping bentgrass. Federal Register 69, 57257-57260, September 24, 2004

APHIS (2004) Environmental impact statement; Petition for deregulation of genetically engineered glyphosate-tolerant creeping bentgrass. Federal Register 69, 67532-67533, November 18, 2004

Phillip B. C. Jones, PhD., J.D.
Spokane, Washington
PhillJones@nasw.org



SYMPOSIUM ON BIOSAFETY OF GMOs: HIGHLIGHTS
Allison Snow

The 8th International Symposium on the Biosafety of Genetically Modified Organisms took place in Montpellier, France, on September 26-30, 2004. Policy officials and scientists from academia, government, industry, and other groups gathered to discuss recent biosafety research and implications for how genetically modified organisms (GMOs) are regulated. The conference was organized by the International Society for Biosafety Research (ISBR; http://www.isbr.info/), which also publishes the scientific journal Environmental Biosafety Research (http://www.edpsciences.org/ebr/).

The symposium was unique in that substantial funding was provided by U.S. and EU government agencies for a North-South Workshop that included participants from 27 developing countries. In all, 45 countries were represented. The meeting also featured a lively public session for dialogue between French citizens and biosafety researchers, and a position paper by Dr. Marion Guillou, President-Director-General of INRA (Institut National de la Recherche Agronomique).

Plenary talks covered the following topics:

  • Commercialization and biosafety aspects of Bt and other insecticidal crops
  • Biosafety aspects of virus-resistant transgenic crops
  • Biosafety issues of the next generation of transgenic crops, including "pharma" crops
  • Strategies for biological confinement in plants
  • Effects of GM plants and GM inoculants on microbial communities
  • Commercialization and biosafety aspects of GM fish
  • Challenges for biosafety research in developing countries—a North-South Workshop
  • GMO regulations world wide
  • Approaches to discourse and communication on biosafety issues

This format offered a fertile environment for discussion and debate about current issues in environmental biosafety research. Several recurring themes emerged from the talks, posters, question-and-answer sessions, and workshops, as summarized below.

1) How can scientific research inform biosafety decisions?
Governmental regulators need clear answers to questions they are facing on a daily basis, but the empirical research needed to address these questions is often expensive, time-consuming, and/or inconclusive. Regulators and industry representatives are often expected to distinguish between what is "nice to know" and what they really need to know as soon as possible. The challenges of getting reliable answers are often magnified by inadequate scientific literature and a lack of scientific expertise, especially in developing countries. Also, as David Quist pointed out in a poster, priorities that are adopted by regulators can constrain the types of questions that are pursued by the scientific community, mainly through available funding, and this could be a disincentive to exploring unanticipated questions related to biosafety. Symposium participants offered suggestions for how to evaluate research priorities, how to form interdisciplinary research teams and communication networks, and how specific findings can be interpreted in the context of formal risk assessments. A lot of constructive discussion focused on the various goals and expectations of scientists, government agencies, the biotech industry, and the public.

2) How reliable are "negative results" indicating no environmental risks of GMOs?
Many presentations provided evidence that currently grown transgenic crops have no known negative effects on non-target species, wild relatives of crop plants, or microbial communities. Marc Fuchs stated that the benefits of virus-resistant transgenic squash far outweigh risks to the environment, and William Muir concluded that transgenic zebrafish sold as pets (GloFishTM) are not expected to be hazardous if they are introduced into aquatic habitats.

While these findings are welcome, researchers also acknowledged that all research methods have certain limitations. Ecological studies that are limited to a few small field sites and/or just a few field seasons could easily miss detecting effects of GMOs that occur over larger geographic scales and timeframes. Some questions, such as the potential for insects to evolve resistance to Bt crops, will only be resolved by post-commercial research and monitoring. Many speakers offered suggestions about how to weigh findings from laboratory experiments, field studies, and modeling exercises, each of which has intrinsic advantages and disadvantages. In addition, several speakers reminded the audience of statistical tests that should be used to minimize the chance of erroneously concluding that there is no effect of a GMO in a given study when in fact there is.

3) How can regulators in developing countries acquire scientific information for environmental risk assessment?
The symposium’s focus on developing and less-industrialized countries highlighted the economic, environmental, and health benefits that are expected to accrue from GMOs within the next decade. At the same time, the unique challenges of carrying out science-based risk assessments in these countries were also emphasized, especially in light of limited resources and emerging requirements of the Cartagena Protocol on Biosafety.

To address these issues, the symposium’s North-South Workshop started with a session on sources of support for biosafety research and explanations of key acronyms (e.g., CBD, UNEP-GEF and NBFs, ICGEB, and USAID-BBI, all of which can be deciphered in the Symposium Proceedings: http://www.isbr.info/). This was followed by series of research updates about studies of gene flow, Bt crops, herbicide resistance, and other topics in various countries. Eliana Fontes and Gabor Lovei discussed the philosophy and approach of the GMO Guidelines Project (http://www.gmo-guidelines.info) in Brazil and Kenya, respectively. Bernal Valverde talked about the possibility that farmers in Costa Rica might be confronted with glyphosate-resistant volunteer rice and weedy rice if anticipated stewardship guidelines are not followed. Atanas Atanassov discussed risk assessment studies in Bulgaria, and V. S. Siva Reddy summarized research on chloroplast transformation in India.

A number of talks presented advances in biosafety research in China, including Baorong Lu’s studies of gene flow in rice and Zhen Zhu’s work on insect-resistant rice. As a more detailed example, Kongming Wu discussed research on the refuge/high dose strategy for delaying of the evolution of resistance in target pests of Bt cotton, which was deregulated in China in 1997. For cotton bollworm, alternate host crops such as wheat, soybean, peanut, and corn can provide refuges for the maintenance of susceptible insects throughout the growing season. However, he reported that some larvae survive on Bt cotton in the late part of the growing season, indicating that a high dose is not being achieved. Also, Wu detected bollworms that were resistant to Cry1Ac at a frequency of ~0.00059 in Shandong Province. It is not known whether this frequency will increase, but yearly resistance monitoring has not detected a breakdown of susceptibility so far. These studies and others like them are crucial for understanding the long-term efficacy of Bt crops.

4) How far have we come and where is the field going?
Speakers often reflected on the accomplishments of the biosafety research community, especially in terms of peer-reviewed publications and capacity building. For example, Kornelia Smalla summarized a decade of research on soil microbial communities in which the effects of GM crops were found to be negligible compared to effects of soil type, plant species, plant developmental stages, or year-to-year variation. Charles Kessler noted that the European Community spent around US$100 million on biosafety research from 1985-2000. He stated that this research did not identify new risks of GM technology beyond the usual uncertainties and outcomes of conventional plant breeding.

Many new directions in biosafety research were discussed at the symposium, and the current focus on a few herbicide-resistant and Bt crops is likely to change. New types of GMOs that are in the pipeline include transgenic fish, insects, cats, livestock, viruses for biological control and "magnifection," pharmaceutical-producing crop plants, forage crops, horticultural crops, and trees. For some of these products, new methods of biological confinement are being investigated. With regard to confining gene flow from crops, speakers discussed the advantages and disadvantages of chloroplast transformation, apomixis, seed sterility, and transgenic mitigation of introgression. To gain a broader overview of some of the latest developments in environmental biosafety research, readers are encouraged to browse through the Symposium Proceedings, with extended abstracts from 61 invited plenary talks and 63 posters (available to the public at the ISBR website: http://www.isbr.info/).

Allison Snow
Ohio State University,
Columbus, OH
snow.1@osu.edu




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