An Efficient Stevia rebaudiana Transformation System and In vitro Enzyme Assays Reveal Novel Insights into UGT76G1 Function.

Stevia rebaudiana (Bertoni) is one of a very few plant species that produce zero calorie, sweet compounds known as steviol glycosides (SG). SGs differ in their sweetness and organoleptic properties depending on the number and positioning of sugar groups on the core steviol backbone. There is great interest of modulating the SG profiles of the Stevia plant to enhance the flavor profile for a given application in the food and beverage industries. Here, we report a highly efficient Agrobacterium-mediated stable transformation system using axillary shoots as the initial explant. Using this system, we generated over 200 transgenic Stevia plants overexpressing a specific isoform of UGT76G1. By comparing the SG profiles among independent transgenic events, we demonstrated that altering UGT76G1 expression can change the ratios of specific SG species. Furthermore, using recombinant proteins produced in E. coli, we show that two closely related UGT76G1 isoforms differ in their substrate specificities, providing new insights into mechanisms underlying the diversity of SG profiles that are observed across Stevia germplasm. Finally, we found evidence suggesting that alternative and/or aberrant splicing may serve to influence the ability of the plant to produce functional UGT76G1 transcripts, and possibly produce enzyme variants within the plant.

Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus.

BACKGROUND: Large T-DNA fragment transfer has long been a problem for Agrobacterium-mediated transformation. Although vector systems, such as the BIBAC series, were successfully developed for the purpose, low transformation efficiencies were consistently observed. RESULTS: To gain insights of this problem in monocot transformation, we investigated the T-strand accumulation of various size of T-DNA in two kinds of binary vectors (one copy vs. multi-copy) upon acetosyringone (AS) induction and explored ways to improve the efficiency of the large T-DNA fragment transfer in Agrobacterium-mediated rice transformation. By performing immuno-precipitation of VirD2-T-strands and quantitative real-time PCR assays, we monitored the accumulation of the T-strands in Agrobacterium tumeficiens after AS induction. We further demonstrated that extension of AS induction time highly significantly improved large-size T-DNA transfer to rice cells. CONCLUSIONS: Our data provide valuable information of the T-strand dynamics and its impact on large T-DNA transfer in monocots, and likely dicots as well.

Cholesterol accumulation by suppression of SMT1 leads to dwarfism and improved drought tolerance in herbaceous plants.

Dwarfism and drought tolerance are 2 valuable traits in breeding of many crops. In this study, we report the novel physiological roles of cholesterol in regulation of plant growth and drought tolerance. Compared with the wild type, sterol-C24-methyltransferase 1 (SMT1) gene transcript was greatly reduced in a bermudagrass mutant with dwarfism and enhanced drought tolerance, accompanied with cholesterol accumulation, elevated transcript levels of a small group of genes including SAMDC, and increased concentrations of putrescine (Put), spermidine (Spd), and spermine (Spm). Knock-down of OsSMT1 expression by RNA interference resulted in similar phenotypic changes in transgenic rice. Moreover, exogenously applied cholesterol also led to elevated transcripts of a similar set of genes, higher levels of Put, Spd, and Spm, improved drought tolerance, and reduced plant height in both bermudagrass and rice. We revealed that it is Spm, but not Spd, that is responsible for the height reduction in bermudagrass and rice. In conclusion, we suggest that cholesterol induces expression of SAMDC and leads to dwarfism and elevated drought tolerance in plants as a result of the promoted Spd and Spm synthesis.

Effects of overexpression of jasmonic acid biosynthesis genes on nicotine accumulation in tobacco.

Nicotine is naturally synthesized in tobacco roots and accumulates in leaves as a defense compound against herbivory attack. Nicotine biosynthesis pathway has been extensively studied with major genes and enzymes being isolated and functionally characterized. However, the molecular regulation of nicotine synthesis has not been fully understood. The phytohormone jasmonic acid (JA) mediates many aspects of plant defense responses including nicotine biosynthesis. In this study, five key genes (AtLOX2, AtAOS, AtAOC2, AtOPR3, AtJAR1) involved in JA biosynthesis from Arabidopsis were individually overexpressed, and a JA-Ile hydrolysis-related gene, NtJIH1, was suppressed by RNAi approach, to understand their effects on nicotine accumulation in tobacco. Interestingly, while transgene expression was high, levels of JA-Ile (the biologically active form of JA) were often significantly reduced. Meanwhile, nicotine content in these transgenic plants did not increase. The research revealed a tightly controlled JA signaling pathway and a complicated regulatory network for nicotine biosynthesis by JA signaling.

Evaluation of parameters affecting switchgrass tissue culture: toward a consolidated procedure for Agrobacterium-mediated transformation of switchgrass (Panicum virgatum).

BACKGROUND: Switchgrass (Panicum virgatum), a robust perennial C4-type grass, has been evaluated and designated as a model bioenergy crop by the U.S. DOE and USDA. Conventional breeding of switchgrass biomass is difficult because it displays self-incompatible hindrance. Therefore, direct genetic modifications of switchgrass have been considered the more effective approach to tailor switchgrass with traits of interest. Successful transformations have demonstrated increased biomass yields, reduction in the recalcitrance of cell walls and enhanced saccharification efficiency. Several tissue culture protocols have been previously described to produce transgenic switchgrass lines using different nutrient-based media, co-cultivation approaches, and antibiotic strengths for selection. RESULTS: After evaluating the published protocols, we consolidated these approaches and optimized the process to develop a more efficient protocol for producing transgenic switchgrass. First, seed sterilization was optimized, which led to a 20% increase in yield of induced calluses. Second, we have selected a N6 macronutrient/B5 micronutrient (NB)-based medium for callus induction from mature seeds of the Alamo cultivar, and chose a Murashige and Skoog-based medium to regenerate both Type I and Type II calluses. Third, Agrobacterium-mediated transformation was adopted that resulted in 50-100% positive regenerated transformants after three rounds (2 weeks/round) of selection with antibiotic. Genomic DNA PCR, RT-PCR, Southern blot, visualization of the red fluorescent protein and histochemical ß-glucuronidase (GUS) staining were conducted to confirm the positive switchgrass transformants. The optimized methods developed here provide an improved strategy to promote the production and selection of callus and generation of transgenic switchgrass lines. CONCLUSION: The process for switchgrass transformation has been evaluated and consolidated to devise an improved approach for transgenic switchgrass production. With the optimization of seed sterilization, callus induction, and regeneration steps, a reliable and effective protocol is established to facilitate switchgrass engineering.

Accumulation of medium-chain, saturated fatty acyl moieties in seed oils of transgenic Camelina sativa.

With its high seed oil content, the mustard family plant Camelina sativa has gained attention as a potential biofuel source. As a bioenergy crop, camelina has many advantages. It grows on marginal land with low demand for water and fertilizer, has a relatively short life cycle, and is stress tolerant. As most other crop seed oils, camelina seed triacylglycerols (TAGs) consist of mostly long, unsaturated fatty acyl moieties, which is not desirable for biofuel processing. In our efforts to produce shorter, saturated chain fatty acyl moieties in camelina seed oil for conversion to jet fuel, a 12:0-acyl-carrier thioesterase gene, UcFATB1, from California bay (Umbellularia californica Nutt.) was expressed in camelina seeds. Up to 40% of short chain laurate (C12:0) and myristate (C14:0) were present in TAGs of the seed oil of the transgenics. The total oil content and germination rate of the transgenic seeds were not affected. Analysis of positions of these two fatty acyl moieties in TAGs indicated that they were present at the sn-1 and sn-3 positions, but not sn-2, on the TAGs. Suppression of the camelina KASII genes by RNAi constructs led to higher accumulation of palmitate (C16:0), from 7.5% up to 28.5%, and further reduction of longer, unsaturated fatty acids in seed TAGs. Co-transformation of camelina with both constructs resulted in enhanced accumulation of all three medium-chain, saturated fatty acids in camelina seed oils. Our results show that a California bay gene can be successfully used to modify the oil composition in camelina seed and present a new biological alternative for jet fuel production.

Control of brown patch (Rhizoctonia solani) in tall fescue (Festuca arundinacea Schreb.) by host induced gene silencing.

KEY MESSAGE: Transgenic tall fescue plants expressing RNAi constructs of essential genes of Rhizoctonia solani were resistant to R. solani. Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species widely used for home lawns and on golf courses in North Carolina and other transition zone states in the US. The most serious and frequently occurring disease of tall fescue is brown patch, caused by a basidiomycete fungus, Rhizoctonia solani. This research demonstrates resistance to brown patch disease achieved by the application of host induced gene silencing. We transformed tall fescue with RNAi constructs of four experimentally determined "essential" genes from R. solani (including genes encoding RNA polymerase, importin beta-1 subunit, Cohesin complex subunit Psm1, and a ubiquitin E3 ligase) to suppress expression of those genes inside the fungus and thus inhibit fungal infection. Four gene constructs were tested, and 19 transgenic plants were obtained, among which 12 plants had detectable accumulation of siRNAs of the target genes. In inoculation tests, six plants displayed significantly improved resistance against R. solani. Lesion size was reduced by as much as 90 %. Plants without RNAi accumulation did not show resistance. To our knowledge, this is the first case that RNAi constructs of pathogen genes introduced into a host plant can confer resistance against a necrotrophic fungus.

Genetic Factors for Enhancement of Nicotine Levels in Cultivated Tobacco.

Nicotine has practical applications relating to smoking cessation devices and alternative nicotine products. Genetic manipulation for increasing nicotine content in cultivated tobacco (Nicotiana tabacum L.) may be of value for industrial purposes, including the possibility of enhancing the efficiency of nicotine extraction. Biotechnological approaches have been evaluated in connection with this objective, but field-based results are few. Here, we report characterization of two genes encoding basic-helix-loop-helix (bHLH) transcription factors (TFs), NtMYC2a and NtMYC2b from tobacco. Overexpression of NtMYC2a increased leaf nicotine levels in T1 transgenic lines approximately 2.3-fold in greenhouse-grown plants of tobacco cultivar 'NC 95'. Subsequent field testing of T2 and T3 generations of transgenic NtMYC2a overexpression lines showed nicotine concentrations were 76% and 58% higher than control lines, respectively. These results demonstrated that the increased nicotine trait was stably inherited to the T2 and T3 generations, indicating the important role that NtMYC2a plays in regulating nicotine accumulation in N. tabacum and the great potential of NtMYC2a overexpression in tobacco plants for industrial nicotine production. Collected data in this study also indicated a negative feedback inhibition of nicotine biosynthesis. Further enhancement of nicotine accumulation in tobacco leaf may require modification of the processes of nicotine transport and deposition.

A photorespiratory bypass increases plant growth and seed yield in biofuel crop Camelina sativa.

BACKGROUND: Camelina sativa is an oilseed crop with great potential for biofuel production on marginal land. The seed oil from camelina has been converted to jet fuel and improved fuel efficiency in commercial and military test flights. Hydrogenation-derived renewable diesel from camelina is environmentally superior to that from canola due to lower agricultural inputs, and the seed meal is FDA approved for animal consumption. However, relatively low yield makes its farming less profitable. Our study is aimed at increasing camelina seed yield by reducing carbon loss from photorespiration via a photorespiratory bypass. Genes encoding three enzymes of the Escherichia coli glycolate catabolic pathway were introduced: glycolate dehydrogenase (GDH), glyoxylate carboxyligase (GCL) and tartronic semialdehyde reductase (TSR). These enzymes compete for the photorespiratory substrate, glycolate, convert it to glycerate within the chloroplasts, and reduce photorespiration. As a by-product of the reaction, CO2 is released in the chloroplast, which increases photosynthesis. Camelina plants were transformed with either partial bypass (GDH), or full bypass (GDH, GCL and TSR) genes. Transgenic plants were evaluated for physiological and metabolic traits. RESULTS: Expressing the photorespiratory bypass genes in camelina reduced photorespiration and increased photosynthesis in both partial and full bypass expressing lines. Expression of partial bypass increased seed yield by 50-57 %, while expression of full bypass increased seed yield by 57-73 %, with no loss in seed quality. The transgenic plants also showed increased vegetative biomass and faster development; they flowered, set seed and reached seed maturity about 1 week earlier than WT. At the transcriptional level, transgenic plants showed differential expression in categories such as respiration, amino acid biosynthesis and fatty acid metabolism. The increased growth of the bypass transgenics compared to WT was only observed in ambient or low CO2 conditions, but not in elevated CO2 conditions. CONCLUSIONS: The photorespiratory bypass is an effective approach to increase photosynthetic productivity in camelina. By reducing photorespiratory losses and increasing photosynthetic CO2 fixation rates, transgenic plants show dramatic increases in seed yield. Because photorespiration causes losses in productivity of most C3 plants, the bypass approach may have significant impact on increasing agricultural productivity for C3 crops.

A novel gateway-compatible binary vector series (PC-GW) for flexible cloning of multiple genes for genetic transformation of plants.

The rapidly advancing field of plant synthetic biology requires transforming plants with multiple genes. This has sparked a growing interest in flexible plant transformation vectors, which can be used for multi-gene transformations. We have developed a novel binary vector series, named the PC-GW series (GenBank: KP826769-KP826773), for Agrobacterium-mediated plant transformation. The PC-GW vectors use the pCAMBIA vector backbone, and contain NPTII, hpt, bar, mCherry or egfp genes as selectable markers for plant transformation. In a modified multiple cloning site (MCS) of the T-DNA region, we have placed the attR1, attR2 and ccdB sequences for rapid cloning of one to four genes by Gateway™-assisted recombination. In addition, we have introduced four meganuclease sites, and other restriction sites for multi-gene vector construction. Finally, we have placed a CaMV 35S promoter and a 35S terminator on the 5' and 3' ends of the MCS. The CaMV 35S promoter is flanked by PstI restriction sites that can be used to replace it with another promoter sequence if needed. The PC-GW vectors provide choices for selectable markers, cloning methods, and can accommodate up to eight gene constructs in a single T-DNA, thereby significantly reducing the number of transformations or crosses needed to generate multi-transgene expressing plants.

Overexpression of ubiquitin-like LpHUB1 gene confers drought tolerance in perennial ryegrass.

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin-like post-translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate-controlled growth chambers. Here, pasture-type and turf-type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.

Plant regeneration and genetic transformation of C. canadensis: a non-model plant appropriate for investigation of flower development in Cornus (Cornaceae).

KEY MESSAGE : Efficient Agrobacterium -mediated genetic transformation for investigation of genetic and molecular mechanisms involved in inflorescence architectures in Cornus species. Cornus canadensis is a subshrub species in Cornus, Cornaceae. It has recently become a favored non-model plant species to study genes involved in development and evolution of inflorescence architectures in Cornaceae. Here, we report an effective protocol of plant regeneration and genetic transformation of C. canadensis. We use young inflorescence buds as explants to efficiently induce calli and multiple adventitious shoots on an optimized induction medium consisting of basal MS medium supplemented with 1 mg/l of 6-benzylaminopurine and 0.1 mg/l of 1-naphthaleneacetic acid. On the same medium, primary adventitious shoots can produce a large number of secondary adventitious shoots. Using leaves of 8-week-old secondary shoots as explants, GFP as a reporter gene controlled by 35S promoter and hygromycin B as the selection antibiotic, a standard procedure including pre-culture of explants, infection, co-cultivation, resting and selection has been developed to transform C. canadensis via Agrobacterium strain EHA105-mediated transformation. Under a strict selection condition using 14 mg/l hygromycin B, approximately 5 % explants infected by Agrobacterium produce resistant calli, from which clusters of adventitious shoots are induced. On an optimized rooting medium consisting of basal MS medium supplemented with 0.1 mg/l of indole-3-butyric acid and 7 mg/l hygromycin B, most of the resistant shoots develop adventitious roots to form complete transgenic plantlets, which can grow normally in soil. RT-PCR analysis demonstrates the expression of GFP transgene. Green fluorescence emitted by GFP is observed in transgenic calli, roots and cells of transgenic leaves under both stereo fluorescence microscope and confocal microscope. The success of genetic transformation provides an appropriate platform to investigate the molecular mechanisms by which the various inflorescence forms are developed in Cornus plants.

Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses.

Plant defense responses can lead to altered metabolism and even cell death at the sites of Agrobacterium infection, and thus lower transformation frequencies. In this report, we demonstrate that the utilization of culture conditions associated with an attenuation of defense responses in monocot plant cells led to highly improved Agrobacterium-mediated transformation efficiencies in perennial ryegrass (Lolium perenne L.). The removal of myo-inositol from the callus culture media in combination with a cold shock pretreatment and the addition of L-Gln prior to and during Agrobacterium-infection resulted in about 84 % of the treated calluses being stably transformed. The omission of myo-inositol from the callus culture media was associated with the failure of certain pathogenesis related genes to be induced after Agrobacterium infection. The addition of a cold shock and supplemental Gln appeared to have synergistic effects on infection and transformation efficiencies. Nearly 60 % of the stably transformed calluses regenerated into green plantlets. Calluses cultured on media lacking myo-inositol also displayed profound physiological and biochemical changes compared to ones cultured on standard growth media, such as reduced lignin within the cell walls, increased starch and inositol hexaphosphate accumulation, enhanced Agrobacterium binding to the cell surface, and less H(2)O(2) production after Agrobacterium infection. Furthermore, the cold treatment greatly reduced callus browning after infection. The simple modifications described in this report may have broad application for improving genetic transformation of recalcitrant monocot species.

Sodium hydroxide pretreatment of genetically modified switchgrass for improved enzymatic release of sugars.

Overcoming biomass recalcitrance to bioconversion is crucial for cellulosic biofuels commercialization. In this study, Alamo switchgrass (Panicum virgatum L.) was genetically transformed to suppress the expression of 4-coumarate-CoA ligase (4CL). The transgenic plants were determined to have lignin content reductions of up to 5.8%. The ratios of acid soluble lignin (ASL) to acid insoluble lignin (AIL) and syringyl/guaiacyl (S/G) in transgenic plants were 21.4-64.3% and 11.8-164.5%, respectively, higher than those of conventional biomass. Both conventional and transgenic plants were pretreated with 0.5%, 1%, and 2% (w/v) NaOH for 15, 30, and 60min at 121°C, followed by enzymatic hydrolysis with commercial cellulases and xylanases. At the optimal conditions, the glucan and xylan conversion efficiency in the best transgenic plants were 16% and 18% higher than the conventional plant, respectively. The results show that down-regulation of 4CL gene promoted enzymatic hydrolysis of plant cell walls following a mild alkali pretreatment.

Dilute sulfuric acid pretreatment of transgenic switchgrass for sugar production.

Conventional Alamo switchgrass and its transgenic counterparts with reduced/modified lignin were subjected to dilute sulfuric acid pretreatment for improved sugar production. At 150 °C, the effects of acid concentration (0.75%, 1%, 1.25%) and residence time (5, 10, 20, 30 min) on sugar productions in pretreatment and enzymatic hydrolysis were investigated, with the optimal pretreatment conditions determined for each switchgrass genotype based on total sugar yield and the amounts of sugar degradation products generated during the pretreatment. The results show that genetic engineering, although did not cause an appreciable lignin reduction, resulted in a substantial increase in the ratio of acid soluble lignin:acid insoluble lignin, which led to considerably increased sugar productions in both pretreatment and enzymatic hydrolysis. At an elevated threshold concentration of combined 5-hydroxyfuranmethal and furfural (2.0 g/L), the overall carbohydrate conversions of conventional switchgrass and its transgenic counterparts, 10/9-40 and 11/5-47, reached 75.9%, 82.6%, and 82.2%, respectively.

Protamine-mediated DNA coating remarkably improves bombardment transformation efficiency in plant cells.

We have developed a method by which remarkably higher efficiencies of transient and stable transformation were achieved in bombardment transformation of plants. Over fivefold increase in transient gus gene expression was achieved when rice or maize suspension cells were bombarded with gold particles coated with plasmid DNA in the presence of protamine instead of the conventional spermidine. A 3.3-fold improvement in stable transformation efficiency was also observed using rice suspension cells with the new coating approach. The coated protamine-plasmid DNA complex resisted degradation by a DNase or by rice cell extract much longer than the spermidine-plasmid DNA complex. The results from this study suggest that protamine protects plasmid DNA longer than spermidine when being delivered inside the cells, probably by forming a nano-scale complex, and thus helps improve the efficiency of particle bombardment-mediated plant transformation.

Activity of the 5' regulatory regions of the rice polyubiquitin rubi3 gene in transgenic rice plants as analyzed by both GUS and GFP reporter genes.

Ubiquitin is an abundant protein involved in protein degradation and cell cycle control in plants and rubi3 is a polyubiquitin gene isolated from rice (Oryza sativa L.). Using both GFP and GUS as reporter genes, we analyzed the expression pattern of the rubi3 promoter as well as the effects of the rubi3 5'-UTR (5' untranslated region) intron and the 5' terminal 27 bp of the rubi3 coding sequence on the activity of the promoter in transgenic rice plants. The rubi3 promoter with the 5'-UTR intron was active in all the tissue and cell types examined and supported more constitutive expression of reporter genes than the maize Ubi-1 promoter. The rubi3 5'-UTR intron mediated enhancement on the activity of its promoter in a tissue-specific manner but did not alter its overall expression pattern. The enhancement was particularly intense in roots, pollen grains, inner tissue of ovaries, and embryos and aleurone layers in maturing seeds. The translational fusion of the first 27 bp of the rubi3 coding sequence to GUS gene further enhanced GUS expression directed by the rubi3 promoter in all the tissues examined. The rubi3 promoter should be an important addition to the arsenal of strong and constitutive promoters for monocot transformation and biotechnology.

Gene expression enhancement mediated by the 5' UTR intron of the rice rubi3 gene varied remarkably among tissues in transgenic rice plants.

Introns are important sequence elements that modulate the expression of genes. Using the GUS reporter gene driven by the promoter of the rice (Oryza sativa L.) polyubiquitin rubi3 gene, we investigated the effects of the 5' UTR intron of the rubi3 gene and the 5' terminal 27 bp of the rubi3 coding sequence on gene expression in stably transformed rice plants. While the intron enhanced GUS gene expression, the 27-bp fused to the GUS coding sequence further augmented GUS expression level, with both varying among different tissues. The intron elevated GUS gene expression mainly at mRNA accumulation level, but also stimulated enhancement at translational level. The enhancement on mRNA accumulation, as determined by realtime quantitative RT-PCR, varied remarkably with tissue type. The augmentation by the intron at translational level also differed by tissue type, but to a lesser extent. On the other hand, the 27-bp fusion further boosted GUS protein yield without affecting mRNA accumulation level, indicating stimulation at translation level, which was also affected by tissue type. The research revealed substantial variation in the magnitudes of intron-mediated enhancement of gene expression (IME) among tissues in rice plants and the importance of using transgenic plants for IME studies.

Transcriptional and post-transcriptional enhancement of gene expression by the 5' UTR intron of rice rubi3 gene in transgenic rice cells.

Introns play a very important role in regulating gene expression in eukaryotes. In plants, many introns enhance gene expression, and the effect of intron-mediated enhancement (IME) of gene expression is reportedly often more profound in monocots than in dicots. To further gain insight of IME in monocot plants, we quantitatively dissected the effect of the 5' UTR intron of the rice rubi3 gene at various gene expression levels in stably transformed suspension cell lines. The intron enhanced the GUS reporter gene activity in these lines by about 29-fold. Nuclear run-on experiments demonstrated a nearly twofold enhancement by the 5' UTR intron at the transcriptional level. RNA analysis by RealTime quantitative RT-PCR assays indicated the intron enhanced the steady state RNA level of the GUS reporter gene by nearly 20-fold, implying a strong role of the intron in RNA processing and/or export. The results also implicated a moderate role of the intron in enhancement at the translational level ( approximately 45%). Moreover, results from a transient assay experiment using a shortened exon 1 sequence revealed an important role of exon 1 of rubi3 in gene expression. It may also hint a divergence in IME mechanisms between plant and animal cells. These results demonstrated transcriptional enhancement by a plant intron, but suggested that post-transcriptional event(s) be the major source of IME.

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases.

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.