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PubMed2005

Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome.

Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in Arabidopsis thaliana by genetic engineering. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry weight of the tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome.

Amino Acids↗

PubMed2003

Engineering tolerance and accumulation of lead and cadmium in transgenic plants.

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.

ATP-Binding Cassette Transporters↗

PubMed2004

Production of coumaroylserotonin and feruloylserotonin in transgenic rice expressing pepper hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase.

Transgenic rice (Oryza sativa) plants were engineered to express a N-(hydroxycinnamoyl)transferase from pepper (Capsicum annuum), which has been shown to have hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase activity, a key enzyme in the synthesis of hydroxycinnamic acid amides, under the control of constitutive maize (Zea mays) ubiquitin promoter. The transgenic rice plants require foliar application of amines to support synthesis of hydroxycinnamic acid amides, suggestive of limiting amine substrates in rice shoots. In addition, when T2 homozygous transgenic rice plants were grown in the presence of amines or phenolic acids, two novel compounds were exclusively identified in the leaves of the transgenic plants. These compounds eluted earlier than p-coumaroyltyramine and feruloyltyramine during HPLC chromatography and were identified as p-coumaroylserotonin and feruloylserotonin by liquid chromatography/mass spectrometry and other methods. To test whether the unpredicted production of serotonin derivatives is associated with the pepper N-(hydroxycinnamoyl)transferase, the substrate specificity of the pepper enzyme was analyzed again. Purified recombinant pepper N-(hydroxycinnamoyl)transferase exhibited a serotonin N-hydroxycinnamoyltransferase (SHT) activity, synthesized p-coumaroylserotonin and feruloylserotonin in vitro, and demonstrated a low K(m) for serotonin. SHT activity was inhibited by 10 to 50 mm tyramine. In addition, SHT activity was predominantly found in the root tissues of wild-type rice in parallel with the synthesis of serotonin derivatives, suggesting that serotonin derivatives are synthesized in the root of rice. This is the first report of SHT activity and the first demonstration, to our knowledge, that serotonin derivatives can be overproduced in vivo in transgenic rice plants that express serotonin N-(hydroxycinnamoyl)transferase.

Acyltransferases↗

PubMed2001

Production of 6-methylsalicylic acid by expression of a fungal polyketide synthase activates disease resistance in tobacco.

Salicylic acid (SA) has been shown to act as a signal molecule that is produced by many plants subsequent to the recognition of potentially pathogenic microbes. Increases in levels of SA often trigger the activation of plant defenses and can result in increased resistance to subsequent challenge by pathogens. We observed that the polyketide 6-methylsalicylic acid (6-MeSA), a compound that apparently is not endogenous to tobacco, can mimic SA. Tobacco leaves treated with 6-MeSA show enhanced accumulation of the pathogenesis-related (PR) proteins PR1, beta-1,3-glucanase, and chitinase and also develop increased resistance to tobacco mosaic virus. We transformed tobacco with 6msas, the 6-methylsalicylic acid synthase (6MSAS) gene from Penicillium patulum, to generate plants that constitutively accumulate 6-MeSA. Analysis of primary transformants and the first generation progeny of 6MSAS tobacco revealed that plants can be engineered to accumulate significant amounts of 6-MeSA as a conjugate. Levels of total 6-MeSA increased with plant age. Increased 6-MeSA accumulation correlated with increased levels of PR1 and chitinase proteins and resulted in enhanced resistance of NN genotype 6MSAS tobacco to tobacco mosaic virus. Our results demonstrate that a multistep biosynthetic pathway can be engineered into plants using a single fungal polyketide synthase gene. The functional expression of 6msas can be used to activate disease resistance pathways that normally are induced by SA.

Chitinases↗

PubMed2006

Genetic engineering of Nicotiana tabacum for reduced nornicotine content.

UNLABELLED: Nornicotine is an undesirable secondary alkaloid in cultivated tobacco, because it serves as a precursor to N'-nitrosonornicotine (NNN), a tobacco-specific nitrosamine with suspected carcinogenic properties. Nornicotine is produced through the oxidative N-demethylation of nicotine by a nicotine N-demethylase enzyme during the senescence and curing of tobacco leaves. While the nornicotine content of most commercial burley tobacco is low, a process termed "conversion" can bestow considerably increased nornicotine levels in a portion of the plants within the population. Previously, we isolated a nicotine N-demethylase gene, designated CYP82E4, and demonstrated that RNAi-induced silencing of CYP82E4 and its close homologues is an effective means for suppressing nicotine to nornicotine conversion. In this study, we used real-time polymerase chain reaction to confirm the central role of CYP82E4 in nicotine N-demethylation by demonstrating that the transcript accumulation of CYP82E4 is enhanced as much as 80-fold in converter vs nonconverter tobacco. We also show the design of an optimized RNAi construct (82E4Ri298) that suppressed nicotine to nornicotine conversion from 98% to as low as 0.8% in a strong converter tobacco line, a rate of nornicotine production that is about 3.6-fold lower than typically detected in commercial varieties. Southern blot analysis showed that a single copy of the RNAi transgene was as effective in suppressing nornicotine accumulation as multiple copies. Greenhouse-grown transgenic plants transformed with the RNAi construct were morphologically indistinguishable from the empty vector or wild-type controls. These results demonstrate that the genetic transformation of tobacco with the 82E4Ri298 construct is an effective strategy for reducing nornicotine and ultimately NNN levels in tobacco. KEYWORDS: Alkaloid; cytochrome P450; gene silencing; nicotine N-demethylase; N'-nitrosonornicotine; plant genetic engineering; metabolic engineering; Nicotiana tabacum L.; real-time PCR; RNA interference; tobacco-specific nitrosamines.

Cytochrome P-450 Enzyme System↗

PubMed1987

In vivo uncoating and efficient expression of foreign mRNAs packaged in TMV-like particles.

The ribonucleocapsids of many plant viruses are extremely stable. The protein coat protects the RNA genome against degradation during the accumulation and spread of progeny virions. Chimeric single-stranded RNA molecules were transcribed in vitro from recombinant plasmids and later encapsidated, in vitro, into ribonucleoprotein particles (pseudoviruses) 60 nanometers long that resembled tobacco mosaic virus. Transcripts encoding an assayable enzyme, chloramphenicol acetyltransferase (CAT), were packaged into pseudovirus particles to assess the utility of this single-stranded RNA delivery system in a wide range of cell types. In all cases, packaged CAT messenger RNA was uncoated and transiently expressed. Significantly higher levels of CAT activity were detected with packaged than with naked CAT messenger RNA after inoculation of plant protoplasts in the presence of polyethylene glycol or abrasive inoculation of intact leaf surfaces. Structural events that lead to the uncoating and expression of CAT messenger RNA showed no cell specificity. This observation may support the view that the comparatively restricted host range of a true plant virus results from events that occur later during the infection cycle.

Acetyltransferases↗

PubMed1996

[Genetic improvement of legumes].

Genetic improvement, a process aimed to obtain populations with a high frequency of desirable phenotypes, comprises both traditional and biotechnology approaches. Through these manipulations it is possible to address any problem, provided it is associated with a gene determined character. In the case of legume crops, some features have been changed such as yield, plant morphology, susceptibility to pathogens and other stress factors which reduce grain quality and output. In Venezuela an example of genetic improvement is represented by the domestication of Canavalia ensiformis (L) DC. working out some problems which limited its use as a crop, such as the shape and growing pattern, to obtain erect plants, and pod length to avoid its contact with the humid soil which accelerates rotting. Regarding the improvement of grain quality, genotypes have been found with low canavanine content.

Fabaceae↗

PubMed1985

Mortality of workers in an automobile engine and parts manufacturing complex.

A proportionate mortality ratio (PMR) study was conducted using data on workers from three local unions representing an integrated automobile factory composed of forge, foundry, and engine (machine and assembly) plants. Ninety four percent of the death certificates were obtained for all active and non-active workers who died during the period 1 January 1970 to 31 December 1979 and were vested in union and company benefit programmes. Observed numbers of deaths were compared with expected numbers based on two standards, the proportionate mortality among men in the United States 1970-9 and among men in Erie County 1975. There was close agreement between the number of observed and expected deaths by either standard of comparison among white auto workers in the forge and foundry plants. Valid analyses of cause specific mortality among non-whites could be conducted for the foundry plant only. Although there was raised PMR for deaths due to diseases of the circulatory system using the Erie County standard, none of the other cause specific PMRs was significant. Although based on small numbers, the risk of cancer of the lung was significantly high in non-whites under age 50 in the foundry (PMR = 2.6; p less than 0.05). The cause specific PMRs for whites in the engine plant were statistically significant for malignant neoplasms (1.2) and all external causes (0.62) based on the US white male standard. Analysis of cancer specific mortality among white men in the machining/assembly plant showed significant excesses for cancer of the digestive system (PMR=1.5), particularly of the liver (PMR=2.6) and pancreas (PMR=1.9); cancers of the respiratory system (PMR=1.4 using the Erie County standard); and cancer of the urinary bladder (PMR=2.3). Workers employed for more than 20 years showed statistically increased mortality ratios for cancers of the digestive system (1.9), particularly cancer of the pancreas (2.3) and cancer of the rectum (2.8). Individuals whose employment began during or before 1950 exhibited increased PMRs for cancers of the digestive organs (1.8), particularly of the pancreas (2.5) and of the bladder (3.4). Workers whose employment began after 1950, on the other hand, exhibited raised PMRs for cancers of the respiratory system (1.5) and of the kidney (3.2). Since the foundry and forge plants did not start production until 1955, mortality associated with those work settings may be greater in the future.

Adult↗

PubMed2001

Ribozyme genes protecting transgenic melon plants against potyviruses.

Potyviruses are the most important viral pathogens of crops worldwide. Under a contract with Gene Shears Pty Limited, we are using ribozyme genes to protect melon plants against two potyviruses: WMV2 and ZYMV. Different polyribozyme genes were designed, built and introduced into melons plants. Transgenic melon plants containing a resistance gene were obtained and their progeny was challenged by the appropriate virus. Most of the genes tested conferred some degree of resistance to the viruses in glasshouse trials. Melon plants from one family containing one gene directed against WMV2 were also field-trialed on small plots under natural infection pressure and were found immune to WMV2. Field trial is in progress for plants containing genes against ZYMV. Some of the ribozyme genes used in the plants were also assayed in a transient expression system in tobacco cells. This enabled us to study the sequence discrimination capacity of the ribozyme in the case of one ribozyme target site. We found that a mutated target GUG (non cleavable) was less susceptible to inhibition by the ribozyme gene than the corresponding wild type target GUA (cleavable). Work is now in progress to incorporate multiple resistance genes in melon plants, in constructs designed in compliance with the evolving European regulations concerning transgenic plants. The use of ribozyme genes to protect plants against viruses provides an alternative to the technologies currently used for protecting crops against viruses, based on the concept of Pathogen Derived Resistance (see for example 14). In the light of concerns expressed by some plant virologists (13) about the use of viral genes in transgenic plants, it may be that ribozyme genes will find many uses in this area of agricultural biotechnology.

Agriculture↗

PubMed1993

Plants transformed with a cistron of a potato virus Y protease (NIa) are resistant to virus infection.

An oligonucleotide carrying signals for translation initiation in plants was engineered upstream to a cDNA clone containing nucleotides 5812-7260 of the potato virus Y (PVY) genome. This fragment contains all but the first 100 5' terminal bases of the cistron encoding one of the PVY proteases (NIa) as well as the first 251 bases of the next cistron (NIb). Nicotiana tabacum cv. SR1 plants were transformed with this fragment. The presence of the NIa sequences in transformed plants was determined by hybridization or PCR, and its expression was ascertained by reverse transcription coupled to PCR. Plants expressing NIa were self-pollinated, and the R1 kanamycin-resistant progeny were rechecked for NIa expression. Several of these plants were found to be resistant to PVY infection, inasmuch as they did not develop symptoms for at least 50 days (the duration of the experiments), and no viral accumulation could be detected in their leaves by ELISA. All of the descendents of resistant homozygous R2 plants were also resistant. Several of the plants transformed with the last three cistrons of PVY (bases 5812-9704; NIa-NIb-coat protein) were also resistant to PVY. None of the transformed plants exhibited resistance to tobacco mosaic virus. Exposure of the plants to 35 degrees C for 48 hr prior to inoculation lowered, but did not abolish, resistance.

Base Sequence↗

PubMed1986

Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene.

A chimeric gene containing a cloned cDNA of the coat protein (CP) gene of tobacco mosaic virus (TMV) was introduced into tobacco cells on a Ti plasmid of Agrobacterium tumefaciens from which tumor inducing genes had been removed. Plants regenerated from transformed cells expressed TMV mRNA and CP as a nuclear trait. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for development of disease symptoms. The seedlings that expressed the CP gene were delayed in symptom development and 10 to 60 percent of the transgenic plants failed to develop symptoms for the duration of the experiments. Increasing the concentration of TMV in the inoculum shortened the delay in appearance of symptoms. The results of these experiments indicate that plants can be genetically transformed for resistance to virus disease development.

DNA↗

PubMed1986

The right border region of pTiT37 T-DNA is intrinsically more active than the left border region in promoting T-DNA transformation.

Deletions of border regions of T-DNA on Agrobacterium Ti plasmid or mini-T plasmid have shown the right border region of pTiT37 T-DNA to be more active than the left border region in promoting T-DNA transformation. In this study we examine the possibility that the apparent difference in activity of left and right border regions may be due to position or orientation differences between the left and right borders with respect to the transferred onc genes. We have constructed eight similar single-border mini-T plasmids that contain a left border segment or a right border segment of pTiT37 T-DNA at various positions with either orientation with respect to the onc genes. We assayed the plant tumor-inducing activity of these mini-T plasmids in Agrobacterium tumefaciens LBA4404 containing the virulence helper plasmid pAL4404. Regardless of the position and orientation of the border-containing segment in the mini-T plasmid, mini-T plasmids with the right border segment were highly virulent, whereas those with the left border segment were only weakly so. These results indicate that the difference in transformational activity between the left and right border regions is intrinsic and not an effect of position or orientation with respect to the onc genes. The pattern of the mini-T plasmid sequences integrated into the plant genome suggests that T-DNA transformation involves the directional transfer of a linear intermediate bounded by the border repeats.

Base Sequence↗

PubMed1997

Yeast 5-aminolevulinate synthase provides additional chlorophyll precursor in transgenic tobacco.

Synthesis of the tetrapyrrole precursor 5-aminolevulinate (ALA) in plants starts with glutamate and is a tRNA-dependent pathway consisting of three enzymatic steps localized in plastids. In animals and yeast, ALA is formed in a single step from succinyl CoA and glycine by aminolevulinate synthase (ALA-S) in mitochondria. A gene encoding a fusion protein of yeast ALA-S with an aminoterminal transit sequence for the small subunit of ribulose bisphosphate carboxylase was introduced into the genome of wild-type tobacco and a chlorophyll-deficient transgenic line expressing glutamate 1-semi-aldehyde aminotransferase (GSA-AT) antisense RNA. Expression of ALA-S in the GSA-AT antisense transgenic line provided green-pigmented co-transformants similar to wild-type in chlorophyll content, while transformants derived from wild-type plants did not show phenotypical changes. The capacity to synthesize ALA and chlorophyll was increased in transformed plants, indicating a contribution of ALA-S to the ALA supply for chlorophyll synthesis. ALA-S activity was detected in plastids of the transformants. Preliminary evidence is presented that succinyl CoA, the substrate for ALA-S, can be synthesized and metabolized in plastids. The transgenic plants formed chlorophyll in the presence of gabaculine, an inhibitor of GSA-AT. Steady-state RNA and protein levels and consequently, the enzyme activity of GSA-AT were reduced in plants expressing ALA-S. In analogy to the light-dependent ALA synthesis attributed to feedback regulation, a mechanism at the level of intermediates or tetrapyrrole end-products is proposed, which co-ordinates the need for heme and chlorophyll precursors and restricts synthesis of ALA by regulating GSA-AT gene expression. The genetically engineered tobacco plants containing the yeast ALA-S activity demonstrate functional complementation of the catalytic activity of the plant ALA-synthesizing pathway and open strategies for producing tolerance against inhibitors of the C5 pathway.

5-Aminolevulinate Synthetase↗

PubMed2000

Analysis of the activity of promoters from two photosynthesis-related genes psaF and petH of spinach in a monocot plant, rice.

The subunit III of photosystem I and ferredoxin-NADP(+)-oxidoreductase are encoded by nuclear genes, namely psaF and petH. The activity of their promoters from spinach has been evaluated in transgenic tobacco earlier. Evaluation of the activity of these Dicotyledoneae-specific promoters has been carried out in a monocot system (i.e. rice) by transient gene expression system, based on electroporation-mediated gene delivery into protoplasts from leaves and roots. It has been found that various promoter deletions show higher activity in leaf protoplasts and elements for quantitative response are widely distributed. Transgenic rice has also been produced with a petH promoter and gus reporter gene construct. Although petH promoter is a weak promoter in comparison to the 35S promoter, it expresses well in green tissues and could be useful for plant genetic engineering.

Bacterial Proteins↗

PubMed2004

[Purification and cloning of an antifungal protein from the rice diseases controlling bacterial strain Paenibacillus polymyxa WY110].

Paenibacillus polymyxa WY110, a plant growth-promoting bacteria strain isolated from rice rhizosphere could suppress the growth of various plant pathogens effectively. With (NH4)2SO4 fractional precipitation, DEAE-Sephadex A-50 chromatography and Sephacryl S-200 chromatography followed by tracks of fraction antagonistic assay and SDS-PAGE, an antifungal protein P2 with in vitro anti-Pyricularia oxyzae activity was isolated and purified. It was showed with antagonistic activity on PDA plates that the growth of Pyricularia oryzae was inhibited by 1.5 microg of P2 protein effectively. N-terminal amino acid residues analysis showed 24 amino acid sequence: H2N-Ala-Asn-Val-Phe-Trp-Glu-Pro-Leu-Ser-Tyr-Tyr-Asn-Pro-Ser-Thr-Trp-Gln-Lys-Ala-Asp-Gly-Tyr-Ser-Asn-. Using this amino acid sequence as a target, the similarity of P2 protein was searched with BlastP program on Internet. It was showed a high homology between the P2 protein and the precursors of beta-1, 3-1, 4-glucanases from Bacillus. The beta-1, 3-1, 4-glucanase activity of P2 protein was identified with the specific substrate lichenan. According to the N-terminal partial sequence of P2 protein and the C-terminal conserved sequence of beta-1, 3-1, 4-glucanase, the primers for both terminals were synthesized. Using the genomic DNA of WY110 as the template, the full-length sequence of the gene encoding P2 was amplified by high fidelity PCR, then cloned into pMD18-T vecter. Sequence analysis showed the 72 nucleotide sequence on 5'-end matched with the known 24 amino acid sequence on N-terminal of P2 protein. The sequence (GenBank Accession Number: AF284449) was 636 bp in length encoding 212 amino acids. Comparing with a beta-1, 3-1, 4-glucanase gene (gluB) from Paenibacillus polymyxa, the sequence homology for nucleotides and deduced amino acids were 84% and 88.7% respectively. The cloning of the gene encoding P2 protein would be a new potential objective gene for plant gene engineering.

Amino Acid Sequence↗

PubMed2006

In situ methods to localize transgenes and transcripts in interphase nuclei: a tool for transgenic plant research.

Genetic engineering of commercially important crops has become routine in many laboratories. However, the inability to predict where a transgene will integrate and to efficiently select plants with stable levels of transgenic expression remains a limitation of this technology. Fluorescence in situ hybridization (FISH) is a powerful technique that can be used to visualize transgene integration sites and provide a better understanding of transgene behavior. Studies using FISH to characterize transgene integration have focused primarily on metaphase chromosomes, because the number and position of integration sites on the chromosomes are more easily determined at this stage. However gene (and transgene) expression occurs mainly during interphase. In order to accurately predict the activity of a transgene, it is critical to understand its location and dynamics in the three-dimensional interphase nucleus. We and others have developed in situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. Furthermore, this approach is useful for studying nuclear organization and the dynamics of genes and chromatin.

Journal Article↗

PubMed2002

Targeting tryptophan decarboxylase to selected subcellular compartments of tobacco plants affects enzyme stability and in vivo function and leads to a lesion-mimic phenotype.

Tryptophan decarboxylase (TDC) is a cytosolic enzyme that catalyzes an early step of the terpenoid indole alkaloid biosynthetic pathway by decarboxylation of L-tryptophan to produce the protoalkaloid tryptamine. In the present study, recombinant TDC was targeted to the chloroplast, cytosol, and endoplasmic reticulum (ER) of tobacco (Nicotiana tabacum) plants to evaluate the effects of subcellular compartmentation on the accumulation of functional enzyme and its corresponding enzymatic product. TDC accumulation and in vivo function was significantly affected by the subcellular localization. Immunoblot analysis demonstrated that chloroplast-targeted TDC had improved accumulation and/or stability when compared with the cytosolic enzyme. Because ER-targeted TDC was not detectable by immunoblot analysis and tryptamine levels found in transient expression studies and in transgenic plants were low, it was concluded that the recombinant TDC was most likely unstable if ER retained. Targeting TDC to the chloroplast stroma resulted in the highest accumulation level of tryptamine so far reported in the literature for studies on heterologous TDC expression in tobacco. However, plants accumulating high levels of functional TDC in the chloroplast developed a lesion-mimic phenotype that was probably triggered by the relatively high accumulation of tryptamine in this compartment. We demonstrate that subcellular targeting may provide a useful strategy for enhancing accumulation and/or stability of enzymes involved in secondary metabolism and to divert metabolic flux toward desired end products. However, metabolic engineering of plants is a very demanding task because unexpected, and possibly unwanted, effects may be observed on plant metabolism and/or phenotype.

Aromatic-L-Amino-Acid Decarboxylases↗

PubMed2004

Peanut stripe potyvirus resistance in peanut (Arachis hypogaea L.) plants carrying viral coat protein gene sequences.

Peanut (Arachis hypogaea L.) lines exhibiting high levels of resistance to peanut stripe virus (PStV) were obtained following microprojectile bombardment of embryogenic callus derived from mature seeds. Fertile plants of the commercial cultivars Gajah and NC7 were regenerated following co-bombardment with the hygromycin resistance gene and one of two forms of the PStV coat protein (CP) gene, an untranslatable, full length sequence (CP2) or a translatable gene encoding a CP with an N-terminal truncation (CP4). High level resistance to PStV was observed for both transgenes when plants were challenged with the homologous virus isolate. The mechanism of resistance appears to be RNA-mediated, since plants carrying either the untranslatable CP2 or CP4 had no detectable protein expression, but were resistant or immune (no virus replication). Furthermore, highly resistant, but not susceptible CP2 T0 plants contained transgene-specific small RNAs. These plants now provide important germplasm for peanut breeding, particularly in countries where PStV is endemic and poses a major constraint to peanut production.

Arachis↗