Transgenic cucumber plants harboring a rice chitinase gene exhibit enhanced resistance to gray mold (Botrytis cinerea)

A rice chitinase cDNA (RCC2) driven by the CaMV 35S promoter was introduced into cucumber (Cucumis sativus L.) through Agrobacterium mediation. More than 200 putative transgenic shoots were regenerated and grown on MS medium supplemented with 100 mg/l kanamycin. Sixty elongated shoots were examined for the presence of the integrated RCC2 gene and subsequently confirmed to have it. Of these, 20 were tested for resistance against gray mold (Botrytis cinerea) by infection with the conidia: 15 strains out of the 20 independent shoots exhibited a higher resistance than the control (non-transgenic plants). Three transgenic cucumber strains (designated CR29, CR32 and CR33) showed the highest resistance against B. cinerea: the spread of disease was inhibited completely in these strains. Chitinase gene expression in highly resistant transgenic strains (CR32 and CR33) was compared to that of a susceptible transgenic strain (CR20) and a control. Different responses for disease resistance were observed among the highly resistant strains. CR33 inhibited appressoria formation and penetration of hyphae. Although CR32 permitted penetration of hyphae, invasion of the infection hyphae was restricted. Furthermore, progenies of CR32 showed a segregation ratio of 3:1 (resistant:susceptible). As the disease resistance against gray mold was confirmed to be inheritable, these highly resistant transgenic cucumber strains would serve as good breeding materials for disease resistance. Received: 31 March 1996 / Revision received: 2 July 1997 / Accepted: 18 July 1997     

Transgenic Arabidopsis thaliana expressing a barley UDP-glucosyltransferase exhibit resistance to the mycotoxin deoxynivalenol

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of small grain cereal crops. FHB causes yield reductions and contamination of grain with trichothecene mycotoxins such as deoxynivalenol (DON). DON inhibits protein synthesis in eukaryotic cells and acts as a virulence factor during fungal pathogenesis, therefore resistance to DON is considered an important component of resistance against FHB. One mechanism of resistance to DON is conversion of DON to DON-3-O-glucoside (D3G). Previous studies showed that expression of the UDP-glucosyltransferase genes HvUGT13248 from barley and AtUGt73C5 (DOGT1) from Arabidopsis thaliana conferred DON resistance to yeast. Over-expression of AtUGt73C5 in Arabidopsis led to increased DON resistance of seedlings but also to dwarfing of transgenic plants due to the formation of brassinosteroid-glucosides. The objectives of this study were to develop transgenic Arabidopsis expressing HvUGT13248, to test for phenotypic changes in growth habit, and the response to DON. Transgenic lines that constitutively expressed the epitope-tagged HvUGT13248 protein exhibited increased resistance to DON in a seed germination assay and converted DON to D3G to a higher extent than the untransformed wild-type. By contrast to the over-expression of DOGT1 in Arabidopsis, which conjugated the brassinosteriod castasterone with a glucoside group resulting in a dwarf phenotype, expression of the barley HvUGT13248 gene did not lead to drastic morphological changes. Consistent with this observation, no castasterone-glucoside formation was detectable in yeast expressing the barley HvUGT13248 gene. This barley UGT is therefore a promising candidate for transgenic approaches aiming to increase DON and Fusarium resistance of crop plants without undesired collateral effects.     

Transgenic indica rice plants harboring a synthetic cry2A* gene of Bacillus thuringiensis exhibit enhanced resistance against lepidopteran rice pests

A novel synthetic cry2A* gene was introduced into the elite indica rice restorer line Minghui 63 by Agrobacterium-mediated transformation. A total of 102 independent transformants were obtained. Among them, 71 transformants were positive cry2A* plants according to PCR analysis. Four highly insect-resistant lines with single-copy insertion (designated as 2A-1, 2A-2, 2A-3, and 2A-4) were selected based on field assessment and Southern blot analysis in the T₁ generation. All four transgenic lines showed Mendelian segregation by seed germination on 1/2 MS medium containing Basta. Homozygous transgenic plants were selected according to germination ratio (100%) in the T₂ generation. Cry2A* protein concentrations were determined in homozygous transgenic lines, their derived hybrids, and their backcross offspring. The Cry2A* protein concentrations of four homozygous transgenic lines ranged from 9.65 to 12.11 μg/g of leaf fresh weight. There was little variation in the hybrids and backcross offspring. Insect bioassays were conducted in both the laboratory and field. All four transgenic lines were significantly resistant to lepidopteran rice pests. These cry2A* transgenic lines can be used to produce insect-resistant hybrids and serve as a resistant source for the development of two-toxin Bt rice.     

Transgenic tobacco plants expressing the geminivirus BL1 protein exhibit symptoms of viral disease.

Bipartite geminiviruses, such as squash leaf curl virus (SqLCV), encode two movement proteins (MPs), BR1 and BL1, that are essential for viral movement in and subsequent infection of the host plant. To elucidate the biochemical functions of these MPs and define their respective contributions to viral infection, we have generated transgenic Nicotiana benthamiana plants expressing SqLCV BR1 and BL1. Transgenic plants expressing BR1 or a truncated BL1 were phenotypically indistinguishable from wild-type N. benthamiana. In contrast, transgenic plants expressing full-length BL1, alone or in combination with BR1, were strikingly abnormal both in their growth properties and phenotypic appearance, with leaves that were mosaic and curled under, thus mimicking typical SqLCV disease symptoms in this host. BL1 was localized to the cell wall and plasma membrane fractions, whereas BR1 was predominantly in the microsomal membrane fraction. These findings demonstrate that expression of BL1 in transgenic plants is sufficient to produce viral disease symptoms, and they further suggest that BL1 and BR1 carry out distinct and independent functions in viral movement.     

Inheritance of freezing resistance in interspecific F1 hybrids of Eucalyptus

Summary The inheritance of freezing resistance in interspecific F₁ hybrid families of Eucalyptus encompassing 27 different species combinations and a range of levels of hardiness was examined. Freezing resistance was assessed by determining the temperatures required to cause either 30% (T30), 40% (T40), or 50% (T50) leakage of electrolytes from excised leaf discs subjected to artificial freezing. Highly significant variation in freezing resistance occurred between species; the maximum difference between parents in any specific combination was over 9°C (E. gunnii x E. globulus). Freezing resistance was inherited in a predominantly additive manner in interspecific hybrids, although there was a tendency towards partial dominance toward the more sensitive species in some combinations (e.g., E. nitens x E. Globulus, E. nitens x E. camaldulensis, E. gunnii x E. globulus). The full expression of this genetic variation appeared to increase with hardiness and in some cases appeared to vary with ontogeny. Estimates of individual narrow-sense heritability of freezing resistance for pure E. nitens families were h ² = 0.66±0.44 and 0.46±0.44. Across all species combinations examined, the heritability of F₁ family means estimated from midparent regression was h ² = 0.76±0.06 and h ² = 0.89±0.06 for T40 and T50 values, respectively. The advantage of using selected parents for interspecific hybridization is demonstrated and the implications of these results for breeding for freezing resistance in Eucalyptus are discussed.     

Mechanisms of resistance to Helicoverpa armigera and introgression of resistance genes into F1 hybrids in chickpea

The noctuid pod borer, Helicoverpa armigera is a major pest of chickpea, and host plant resistance is an important component for managing this pest. We evaluated a set of diverse chickpea genotypes with different levels of resistance to H. armigera, and their F₁ hybrids for oviposition non-preference, antibiosis, and tolerance components of resistance under uniform insect infestation under greenhouse/laboratory conditions. The genotypes ICC 12476, ICC 12477, ICC 12478, ICC 12479, and ICC 506EB were non-preferred for oviposition under no-choice, dual-choice, and multi-choice conditions, and also suffered lower leaf damage in no-choice tests as compared to the susceptible check, ICCC 37. Antibiosis expressed in terms of low larval weights was observed in insects reared on ICC 12476, ICC 12478, and ICC 506EB. Weight gain by the third-instars was also low on ICC 12476, ICC 12477, ICC 12478, ICC 12479, and ICC 506EB at the podding stage. Non-preference for oviposition and antibiosis (poor larval growth) were also expressed in hybrids based on ICC 12477, ICC 12476, ICC 12478, ICC 12479, and ICC 506EB as compared to the hybrids based on the susceptible check, ICCC 37, indicating that oviposition non-preference and antibiosis in the F₁ hybrids is influenced by the parent genotype. Loss in grain yield was lower in ICC 12477, ICC 12478, ICC 12479, and ICC 506EB compared to that on ICCC 37. The genotypes ICC 12477, ICC 12478, ICC 12479, and ICC 506EB showing antixenosis, antibiosis, and tolerance mechanism of resistance to H. armigera can be used for developing chickpea cultivars for resistance to this pest.     

Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase.

Three types of mutation were introduced into the sequence encoding the GDD motif of the putative replicase component of potato virus X (PVX). All three mutations rendered the viral genome completely noninfectious when inoculated into Nicotiana clevelandii or into protoplasts of Nicotiana tabacum (cv. Samsun NN). In order to test whether these negative mutations could inactivate the viral genome in trans, the mutant genes were expressed in transformed N.tabacum (cv. Samsun NN) under control of the 35S RNA promoter of cauliflower mosaic virus and the transformed lines were inoculated with PVX. In 10 lines tested in which the GDD motif was expressed as GAD or GED there was no effect on susceptibility to PVX. In two of four lines transformed to express the ADD form of the conserved motif, the F1 and F2 progeny plants were highly resistant to infection by PVX, although only to strains closely related to the source of the transgene. The resistance was associated with suppression of PVX accumulation in the inoculated and systemic leaves and in protoplasts of the transformed plants, although some low level viral RNA production was observed in the inoculated but not the systemic leaves when the inoculum was as high as 100 or 250 micrograms/ml PVX RNA. These results suggest for a plant virus, as reported previously for Q beta phage, that virus resistance may be engineered by expression of dominant negative mutant forms of viral genes in transformed cells.     

Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.     

Transgenic plants of Petunia hybrida harboring the CYP2E1 gene efficiently remove benzene and toluene pollutants and improve resistance to formaldehyde

The CYP2E1 protein belongs to the P450 enzymes family and plays an important role in the metabolism of small molecular and organic pollutants. In this study we generated CYP2E1 transgenic plants of Petunia using Agrobacterium rhizogenes K599. PCR analysis confirmed that the regenerated plants contained the CYP2E1 transgene and the rolB gene of the Ri plasmid. Southern blotting revealed the presence of multiple copies of CYP2E1 in the genome of transgenic plants. Fluorescent quantitative PCR revealed exogenous CYP2E1 gene expression in CYP2E1 transgenic plants at various levels, whereas no like expression was detected in either GUS transgenic plants or wild-types. The absorption of benzene and toluene by transgenic plants was analyzed through quantitative gas chromatography. Transgenic plants with high CYP2E1 expression showed a significant increase in absorption capacity of environmental benzene and toluene, compared to control GUS transgenic and wild type plants. Furthermore, these plants also presented obvious improved resistance to formaldehyde. This study, besides being the first to reveal that the CYP2E1 gene enhances plant resistance to formaldehyde, also furnishes a new method for reducing pollutants, such as benzene, toluene and formaldehyde, by using transgenic flowering horticultural plants.     

Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress

Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B(5)). Here we show that Vanin-1(-/-) mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body gamma-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1(-/-) mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1(-/-) mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers.     

Transgenic resistance to Mirafiori lettuce virus in lettuce carrying inverted repeats of the viral coat protein gene

Mirafiori lettuce virus (MiLV), a plant RNA virus belonging to the genus Ophiovirus, is considered to be a causal agent of lettuce big-vein disease. In this study, inverted repeats of a fragment of the coat protein (CP) gene of MiLV in a binary vector pBI121 were transferred via Agrobacterium tumefaciens-mediated transformation into lettuce (Lactuca sativa L.) in order to generate MiLV-resistant lettuce. Forty T₁ lines were analyzed for resistance to MiLV by detecting MiLV in leaves, and two lines (lines 408 and 495) were selected as resistant to MiLV. Both lines were susceptible to Lettuce big-vein associated virus (LBVaV), and line 495 showed higher resistance to MiLV than line 408. Further analysis indicated that line 495 showed resistance to big-vein symptoms expression. Small interfering RNA (siRNA) molecules derived from the transgene were detected in plants of line 495. MiLV was detected in roots but not in leaves of line 495 plants after MiLV inoculation, suggesting that resistance to MiLV is less effective in roots than in leaves.     

CSF-1-induced resistance to viral infection in murine macrophages

Murine peritoneal thioglycollate-elicited macrophages were cultured for 3 days in the presence or absence of highly purified human macrophage colony stimulating factor (CSF-1). The cells were then challenged with vesicular stomatitis virus (VSV) for 24 hr. Ability to resist viral infection was measured in two ways. First, macrophage viability after infection with VSV was measured by washing to remove dead cells, staining the remaining cells with crystal violet, and reading absorbance. Second, a yield reduction assay was used to measure viral replication in the macrophage cultures. Cells treated with CSF-1 (500 to 2000 U/ml) and infected with VSV looked similar microscopically to uninfected cells and had absorbance values twofold to threefold higher than those of infected cultures not treated with CSF-1. The CSF-1-treated cultures also had a virus titer one log lower than that of the untreated cultures. Treatment with partially purified murine CSF-1 induced a similar reduction in virus titer, whereas other murine CSF tested (purified murine GM-CSF, lung-conditioned medium that contains GM-CSF and G-CSF, and WEHI-3B-conditioned medium as a source of IL 3) had little to no effect on virus titer. Antibody to murine IFN-alpha/beta added to the macrophage cultures inhibited the protective effect of CSF-1, indicating that the CSF-1 effect was due to induction of endogenous IFN. Treatment with lipopolysaccharide (1 ng/ml) had some protective effect, which was blocked with polymyxin B. Polymyxin B did not inhibit the effect of CSF-1.     

Extreme resistance to cucumber mosaic virus (CMV) in transgenic tomato expressing one or two viral coat proteins

For the production of broad commercial resistance to cucumber mosaic virus (CMV) infection, tomato plants were transformed with a combination of two coat protein (CP) genes, representing both subgroups of CMV. The CP genes were cloned from the CMV-D strain and Italian CMV isolates (CMV-22 of subgroup I and CMV-PG of subgroup II) which have been shown to produce severe disease symptoms. Four plant transformation vectors were constructed: pMON18774 and pMON18775 (CMV-D CP), pMON18831 (CMV-PG CP) and pMON18833 (CMV-22 CP and CMV-PG CP). Transformed R0 plants were produced and lines were selected based on the combination of three traits: CMV CP expression at the R0 stage, resistance to CMV (subgroup I and/or II) infection in growth chamber tests in R1 expressing plants, and single transgene copy, based on R1 segregation. The results indicate that all four vector constructs generated plants with extremely high resistant to CMV infection. The single and double gene vector construct produced plants with broad resistance against strains of CMV from both subgroups I and II at high frequency. The engineered resistance is of practical value and will be applied for major Italian tomato varieties. This revised version was published online in June 2006 with corrections to the Cover Date.