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.     

Characterization of cymbidium mosaic virus coat protein gene and its expression in transgenic tobacco plants

Cymbidium mosaic virus (CyMV) is the most prevalent virus infecting orchids. Here, we report the isolation of partial cDNA clones encoding the genomic RNA of CyMV. Like most of the polyadenylated monopartite positive-strand RNA viruses, the open reading frame (ORF) coding for the viral coat protein (CP) is located at the 3 end. The ORF predicts a polypeptide chain of 220 amino acids with a molecular weight of 23 600. Sequence comparison of this ORF to the CP sequences of potato virus X(PVX) and white clover mosaic virus (WCIMV) revealed a strong amino acid homology in the mid-portion of the CP, but the overall homology was low. The CyMV CP gene was placed downstream of a cauliflower mosaic virus 35S promoter and the chimaeric gene was transferred into Nicotiana benthamiana. Transgenic plants expressing the CyMV CP were protected against CyMV infection.     

Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus,zucchini yellow mosaic virus,and watermelon mosaic virus-2 is resistant to these three viruses in the field

Cantaloupe line CZW-30 containing coat protein gene constructs of cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic virus 2 potyvirus (WMV-2) was investigated in the field over two consecutive years for resistance to infections by CMV, ZYMV, and/or WMV-2. Resistance was evaluated under high disease pressure achieved by mechanical inoculations and/or natural challenge inoculations by indigenous aphid vectors. Across five different trials, homozygous plants were highly resistant in that they never developed systemic symptoms as did the nontransformed plants but showed few symptomatic leaves confined close to the vine tips. Hemizygous plants exhibited a significant delay (2–3 weeks) in the onset of disease compared to control plants but had systemic symptoms 9–10 weeks after transplanting to the field. Importantly, ELISA data revealed that transgenic plants reduced the incidence of mixed infections. Only 8% of the homozygous and 33% of the hemizygous plants were infected by two or three viruses while 99% of the nontransformed plants were mixed infected. This performance is of epidemiological significance. In addition, control plants were severely stunted (44% reduction in shoot length) and had poor fruit yield (62% loss) compared to transgenic plants, and most of their fruits (60%) were unmarketable. Remarkably, hemizygous plants yielded 7.4 times more marketable fruits than control plants, thus suggesting a potential commercial performance. This is the first report on extensive field trials designed to assess the resistance to mixed infection by CMV, ZYMV, and WMV-2, and to evaluate the yield of commercial quality cantaloupes that are genetically engineered.     

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP⁺) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP⁺ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP⁺ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.     

Satellite-RNA-mediated resistance to cucumber mosaic virus in transgenic plants of hot pepper (Capsicum annuum cv. Golden Tower)

We previously established a system of in vitro regeneration and Agrobacterium-mediated transformation for hot pepper plants. The level of protection against cucumber mosaic virus in the progeny of the transgenic hot pepper plants that express cucumber mosaic virus (CMV) satellite RNA was investigated. The transgenic hot pepper plants were self-fertilized, and their progeny were tested for stable inheritance and expression of the cDNA of CMV satellite RNA. Polymerase chain reaction and RNA gel blot analyses showed that the introduced gene was stably transmitted and expressed in the progeny. Symptom attenuation in the offspring was confirmed upon inoculation with CMV-Y or CMV-Korea (CMV-Kor) strains. Received: 30 September 1996 / Revision received: 5 May 1997 / Accepted: 22 May 1997     

Production of Bean yellow mosaic virus resistant subterranean clover (Trifolium subterraneum) plants by transformation with the virus coat protein gene

Transgenic lines of subterranean clover were constructed that contained three different Bean yellow mosaic virus (BYMV) coat protein (CP) gene constructs; full-length CP, the core region of the CP, and full-length CP plus the 3′ untranslated region of the viral genome. Transgenic plants containing the full-length and core CP gene constructs showed high and moderate levels of BYMV resistance. Resistance was measured as a lack or amelioration of viral disease symptoms, which was correlated with a reduction in virus levels and yield loss. A range of different resistance phenotypes was observed. They included reduced infection rates, delay and reduction in local lesion development, and delay and reduction in severity of systemic symptom development. Resistance levels were not correlated with transgene mRNA levels and no transgene-encoded protein was detected in any of the transgenic lines. This is the first example of genetically engineered virus resistance in a clover.     

Rapid production and field testing of homozygous transgenic tobacco lines with virus resistance conferred by expression of satellite RNA and coat protein of cucumber mosaic virus

A procedure for the fast production of homozygotic transgenic plants was developed. Leaf discs of haploid tobacco plants from anther cultures were transformed with a chimaeric vector containing coat protein (CP) and satellite RNA (Sat-RNA) genes from cucumber mosaic virus (CMV). One-hundred-and-twelve Kanamycin-resistant transformed haploid plants were subjected to selection based on the expression of both CP and Sat-RNA. Eighty-nine transgenic plants expressing both genes were selected and tested for their resistance to CMV by inoculation with high concentration of CMV (200 g ml^–1). Only five plants showed no symptoms of viral infection 30 days after inoculation. These plants were then diploidized by colchicine treatment. Three homozygous diploid lines with high levels of resistance to CMV were obtained after only one generation. The three transgenic lines were further tested under field conditions. The results showed that the progenies of these transgenic lines were homozygous and were highly resistant to CMV under natural field infection and manual inoculation conditions.     

Cucumber mosaic virus movement protein affects sugar metabolism and transport in tobacco and melon plants

In addition to its influence on plasmodesmal function, tobacco mosaic virus movement protein (TMV‐MP) causes an alteration in carbon metabolism in source leaves and in resource partitioning among the various plant organs. The present study was aimed at characterizing the influence of cucumber mosaic virus (CMV)‐MP on carbohydrate metabolism and transport in both tobacco and melon plants. Transgenic tobacco plants expressing the CMV‐MP had reduced levels of soluble sugars and starch in their source leaves and a significantly reduced root‐to‐shoot ratio in comparison with control plants. A novel virus‐vector system was employed to express the CMV‐coat protein (CP), the CMV‐MP or the TMV‐MP in melon plants. This set of experiments indicated that the viral MPs cause a significant elevation in the proportion of sucrose in the phloem sap collected from petioles of source leaves, whereas this sugar was at very low levels or even absent from the sap of control melon plants. The mode by which the CMV‐MP exerts its effect on phloem‐sap sugar composition is discussed in terms of possible alterations in the mechanism of phloem loading.     

苜蓿花叶病毒外壳蛋白基因对红三叶的遗传转化及转基因植株的抗病性分析

以红三叶(Trifolium pratense L．)子叶为转化体,用农杆菌介导法将外源的苜蓿花叶病毒外壳蛋白基因AMV4转入红三叶,经过筛选、分化和再生,得到了具有卡拉霉素抗性的转基因植株。对这些植株进行PCR、Southern印迹杂交和Northern杂交分析,结果表明,外源目的基因已经整合到红三叶基因组中并且得到了表达。对Northern分析呈阳性的植株进行了抗病性检测,结果表明,表达苜蓿花叶病毒外壳蛋白基因的植株病症减轻,发病率、病情指数及病毒积累量都明显低于对照,有的甚至不表现症状,达到了免疫的程度。     

The cis-expression of the coat protein of turnip mosaic virus is essential for viral intercellular movement in plants

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant-infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double-fluorescent (green and mCherry) protein-tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N-terminus (amino acids 1–97), the core (amino acids 98–245), and the C-terminus (amino acids 246–288). We found that deletion of CP or its segments amino acids 51–199, amino acids 200–283, or amino acids 265–274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6–50 in the N-terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell-to-cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans-expression of the wild-type CP either by TuMV or through genetic transformation-based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis-expressed CP is functional.     

Mutation of a family 8 glycosyltransferase gene alters cell wall carbohydrate composition and causes a humidity-sensitive semi-sterile dwarf phenotype in Arabidopsis

Incompatible plant-pathogen interactions result in the rapid cell death response known as hypersensitive response (HR) and activation of host defense related genes. To understand the cellular mechanism controlling defense response better, a novel pathogenesis-related (PR) gene and putative cell wall protein gene, CaTin2, was isolated through differential screening of a hot pepper cDNA library and characterized. CaTin2 gene was locally and systemically induced in hot pepper plants upon TMV-P₀ inoculation which induces HR. However, CaTin2 gene wasn't regulated by bacterial HR-specific signal pathway. The full-length cDNA for CaTin2, which is 864 nucleotides long, contained the open reading frame of 200 amino acids including cell wall targeting sequences of 26 amino acids. CaTin2 gene has no sequence similarity with other cell wall protein genes except the signal sequence and exists as only one copy in hot pepper genome. CaTin2 gene contains repeated helix-turn-helix motif consisting of 39 amino acids. CaTin2 mRNA accumulation was induced in response to various treatments such as ethylene, SA, MeJA, ABA, methyl viologen, NaCl and wounding at early time points. Subcelluar localization of CaTin2 was confirmed in the cell wall in hot pepper leaves by making CaTin2::smGFP fusion protein. The transgenic plants overexpressing CaTin2 cDNA were resistant to TMV and CMV inoculation. From these results, CaTin2 gene may encode a virus-related new cell wall protein member.     

Alteration in carbon partitioning induced by the movement protein of tobacco mosaic virus originates in the mesophyll and is independent of change in the plasmodesmal size exclusion limit

The influence of the 30 kDa movement protein of tobacco mosaic virus (TMV-MP) on carbon partitioning in trans-genie tobacco plants (Nicotiana tabacum cv. Xanthi) expressing the TMV-MP was investigated. Using reciprocal grafting of transgenic tobacco plants expressing this movement protein and vector control plants, as well as transgenic tobacco plants expressing the TMV-MP in phloem cells only, we showed that the interactive site involved in carbon allocation to roots is localized to the mesophyll tissue. Biomass partitioning experiments conducted on transgenic plants, in which various deletion mutant forms of the TMV-MP (two of which included deletions in the domain responsible for increasing the size exclusion limit) were expressed, revealed that the TMV-MP exerts its influence on carbon allocation via a mechanism that is completely independent of the TMV-MP-induced increase in the plasmodesmal size exclusion limit. Furthermore, small N- and C-terminal deletions in the MP revealed the complexity of the interactions likely to be involved between the MP and an endogenous regulatory mechanism. We propose that the TMV-MP interferes with an endogenous signal transduction pathway that involves macromolecular trafficking through plasmodesmata to regulate biomass partitioning between the source and various sink tissues.     

Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P₀. Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P₀ and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPK _c 1) gene was increased specifically in the incompatible interaction with TMV-P₀. The expression of CaPK _c 1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPK _c 1 responds to several defense-related abiotic stresses in addition to TMV infection. The nucleotide sequence data reported in this paper were submitted to the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number DQ114474.