Resistance to Cucumber mosaic virus in Gladiolus plants transformed with either a defective replicase or coat protein subgroup II gene from Cucumber mosaic virus

Transgenic Gladiolus plants that contain either Cucumber mosaic virus (CMV) subgroup I coat protein, CMV subgroup II coat protein, CMV replicase, a combination of the CMV subgroups I and II coat proteins, or a combination of the CMV subgroup II coat protein and replicase genes were developed. These plants were multiplied in vitro and challenged with purified CMV isolated from Gladiolus using a hand-held gene gun. Three out of 19 independently transformed plants expressing the replicase gene under control of the duplicated CaMV 35S promoter were found to be resistant to CMV subgroup I. Three out of 21 independently transformed plants with the CMV subgroup II coat protein gene under control of the Arabidopsis UBQ3 promoter were resistant to CMV subgroup II. Eighteen independently transformed plants with either the CMV subgroup I coat protein or a combination of CMV subgroups I and II coat proteins were challenged and found to be susceptible to both CMV subgroups I or II. Virus resistant plants with the CMV replicase transgene expressed much lower RNA levels than resistant plants expressing the CMV subgroup II coat protein. This work will facilitate the evaluation of virus resistance in transgenic Gladiolus plants to yield improved floral quality and productivity.     

Susceptibility of some Lycopersicon species and varieties to cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV)

Susceptibility of 33 Lycopersicon species and varieties to Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) were studied. Plants were mechanically inoculated with the C/U1 strain of TMV and U/246 strain of CMV. Virus infection was checked by symptomatology, DAS ELISA and back inoculation (biotest). All the studied Lycopersicon species and varieties were susceptible to TMV-C/U1. L. esculentum Mill. convar. infiniens Lehm. var. flammatum Lehm., L. esculentum Mill. convar. fruticosum Lehm. var. speciosum Lehm. and L. esculentum Mill. convar. infiniens Lehm. var. validum Bail. showed extreme resistance to CMV-U/246. The other 30 species and varieties were susceptible to CMV-U/246. New compatible and incompatible host-virus relations have been reported. The extreme resistant Lycopersicon varieties could be used as resistance sources in tomato breeding.     

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.     

Transgenic F1 hybrids harboring a defective viral replicase exhibit high resistance to CMV in the field

Cucumber mosaic virus (CMV) epidemics occur worldwide in tomato fields, resulting in severe yield losses. Presently, no resistant commercial cultivars are available, and therefore we consider genetic transformation as an attractive alternative to achieve resistance against this disease.     

烟草花叶病毒复制酶介导抗性的研究进展

在抗病毒植物基因工程中,利用病毒的复制酶基因是一种很有前途的方法。本对烟草花叶病毒（TMV）的基因组结构及其编码的蛋白的功能作了简介,同时较详细地阐述了由TMV复制本科的通读部分、全长复制酶以及突变或缺失的复制酶介导的对病毒抗性的研究进展。     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

QTLs involved in the restriction of cucumber mosaic virus (CMV) long-distance movement in pepper

Partial restriction of cucumber mosaic virus (CMV) long-distance movement originating from the Capsicum annuum inbred line ’Vania’ was assessed in a doubled-haploid progeny using two screening methods: the first allowed one to assess the resistance of adult plants decapitated above the fourth leaf and inoculated on the third leaf using a common CMV strain, and the second allowed one to assess CMV resistance to long-distance movement on seedlings inoculated using an atypical CMV strain. For both resistance tests, the behavior of the F₁ hybrid between ’Vania’ and the susceptible line ’H3’ indicated that partial resistance is inherited as a dominant trait. Phenotypic data from the two screening methods were correlated but the one performed on seedlings was much more severe. A subset of 184 molecular markers well-distributed over the pepper genome was selected for QTL mapping using the composite interval mapping (CIM) method. A total of seven genomic regions, including one major effect and several minor effect QTLs, were shown to be associated with partial restriction of CMV long-distance movement. These results are compared with those already obtained in pepper and also in other solanaceous crops, potato and tomato. Received: 22 March 2001 / Accepted: 9 July 2001     

Natural resistance to cucumber mosaic virus in lupin species

Ten species of lupins (Lupinus spp.) were tested for resistance to cucumber mosaic cucumovirus (CMV) in field experiments where inoculation was by naturally-occurring aphid vectors, and in the glasshouse by sap or graft-inoculation. L. albus and six species of ‘rough-seeded’ lupins did not become infected with CMV either under intense inoculum pressure in the field or when graft-inoculated. Two L. hispanicus, 17 L. luteus and four L. mutabilis genotypes became infected with CMV in the field, but no infection was detected in L. hispanicus P26858 or seven L. luteus genotypes. CMV was detected at seed transmission rates of 0.2–16% in seedlings of infected L. luteus, differences in levels of seed transmission between genotypes being significant and relatively stable from year to year. Graft-inoculation of CMV to plants of six genotypes of L. luteus in which no infection was found in the field induced a systemic necrotic reaction suggesting that the resistance they carry is due to hypersensitivity. In L. hispanicus accessions P26849, P26853 and P26858, CMV sub-group II isolate SN caused necrotic spots in inoculated leaves without systemic movement, while sub-group I isolate SL infected them systemically without necrosis. Another sub-group I and two other sub-group II isolates behaved like SL in P26849 and P26853 but infected only inoculated leaves of P26858. This suggests that two strain specific hypersensitive resistance specificities are operating against CMV in L. hispanicus. When plants of L. luteus genotypes that gave hypersensitive reactions on graft-inoculation were inoculated with infective sap containing two sub-group I and seven sub-group II isolates, they all responded like L. hispanicus P26858. A strain group concept is proposed for CMV in lupins based on the two hypersensitive specificities found: strain group 1 represented by isolate SN which induces hypersensitivity with both specificities, strain group 2 represented by the three isolates which induced hypersensitivity only with the specificity present in L. luteus and L. hispanicus P26858, strain group 3 by as yet hypothetical isolates that induce hypersensitivity only in presence of the specificity in L. hispanicus P26849 and P26853 that responded just to isolate SN, and strain group 4 by isolate SL which overcomes both specificities. When F₂ progeny plants from crosses between hypersensitive and susceptible L. luteus parents were inoculated with isolate SN, the resistance segregated with a 3:1 ratio (hypersensitive:susceptible), suggesting that a single dominant hypersensitivity gene, Ncm-1, is responsible. As gene Ncm-1 had broad specificity and was not overcome by any of the five CMV isolates from lupins tested, it is valuable for use in breeding CMV resistant L. luteus cultivars.     

Autonomous cell death, temperature sensitivity and the genetic control associated with resistance to cucumber mosaic virus (CMV) in diploid potatoes (Solanum spp.)

Cucumber mosaic virus (CMV) is a commonly occurring plant virus that causes severe damage in many crops, including the diploid crop species tomato and pepper (Lycopersicon spp. and Capsicum spp., respectively) of the family Solanaceae, but it is neither common nor economically important in cultivated potatoes (Solanum tuberosum; Solanaceae). Resistance to CMV was examined in two diploid (2n=2x=24), highly heterozygous potato populations (Solanum spp.; Solanaceae) consisting of 76 and 126 progeny. Resistance to long-distance transport of CMV controlled by one locus with a major effect and functional at a low temperature (18°C) but overcome at a high temperature (28°C) was identified in one population. In the other population, resistance was controlled by two loci with major effects. In both populations, additional genes with minor effects were probably also involved. Induced resistance to CMV, associated with autonomously developing cell death lesions (Anl) previously not known in potato, was expressed in one parental line. The mechanisms of resistance to CMV may be associated with an inherent or developmental lack of host factors required for compatible CMV-host interactions in viral long distance transport and/or inability of CMV to efficiently suppress the host gene silencing mechanism in potatoes. Polyploidy (gene dose) and high heterozygosity (multiple homologous genes) of potato cultivars may be significant in conferring the durable resistance to CMV. These data provide explanations why CMV is not common and economically important in cultivated potatoes, even though CMV commonly occurs in other crops, weeds and wild plants in potato production areas. Received: 11 February 1999 / Accepted: 25 March 1999     

Resistance to wheat streak mosaic virus in transgenic wheat expressing the viral replicase (NIb) gene

Wheat (Triticum aestivum L. cv. Hi-Line) immature embryos were transformed with the replicase gene (NIb) of wheat streak mosaic virus (WSMV) by the biolistic method. Six independent transgenic plant lines were analyzed for transgene expression and for resistance to mechanical inoculation of WSMV at R3 or R4 generation. Four out of the six lines showed various degree of resistance to WSMV. These lines had initially milder symptoms than controls, and the new growth ranged from milder symptoms, a substantial delay in symptom development, or asymptomatic. Two lines displayed higher resistance with very mild virus symptoms after inoculation and the new growth of 72% and 32% plants from these lines were asymptomatic and had no detectable virus through the plant life cycle. Interestingly, five out of the six transgenic lines had no detectable transgene mRNA expression by RNA gel blot hybridization. The only line that had detectable transgene mRNA did not show delay in the symptom development but had overall milder symptom to the virus.     

Tomato lines segregation for resistance to cucumber mosaic virus

15 lines were bred by interspecific hybridisation and I1 to I6 generation of three of them were tested for resistance to CMV. In spite of the selection by CMV resistance in the progenies the number of the resistant plants did not always increase. The progenies having 100 % symptomless plants for two or more consecutive years were not selected in the studied lines. A large spectrum of variations in the percentage of symptomless plants in the progenies per year and the presence of disease and symptomless parts in one plant were established. These results are possibly due to the complex mechanism of inheritance of CMV resistance as well as to the influence of environmental factors on the expression of the resistance observed     

Phosphorylation of cucumber mosaic virus RNA polymerase 2a protein inhibits formation of replicase complex

The 2a (polymerase) protein of cucumber mosaic virus (CMV) was shown to be phosphorylated both in vivo and in vitro. In vitro assays using 2a protein mutants and tobacco protein kinases showed that the 2a protein has at least three phosphorylation sites, one of which is located within the N-terminal 126 amino acid region. This region is essential and sufficient for interaction with the CMV 1a protein. When phosphorylated in vitro, the 2a protein N-terminal region failed to interact with the 1a protein. Since the 1a-2a interaction is essential for the replication of CMV, this suggests that phosphorylation of the N-terminal region of the 2a protein negatively modulates the interaction in vivo, and may have a regulatory role acting directly in viral infection.     

Resistance phenotypes of transgenic tobacco plants expressing different cucumber mosaic virus (CMV) coat protein genes

Tobacco plants expressing a transgene encoding the coat protein (CP) of a subgroup I strain of cucumber mosaic cucumovirus (CMV), I17F, were not resistant to strains of either subgroup I or II. In contrast, the expression of the CP of a subgroup II strain, R, conferred substantial resistance, but only towards strains of the same subgroup. When protection was observed, the levels of resistance were similar when plants were inoculated with either virions or viral RNA, but resistance was more effective when plants were inoculated with viruliferous aphids. Resistance was not dependent on inoculum strength and was expressed as a recovery phenotype not yet described for plants expressing a CMV CP gene. Recovery could be observed either early in infection (less than one week after inoculation) or later (4 to 5 weeks after inoculation). In plants showing early recovery, mild symptoms were observed on the inoculated leaves, and in some cases symptoms developed on certain lower systemically infected leaves, but the upper leaves were symptomless and virus-free. Late recovery corresponded to the absence of both symptoms and virus in the upper leaves of plants that were previously fully infected. Northern blot analyses of resistant plants suggested that a gene silencing mechanism was not involved in the resistance observed.     

Genetic control of resistance to cucumber mosaic virus in Cucurbita pepo

Segregation ratios in the F₂s of crosses between courgette cultivars and the pumpkin cv. Cinderella indicated that the resistance of the latter to cucumber mosaic virus (CMV), expressed as a failure to develop systemic symptoms, was controlled by two unlinked recessive genes. However, data from the backcross generations were not consistent with this. Biometrical analysis showed significant gene interactions, possibly between the genes for CMV resistance and the background genotype determining plant vigour and a gene dosage effect for resistance. The resistance has been successfully backcrossed into courgette breeding material.     

Generation of a murine single chain Fv (scFv) antibody specific for cucumber mosaic virus (CMV) using a phage display library

With the long-term goal of generating CMV-resistant transgenic plants using antibody genes, a single-chain variable fragment (scFv) antibody that binds to the cucumber mosaic virus was isolated from a scFv phage display library by four rounds of affinity selection with CMV-Mf as an antigen. The scFv has the identical binding specificity to CMV as a monoclonal antibody that is generated by the hybridoma fusion technique, and recognized purified preparations of CMV isolates belonging to either subgroup I or II in immunoblotting. The nucleotide sequences of the recombinant antibody showed that a heavy chain variable region (V(H)) gene belonged to the VH3 subgroup and the kappa light chain variable region (V kappa) came from the Vkappa4 subgroup. Our results demonstrate that the scFv phage display library, an alternative approach to the traditional hybridoma fusion technique, has a potential applicability in the study of plant virus and plant pathology.     

High-resolution mapping of zym, a recessive gene for Zucchini yellow mosaic virus resistance in cucumber

Key message

Using a high-resolution mapping approach, we identified a candidate gene for ZYMV resistance in cucumber. Our findings should assist the development of high-versatility molecular markers for MAS for ZYMV resistance.

Abstract

Zucchini yellow mosaic virus (ZYMV) causes significant disease, which leads to fruit yield loss in cucurbit crops. Since ZYMV resistance is often inherited recessively in cucumber, marker-assisted selection (MAS) is a useful tool for the development of resistant cucumber cultivars. Using 128 families of an F_2:3 population derived from a cross between susceptible ‘CS-PMR1’ and resistant ‘A192-18’ cucumber inbred lines, we confirmed that ZYMV resistance is conferred by a single recessive locus: zym ^A192-18 . We constructed a cucumber genetic linkage map that included 125 simple sequence repeat (SSR) markers segregating into 7 linkage groups (chromosomes). The zym ^A192-18 locus was mapped to chromosome 6, at genetic distances of 0.9 and 1.3 cM from two closely linked SSR markers. For high-resolution genetic mapping, we identified new molecular markers cosegregating with the zym ^A192-18 locus; using cucumber genomic and molecular marker resources and screening an F₂ population of 2,429 plants, we narrowed down the zym ^A192-18 locus to a <50-kb genomic region flanked by two SSR markers, which included six candidate genes. Sequence analysis of the candidate genes’ coding regions revealed that the vacuolar protein sorting-associated protein 4-like (VPS4-like) gene had two SNPs between the parental lines. Based on SNPs of the VPS-4-like gene, we developed zym ^A192-18 -linked DNA markers and found that genotypes associated with these markers were correlated with the ZYMV resistance phenotype in 48 cucumber inbred lines. According to our data, the gene encoding VPS4-like protein is a candidate for the zym ^A192-18 locus. These results may be valuable for MAS for ZYMV resistance in cucumber.     

Expression of the gene encoding the coat protein of cucumber mosaic virus (CMV) strain WL appears to provide protection to tobacco plants against infection by several different CMV strains.

The gene (cp) encoding the coat protein (CP) of cucumber mosaic virus (CMV) strain WL (CMV-WL, which belongs to CMV subgroup II) was custom polymerase chain reaction (CPCR)-engineered for expression as described by Slightom [Gene 100 (1991) 251-255]. CPCR amplification was used to add 5'- and 3'-flanking NcoI sites to the CMV-WL cp gene, and cp was cloned into the expression vector, pUC18cpexp. This CMV-WL cp expression cassette was transferred into the genome of tobacco (Nicotiana tabacum cv. Havana 423) via the Agrobacterium T-DNA transfer mechanism. R0 plants that express the CMV-WL cp gene were subcloned, propagated, and challenge-inoculated with CMV-WL. Several R0 plant lines showed excellent protection against CMV-WL infection; however, plants found to accumulate the highest CP levels did not show the highest degree of protection. Thus in our case, CP levels appear not to be a useful predictor of the degree of protection. Plants from the best protected CMV-WL cp gene-expressing R0 tobacco lines were also inoculated with CMV strains belonging to the other major CMV subgroup (subgroup I), CMV-C and CMV-Chi, and compared in a parallel experiment with a transgenic tobacco plant line that expresses the CMV-C cp gene. Plants expressing the CMV-WL cp gene appeared to show a broader spectrum of protection against infection by the various CMV strains than plants expressing the CMV-C cp gene.     

Resistance to cucumber mosaic virus in potato

Reactions to two subgroup I isolates (Fny-CMV and Pf-CMV) and two subgroup II isolates (A9-CMV and LS-CMV) of cucumber mosaic virus (CMV) were studied in three non tuber-bearing wild potato species (Solanum spp.) of the series Etuberosa, and in two tuber-bearing interspecific potato hybrids and four potato cultivars using graft-inoculation. Three classes of phenotypic reactions (susceptible, hypersensitive, extreme resistance) were observed in the tuber-bearing genotypes. Susceptible genotypes developed mosaic or severe mosaic with leaf malformation and had high CMV titres. Hypersensitive genotypes developed either top necrosis or vein necrosis and/or necrotic spots on apical leaves, and had low CMV titres. Extremely resistant genotypes had no symptoms and no CMV was detected. The hybrid 87HW13.7 (S. tuberosum×S. multidissectum) developed top necrosis specific to infection with Fny-CMV. The hybrid ‘A6’ (S. demissum×S. tuberosum cv. Aquila) was hypersensitive to all CMV isolates tested. Extreme resistance was not functional against all CMV isolates. Neither hypersensitivity nor extreme resistance were related to the CMV subgroup.