Field resistance of transgenic burley tobacco lines and hybrids expressing the tobacco vein mottling virus coat protein gene

Fifty transgenic lines expressing the tobacco vein mottling virus (TVMV) coat protein (CP) gene in five genetic backgrounds were evaluated under field conditions for response to mechanic inoculation with TVMV, tobacco etch virus (TEV) and potato virus Y (PVY). TVMV CP transgenic lines conferred resistance to TVMV, TEV and PVY under field conditions. Combining two strategies, coat protein-mediated resistance (CPMR) coupled with an endogenous resistance gene (Virgin A Mutant, VAM) significantly extended the range and magnitude of virus resistance and provided a potential valuable new source of protection against potyviruses. CP transgenic lines lacking the VAM gene had high resistance to TEV, medium resistance to PVY, and a recovery phenotype to TVMV. A series of hybrids involving transgenic lines were generated and tested under field conditions for response to virus inoculation. One copy of TVMV-CP gene presented in lines homozygous for the VAM gene provided effective resistance to all three potyviruses. These studies also suggested that selection of a suitable recipient genotype was critical and that field evaluation was necessary in order to select elite resistant transgenic lines. Engineering viral CP genes into genotypes possessing some level of virus resistance could be critical to achieve an effective level of resistance.     

Combining a host gene and a tobacco vein mottling virus coat protein gene for broad and effective resistance to potyviruses in tobacco (Nicotiana tabacum L.)

Over 100 transgenic tobacco lines in five genetic backgrounds were transformed with the tobacco vein mottling virus (TVMV) coat protein (CP) gene. Transgenic lines were initially tested for their reaction to inoculation with a TVMV systemic strain (TVMV-S) and a potato virus Y common strain (PVY-O). Of the 104 TVMV CP lines 60% were classified as resistant to PVY-O, whereas only 30% of these same lines were resistant to TVMV-S. A subset of six PVY-O-resistant transgenic lines and four control lines were tested for their reaction to a local isolate of TVMV, tobacco etch virus (TEV) and five isolates of PVY. The same ten lines were also tested for responses to a serial dilution of inoculum for two PVY isolates, PVY-KY1 and PVY-NN. Transgenic lines carrying an endogenous resistance gene known as Virgin A mutant (VAM) had greater resistance and a broader spectrum of resistance than did transgenic lines without the VAM gene. This additive effect of the endogenous resistance gene and coat protein-mediated resistance (CPMR) was not overcome by the highest inoculum concentration. The results indicate that the additive effect of the VAM gene and CPMR could extend the effectiveness of CPMR in controlling potiviruses. These findings could have important implications for plant improvement programs using CPMR against potyvirus diseases.     

Expression of Multiple Virus-derived Resistance Determinants in Transgenic Plants Does Not Lead to Additive Resistance Properties

Plants can be protected against infection by potyviruses by expressing different portions of potyviral genomes as transgenes. This strategy has proven effective with several potyvirus genes, including the Nla, Nlb, and coat protein coding regions. Given the effectiveness of separate potyvirus coding regions as determinants of resistance, we tested the hypothesis that combinations of potyvirus coding regions would provide additively greater protection of plants against potyviruses. For this, we compared transgenic plant lines that expressed either the coat protein (CP) or the Nla+Nlb+coat protein (NNC) coding regions from tobacco vein mottling virus (TVMV). We found that plants that carry the NNC gene combination were invariably less resistant to TVMV than were lines that contain a CP gene alone. Additionally, we found that NNC lines displayed virtually no resistance to tobacco etch virus (TEV), in contrast to the CP lines. We conclude that combining more than one virus-derived resistance determinant in a single construct is detrimental to the production of virus-resistant plants.     

CMV抑制PVYN CP基因沉默及其介导的抗病性

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T,代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVY^N CP基因的沉默,而且CMV对PVY^N CP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVY^N CP基因沉默则未受到影响。采用ELISA方法对CMV PVY^N复合接种的转基因植株进行PVY^N检测,结果表明,接种叶及下部叶没有检测到PVY^N,植株叶片对PVY^N表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVY^N,植株叶片对PVY^N表现为感病。该文报道了在表达PVY^N CP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。     

Strong resistance to potato tuber necrotic ringspot disease in potato induced by transformation with coat protein gene sequences from an NTN isolate of Potato virus Y

The potato cv. Igor is susceptible to infection with Potato virus Y (PVY) and in Slovenia it has been so severely affected with NTN isolates of PVY causing potato tuber necrotic ringspot disease (PTNRD) that its cultivation has ceased. Plants of cv. Igor were transformed with two transgenes that contained coat protein gene sequence of PVY^NTN. Both transgenes used PVY sequence in a sense (+) orientation, one in native translational context (N‐CP), and one with a frame‐shift mutation (FS‐CP). Although most transgenic lines were susceptible to infection with PVY^NTN and PVY^O, several lines showed resistance that could be classified into two types. Following manual or graft inoculation, plants of partially resistant lines developed some symptoms in foliage and tubers, and virus titre in the foliage, estimated by ELISA, was low or undetectable. In highly resistant (R) lines, symptoms did not develop in foliage and on tubers, and virus could not be detected in foliage by ELISA or infectivity assay. Four lines from 34 tested (two N‐CP and two FS‐CP) were R to PVY^NTN and PVY^O and one additional line was R to PVY^O. When cv. Spey was transformed with the same constructs, they did not confer strong resistance to PVY^O.     

改造的马铃薯Y病毒复制酶基因介导高度抗病性

提取马铃薯Y病毒中国分离株(PVY—c)的mRNA作为模板,随机六聚脱氧核苷酸和寡聚dT为引物合成了单链cDNA。通过聚合酶链式反应(PcR)获得了PVY—C的核内含体b(Nib)全长cDNA克隆。在对其进行全序列分析的基础上,构建了PVY—CNIb基因全长．5’端缺失381个碱基和Nib反义RNA三种不同形式高等植物表达载体。在土壤农杆菌LBA4404的介导下,转化烟草生产品种NC89,获得了所有三种表达载体的转基因植株。通过分子生物学检测和抗性分析发现不同形式的Nib基因序列的转基因植株对马铃薯Y病毒表现不同程度的抗性。其中,以5’端缺失的Nlb的基因转化植株表现最好,从总共20个这类转化株系中筛选到4个株系至少在100μg／m1 PVY—C接种浓度下,表现完全的抗病效果。从总共39个全长Nib基因转化株系中,仅有一个株系,在100μg／ml PVY—c的攻毒接种下具有完全的抗病性。所有33个Nib基因反义RNA的转化植株中,无一株系表现完全的抗病效果,但是有部分株系能不同程度地延缓或减轻发病程度,并有部分植株在发病后50d左右有恢复健康的趋势。虽然能够在上述3种形式的Nib基因序列的转基因植物中检测到相应的RNA的转录产物,但是均未能检测到其相应的蛋白表达产物。     

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.     

Expression of the PVYO coat protein (CP) under the control of the PVX CP gene leader sequence: protection under greenhouse and field conditions against PVYO and PVYN infection in three potato cultivars

Coat protein-mediated resistance (CPMR), resistance conferred as a result of the expression of viral coat proteins in transgenic plants, has been illustrated to be an effective way of protecting plants against several plant viruses. Nonetheless, consistent protection has not been achieved for transgenic plants expressing the coat protein of potato virus Y (PVY), the type member of the potyvirus family. In this report, three different potato cultivars were transformed with a chimeric construct consisting of the capsid protein (CP) coding sequences of PVY flanked by the AUG codon and the translational enhancer from the coat protein gene of potato virus X (PVX). These cultivars were shown to express high levels of PVY CP and confer a high degree of protection against PVY^o and PVY^N under both greenhouse and field conditions. In addition, transgenic plants infected with potato virus A (PVA), a related potyvirus, exhibited a delay in virus accumulation, which could be easily overcome with increasing virus concentrations. Received: 26 October 1995 / Accepted: 14 June 1996     

Distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants.

Tobacco etch potyvirus engineered to express the reporter protein beta-glucuronidase (TEV-GUS) was used for direct observation and quantitation of virus translocation in plants. Four TEV-GUS mutants were generated containing capsid proteins (CPs) with single amino acid substitutions (R154D and D198R), a double substitution (DR), or a deletion of part of the N-terminal domain (delta N). Each modified virus replicated as well as the parental virus in protoplasts, but was defective in cell-to-cell movement through inoculated leaves. The R154D, D198R and DR mutants were restricted essentially to single, initially infected cells. The delta N variant exhibited slow cell-to-cell movement in inoculated leaves, but was unable to move systemically due to a lack of entry into or replication in vascular-associated cells. Both cell-to-cell and systemic movement defects of each mutant were rescued in transgenic plants expressing wild-type TEV CP. Cell-to-cell movement, but not systemic movement, of the DR mutant was rescued partially in transgenic plants expressing TEV CP lacking the C-terminal domain, and in plants expressing CP from the heterologous potyvirus, potato virus Y. Despite comparable levels of accumulation of parental virus and each mutant in symptomatic tissue of TEV CP-expressing transgenic plants, virions were detected only in parental virus- and delta N mutant-infected plants, as revealed using three independent assays. These data suggest that the potyvirus CP possesses distinct, separable activities required for virion assembly, cell-to-cell movement and long-distance transport.     

转不可翻译PVYN CP基因烟草的抗病性分析

我们曾报道表达不可翻译PVY~N CP基因的转基因烟草抗病性是由RNA介导的,其抗病性类似于转录后的基因沉默(PTGS)。本研究以这类不同抗性的Tn代转基因烟草植株为材料,对自交后的T1代转基因植株的遗传和抗病性进行了分析,并选取部分T_1代抗病株系自交留种。对T_2代RNA介导抗病性转基因植株进行了分子分析和一系列抗病性研究。结果表明,含1-2个转基因拷贝的T_0代感病植株,在T_1代中的Km抗性分离符合单位点插入的3∶1的遗传规律;含3个或3个以上转基因拷贝的T_0代中抗或高抗植株,在T_1代中的Km抗性分离符合多位点插入的15∶1或63∶1的遗传规律。大多数T_1、T_2代转基因植株的抗病性与转基因拷贝数成正相关,转基因在T_1、T_2代植株中能够转录表达,且转基因植株之间转基因mRNA在细胞质中的积累水平与转基因植株的抗病性成负相关。转基因植株的抗病性能够在T_1、T_2代中遗传,且T_2代转基因植株的抗病性具有以下特征:1)既抗病毒粒体又抗病毒RNA的侵染,且这种抗病性不受接种物剂量的影响;2)抗病谱较窄,只对PVY的某些株系具有高度抗病性;3)与传毒方式无关,既抗摩擦接种又抗带毒蚜虫接种;4)与植株的发育阶段没有关系。     

Resistance to Vanilla Necrosis Potyvirus in Transgenic Nicotiana benthamiana Plants Containing the Virus Coat Protein Gene

The full-length vanilla necrosis potyvirus (VNV) coat protein (CP) gene was introduced into Nicotiana benthamiana plants via Agrobacterium tumefaciens-mediated transformation. Four constructs contained either: sense (+) CP sequence, antisense (-) CP sequence, sense CP sequence with a Kozak's consensus ATG resulting in a change in the first amino acid, or antisense CP sequence with the Kozak's modification. When mechanically inoculated with a high concentration of VNV, one of the plant lines containing the full-length sense CP gene was highly resistant to virus infection. Plants from the resistant lines expressed the CP at a relatively low level compared to susceptible lines containing the same construct. Plants containing the other three constructs were either susceptible or showed delayed symptom expression.     

Recombination with coat protein transgene in a complementation system based on Cucumber mosaic virus (CMV)

In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an Nsi I site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants expressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination be