Small-scale field tests with transgenic potato,cv. Bintje,to test resistance to primary and secondary infections with potato virus y

The coat protein (CP) gene of the potato virus Y (PVY) strain N605 has been cloned into a plant binary expression vector and introduced into the potato variety Bintje. The transformed lines, Bt6, that contained two copies of the CP gene showed complete resistance to the homologous strain PVY-N605 and a good resistance to the related strain PVY-O803 in the greenhouse. The good resistance of Bt6 to primary and secondary infections by PVY was confirmed in two successive field tests where the virus was transmitted by its natural aphid vector.     

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.     

High resistance to plum pox virus (PPV) in transgenic plants containing modified and truncated forms of PPV coat protein gene

Two modified plum pox virus (PPV) coat protein (CP) gene constructs, designed to reduce putative biological risks associated with heteroen capsidation, were integrated into Nicotiana benthamiana plants. The first one contained a deletion of the nucleotides encoding for the DAG amino acid triplet involved in virus aphid-transmission. In the second one, the first 420 nucleotides of the PPV CP gene were removed. We present here the analysis and the selection throughout the generations of PPV-resistant transgenic lines containing these constructs. In most of the lines, a recovery phenotype was observed and was associated with a down-regulation of the transgene products (RNA or protein). We also describe two lines that were highly resistant to PPV. This immunity was correlated with a high number of transgene copies (at least three) and with low or undetectable transgene RNA levels. No heterologous protection was observed against other potyviruses. These characteristics indicate that the described resistance against PPV was RNA-mediated and can be classified as a 'sense suppression' or homology-dependent resistance. Moreover, the production of a highly resistant line containing the PPV CP gene with one third of its 5 end deleted indicated that this region is not necessary to trigger the plant resistance mechanism(s)     

黑龙江马铃薯Y病毒P1基因的特点北大核心CSCD

【目的】本研究通过对不同PVY分离物基因的测序及分析,从而了解PVY株系的多样性,进而对PVY病毒的分子检测及防治提供重要的资料和参考。【方法】本研究针对黑龙江15个马铃薯Y病毒样品的P1基因进行克隆测序和进化树分析。【结果】经比对分析,样品被分成两组,有10个样品的基因类型高度同源,且相对保守,是本地区的优势群组,无论是与国内其它地区样品比较还是与国外样品比较,其亲缘关系都有一定距离;而另一组中的5个样品的P1基因与本地优势组群有较大差异,且这5个样品间也有一定的差异,并与国内其它地区和国外一些样品的P1基因序列比较接近。通过比对Gen Bank中已上传的序列提供的PVY株系的信息,得知本次试验的P1基因与PVY^(NTN-NW)株系是相似的,且这15个样品与国内其他样品一样都是由PVY^N株系演变而来。【结论】由P1基因分析表明,PVY受环境影响较大,黑龙江10个样品的PVY在长期的进化中产生了具有地方特点的变化,而后来的5个样品说明中国大部分PVY有可能是跟随国外品种资源的引进进入,同时PVY也随国内不同区域间资源交流和种薯调运而传播。     

Pathogen-derived resistance provides papaya with effective protection against papaya ringspot virus

Transgenic Carica papaya plants (cv. Sunset, R0 clone 55-1) carrying the coat protein gene of papaya ringspot virus (strain HA 5-1) remained symptomless and ELISA-negative for 24 months after inoculation with Hawaiian strains of papaya ringspot virus under field conditions. Non-transgenic and transgenic control plants lacking the coat protein gene developed disease symptoms within one month after manual inoculation or within four months when natural aphid populations were the inoculum vectors. Mean trunk diameter was significantly greater in cloned 55-1 plants compared with virus-infected controls (14.7 cm versus 9.3 cm after 18 months). Fruit brix, plant morphology, and fertility of 55-1 plants were all normal, and no pleiotropic effects of the coat protein gene were observed. These results indicate that pathogen-derived resistance can provide effective protection against a viral disease over a significant portion of the crop cycle of a perennial species.     

转基因植物中RNA介导的病毒抗性研究进展

利用病毒核酸序列培育抗病毒的转基因植物是一个重要的抗病毒基因工程策略。虽然很多种病毒的不同核酸序列已被使用并证明转基因植物有不同程度的抗病毒效果，但其抗病机制大多不清楚。目前至少有两种明显不同的抗病机制类型：一种是要求病毒编码的蛋白质的表达；另一种是仅仅依靠转基因的ＴＮＡ转录。本文综述了这种ＲＮＡ介导的抗性特点、分子生物学、抗病机制，以及与共抑制的相似性，并对ＲＮＡ介导的病毒抗性的意义加以讨论。     

An Ry-mediated resistance response in potato requires the intact active site of the NIa proteinase from potato virus Y

Ry confers extreme resistance to all strains of potato virus Y (PVY). To identify the elicitor of the Ry-mediated resistance against PVY in potato, we expressed each of the PVY-encoded proteins in leaves of PVY-resistant (Ry) and -susceptible (ry) plants. For most of the proteins tested, there was no evident response. However, when the NIa proteinase was expressed in leaves of Ry plants, there was a hypersensitive response (HR). Proteinase active site mutants failed to induce the Ry-mediated response. The HR was also induced by the NIa proteinase from pepper mottle virus (PepMoV), which has the same cleavage specificity as the PVY enzyme, but not by the tobacco etch virus (TEV) or the potato virus A (PVA) proteinases that cleave different peptide motifs. Based on these results, we propose that Ry-mediated resistance requires the intact active site of the NIa proteinase. Although the structure of the active proteinase could have elicitor activity, it is possible that this proteinase releases an elicitor by cleavage of a host-encoded protein. Alternatively, the proteinase could inactivate a negative regulator of the Ry-mediated resistance response.     

Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene

Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat‐growing areas in China. Because it is vectored by the fungus‐like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co‐transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12‐1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12‐1 showed broad‐spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild‐type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus‐derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad‐spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV.     

Transfer of resistance to potato leafroll virus, potato virus Y and potato virus X from Solarium brevidens to S. tuberosum through symmetric and designed asymmetric somatic hybridisation

Resistance to potato leafroll virus (PLRV), potato virus Y (PVY^o) and potato virus X (PVX) was studied in symmetric and asymmetric somatic hybrids produced by electrofusion between Solanum brevidens (2n=2×=24) and dihaploid S. tuberosum (2n=2×=24), and also in regenerants (B-hybrids) derived through protoplast culture from a single somatic hybrid (chromosome number 48). All of the somatic hybrids between 5. brevidens and the two dihaploid lines of potato cv. Pito were extremely resistant to PLRV and PVY^oand moderately resistant to PVX, irrespective of their chromosome number and ploidy level (tetraploid or hexaploid). Most (56%) of the asymmetric hybrids of irradiated S. brevidens and the dihaploid line of potato cv. Pentland Crown (PDH40) had high titres of PVY^osimilar to those of PDH40, whereas the rest of the hybrids had PVY^otitres less than a tenth of those in PDH40. Three B-hybrids had a highly reduced chromosome number (27, 30 and 34), but were however as resistant to PLRV, PVY^oand PVX as 5. brevidens. Two asymmetric hybrids and one B-hybrid were extremely resistant to PLRV but susceptible to both PVY and PVX. The results suggested that resistance to PLRV in 5. brevidens is controlled by a gene or genes different from those controlling resistance to PVY and PVX, and the gene(s) for resistance to PVY and PVX are linked in S. brevidens.     

Coat protein gene-mediated protection in Lactuca sativa against lettuce mosaic potyvirus strains

Lettuce mosaic potyvirus (LMV) can be very destructive on lettuce crops worldwide. The LMV strain 0 (LMV-0) coat protein (CP) gene was engineered for expression in plants. It was introduced into three susceptible cultivars of Lactuca sativa using an improved procedure for transformation and regeneration of lettuce, by co-cultivation of leaf explants with Agrobacterium tumefaciens. Several transformants accumulated detectable levels of LMV CP. The R1 progeny of twelve R0 transformants (four plants per cultivar) with T-DNA integration at one single locus, was studied for protection against LMV. The progeny from five R0 transformants showed resistance to LMV-0, with the effectiveness of resistance depending on the development stage of the plants at the time of inoculation. The R1 and R2 progeny from one of these R0 transformants, Cocarde-9a, were more extensively analysed. The homozygous but not the hemizygous R1 plants displayed protection to LMV-0. The R2 progeny from one homozygous R1 plant were shown to be resistant to infection by LMV-0 and other LMV strains. As previously observed in other cases of potyvirus sequence-mediated protection, a phenomenon of recovery was observed in some plants, as well as complete resistance. However, this recovery phenotype was not always maintained, as opposed to the previous described cases, leading to a late progression of viral infection.     

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.     

Resistance to potato virus Y and potato virus X in Solanurn brevidens

Although Solanum brevidens could be infected with potato virus X (PVX), potato virus Y⁰ (PVY⁰) and PVY^N, no symptoms of infection were apparent and tests by double antibody sandwich ELISA, electron microscopy and sap transmission to local lesion test plants indicated that the titres of PVX were less than a tenth of those of PVY⁰ and PVY^N were less than a hundredth of those in infected plants of PDH40, a susceptible dihaploid clone of S. tuberosum cv. Pentland Crown. Furthermore, PVY⁰- and PVY^N- infected leaves of S. brevidens were a poor source of inoculum in aphid transmission tests. The possibility of a common mechanism and genetic basis of resistance to PVY, PVX and potato leaf roll virus in S. brevidens is discussed.     

Accumulation of potato virus Y is enhanced in Solatium brevidens also infected with tobacco mosaic virus or potato spindle tuber viroid

The accumulation of potato virus Y?(PVY?) and potato leaf roll virus (PLRV) was studied in plants of Solanum brevidens co-infected with each of six viruses or a viroid. Virus could not be detected by ELISA in plants of S. brevidens infected solely with PVY. However, accumulation of PVY was increased c. 1000-fold in plants doubly infected with tobacco mosaic virus or potato spindle tuber viroid (PSTVd). PVY titres in doubly infected plants of S. brevidens were between 1% and 0.1% of those found in the PVY-susceptible interspecific Solanum hybrid DTO-33. Double infections of 5. brevidens by PVY and alfalfa mosaic virus or potato viruses M, S, T or X did not significantly enhance PVY accumulation. Accumulation of PLRV was not enhanced in plants co-infected with any of the six viruses or PSTVd.     

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.     

Limited degree of serological variability in pepper strains of potato virus Y as revealed by analysis with monoclonal antibodies

The degree of serological variability among pepper strains of potato virus Y (PVY) was assessed through the analysis of samples of infected pepper collected in three main pepper producing regions of Spain. Samples corresponding to the period 1980–1991 were analysed by ELISA with five different monoclonal antibodies (MAbs) produced against potato strains of the virus. The results obtained show a limited degree of epitope variability among pepper PVY-isolates, since only eight out of 32 possible serological profiles were found. Most isolates are not recognised by a MAb directed towards an epitope reported to be present in all potato-PVY isolates. The overall serological behaviour of pepper isolates with these MAbs places them as closer to the group O, of the three groups into which the potato isolates of PVY have been subdivided.     

Potato virus Y NIa protease activity is not sufficient for elicitation of Ry-mediated disease resistance in potato

Ry confers extreme resistance (ER) to all strains of potato virus Y (PVY). In previous work, we have shown that the protease domain of the nuclear inclusion a protease (NIaPro) from PVY is the elicitor of the Ry-mediated resistance and that integrity of the protease active site is required for the elicitation of the resistance response. Two possibilities arise from these results: first, the structure of the active protease has elicitor activity; second, NIa-mediated proteolysis is required to elicit the resistance response. To resolve these possibilities, the NIaPro from PVY was randomly mutagenised and the clones obtained were screened for elicitation of cell death as an indicator of resistance and proteolytic activity. We did not find any mutants that had retained the ability to elicit cell death but had lost protease activity, as measured by processing of the NIa cleavage site in the viral genome. This was consistent with the idea that protease activity is necessary for elicitor activity. However, protease activity was not sufficient because we found three elicitor-defective mutants in which there was a high level of protease activity in this assay.     

Engineering virus resistance using a modified potato gene

Natural mutations in translation initiation factor eIF4E confer resistance to potyviruses in many plant species. Potato is a staple food crop plagued by several potyviruses, yet to date no known eIF4E-mediated resistance genes have been identified. In this study, we demonstrate that transgenic expression of the pvr1(2) gene from pepper confers resistance to Potato virus Y (PVY) in potato. We then use this information to convert the susceptible potato ortholog of this allele into a de novo allele for resistance to PVY using site-directed mutagenesis. Potato plants overexpressing the mutated potato allele are resistant to virus infection. Resistant lines expressed high levels of eIF4E mRNA and protein. The resistant plants showed growth similar to untransformed controls and produced phenotypically similar tubers. This technique disrupts a key step in the viral infection process and may potentially be used to engineer virus resistance in a number of economically important plant-viral pathosystems. Furthermore, the general public may be more amenable to the 'intragenic' nature of this approach because the transferred coding region is modified from a gene in the target crop rather than from a distant species.     

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.