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We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA. 相似文献
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Linyu Liu Haiyan Wang Yan Fu Wen Tang Pingjuan Zhao Yanli Ren Zhixin Liu Kunxin Wu Xiuchun Zhang 《Molecular Plant Pathology》2023,24(2):154-166
Most plant viruses encode suppressors of RNA silencing (VSRs) to protect themselves from antiviral RNA silencing in host plants. The capsid protein (CP) of Turnip crinkle virus (TCV) is a well-characterized VSR, whereas SUPPRESSOR OF GENE SILENCING 3 (SGS3) is an important plant-encoded component of the RNA silencing pathways. Whether the VSR activity of TCV CP requires it to engage SGS3 in plant cells has yet to be investigated. Here, we report that TCV CP interacts with SGS3 of Arabidopsis in both yeast and plant cells. The interaction was identified with the yeast two-hybrid system, and corroborated with bimolecular fluorescence complementation and intracellular co-localization assays in Nicotiana benthamiana cells. While multiple partial TCV CP fragments could independently interact with SGS3, its hinge domain connecting the surface and protruding domains appears to be essential for this interaction. Conversely, SGS3 enlists its N-terminal domain and the XS rice gene X and SGS3 (XS) domain as the primary CP-interacting sites. Interestingly, SGS3 appears to stimulate TCV accumulation because viral RNA levels of a TCV mutant with low VSR activities decreased in the sgs3 knockout mutants, but increased in the SGS3-overexpressing transgenic plants. Transgenic Arabidopsis plants overexpressing TCV CP exhibited developmental abnormalities that resembled sgs3 knockout mutants and caused similar defects in the biogenesis of trans-acting small interfering RNAs. Our data suggest that TCV CP interacts with multiple RNA silencing pathway components that include SGS3, as well as previously reported DRB4 (dsRNA-binding protein 4) and AGO2 (ARGONAUTE protein 2), to achieve efficient suppression of RNA silencing-mediated antiviral defence. 相似文献
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RNA silencing technology was used to confer resistance to cucumber green mottle mosaic virus (CGMMV). Nicotiana benthamiana was transformed with a transgene designed to produce an inverted repeat RNA containing CGMMV-coat protein gene (CP) sequences, which were separated by an intron sequence, under the control of the cauliflower mosaic virus 35S promoter. We
attempted to confirm the resistance of seven independent transgenic lines; five lines showed resistance to CGMMV infection.
The systemic spread of virus was prevented after the inoculation of CGMMV, and the CP-specific short interfering RNA (siRNA) was detected in resistant lines. Thus, the resistance against CGMMV through RNA silencing
is strong and efficient. 相似文献
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Coat-protein-mediated resistance to tobacco mosaic virus: discovery mechanisms and exploitation 总被引:7,自引:0,他引:7
Beachy RN 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1999,354(1383):659-664
In 1986 we reported that transgenic plants which accumulate the coat protein of tobacco mosaic virus (TMV) are protected from infection by TMV, and by closely related tobamoviruses. The phenomenon is referred to as coat-protein-mediated resistance (CP-MR), and bears certain similarities to cross protection, a phenomenon described by plant pathologists early in this century. Our studies of CP-MR against TMV have demonstrated that transgenically expressed CP interferes with disassembly of TMV particles in the inoculated transgenic cell. However, there is little resistance to local, cell-to-cell spread of infection. CP-MR involves interaction between the transgenic CP and the CP of the challenge virus, and resistance to TMV is greater than to tobamo viruses that have CP genes more distantly related to the transgene. Using the known coordinates of the three-dimensional structure of TMV we developed mutant forms of CP that have stronger inter-subunit interactions, and confer increased levels of CP-MR compared with wild-type CP. Similarly, it is predicted that understanding the cellular and structural basis of CP-MR will lead to the development of variant CP transgenes that each can confer high levels of resistance against a range of tobamoviruses. 相似文献
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<Emphasis Type="Italic">Soybean dwarf virus</Emphasis>-resistant transgenic soybeans with the sense coat protein gene 总被引:2,自引:1,他引:1
Tougou M Yamagishi N Furutani N Shizukawa Y Takahata Y Hidaka S 《Plant cell reports》2007,26(11):1967-1975
We transformed a construct containing the sense coat protein (CP) gene of Soybean dwarf virus (SbDV) into soybean somatic embryos via microprojectile bombardment to acquire SbDV-resistant soybean plants. Six independent T(0) plants were obtained. One of these transgenic lines was subjected to further extensive analysis. Three different insertion patterns of Southern blot hybridization analysis in T(1) plants suggested that these insertions introduced in T(0) plants were segregated from each other or co-inherited in T(1) progenies. These insertions were classified into two types, which overexpressed SbDV-CP mRNA and accumulated SbDV-CP-specific short interfering RNA (siRNA), or repressed accumulation of SbDV-CP mRNA and siRNA by RNA analysis prior to SbDV inoculation. After inoculation of SbDV by the aphids, most T(2) plants of this transgenic line remained symptomless, contained little SbDV-specific RNA by RNA dot-blot hybridization analysis and exhibited SbDV-CP-specific siRNA. We discuss here the possible mechanisms of the achieved resistance, including the RNA silencing. 相似文献
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Ying‐Juan Chen Feng Zhu Jian Liu Xing‐Guang Deng Xiao‐Jun Pu Li‐Juan Chen Tian‐Lin Sun Wei‐Kai Bao De‐Hui Xi Hong‐Hui Lin 《Journal of Phytopathology》2014,162(11-12):758-769
Cross‐protection has been used successfully and commercially to control a range of virus diseases for which the selection of suitable mild strains of plant viruses is necessary. Turnip crinkle virus (TCV) is highly pathogenic on Arabidopsis plants and its silencing suppressor‐defective mutant, TCVΔCP, can induce highly localized RNA silencing which is differs from that of other protective strains. We found that TCVΔCP provides some protection against wild‐type TCV but lacks complete protection, and the relative locations of the protective virus and challenge virus affect the degree of cross‐protection. However, similar cross‐protection afforded by TCVΔCP is not observed in Nicotiana benthamiana plants. As expected, TCVΔCP pre‐infected Arabidopsis plants fail to protect against infection with the unrelated Cucumber mosaic virus, strain Fhy. It appears that cross‐protection afforded by TCVΔCP requires that the challenge virus be very similar in sequence, which is a characteristic of RNA silencing. In order to investigate whether the protection is associated with the highly localized RNA silencing, mutant plants involved in key silencing pathway genes of RNA silencing machinery, including dcl2, dcl4 and triple dcl2/dcl3/dcl4 mutants were used. The results demonstrate that cross‐protection afforded by TCVΔCP is dependent on host RNA silencing, and both DCL2 and DCL4 play important roles in this process. 相似文献
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dsRNA-mediated resistance to Beet Necrotic Yellow Vein Virus infections in sugar beet (Beta vulgaris L. ssp. vulgaris) 总被引:1,自引:0,他引:1
Britt-Louise Lennefors Eugene I. Savenkov Jan Bensefelt Elisabeth Wremerth-Weich Petra van Roggen Stig Tuvesson Jari P. T. Valkonen Jan Gielen 《Molecular breeding : new strategies in plant improvement》2006,18(4):313-325
Rhizomania, one of the most devastating diseases in sugar beet, is caused by Beet Necrotic Yellow Vein Virus (BNYVV) belonging to the genus Benyvirus. Use of sugar beet varieties with resistance to BNYVV is generally considered as the only way to maintain a profitable yield on rhizomania-infested fields. As an alternative to natural resistance, we explored the transgenic expression of viral dsRNA for engineering resistance to rhizomania. Transgenic plants expressing an inverted repeat of a 0.4 kb fragment derived from the BNYVV replicase gene displayed high levels of resistance against different genetic strains of BNYVV when inoculated using the natural vector, Polymyxa betae. The resistance was maintained under high infection pressures and over prolonged growing periods in the greenhouse as well as in the field. Resistant plants accumulated extremely low amounts of transgene mRNA and high amounts of the corresponding siRNA in the roots, illustrative of RNA silencing as the underlying mechanism. The transgenic resistance compared very favourably to natural sources of resistance to rhizomania and thus offers an attractive alternative for breeding resistant sugar beet varieties. 相似文献
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De Bolle MF Butaye KM Goderis IJ Wouters PF Jacobs A Delauré SL Depicker A Cammue BP 《Plant molecular biology》2007,63(4):533-543
Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression
of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene
expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why
chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs
on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs
do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results
show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have
an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene
expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression
in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.
Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work 相似文献
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Chen Yuh-Kun Lohuis Dick Goldbach Rob Prins Marcel 《Molecular breeding : new strategies in plant improvement》2004,14(3):215-226
The application of RNA-mediated resistance against Cucumber mosaic virus (CMV) by using single transgene constructs generally results in only a small portion of resistant individuals. Inverted repeat constructs encoding self-complementary double-stranded RNA have been demonstrated a potential way to obtain RNA-mediated resistance at high efficiency. To test this observation as a possible method for high frequency induction of CMV resistance, Nicotiana benthamiana plants were transformed with transgenes designed to produce double strand RNA molecules of CMV RNA 2 or coat protein (CP) gene sequences. Seventy-five percent of the tested R0 plants transformed with an RNA 2-derived inverted repeat construct (1534 nt CMV sequence) showed extreme resistance to CMV, while a lower percentage of resistance (30%) was observed in R0 lines transformed with a similar construct of a shorter viral RNA 2 sequence (490 nt). The resistance level conferred by CP sequences was also efficient by using a dsRNA construct, reaching a level of 50%. Self-pollinated (S1) progenies obtained from most resistant R0 plants all showed resistance levels of 100%, perfectly correlating with the expression of transgenic siRNAs. The results indicate that the use of inverted repeat viral transgenes is a highly efficient approach to obtain CMV resistant transgenic plants. Consequently, only a handful of transgenic plants will have to be generated using such constructs for successful resistance, which enables the implementation of this protocol for crops that are difficult to transform, such as ornamental plants in which CMV is an important pathogen. 相似文献
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Przemyslaw Lehmann Carol E. Jenner Edward Kozubek Andrew J. Greenland John A. Walsh 《Molecular breeding : new strategies in plant improvement》2003,11(2):83-94
Oilseed rape (Brassica napus) lines transformedwith the coat protein (CP) gene of Turnip mosaic virus(TuMV) were used to determine the effectiveness of resistance to TuMV mediatedby CP RNA or coat protein. Lines with one, two, or more copies of transgeneswere produced. T2 and T3 lines containing the CP genewitha functional start codon synthesised coat protein and showed high, but variablelevels of resistance to TuMV (21–96% resistant plants per line). TheT1 and T2 progeny of all lines carrying the CP gene withamutated start codon so that RNA but not protein was expressed, were assusceptible to TuMV as controls. Thus, in these experiments we were able toinduce CP-mediated resistance, but not RNA-mediated resistance. 相似文献
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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. 相似文献
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Vassilakos N Bem F Tzima A Barker H Reavy B Karanastasi E Robinson DJ 《Transgenic research》2008,17(5):929-941