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。     

The use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants

As the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (TEV) and potato virus Y (PVY), in transgenic tobacco plants. Tobacco lines expressing the foreign gene at varying levels were examined for resistance against TEV and PVY infection. There was a clear, direct correlation between the level of oryzacystatin message, inhibition of papain (a cysteine proteinase), and resistance to TEV and PVY in all lines tested. The inhibitor was ineffective against tobacco mosaic virus (TMV) infection because processing of this virus does not involve cysteine proteinases. These results show that plant cystatins can be used against different potyviruses and potentially also against other viruses, whose replication involves cysteine proteinase activity.     

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.     

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.     

Spontaneous short-range silencing of a GFP transgene in Nicotiana benthamiana is possibly mediated by small quantities of siRNA that do not trigger systemic silencing

A green fluorescent protein (GFP) transgene under the control of the 35S cauliflower mosaic virus (CaMV) promoter was introduced by Agrobacterium-mediated transformation into Nicotiana benthamiana to generate fourteen transgenic lines. Homozygous lines that contained one or two copies of the transgene showed great variation of GFP expression under ultraviolet (UV) light, which allowed classification into three types of transgenic plants. Plants from more than half of the transgenic lines underwent systemic RNA silencing and produced short interfering RNA (siRNA) as young seedlings, while plants of the remaining lines developed, in a spontaneous manner, defined GFP-silenced zones on their leaves, mostly in the form of circular spots that expanded to about 4-7 mm in size. In some of the latter lines, the GFP-silenced spots remained stable, but no systemic silencing occurred. Here we characterize this phenomenon, which we term spontaneous short-range silencing (SSRS). Biochemical analysis of silenced spot tissue did not reveal detectable levels of siRNA. However, agro-infiltration with the suppressor proteins P19 of cymbidium ring spot virus (CymRSV), HC-Pro of tobacco etch virus (TEV), and crosses to a P19 transgenic line, nevertheless suggests that low concentrations of siRNA may have a functional role in the locally silenced zone. We propose that small alterations in the steady-state concentration of siRNAs and their cognate mRNA are decisive with regard to whether silencing remains local or spreads in a systemic manner.     

转不可翻译PVY^N 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T0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T1代抗病株系自交留种。对T2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T0代感病植株,在T1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T0代中抗或高抗植株,在T1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T1、T2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T1、T2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T1、T2代中遗传,且T2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。     

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。     

Host-Specific Involvement of the HC Protein in the Long-Distance Movement of Potyviruses

Plum pox virus (PPV) is a member of the Potyvirus genus that, in nature, infects trees of the Prunus genus. Although PPV infects systemically several species of the Nicotiana genus, such as N. clevelandii and N. benthamiana, and replicates in the inoculated leaves of N. tabacum, it is unable to infect systemically the last host. The long-distance movement defect of PPV was corrected in transgenic tobacco plants expressing the 5"-terminal region of the genome of tobacco etch virus (TEV), a potyvirus that infects systemically tobacco. The fact that PPV was unable to move to upper noninoculated leaves in tobacco plants transformed with the same TEV transgene, but with a mutation in the HC protein (HC-Pro)-coding sequences, identifies the multifunctional HC-Pro as the complementing factor, and strongly suggests that a defect in an HC-Pro activity is responsible for the long-distance movement defect of PPV in tobacco. Whereas PPV HC-Pro strongly intensifies the symptoms caused by potato virus X (PVX) in the PPV systemic hosts N. clevelandii and N. benthamiana, it has no apparent effect on PVX pathogenicity in tobacco, supporting the hypothesis that long-distance movement and pathogenicity enhancement are related activities of the potyviral HC proteins. The movement defect of PPV in tobacco could also be complemented by cucumber mosaic virus in a mixed infection, demonstrating that at least some components of the long-distance machinery of the potyviruses are not strictly virus specific. A general conclusion of this work is that the HC-Pro might be a relevant factor for controlling the host range of the potyviruses.