Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing

In this study we applied RNA silencing to engineer potato plants that are resistant to potato virus Y (PVY). We expressed double-stranded (ds) RNA derived from the 3 terminal part of the coat protein gene of PVY, which is highly conserved in sequence amongst different PVY isolates, in transgenic potatoes of the commercial variety Spunta. Transgenic plants were analyzed for generation of transgene-derived short interfering RNAs (siRNAs) prior to virus inoculation. Twelve of fifteen transgenic lines produced siRNAs and were highly resistant to three strains of PVY, each belonging to three different subtypes of the virus (PVY^N, PVY^O and PVY^NTN). Infection of transgenic plants with Potato virus X (PVX) simultaneously or prior to the challenge with PVY did not interfere with PVY-resistance.Anastasia Missiou: M.A. and K.K. have contributed equally to this workKriton Kalantidis: M.A. and K.K. have contributed equally to this work     

重组马铃薯X病毒载体介导的烟草γ-微管蛋白基因沉默

用重组马铃薯X病毒(potato virus X,PVX)载体将γ-微管蛋白反义基因导入烟草(Nicotiana tabacum var.Samsun NN),得到了γ-微管蛋白基因沉默的烟草植株。它与侵染PVX空载体的正对照相比有明显的差异:不同形态的叶片分层交替生长;到生殖期所有的花苞都提前脱落;小孢子不能发育到四分体阶段。沉默的形态反应主要起始于顶端幼嫩组织。在沉默过程中除存在基因沉默及恢复现象外还出现靶基因水平的陡然升高,甚至有时会明显反超过正常对照水平。     

Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase.

Three types of mutation were introduced into the sequence encoding the GDD motif of the putative replicase component of potato virus X (PVX). All three mutations rendered the viral genome completely noninfectious when inoculated into Nicotiana clevelandii or into protoplasts of Nicotiana tabacum (cv. Samsun NN). In order to test whether these negative mutations could inactivate the viral genome in trans, the mutant genes were expressed in transformed N.tabacum (cv. Samsun NN) under control of the 35S RNA promoter of cauliflower mosaic virus and the transformed lines were inoculated with PVX. In 10 lines tested in which the GDD motif was expressed as GAD or GED there was no effect on susceptibility to PVX. In two of four lines transformed to express the ADD form of the conserved motif, the F1 and F2 progeny plants were highly resistant to infection by PVX, although only to strains closely related to the source of the transgene. The resistance was associated with suppression of PVX accumulation in the inoculated and systemic leaves and in protoplasts of the transformed plants, although some low level viral RNA production was observed in the inoculated but not the systemic leaves when the inoculum was as high as 100 or 250 micrograms/ml PVX RNA. These results suggest for a plant virus, as reported previously for Q beta phage, that virus resistance may be engineered by expression of dominant negative mutant forms of viral genes in transformed cells.     

Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing

Tobacco plants transformed with the RNA polymerase (RdRp) gene of potato virus X (PVX) that are extremely resistant to infection by potato virus X have previously been described. The PVX-resistant plants accumulated the RdRp protein at a lower level than fully susceptible plants transformed with the same RdRp construct. In this paper the difference between the PVX-resistant and susceptible transformed plants is investigated and it is demonstrated that there are three associated phenotypes of the RdRp transgene that vary in parallel between transformed lines. These phenotypes are: accumulation of the transgenic RdRp RNA at a low level; strain-specific resistance to PVX; and the ability of the transgene to trans -inactivate homologous transgenes. This gene-silencing potential of the transgenes conferring PVX resistance was illustrated by analysis of progeny from a cross between a transformant that was extremely resistant to PVX and a second PVX-susceptible transformant. In other transformants, in which the resistance was less extreme, the same three phenotypes were associated but in a transgene dosage-dependent manner. The same association of strain-specific resistance and low-level accumulation of the transgenic RdRp RNA was observed with plants that were transformed with mutant or wild-type versions of the RdRp gene. Strain-specific resistance was also produced in plants transformed with untranslatable versions of the RdRp transgene. Based on these data it is proposed that homology-dependent gene silencing and transgenic resistance to PVX may be due to the same RNA-based mechanism. An undefined genomic feature is proposed to account for the variation in the resistance and trans -inactivation phenotypes of different transformants. It is further proposed that this genome feature influences a cytoplasmic mechanism that degrades RNA with sequence homology to the silencing transgene.     

Accumulation of tyrosol glucoside in transgenic potato plants expressing a parsley tyrosine decarboxylase

As part of the response to pathogen infection, potato plants accumulate soluble and cell wall-bound phenolics such as hydroxycinnamic acid tyramine amides. Since incorporation of these compounds into the cell wall leads to a fortified barrier against pathogens, raising the amounts of hydroxycinnamic acid tyramine amides might positively affect the resistance response. To this end, we set out to increase the amount of tyramine, one of the substrates of the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction, by placing a cDNA encoding a pathogen-induced tyrosine decarboxylase from parsley under the control of the 35S promoter and introducing the construct into potato plants via Agrobacterium tumefaciens-mediated transformation. While no alterations were observed in the pattern and quantity of cell wall-bound phenolic compounds in transgenic plants, the soluble fraction contained several new compounds. The major one was isolated and identified as tyrosol glucoside by liquid chromatography-electrospray ionization-high resolution mass spectrometry and NMR analyses. Our results indicate that expression of a tyrosine decarboxylase in potato does not channel tyramine into the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction but rather unexpectedly, into a different pathway leading to the formation of a potential storage compound.     

PVY-resistant transgenic potato plants expressing an anti-NIa protein scFv antibody

A synthetic gene encoding a single chain Fv fragment of an antibody directed against the nuclear inclusion a (NIa) protein of potato virus Y (PVY) was used to transform two commerical potato cultivars (Claustar and BF15). The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. Immunoblot analysis showed that most of the resulting transgenic plants accumulate high levels of the transgenic protein. Furthermore, a majority of the selected transgenic lines showed an efficient and complete protection against the challenge virus after mechanical inoculation with PVY^o strain. Two transgenic lines showed an incomplete resistance with delayed appearance of symptoms accompanied by low virus titers, whereas one line developed symptoms during the first days after inoculation but recovered rapidly, leading to a low virus accumulation rate. These results confirm that expression of scFv antibody is able to inhibit a crucial step in the virus multiplication, such as polyprotein cleavage is a powerful strategy for engineered virus resistance. It can lead to a complete resistance that was not obtained previously by expression of scFv directed against the viral coat protein.     

转基因植物转录后基因沉默的克服对策

人们对植物进行遗传转化的目的是让转基因在植物基因组中能够稳定整合并在当代及其子代中能够有效、稳定的表达 ,但是由于多种因素的影响 ,转基因在植物中的表达并不很理想。自从 1 986年Ｐｅｅｒｂｏｔｔｅ报道转基因烟草中转基因发生沉默以来 ,很多学者也都发现大量的转基因植株不能正常表达[1]。经过多年的研究 ,发现导致转基因沉默的机理有多种 ,根据其作用机理和水平的不同 ,人们通常把转基因沉默分为[2 ,3]:位置依赖性基因沉默 (Ｐｏｓｔｉｏｎ -ｄｅｐｅｎｄｅｎｔｇｅｎｅｓｉｌｅｎｃｉｎｇ ,ＰＤＧＳ)、转录水平的基因沉默 (Ｔｒ…     

RNA silencing movement in plants.

Higher eukaryotes have developed a mechanism of sequence-specific RNA degradation which is known as RNA silencing. In plants and some animals, similar to the nematode Caenorhabditis elegans, RNA silencing is a non-cell-autonomous event. Hence, silencing initiation in one or a few cells leads progressively to the sequence-specific suppression of homologous sequences in neighbouring cells in an RNA-mediated fashion. Spreading of silencing in plants occurs through plasmodesmata and results from a cell-to-cell movement of a short-range silencing signal, most probably 21-nt siRNAs (short interfering RNAs) that are produced by one of the plant Dicer enzymes. In addition, silencing spreads systemically through the phloem system of the plants, which also translocates metabolites from source to sink tissues. Unlike the short-range silencing signal, there is little known about the mediators of systemic silencing. Recent studies have revealed various and sometimes surprising genetic elements of the short-range silencing spread pathway, elucidating several aspects of the processes involved. In this review we attempt to clarify commonalities and differences between the individual silencing pathways of RNA silencing spread in plants.     

Patterns of phenylpropanoids in non-inoculated and potato virus Y-inoculated leaves of transgenic tobacco plants expressing yeast-derived invertase

The patterns of secondary metabolites in leaves of yeast invertase-transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were analyzed. Plants expressing cytosolic yeast-derived invertase (cytInv) or apoplastic (cell wall associated) yeast invertase (cwInv) showed a characteristic phytochemical phenotype compared to untransformed controls (wild-type plants). The level of phenylpropanoids decreased in the cytInv plants but increased in the cwInv plants, which showed an induced de novo synthesis of a caffeic acid amide, i.e. N-caffeoylputrescine. In addition, the level of the coumarin glucoside scopolin was markedly enhanced. Increased accumulation of scopolin in the cwInv plants is possibly correlated with the induction of defense reactions and the appearance of necrotic lesions similar to the hypersensitive response caused by avirulent pathogens. This is consistent with results from potato virus Y-infected plants. Whereas there was no additional increase in the coumarins in leaves following infection in cwInv plants, wild-type plants showed a slight increase and cytInc a marked increase.