Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing

Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana benthamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. benthamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing.     

Characterization of the recombinant forms arising from a Potato virus X chimeric virus infection under RNA silencing pressure

Recombination is a frequent phenomenon in RNA viruses whose net result is largely influenced by selective pressures. RNA silencing in plants acts as a defense mechanism against viruses and can be used to engineer virus resistance. Here, we have investigated the influence of RNA silencing as a selective pressure to favor recombinants of PVX-HCT, a chimeric Potato virus X (PVX) vector carrying the helper-component proteinase (HC-Pro) gene from Plum pox virus (PPV). All the plants from two lines expressing a silenced HC-Pro transgene were completely resistant to PPV. However a significant proportion became infected with PVX-HCT. Analysis of viral RNAs accumulating in silenced plants revealed that PVX-HCT escaped silencing-based resistance by removal of the HC-Pro sequences that represented preferential targets for transgene-promoted silencing. The virus vector also tended to lose the HC-Pro insert when infecting transgenic plants containing a nonsilenced HC-Pro transgene or wild-type (wt) Nicotiana benthamiana plants. Nevertheless, loss of HC-Pro sequences was faster in nonsilenced transgenic plants than in wt plants, suggesting the transgene plays a role in promoting a higher selective pressure in favor of recombinant virus versions. These results indicate that the outcome of recombination processes depends on the strength of selection pressures applied to the virus.     

Transient expression of HPV16 E7 peptide (aa 44-60) and HPV16 L2 peptide (aa 108-120) on chimeric potyvirus-like particles using Potato virus X-based vector

The optimized expression of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV16) was developed. Epitope derived from minor capsid protein L2 was expressed as N-terminal fusion with ACP while an epitope derived from E7 oncoprotein was fused to its C-terminus. The construct was cloned into Potato X potexvirus (PVX) based vector and transiently expressed in plants using Agrobacterium tumefaciens mediated inoculation. To increase the level of expressed protein the transgenic Nicotiana benthamiana plants expressing Potato virus A HC-Pro gene and transgenic Nicotiana tabacum, cv. Petit Havana SR1 carrying Potato virus A P3 protein gene were tested. Synergistic infection of host plants with PVX carrying the construct and Potato virus Y(O) (PVY(O)) increased the expression of L2ACPE7 in N. tabacum and in transgenic N. benthamiana carrying potyviral HC-Pro gene as compared to control plants infected with L2ACPE7 only.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

The ORF1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants

Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided in trans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.     

Identification of secret agent as the O-GlcNAc transferase that participates in Plum pox virus infection

Serine and threonine of many nuclear and cytoplasmic proteins are posttranslationally modified with O-linked N-acetylglucosamine (O-GlcNAc). This modification is made by O-linked N-acetylglucosamine transferases (OGTs). Genetic and biochemical data have demonstrated the existence of two OGTs of Arabidopsis thaliana, SECRET AGENT (SEC) and SPINDLY (SPY), with at least partly overlapping functions, but there is little information on their target proteins. The N terminus of the capsid protein (CP) of Plum pox virus (PPV) isolated from Nicotiana clevelandii is O-GlcNAc modified. We show here that O-GlcNAc modification of PPV CP also takes place in other plant hosts, N. benthamiana and Arabidopsis. PPV was able to infect the Arabidopsis OGT mutants sec-1, sec-2, and spy-3, but at early times of the infection, both rate of virus spread and accumulation were reduced in sec-1 and sec-2 relative to spy-3 and wild-type plants. By matrix-assisted laser desorption ionization-time of flight mass spectrometry, we determined that a 39-residue tryptic peptide from the N terminus of CP of PPV purified from the spy-3 mutant, but not sec-1 or sec-2, was O-GlcNAc modified, suggesting that SEC but not SPY modifies the capsid. While our results indicate that O-GlcNAc modification of PPV CP by SEC is not essential for infection, they show that the modification has a role(s) in the process.     

Coat protein mediated resistance to Plum Pox Virus in Nicotiana clevelandii and N. benthamiana

Transgenic Nicotiana benthamiana and N. clevelandii plants expressing the coat protein of Plum Pox Virus under the control of the 35S promoter from Cauliflower Mosaic Virus were engineered by Agrobacterium tumefaciens mediated transformation. The phenomenon of virus resistance was observed at different levels when transgenic plants, expressing the coat protein and control plants were compared after challenge infection with Plum Pox Virus. N. clevelandii coat protein transgenic plants circumvent virus accumulation. After an initial increase in virus titer similar to the control plants, some coat protein expressing plants showed a reduced accumulation of virus and inhibition of the systemic spread, characterized by decrease of the virus titer and formation of new symptomless leaves. In other N. clevelandii coat protein expressing plants virus accumulation was inhibited and disease symptoms never appeared. N. benthamiana coat protein expressing plants were also protected. After a temporary virus accumulation, virus titer decreased without the appearance of symptoms with the exception of a few plants, which showed a delay of thirty days in the development of symptoms post challenge infection.Abbreviations PPV Plum Pox Virus - CP coat protein - CaMV Cauliflower Mosaic Virus - CP+ coat protein expressing plant - CP– control plant = non coat protein expressing plant - TMV Tobacco Mosaic Virus - NPTII neomycin phosphotransferaseII - IBA indole-3-butyric acid - BAP 6-benzylaminopurine; - MS Murashige Skoog - ELISA enzyme linked immunosorbent assay     

Site-directed mutagenesis and generation of chimeric viruses by homologous recombination in yeast to facilitate analysis of plant-virus interactions

A yeast homologous recombination system was used to generate mutants and chimeras in the genome of Potato leafroll virus (PLRV). A yeast-bacteria shuttle vector was developed that allows mutants and chimeras generated in yeast to be transformed into Escherichia coli for confirmation of the mutations and transformed into Agrobacterium tumefaciens to facilitate agroinfection of plants by the mutant PLRV genomes. The advantages of the system include the high frequency of recovered mutants generated by yeast homologous recombination, the ability to generate over 20 mutants and chimeras using only two restriction endonuclease sites, the ability to introduce multiple additional sequences using three and four DNA fragments, and the mobilization of the same plasmid from yeast to E. coli, A. tumefaciens, and plants. The wild-type PLRV genome showed no loss of virulence after sequential propagation in yeast, E. coli, and A. tumefaciens. Moreover, many PLRV clones with mutations generated in the capsid protein and readthrough domain of the capsid protein replicated and moved throughout plants. This approach will facilitate the analysis of plant-virus interactions of in vivo-generated mutants for many plant viruses, especially those not transmissible mechanically to plants.     

Production of siRNA targeted against TYLCV coat protein transcripts leads to silencing of its expression and resistance to the virus

The coat protein (CP) of Tomato yellow leaf curl virus (TYLCV), encoded by the v1 gene, is the only known component of the viral capsid. In addition, the CP plays a role in the virus transport into the host cell nucleus where viral genes are replicated and transcribed. In this study, we analyzed the effect of small interfering double-stranded RNAs (siRNAs), derived from an intron-hairpin RNA (ihpRNA) construct and targeting the v1 gene product, on CP accumulation. Transient assays involving agroinfiltration of the CP-silencing construct followed by infiltration of a fused GFP-CP (green fluorescent protein-coat protein) gene showed down-regulation of GFP expression in Nicotiana benthamiana. Some of the transgenic tomato plants (cv. Micro-Tom), expressing the siRNA targeted against the TYLCV CP gene, did not show disease symptoms 7 weeks post-inoculation with the virus, while non-transgenic control plants were infected within 2 weeks post inoculation. The present study demonstrates, for the first time, that siRNA targeted against the CP of TYLCV can confer resistance to the virus in transgenic tomato plants, thereby enabling flowering and fruit production.     

Rice dwarf phytoreovirus segment S6-encoded nonstructural protein has a cell-to-cell movement function

Rice dwarf virus (RDV) is a member of the genus Phytoreovirus, which is composed of viruses with segmented double-stranded RNA genomes. Proteins that support the intercellular movement of these viruses in the host have not been identified. Microprojectile bombardment was used to determine which open reading frames (ORFs) support intercellular movement of a heterologous virus. A plasmid containing an infectious clone of Potato virus X (PVX) defective in cell-to-cell movement and expressing either beta-glucuronidase or green fluorescent protein (GFP) was used for cobombardment with plasmids containing ORFs from RDV gene segments S1 through S12 onto leaves of Nicotiana benthamiana. Cell-to-cell movement of the movement-defective PVX was restored by cobombardment with a plasmid containing S6. In the absence of S6, no other gene segment supported movement. Identical results were obtained with Nicotiana tabacum, a host that allows fewer viruses to infect and spread within its tissue. S6 supported the cell-to-cell movement of the movement-defective PVX in sink and source leaves of N. benthamiana. A mutant S6 lacking the translation start codon did not complement the cell-to-cell movement of the movement-defective PVX. An S6 protein product (Pns6)-enhanced GFP fusion was observed near or within cell walls of epidermal cells from N. tabacum. By immunocytochemistry, unfused Pns6 was localized to plasmodesmata in rice leaves infected with RDV. S6 thus encodes a protein with characteristics identical to those of other viral proteins required for the cell-to-cell movement of their genome and therefore is likely required for the cell-to-cell movement of RDV.     

A search for evidence of virus/transgene interactions in potatoes transformed with the potato leafroll virus replicase and coat protein genes

A search was conducted to detect evidence for interactions between potato leafroll virus (PLRV)-derived transgenes expressed in Russet Burbank potato and viruses to which the transgenic plants were exposed and by which they were infected. More than 25000 plants in 442 lines transformed with 16 different coat protein gene (CP) constructs and nearly 40000 plants in 512 lines transformed with seven different replicase gene (Rep) constructs of PLRV were exposed to field infection over a 6-year period. These plants were individually inspected for type and severity of virus symptoms. Heterologous viruses found infecting the plants were identified and examined for alterations in transmission characteristics, serological affinity, host range, and symptoms. Selected isolates of PLRV from field-infected plants were examined for unusual symptoms produced in diagnostic hosts and for alteration in sedimentation properties in density gradient tubes. Viruses that were propagated in selected transgenic lines in a greenhouse were examined for similar alterations. Transmission characteristics and serological properties were not altered when they replicated in potatoes containing CP constructs in the field or greenhouse. Potato plants expressing CP or Rep constructs of PLRV were not infected in the field or in the greenhouse with viruses that do not normally infect potato. New viruses or viruses with altered sedimentation characteristics, symptoms, or host range were not detected in field-exposed or greenhouse-inoculated potato plants expressing CP or Rep gene constructs of PLRV.     

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.     

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