Peanut Clump Virus Research Articles

Retraction statement: 'Identification, subcellular localization and some properties of a cysteine-rich suppressor of gene silencing encoded by peanut clump virus' by Patrice Dunoyer, Sébastien Pfeffer, Christiane Fritsch, Odile Hemmer, Olivier Voinnet and K. E. Richards (https://onlinelibrary.wiley.com/doi/full/10.1046/j.0960-7412.2001.01242.x) and 'Correction' by Dunoyer et al. (https://onlinelibrary.wiley.com/doi/10.1111/tpj.12884).

The above original article, first published online on 01 March 2002 in Wiley Online Library (wileyonlinelibrary.com), and in volume 29, pp. 555-567, has been retracted by agreement between the following authors: Patrice Dunoyer, Sébastien Pfeffer, Christiane Fritsch, Odile Hemmer, Olivier Voinnet and K. E. Richards, the journal Editor-in-Chief, Lee Sweetlove, and John Wiley & Sons Ltd. This notice updates and replaces the article and its correction notice, published on 10 June 2015 and in volume 82, pp. 1076-1077. In the correction notice, updated versions of Figures 2, 3 and 6 were published. It has since come to light that an image provided for the correction notice (Fig 2C) contained evidence of inappropriate manipulation. Additional inconsistencies in the representation of the data shown in Figures 3 and 4 have also become apparent. Patrice Dunoyer and Sébastien Pfeffer take full responsibility for the described manipulation and inconsistencies that were made without the consent or knowledge of any of the other co-authors. The authors consider that the integrity of the paper has been compromised and that the most appropriate course of action is a retraction of the article.

Estimation of the functions of viral RNA silencing suppressors by apple latent spherical virus vector.

Apple latent spherical virus (ALSV) is a latent virus with wide host range of plant species. In the present study, we prepared ALSV vectors expressing RNA silencing suppressors (RSSs) from eight plant viruses: P19 of carnation Italian ring spot virus (tombusvirus), 2b of peanut stunt virus (cucumovirus), NSs of tomato spotted wilt virus (tospovirus), HC-Pro of bean yellow mosaic virus (potyvirus), γb of barley stripe mosaic virus (hordeivirus), P15 of peanut clump virus (pecluvirus), P1 of rice yellow mottle virus (sobemovirus), or P21 of beet yellows virus (closterovirus). These vectors were inoculated to Nicotiana benthamiana to investigate the effects of RSSs on the virulence and accumulation of ALSV. Among the vectors, ALSV expressing NSs (ALSV-NSs) developed severe mosaic symptoms in newly developed leaves followed by plant death. Infection of ALSV-γb induced characteristic concentric ringspot symptoms on leaves, and plants infected with ALSV-HC-Pro showed mosaic and dwarf symptoms. Infection of the other five ALSV vectors did not show symptoms. ELISA and immunoblot assay indicated that virus titer increased in leaves infected with ALSV-NSs, γb, HC-Pro, or P19. RT-qPCR indicated that the amount of ALSV in plants infected with ALSV-NSs was increased by approximately 45 times compared with that of wtALSV without expression of any RSS. When ALSV-P19, NSs, or HC-Pro was inoculated to Cucumis sativus plants, none of these ALSV vectors induced symptoms, but accumulation of ALSV in plants infected with ALSV-NSs was increased, suggesting that functions of RSSs on virulence and accumulation of ALSV depend on host species.

Neutralization of mobile antiviral small RNA through peroxisomal import.

In animals, certain viral proteins are targeted to peroxisomes to dampen the antiviral immune response mediated by these organelles1-3. In plants, RNA interference (RNAi) mediated by small interfering (si)RNA is the main antiviral defence mechanism. To protect themselves against the cell- and non-cell autonomous effects of RNAi, viruses produce viral suppressors of RNA silencing (VSR)4, whose study is crucial to properly understand the biological cycle of plant viruses and potentially find new solutions to control these pathogens. By combining biochemical approaches, cell-specific inhibition of RNAi movement and peroxisome isolation, we show here that one such VSR, the peanut clump virus (PCV)-encoded P15, isolates siRNA from the symplasm by delivering them into the peroxisomal matrix. Infection with PCV lacking this ability reveals that piggybacking of these VSR-bound nucleic acids into peroxisomes potentiates viral systemic movement by preventing the spread of antiviral siRNA. Collectively, these results highlight organellar confinement of antiviral molecules as a novel pathogenic strategy that may have its direct counterpart in other plant and animal viruses.

Acquisition and Transmission of Peanut clump virus by Polymyxa graminis on Cereal Species

The objective of this study was to investigate the specificity of the interactions between Polymyxa graminis, Peanut clump virus (PCV), and cereals, particularly the acquisition and the transmission of the virus by three P. graminis formae speciales. A new strategy has been developed: it involves using sugarcane as the common host for both the virus and its vector in order to produce the viruliferous zoospores of P. graminis f. sp. subtropicalis, temperata, and tropicalis that were then inoculated on cereal species. This experiment enabled the role of P. graminis f. sp. tropicalis and subtropicalis zoospores in PCV transmission to be demonstrated. The efficiency of this transmission was shown to vary, depending on the P. graminis special forms. Interestingly, the high transmission of the PCV isolate from Burkina Faso by an isolate of P. graminis f. sp. tropicalis from Niger on pearl millet suggests that there is a coevolution mechanism in this pathosystem. The study also provides evidence that the host plant species in which Polymyxa zoospores are produced could affect the infectivity of the vector. Finally, using Polymyxa quantitation by quantitative reverse-transcription polymerase chain reaction and in situ observations of the virus, the study demonstrates the independence of the development of PCV and its vector in the host plants.

Polymyxa graminis . [Distribution map

Abstract A new distribution map is provided for Polymyxa graminis Ledingham. Cercozoa: Plasmodiophorida. Hosts: groundnut ( Arachis hypogaea ), oats ( Avena sativa ), barley ( Hordeum vulgare ), rice ( Oryza sativa ), rye ( Secale cereale ), wheat ( Triticum aestivum ), millet ( Panicum miliaceum ) and sorghum ( Sorghum bicolor ). Information is given on the geographical distribution in Europe (Belgium, France, Mainland France, Germany, Greece, Mainland Greece, Italy, Mainland Italy, Netherlands, Spain, Mainland Spain, UK, England and Wales), Asia (China, Anhui, Gansu, Henan, Hubei, Hunan, Jiangsu, Jiangxi, Shaanxi, Shandong, Shanxi, Sichuan, Zhejiang, India, Andhra Pradesh, Gujarat, Punjab, Rajasthan, Iran, Japan, Honshu, Lebanon, Pakistan, Syria, Turkey), Africa (Burkina Faso, Cote d'Ivoire, Mali, Niger, Senegal), North America (Canada, Manitoba, Ontario, Prince Edward Island, USA, Arkansas, Florida, Georgia, Illinois, Iowa, Kansas, Kentucky, Maryland, Missouri, Nebraska, New Mexico, New York, Oklahoma, Pennsylvania, South Carolina, Virginia, Washington), South America (Bolivia, Brazil, Rio Grande do Sul, Colombia), Oceania (New Zealand). Vector of numerous plant viruses, including Soil-borne wheat mosaic virus, Wheat yellow mosaic virus, Wheat spindle streak mosaic virus, Barley yellow mosaic virus, Oat mosaic virus, Rice stripe necrosis virus and Peanut clump virus. Several viruses transmitted by P. graminis cause major widespread diseases on barley, wheat and groundnut. P. graminis is presumed to be present in all fields infested with the viral diseases it transmits, but is also present in virus-free soils. The distribution of the viruses transmitted by P. graminis is greater than the confirmed distribution of P. graminis shown on the map.

Targeting highly conserved 3′-untranslated region of pecluviruses for sensitive broad-spectrum detection and quantitation by RT-PCR and assessment of phylogenetic relationships

The 3′-end region of many virus isolates has been shown to possess conserved sequences in addition to the presence of numerous genomic and subgenomic RNAs. Utilizing these sequences, a broad-spectrum reverse transcription-polymerase chain reaction protocol has been developed to detect all the known Indian peanut clump virus and Peanut clump virus isolates, that cause peanut clump diseases in West Africa and India. The primers were targeted at the highly conserved 3′-untranslated regions of the PCV RNA-1 and RNA-2. The conservation was confirmed by sequencing these untranslated regions of RNA-1 for six isolates and RNA-2 for one isolate. The conserved structure of the RNA-1 and RNA-2 was observed and the importance of this region for the virus survival was confirmed. The primers were also designed for virus quantitation using a Taqman ®-based real-time RT-PCR. The use of RT-PCR and real-time quantitative RT-PCR improved the sensitivity of PCV detection compared to ELISA. RT-PCR also led to the detection of IPCV and PCV on two new natural hosts: Oldenlandia aspera and Vigna subterranea. Real-time RT-PCR is considered to be an ideal tool for identifying resistant sources to both IPCV and PCV.

Seed, soil and vegetative transmission contribute to the spread of pecluviruses in Western Africa and the Indian sub-continent

Peanut clump and sugarcane red leaf mottle diseases are caused by viruses of the genus Pecluvirus. Indian peanut clump virus occurs in the Indian sub-continent and Peanut clump virus in West Africa. A feature of these viruses is that they are both seed and soil transmitted. Both modes of transmission contribute to long-term persistence and field spread. Data on seed transmission in pearl millet, virus movement within the plant and virus diversity based on RNA-1 partial sequences are presented. This study emphasizes that pecluviruses are also viruses of cereals infecting sorghum and pearl millet, and highlights a correlation between the countries cultivating these two crops and the virus distribution. Ways of controlling pecluviruses and their vector, Polymyxa graminis, taking into account the virus dissemination routes, are proposed.

Role of tRNA-like structures in controlling plant virus replication

Transfer RNA-like structures (TLSs) that are sophisticated functional mimics of tRNAs are found at the 3′-termini of the genomes of a number of plant positive strand RNA viruses. Three natural aminoacylation identities are represented: valine, histidine, and tyrosine. Paralleling this variety in structure, the roles of TLSs vary widely between different viruses. For Turnip yellow mosaic virus, the TLS must be capable of valylation in order to support infectivity, major roles being the provision of translational enhancement and down-regulation of minus strand initiation. In contrast, valylation of the Peanut clump virus TLS is not essential. An intermediate situation seems to exist for Brome mosaic virus, whose RNAs 1 and 2, but not RNA 3, need to be capable of tyrosylation to support infectivity. Other known roles for certain TLSs include: (i) the recruitment of host CCA nucleotidyltransferase as a telomerase to maintain intact 3′ CCA termini, (ii) involvement in the encapsidation of viral RNAs, and (iii) presentation of minus strand promoter elements for replicase recognition. In the latter role, the promoter elements reside within the TLS but are not functionally dependent on tRNA mimicry. The phylogenetic distribution of TLSs indicates that their evolutionary history includes frequent horizontal exchange, as has been observed for protein-coding regions of plant positive strand RNA viruses.

Nonregulated expression of TGBp3 of hordei-like viruses but not of potex-like viruses inhibits beet necrotic yellow vein virus cell-to-cell movement

Plant viruses containing a Triple Gene Block (TGB) movement protein gene cassette fall into two classes. We have shown previously that the third TGB protein (TGBp3) of beet necrotic yellow vein virus (BNYVV; Class 1) and peanut clump virus (Class 1) inhibit BNYVV intercellular movement when expressed from a co-inoculated BNYVV RNA 3-based replicon. Here we show that autonomous expression of TGBp3's of four other Class 1 viruses of various genera also inhibits BNYVV movement. No such effect was observed for four Class 2 virus TGBp3's, suggesting that the roles of Class 1 and 2 TGBp3's in movement differ significantly.

Development of Molecular Detection of Three Species of Seed-Transmissible Viruses Useful for Plant Quarantine

【Three pairs of specific primers were developed for rapid and precise RT-PCR detection of three seed-transmissible viruses, namely Peanut clump virus (PCV, Pecluvirus), White clover mosaic virus (WCIMV, Potexvirus) and Carrot red leaf virus (CaRLV, Luteovirus). Each primer set was found in conserved region through multiple sequence alignment in the DNAMAN. Total nucleic acids extracted from PCV-, WCMV-, and CaRLV-infected seeds and healthy plants were used for RT-PCR detection using each virus-specific primer, Sizes of PCV, WCIMV, and CaRLV PCR products were 617bp (PCV-uni5 and PCV-uni3 primers), 561bp (WCMV-CP5 and WCMV-CP3 primers), and 626bp (CL1-UP and CL2-DN primers); which corresponded to the target sizes. Nucleotides sequences of each amplified cDNA were confirmed which belonged to the original virus. This study suggests that these virus-specific primer sets can specifically amplify viral sequences in infected seeds. Thus, they can be used for specific detection of three viruses (PCV, WCMV and CaRLV) from imported seed samples for plant quarantine service.】

Mapping of viral RNA sequences required for assembly of peanut clump virus particles.

RNA sequences required for assembly into rod-shaped virions of RNA-1 and RNA-2 of Peanut clump virus (PCV) were mapped by testing the ability of different RNA-1 and -2 deletion mutants to be encapsidated in vivo in an RNase-resistant form. Encapsidation of RNA-1 was found to require a sequence domain in the 5'-proximal part of the P15 gene, the 3'-proximal gene of RNA-1. On the other hand, the subgenomic RNA which encodes P15 was not encapsidated, suggesting that other features of RNA-1 are important as well. Two sequences which could drive encapsidation of RNA-2 deletion mutants were located. One was in the 5'-proximal coat protein gene and the other in the P14 gene near the RNA 3' terminus. There were no obvious sequence homologies between the different assembly initiation sequences.

Dynamics of Polymyxa graminis and Indian peanut clump virus (IPCV) infection on various monocotyledonous crops and groundnut during the rainy season

The progress of Indian peanut clump virus (Hyderabad isolate; IPCV‐H) and its vector Polymyxa graminis in various monocotyledonous crops and groundnut was studied during the 1994, 1995 and 1996 rainy seasons in a naturally infested field in India. The roles of rainfall and temperature in the dynamics of infection by both the virus and its vector were analysed by exposing young seedlings for short periods in the field. Of the host crops studied, wheat, followed by barley, showed the highest virus incidence, although P. graminis was rarely observed in roots of wheat and was not detected in those of barley. The roots of maize, pearl millet and sorghum plants infected by P. graminis showed intense colonization by sporosori. IPCV accumulated in systemically infected maize plants; the sorghum and pearl millet cultivars studied showed a transient presence of IPCV‐H. Rice was seldom infected by the virus and P. graminis was not detected in its roots. Groundnut was a systemic host for the virus, although during these experiments no P. graminis was found in its roots. Groundnut appeared to be susceptible to infection, mostly in the early stages of crop development, and the rate of IPCV‐H transmission in groundnut seeds was highest (13%) for plants infected when young. The seed‐transmission rate quickly decreased in plants showing symptoms 1 month after sowing. Time of infection had little influence on groundnut pod yield, which was always reduced by >60% in infected plants. There was some evidence that the quantity and distribution of rainfall influenced the incidences of IPCV‐H and P. graminis: high rainfall resulted in high incidences of the virus and P. graminis, and a weekly rainfall of 14 mm was sufficient for P. graminis to initiate infection. Temperatures prevailing during the rainy season ranged from 23 to 30°C and were found to be conducive to natural virus transmission. These results suggest measures to be explored for controlling peanut clump disease.

Identification, subcellular localization and some properties of a cysteine-rich suppressor of gene silencing encoded by peanut clump virus.

In plants, post-transcriptional gene silencing (PTGS) is part of a defence mechanism against virus infection. Several plant viruses have been shown to encode proteins which can counteract PTGS. In this paper it is demonstrated that P15 of peanut clump pecluvirus (PCV) has anti-PTGS activity. P15 is a small cysteine-rich protein with no sequence similarity to previously described PTGS-suppressor proteins which has several novel properties. It possesses four C-terminal proximal heptad repeats that can potentially mediate a coiled-coil interaction and is targeted to peroxisomes via a C-terminal SKL motif. The coiled-coil sequence is necessary for the anti-PTGS activity of P15, but the peroxisomal localization signal is not, although it is required for efficient intercellular movement of the virus.

Molecular characterization and detection of European isolates of Soil‐borne wheat mosaic virus

Sequencing of a recently identified isolate of Soil‐borne wheat mosaic virus (SBWMV) from the UK confirmed its identity as a European strain of the species and provided further evidence for taxonomic divisions in the group. Two RT–PCR protocols were developed for the detection of all SBWMV strains and for the specific detection of the European SBWMV strain, and were tested successfully on 21 isolates of SBWMV from a range of countries. Both protocols worked well using either purified total RNA in one‐ or two‐step RT‐PCR, or immunocapture (IC) RT–PCR. The sensitivity of IC RT‐PCR was 100 times greater than ELISA. Neither set of primers produced any PCR product with either Wheat spindle streak mosaic virus or Wheat yellow mosaic virus which are frequently associated with SBWMV, or with the related viruses Indian peanut clump virus, Potato mop‐top virus, Beet soil‐borne virus and Beet necrotic yellow vein virus. This new diagnostic protocol will improve disease management by enabling correct identification of the causal pathogen and earlier detection than is possible serologically.

Peanut clump virus RNA-1-encoded P15 regulates viral RNA accumulation but is not abundant at viral RNA replication sites.

RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5'-triphosphate (BrUTP). BrUTP labeling also occurred in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels.

An efficient method for the production of transgenic plants of peanut ( Arachis hypogaea L .) through Agrobacterium tumefaciens-mediated genetic transformation

Cotyledon explants from mature peanut seeds ( Arachis hypogaea L.) were optimized to obtain adventitious shoot buds with high frequencies (>90%). Efficient transformation of these cotyledons by using Agrobacterium tumefaciens strain C58 carrying neomycin phosphotransferase II ( nptII) and ß-glucuronidase ( GUS; uidA), or coat protein gene of the Indian peanut clump virus (IPCVcp) and nptII on binary vectors (pBI121; pROKII:IPCVcp) led to the production of a large percentage (55%) of transgenic plants. Transformed individuals were obtained through selection on medium containing 125 mg l −1 kanamycin. A large number of independently transformed plants (over 75) were successfully transplanted to the glasshouse. Integration of the transgenes and stable genetic transformants in the progeny were assessed by PCR amplification of 700-bp fragment of nptII and 585-bp of IPCVcp genes, and Southern blot hybridizations in the T1 generation of transgenic plants. Analysis of 35 transgenic plants of T1 generation from the progeny of a single transformation event suggested the segregation of a single copy insert in a 3:1 Mendelian ratio. On an average, 120–150 days were required between the initiation of explant transformation and transfer of rooted plants to the greenhouse. The cotyledon regeneration system proved to be an excellent vehicle for the production of a large number of independently transformed peanut plants. Shoot formation was rapid and prolific, and a large proportion of these shoots developed into fertile plants. The method reported here provides new opportunities for the crop improvement of peanut via genetic transformation.

The valine anticodon and valylatability of Peanut clump virus RNAs are not essential but provide a modest competitive advantage in plants.

The role of valine aminoacylation of the two genomic RNAs of Peanut clump virus (PCV) was studied by comparing the amplification in vivo of RNAs with GAC, GDeltaC, or CCA anticodons in the tRNA-like structure (TLS) present at the 3' end of each viral RNA. The PCV RNA1 TLS of isolate PCV2 possesses a GAC anticodon and is capable of highly efficient valylation, whereas the RNA2 TLS has a GDeltaC anticodon that does not support valylation. The presence in RNA1 of GDeltaC or CCA anticodons that conferred nonvalylatability resulted in about 2- to 4-fold and a 14- to 24-fold reduction, respectively, in RNA accumulations in tobacco BY-2 protoplasts inoculated with the RNA1 variants together with wild-type RNA2(GDeltaC). No differences in RNA levels were observed among protoplasts inoculated with the three variant RNA2s in the presence of wild-type RNA1(GAC). All combinations of valylatable and nonvalylatable RNAs 1 and 2 were similarly infectious in Nicotiana benthamiana plants, and viral RNAs accumulated to similar levels; all input TLS sequences were present unchanged in apical leaves. In direct competition experiments in N. benthamiana plants, however, both RNA1 and RNA2 with GAC valylatable anticodons outcompeted the nonvalylatable variants. We conclude that valylation provides a small but significant replicational advantage to both PCV RNAs. Sequence analysis of the TLS from RNA2 of a second PCV isolate, PO2A, revealed the presence of an intact GAC valine anticodon, suggesting that the differential valylation of the genomic RNAs of isolate PCV2 is not a general characteristic of PCV.

The nucleotide sequence of Indian peanut clump virus RNA 2: sequence comparisons among pecluviruses.

The RNA-2 molecule of an isolate of the L serotype of Indian peanut clump virus (IPCV) was shown to consist of 4,290 nucleotides with five open reading frames (ORF). The arrangement of the ORFs resembled that in RNA-2 of Peanut clump virus (PCV) from West Africa. The proteins encoded by the ORFs in IPCV-L RNA are between 32% and 93% identical to those encoded by PCV RNA. Partial sequence data for the RNA-2 of isolates of the H and T serotypes of IPCV show that the coat and P40 proteins encoded by the 5'-most ORFs of RNA-2 of IPCV-L, IPCV-H and IPCV-T are as similar to each other as any is to the corresponding proteins of PCV. A conserved motif 'F-E-x6-W' is present near the C-termini of the coat proteins of all three IPCV serotypes and of PCV, as it is in the coat proteins of other viruses that have rod-shaped particles, such as Tobacco mosaic virus and Tobacco rattle virus. The results support the distinction of IPCV and PCV as separate virus species, but also raise the question of how the serotypes of IPCV should be classified.

Virus-like particles assemble in plants and bacteria expressing the coat protein gene of Indian peanut clump virus

cDNA copies of the coat protein (CP) gene of Indian peanut clump virus (IPCV)-H were introduced into cells of Nicotiana benthamiana or Escherichia coli by transformation with vectors based on pROKII or pET respectively. In both plant and bacterial cells, IPCV CP was expressed and assembled to form virus-like particles (VLP). In plant extracts, the smallest preponderant particle length was about 50 nm. Other abundant lengths were about 85 and about 120 nm. The commonest VLP length in bacterial extracts was about 30 nm. Many of the longer VLP appeared to comprise aggregates of shorter particles. The lengths of the supposed 'monomer' VLP corresponded approximately to those expected for encapsidated CP gene transcript RNA. Immunocapture RT-PCR, using primers designed to amplify the CP gene, confirmed that the VLP contained RNA encoding IPCV-H CP. The results show that encapsidation does not require the presence of the 5'-terminal untranslated sequence of the virus RNA and suggest that if there is an 'origin of assembly' motif or sequence, it lies within the CP gene. When transgenic plants expressing IPCV-H CP were inoculated with IPCV-L, a strain that is serologically distinct from IPCV-H, the virus particles that accumulated contained both types of CP.

The First Triple Gene Block Protein of Peanut Clump Virus Localizes to the Plasmodesmata during Virus Infection

The subcellular localization of the first triple gene block protein (TGBp1) of peanut clump pecluvirus (PCV) was studied by subcellular fractionation and immunogold cytochemistry using TGBp1-specific antibodies raised against a fusion protein expressed in and purified from bacteria. In the inoculated and apical leaves of virus-infected Nicotiana benthamiana, TGBp1 localized to the cell wall and P30 fractions. Electron microscopy of immunogold-decorated ultrathin sections of the infected leaf tissue revealed TGBp1-specific labeling of the plasmodesmata joining mesophyll cells. In longitudinal sections of the plasmodesmata, the TGBp1-specific labeling was most commonly associated with the plasmodesmal collar region. In transgenic N. benthamiana, which constitutively expressed TGBp1, no TGBp1-specific immunogold labeling of plasmodesmata was observed, but plasmodesmata were gold decorated when the transgenic plants were infected with a TGBp1-defective PCV mutant, indicating that factors induced by the virus infection target and/or anchor the transgene TGBp1 to the plasmodesmata.