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Stem cells are essential for the development and organ regeneration of multicellular organisms, so their infection by pathogenic viruses must be prevented. Accordingly, mammalian stem cells are highly resistant to viral infection due to dedicated antiviral pathways including RNA interference (RNAi). In plants, a small group of stem cells harbored within the shoot apical meristem generate all postembryonic above-ground tissues, including the germline cells. Many viruses do not proliferate in these cells, yet the molecular bases of this exclusion remain only partially understood. Here, we show that a plant-encoded RNA-dependent RNA polymerase, after activation by the plant hormone salicylic acid, amplifies antiviral RNAi in infected tissues. This provides stem cells with RNA-based virus sequence information, which prevents virus proliferation. Furthermore, we find RNAi to be necessary for stem cell exclusion of several unrelated RNA viruses, despite their ability to efficiently suppress RNAi in the rest of the plant. This work elucidates a molecular pathway of great biological and economic relevance and lays the foundations for our future understanding of the unique systems underlying stem cell immunity.

Members of the genus Emaravirus, including Raspberry leaf blotch virus (RLBV), are enveloped plant viruses with segmented genomes of negative-strand RNA, although the complete genome complement for any of these viruses is not yet clear. Currently, wheat mosaic virus has the largest emaravirus genome comprising eight RNAs. Previously, we identified five genomic RNAs for RLBV; here, we identify a further three RNAs (RNA6-8). RNA6-8 encode proteins that have clear homologies to one another, but not to any other emaravirus proteins. The proteins self-interacted in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) experiments, and the P8 protein interacted with the virus nucleocapsid protein (P3) using BiFC. Expression of two of the proteins (P6 and P7) using potato virus X led to an increase in virus titre and symptom severity, suggesting that these proteins may play a role in RLBV pathogenicity; however, using two different tests, RNA silencing suppression activity was not detected for any of the RLBV proteins encoded by RNA2-8.

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form β-strands, namely β1 and β2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the β1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within β1 without preventing the membrane association or movement function of P4.

The specificity of the interaction between the coat protein (CP) and 2b nematode-transmission helper protein of two isolates, PpK20 and PaY4, of Tobacco rattle virus (TRV) that differ in their transmission characteristics was investigated. Yeast two-hybrid experiments identified a central domain of the CP that promoted CP:CP interaction but inhibited CP:2b interaction. Deletion of a conserved coiled-coil domain from the 2b protein prevented its interaction with CP, whereas deletion of N- and C-terminal domains of the 2b protein greatly enhanced its interaction with CP. A C-terminal flexible domain of the CP was also shown to be important for interaction with the 2b protein. However, this domain was not sufficient to direct isolate-specific interaction of these proteins either in yeast or via a chimeric TRV in planta. Although these two TRV isolates are both transmitted by a shared vector trichodorid nematode, Paratrichodorus pachydermus, the CP from isolate PpK20 did not interact with the 2b protein from isolate PaY4, and vice versa, suggesting that in the field mixed infections of TRV are unlikely to cause promiscuous transmission by alternative trichodorid nematode species.

Transmission of the tobraviruses Tobacco rattle virus (TRV) and Pea early-browning virus (PEBV) by trichodorid vector nematodes requires the viral coat protein (CP) and the 2b protein, a nonstructural protein encoded by RNA2, the smaller of the two viral genomic RNAs. It is hypothesized that the 2b protein functions by interacting with a small, flexible domain located at the C-terminus of the CP, forming a bridge between the virus particle and the internal surface of the vector nematode feeding apparatus. Antibodies specific for the 2b protein of PEBV or TRV did not bind to virus particles that were adsorbed to electron microscope grids and were not able to trap virus particles from extracts of infected plants. However, electron microscopy of thin sections of plants infected with PEBV probed with 2b-specific antibodies which were further gold-labeled showed that the 2b protein localizes exclusively to virus particles. Similarly, immunogold localization studies showed that the 2b protein of TRV isolate PaY4 is associated only with TRV PaY4 virus particles. When a recombinant TRV encoding the PaY4 2b protein and the CP from TRV isolate PpK20 was examined, the 2b protein could not be detected by Western blotting and in IGL experiments was not associated with virus particles. These results suggest that in the absence of a specific interaction between compatible CP and 2b proteins, the 2b protein does not accumulate.

Analysis of RNA2 of TRV PaY4 showed it to be recombinant, carrying 3'-terminal sequences derived from RNA1. Virus produced using an infectious cDNA clone of PaY4 RNA2 was nematode transmissible, demonstrating that natural TRV recombinant isolates are not necessarily defective. Mutations introduced into PaY4 RNA2 showed that the 2b gene, but not the 2c gene, is required for transmission by both Paratrichodorus pachydermus and P. anemones nematodes. Experiments examined whether infection of plants with two different virus clones would impact upon nematode transmission of either virus. Simultaneous inoculation with TRV clones expressing green or red fluorescent proteins revealed that mixing of the two virus populations did not occur, although, in roots, adjacent cells were found containing green- or red-tagged viruses. Subsequently, in similar experiments it was found that a TRV PaY4 2b mutant was transmitted when combined with wild-type TRV PaY4. Also, transmission of a 2b mutant of an in vitro TRV/PEBV recombinant virus (TRV-C1) occurred after coinfection with wild-type virus. Thus, the tobravirus 2b transmission protein is trans-acting. Although TRV PaY4 and TRV PpK20 are both transmitted by P. pachydermus, a 2b mutant of TRV PaY4 was not transmitted when coinoculated to plants with TRV PpK20.

The complete nucleotide sequence of RNA2 of the E116 isolate of Dutch pea early-browning virus (PEBV-D) was obtained from overlapping cDNA clones. The RNA was found to encode three open reading frames corresponding to, in 5' to 3' order, the coat protein, the 2b nematode transmission protein and the C-terminal part of the cysteine-rich 1b protein derived from RNA1. The 3' non-coding region of PEBV-D RNA2 was also shown to be derived from RNA1. This is the first demonstration that recombination of PEBV occurs in nature. Comparison of the amino acid sequences of the PEBV-D RNA2 proteins with those of British PEBV and several isolates of tobacco rattle virus reveals complex patterns of mixing of the genomes of these two viruses.

Although sequence data have been obtained for several tobravirus isolates, only two of these isolates are nematode-transmissible. Tobacco rattle virus (TRV) PpK20 is transmitted by Paratrichodorus pachydermus, whereas pea early-browning virus (PEBV) TpA56 is transmitted by Trichodorus primitivus. To clarify whether differences in the genome structure of these isolates are relevant to the specificity of interactions with particular vector nematodes, or merely reflect a taxonomic difference between TRV and PEBV, we have sequenced RNA2 of a new isolate of TRV (TpO1) that is transmitted by the same vector nematode as PEBV TpA56 but is not transmitted by the nematode vector of TRV PpK20. TRV TpO1 RNA2 encodes, in 5' to 3' order, a coat protein (CP), a 9K protein, a 2b (29K) protein and a 2c (18K) protein. Amino acid sequence comparison shows that both the CP and 2b proteins of TRV TpO1 resemble more closely the analogous proteins from PEBV TpA56 than those from TRV PpK20. Also, the TRV TpO1 9K protein has similarities with the PEBV 9K protein whereas this protein is lacking in TRV PpK20.

Pea early browning virus (PEBV) is transmitted between plants by root-feeding trichodorid nematodes. Mutagenesis studies have implicated two non-structural viral proteins in the transmission process. These two proteins [the 29 kDa ('29K') protein and the 23K protein] were expressed in bacteria and used to raise antibodies. In Western blotting experiments, the antibodies detected both of these virus proteins in leaves and roots of infected Nicotiana bethamiana and N. clevelandii plants. Periodate treatment of proteins transferred to nitrocellulose membranes suggested that the PEBV 23K protein may be glycosylated. A PEBV mutant was constructed lacking the complete 23K coding sequence. The mutant was able systemically to infect Nicotiana spp. but caused striking chlorotic ringspot leaf symptoms and stunting of both leaves and roots. These symptoms were absent in plants doubly-infected with the mutant and wild-type PEBV. The 23K gene deletion mutant was transmitted by nematodes at a much reduced frequency compared to wild-type virus, indicating that the 23K protein is involved in but not essential for vector transmission. Western immuno-blot and ELISA experiments revealed that the reduction in the nematode-transmissibility of PEBV carrying mutations in the 23K gene did not result from interference in the expression of the 29K transmission protein or from gross changes in the titre of virus in the roots of infected plants.

Pea early browning virus (PEBV) is a member of the genus, Tobravirus. It is transmitted by soil-inhabiting trichodorid nematodes and through seeds from diseased plants. By introducing mutations into the PEBV genome, we have studied the viral determinants of seed transmission in pea. Neither deleting a portion of the genome containing the three nonstructural genes in RNA2 nor the interuption of any of the three genes individually prevented PEBV seed transmission. However, a comparison of two PEBV isolates indicated a minor role for RNA2 or its products. In contrast, the removal of the coding sequence of the 12K gene in RNA1 almost completely abolished viral seed transmission. The virus lacking the 12K gene caused more severe symptoms on leaves and pods, and accumulated to a higher level than the wild-type virus in both types of tissues. However, the 12K deletion mutant accumulated poorly in anthers and carpels, and could not be detected in pollen grains and ovules. These results suggest that the 12K gene is involved in the infection of the gametic cells and hence the seed transmission of PEBV in pea.

Mutations were introduced into four genes encoded by RNA2 of pea early browning virus (PEBV) to determine their possible involvement in the transmission of this virus by nematodes. Deletion of 28 amino acids from the C-terminus of the coat protein abolished the formation of virus particles. Deletion of 15 amino acids at the C-terminus did not affect particle formation but did abolish nematode transmission. In contrast, deletion of 13 amino acids immediately preceding the 16 C-terminal residues did not affect particle formation and decreased rather than abolished nematode transmission. A deletion in the gene encoding a 29-kDa protein and a frameshift mutation in the gene encoding a 23-kDa protein both abolished transmission without affecting virus particle formation. Mutations in an ORF encoding a 9-kDa protein, which is located on the genome between the coat protein gene and 29K gene, gave conflicting results. Removal of the AUG initiation codon from the 9K ORF had no effect on transmission, whereas the introduction of a frameshift mutation, which would prematurely terminate expression of the putative 9-kDa protein, decreased the frequency of transmission. The results show that the coat protein and probably all three of the other RNA2-encoded proteins play a role in the transmission of PEBV by vector nematodes.

In northern blots, cDNA probes prepared to soil-borne wheat mosaic virus (SBWMV) RNA-1 and RNA-2 hybridized to RNA-1 and RNA-2, respectively, from a UK isolate of oat golden stripe virus (OGSV), as well as to their homologous RNAs. RT-PCR was used to amplify, clone and sequence a region of about 750 nucleotides spanning the capsid protein gene and part of the readthrough protein on RNA-2 from OGSV, a French isolate of SBWMV and two stable deletion mutants (Lab1 and Okl-7) of SBWMV isolates from Nebraska and Oklahoma respectively. There was very high (96.7-99.1%) nucleotide homology between all these sequences and the wild-type SBWMV sequences from Nebraska and Oklahoma. OGSV was more similar to SBWMV from France and Nebraska than were any of the isolates to SBWMV from Oklahoma. Of the few differences in the deduced amino acid sequences of the capsid proteins from the different isolates, OGSV differed from all SBWMV isolates only in one amino acid (isoleucine for valine at position 88). The high degree of similarity suggests that OGSV may best be classified as an oat strain of SBWMV.

A rapid method was developed using long template (LT)-PCR to amplify the complete RNA2 of isolates of TRV for which no sequence data are available. The amplification makes use of a 5' terminal oligonucleotide which contains degeneracies corresponding to the sequences of several different TRV isolates, and a 3' oligonucleotide which is complementary to a sequence present in all known isolates. This method was used to show the high degree of sequence homology existing in the terminal regions of two uncharacterised TRV isolates (TPO3 and PAY4), and revealed the deletion of an 80-nucleotide sequence in the 5' terminal region of TPO3 RNA2.

Full-length cDNA clones of both RNAs of pea early browning virus have been constructed. Synthetic transcripts derived in vitro from these clones are infectious when inoculated onto plants. Electron microscopy revealed the presence of virions in transcript-inoculated plants, and both purified RNA and virions isolated from such plants could be used to infect other plants. Transcripts of RNA1 alone were able to replicate and spread systemically which is a characteristic of members of the tobravirus group of plant viruses.

The article discusses a study that examines the interaction of translation elongation factor eEF1A with the ORF6 protein of tobacco mosaic virus (TMV) strain L. It found that the interaction between eEF1A and the secondary structure element in the 3'-terminal region of genomic RNA of the West Nile virus promotes the synthesis of viral RNA chains of negative polarity. It demonstrates the possibility of homotypic interactions of ORF6-L protein molecules.