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Development of CRISPR/Cas9 transient gene editing screening tools in plant biology has been hindered by difficulty of delivering high quantities of biologically active single guide RNAs (sgRNAs). Furthermore, it has been largely accepted that in vivo generated sgRNAs need to be devoid of extraneous nucleotides, which has limited sgRNA expression by delivery vectors. Here, we increased cellular concentrations of sgRNA by transiently delivering sgRNAs using a Tobacco mosaic virus-derived vector (TRBO) designed with 5′ and 3′ sgRNA proximal nucleotide-processing capabilities. To demonstrate proof-of-principle, we used the TRBO-sgRNA delivery platform to target GFP in Nicotiana benthamiana (16c) plants, and gene editing was accompanied by loss of GFP expression. Surprisingly, indel (insertions and deletions) percentages averaged nearly 70% within 7 d postinoculation using the TRBO-sgRNA constructs, which retained 5′ nucleotide overhangs. In contrast, and in accordance with current models, in vitro Cas9 cleavage assays only edited DNA when 5′ sgRNA nucleotide overhangs were removed, suggesting a novel processing mechanism is occurring in planta. Since the Cas9/TRBO-sgRNA platform demonstrated sgRNA flexibility, we targeted the N. benthamiana NbAGO1 paralogs with one sgRNA and also multiplexed two sgRNAs using a single TRBO construct, resulting in indels in three genes. TRBO-mediated expression of an RNA transcript consisting of an sgRNA adjoining a GFP protein coding region produced indels and viral-based GFP overexpression. In conclusion, multiplexed delivery of sgRNAs using the TRBO system offers flexibility for gene expression and editing and uncovered novel aspects of CRISPR/Cas9 biology.

The objective of this study was to determine the contribution of different ARGONAUTE proteins in Nicotiana benthamiana (NbAGOs) to the defense against silencing sensitive GFP-expressing viral constructs based on Tomato bushy stunt virus (TBSV) (Tombusvirus), Sunn-hemp mosaic virus (Tobamovirus), and Foxtail mosaic virus (Potexvirus). Upon Tobacco rattle virus (TRV)-mediated down-regulation of NbAGO1, 4, 5, or 6, no effects were noted on susceptibility to any virus construct, whereas knockdown of NbAGO2 specifically prevented silencing of P19-defective TBSV (TGdP19). Down-regulation of a new gene referred to as NbAGO5L showed some reduced silencing for TGdP19 but not for the other two virus constructs, whereas silencing of NbAGO7 gave rise to a subtle increase in susceptibility to all three viruses. Co-infiltrating different TRV-NbAGO constructs simultaneously did not enhance virus susceptibility. However, an unexpected finding was that whenever the TRV-NbAGO1 construct was present, this compromised silencing of genes targeted by co-infiltrated constructs, as shown upon co-infiltration of TRV-NbAGO1 with either TRV-NbAGO2 or TRV-Sul (targeting Magnesium chelatase I). Only after a prolonged period (approximately 2 months) did TRV-Sul-mediated systemic bleaching occur in these co-infected plants, suggesting that TRV-NbAGO1 hinders the silencing ability of other TRV-NbAGO constructs. In conclusion, this study revealed new antiviral NbAGOs and dominant effects of silencing NbAGO1.

ARGONAUTE proteins (AGOs) are known to be key components of the RNA silencing mechanism in eukaryotes that, among other functions, serves to protect against viral invaders. Higher plants encode at least 10 individual AGOs yet the role played by many in RNA silencing-related antiviral defense is largely unknown, except for reports that AGO1, AGO2, and AGO7 play an antiviral role in Arabidopsis (Arabidopsis thaliana). In the plant virus model host Nicotiana benthamiana, Tomato bushy stunt virus (TBSV) P19 suppressor mutants are very susceptible to RNA silencing. Here, we report that a N. benthamiana AGO (NbAGO) with similarity to Arabidopsis AGO2, is involved in antiviral defense against TBSV. The activity of this NbAGO2 is shown to be directly associated with anti-TBSV RNA silencing, while its inactivation does not influence silencing of transiently expressed transgenes. Thus, the role of NbAGO2 might be primarily for antiviral defense.

An international workshop on ‘‘Induction and Suppression of RNA Silencing: Insights from Plant Viral Infections’’ was sponsored by the United States-Israel Binational Agricultural Research and Development Fund (BARD) and organized in Eilat, Israel in March 2010. The focus of this workshop was on molecular mechanisms employed by viruses or their hosts, and their interactions, for the regulation of virus-induced silencing and suppression. Several of the talks also served as potent reminders of scientific hubris and the need to be attentive to earlier results, both for analyses and perspective regarding new findings.

RNA silencing is a common strategy shared by eukaryotic organisms to regulate gene expression, and also operates as a defense mechanism against invasive nucleic acids such as viral transcripts. The silencing pathway is quite sophisticated in higher eukaryotes but the distinct steps and nature of effector complexes vary between and even within species. To counteract this defense mechanism viruses have evolved the ability to encode proteins that suppress silencing to protect their genomes from degradation. This review focuses on our current understanding of how individual components of the plant RNA silencing mechanism are directed against viruses, and how in turn virus-encoded suppressors target one or more key events in the silencing cascade.

Tomato bushy stunt virus and its cell-to-cell movement protein (MP; P22) provide valuable tools to study trafficking of macromolecules through plants. This study shows that wild-type P22 and selected movement-defective P22 amino acid substitution mutants were equivalent for biochemical features commonly associated with MPs (i.e. RNA binding, phosphorylation, and membrane partitioning). This generated the hypothesis that their movement defect was caused by improper interaction between the P22 mutants and one or more host factors. To test this, P22 was used as bait in a yeast (Saccharomyces cerevisiae) two-hybrid screen with a tobacco (Nicotiana tabacum) cDNA library, which identified a new plant homeodomain leucine-zipper protein that reproducibly interacted with P22 but not with various control proteins. These results were confirmed with an independent in vitro binding test. An mRNA for the host protein was detected in plants, and its accumulation was enhanced upon Tomato bushy stunt virusinfection of two plant species. The significance of this interaction was further demonstrated by the failure of the homeodomain protein to interact efficiently with two of the well-defined movement-deficient P22 mutants in yeast and in vitro. This is the first report, to our knowledge, that a new plant homeodomain leucine-zipper protein interacts specifically and in a functionally relevant manner with a plant virus MP.

While browsing this special Plant Physiology issue on the Diversity of Plant Systems, one must be impressed, albeit perhaps not surprised, by the numerous examples showing that research on various plants often leads to fascinating discoveries in several plant science subdisciplines. This Update aims

Tomato bushy stunt virus (TBSV) encodes a small gene (p19) nested within the cell-to-cell movement gene (p22), and their translation yields two proteins with separate activities for virus spread and symptom induction. The objective of this study was to determine the biological relevance associated with the translational mechanism responsible for expression of the nested p22 and p19 genes. Introduction of site-specific mutations to optimize the translational start site context of p22 caused a substantial shift in the ratio of the two proteins, mainly because it dramatically reduced the otherwise abundant levels of p19 protein accumulation in vitro and in vivo. Changes in the dosage or ratios of p22 and p19 proteins failed to noticeably affect virus replication or movement in Nicotiana spp. that support a systemic infection. In contrast, bio-assays with hypersensitive Nicotiana hosts illustrated that a substantially elevated p22/p19 protein ratio increased the size of p19 protein-mediated lesions whereas those induced by the p22 protein tended to be smaller. The reduced levels of p19 protein prevented the onset of a lethal apical necrosis in systemically infected Nicotiana benthamiana plants. Furthermore, the increased p22/p19 protein ratio impaired the ability of TBSV to systemically invade spinach plants. These results suggest that control of tombusvirus p22 and p19 protein ratios and dosage through context-dependent leaky scanning provides a co-translational mechanism to coordinate their biological activities.

This commentary relates to the work by M. Borja et al. (M. Borja, T. Rubio, H. B. Scholthof, and A. O. Jackson, MPMI 12:153-162, 1999) that shows that wild-type virus can be restored frequently by double recombination events between a tomato bushy stunt virus mutant with deletions inactivating the coat protein gene and a coat protein transgene. Here, we focus on evidence suggesting that new viruses might evolve via recombination with transgenes used for disease resistance, and discuss the potential effects of widespread use of these sources of resistance on virus evolution. We argue that the benefits arising from using transgenic sources of resistance for virus disease control outweigh potential negative consequences of evolution of novel hybrid viruses with destructive disease potential.

Tomato bushy stunt virus (TBSV) is a small isometric virus that contains a single-stranded RNA genome with five major genes. In this study, we have analyzed the importance of an additional small sixth open reading frame (ORF) of 207 nucleotides, designated pX, which resides at the 3' end of the genome. Bioassays showed that deletions or additions of nucleotides at the 5' end of the pX gene that were designed to disrupt the ORF, or site-specific inactivation of its start codon, all gave rise to TBSV mutants which were unable to accumulate to detectable levels in cucumber or Nicotiana benthamiana protoplasts. Although these results suggested a role for the putative pX protein, introduction of a premature stop codon in the pX gene had no strong negative effect. However, a comparable mutation that affected the same nucleotides without changing the predicted amino acid sequence greatly reduced RNA accumulation. Therefore, we hypothesize that cis-acting RNA sequences within the pX gene, rather than the predicted protein influence genome accumulation. The requirement of the cis-acting pX ORF sequences appears to be host-dependent because comparisons revealed that subtle pX gene mutations that prohibited accumulation of TBSV RNA in cucumber or N. benthamiana, failed to interfere substantially with replication in Chenopodium quinoa protoplasts or plants. Irrespective of the host, the cis-acting pX gene sequences were dispensable on replicase-deficient RNAs that require helper TBSV for replication in trans. In addition, the pX gene was not essential for in vitro translation of replicase proteins from genomic RNA. These results suggest that neither translation nor polymerase activity of the replicase proteins require pX gene sequences. However, it is possible that very early in the replication cycle of genomic RNA in vivo, the pX gene cis-acting element is essential for some other unidentified function which involves interaction with one or more host components whose composition varies slightly between different plants.

Post-transcriptional gene silencing is commonly observed in polyploid species and often poses a major limitation to plant improvement via biotechnology. Five plant viral suppressors of RNA silencing were evaluated for their ability to counteract gene silencing and enhance the expression of the Enhanced Yellow Fluorescent Protein (EYFP) or the β-glucuronidase (GUS) reporter gene in sugarcane, a major sugar and biomass producing polyploid. Functionality of these suppressors was first verified in Nicotiana benthamiana and onion epidermal cells, and later tested by transient expression in sugarcane young leaf segments and protoplasts. In young leaf segments co-expressing a suppressor, EYFP reached its maximum expression at 48-96 h post-DNA introduction and maintained its peak expression for a longer time compared with that in the absence of a suppressor. Among the five suppressors, Tomato bushy stunt virus-encoded P19 and Barley stripe mosaic virus-encoded γb were the most efficient. Co-expression with P19 and γb enhanced EYFP expression 4.6-fold and 3.6-fold in young leaf segments, and GUS activity 2.3-fold and 2.4-fold in protoplasts compared with those in the absence of a suppressor, respectively. In transgenic sugarcane, co-expression of GUS and P19 suppressor showed the highest accumulation of GUS levels with an average of 2.7-fold more than when GUS was expressed alone, with no detrimental phenotypic effects. The two established transient expression assays, based on young leaf segments and protoplasts, and confirmed by stable transgene expression, offer a rapid versatile system to verify the efficiency of RNA silencing suppressors that proved to be valuable in enhancing and stabilizing transgene expression in sugarcane.

▪ Abstract The development of plant virus gene vectors for expression of foreign genes in plants provides attractive biotechnological tools to complement conventional breeding and transgenic methodology. The benefits of virus-based transient RNA and DNA replicons versus transgenic gene expression include rapid and convenient engineering coupled with flexibility for expeditious application in various plant species. These characteristics are especially advantageous when very high levels of gene expression are desired within a short time, although instability of the foreign gene in the viral genome can present some problems. The strategies that have been tested for foreign gene expression in various virus-based vectors include gene replacement, gene insertion, epitope presentation, use of virus controlled gene expression cassettes, and complementation. Recent reports of the utililization of virus vectors for foreign gene expression in fundamental research and biotechnology applications are discussed.

In this study, we analyzed the influence of two nested genes (p19 and p22) of tomato bushy stunt virus (TBSV) on disease symptoms in systemically infected plants and in local lesion hosts. The contribution of individual genes was determined by bioassays with an infectious clone of wild-type TBSV, with p19/p22 mutant derivatives, and by expression of individual TBSV genes from a heterologous potato virus X (PVX) vector. Our results showed that TBSV genes could be expressed at high levels from the PVX vector. The subcellular localization of these proteins as well as the ability of PVX-expressed p22 to trans complement TBSV cell-to-cell movement defective mutants indicate that the exogenously expressed proteins are functionally active. Inoculation studies with TBSV mutants and the PVX derivatives demonstrated that p19 induced a generalized necrosis upon systemic infection of Nicotiana benthamiana and N. clevelandii. In addition, p19 elicited the formation of local necrotic lesions in N. tabacum; however, in N. glutinosa and N. edwardsonii, the local lesion response was activated by p22. These results show that the p19 and p22 proteins of TBSV are important symptom determinants and that closely related plant species may contain different resistance genes that selectively respond to individual TBSV proteins.

Passage of cucumber necrosis virus (CNV) containing defective interfering (DI) RNAs through cucumber plants decreased the accumulation of DI RNAs to undetectable levels. Subsequent passages in two Nicotiana species (Nicotiana benthamiana or N. clevelandii) resulted in the appearance of DI RNA species that were larger than the DI RNAs observed during exclusive serial passages of CNV through the Nicotiana species. Sequence analysis of cloned cDNAs corresponding to the two DI RNA populations indicated that the smaller CNV-DI RNAs contained the four conserved regions (I through IV) of the genome typical of tombusvirus DI RNAs, whereas the larger DI RNAs were of similar organization but had a direct repeat of the middle portion of the molecule. This result suggests that the host has an influence on the type of DI RNA that accumulates during consecutive high multiplicity of infection passages. A comparative analysis of deletions targeting the individual conserved regions in both CNV and tomato bushy stunt virus (TBSV) DI RNAs revealed that only region III was completely dispensable for accumulation of either DI RNA species. More refined deletion analyses in regions I and II indicated that smaller segments of 75 and 35 nucleotides (nt), respectively, could be deleted without abolishing infectivity. The dispensable sequences in region II of both TBSV and CNV DI RNAs mapped to the top portion of a putative stem-loop structure. These studies indicate that both essential and nonessential sequences are conserved in DI RNAs The essential sequences in regions I, II, and IV likely contain important cis-acting elements, whereas nonessential regions such as region III may play secondary roles such as optimally spacing cis-acting elements or maintaining the DI RNA at an overall size that is stable.

Plant viruses use several strategies to transport their nucleic acid genomes throughout the plants. Regardless of the movement mechanism, a universal major block to uninterrupted viral trafficking is the induction of antiviral silencing that degrades viral RNA. To counteract this defense, viruses encode suppressors that block certain steps in the RNA silencing pathway, and consequently these proteins allow viral spread to proceed. There is a constant battle between plants and viruses and sometimes viruses will succeed and invade the plants and in other cases the RNA silencing mechanism will override the virus. A key role in the silencing versus suppression conflict between plants and viruses is played by one or more members of the Argonaute protein (AGO) family encoded by plants. Here we review the mechanisms and effects of antiviral silencing with an emphasis on the contribution of AGOs, especially the recently discovered role of AGO2.

Plant viruses have an enormous negative impact on agricultural crop production throughout the world, and, consequently, agronomists and plant pathologists have devoted considerable effort toward controlling virus diseases during this century. Prior to the advent of genetic engineering, traditional plant breeding methodology was sometimes successfully applied to develop resistance to viruses of agronomically important crops. In addition, standard techniques of plant pathology, including quarantine, eradication, crop rotation, and certified virus-free stock, have been important tools to control virus diseases, although each has disadvantages, such as expense, questionable effectiveness, and lack of reliability on a yearly basis. The prospects for pathogen-mediated intervention in virus disease development were first realized in 1929 when H.H. McKinney demonstrated that tobacco could be protected from infection by a severe strain of TMV by prior inoculation with

Plant pararetroviruses, such as caulimoviruses, and animal retroviruses have in common the presence of a highly conserved arrangement of cysteines and a histidine in the precursor of the capsid protein. The composition of these amino acids resembles a zinc finger element, a structure that is common to a class of eukaryotic proteins that regulate gene expression. The role of the putative zinc finger in the life-cycle of caulimoviruses was investigated by introducing specific mutations in the coat protein coding region of a cloned and infectious form of figwort mosaic virus, a caulimovirus. This mutated viral genome, which no longer encoded the conserved cysteine and histidine residues, was not infectious in plants. Transient expression assays in protoplasts showed that expression of a reporter gene inserted at different places in the genome was not detectably influenced by the coat protein or its putative zinc finger. It appears that the zinc finger-like element of caulimoviruses is not involved in the regulation of gene expression. These observations support a model which predicts a function of the zinc finger in specific recognition and packaging of viral RNA into virions prior to reverse transcription.

Gene expression of figwort mosaic virus (FMV), a caulimovirus, was investigated by electroporation of Nicotiana edwardsonii cell suspension protoplasts with cloned viral constructs in which a reporter gene was inserted at various positions on the genome. The results showed that the genome of FMV contains two promoters; one is used for the production of a full-length RNA and another initiates synthesis of a separate monocistronic RNA for gene VI. Evidence is provided that the full-length transcript, the probable template for reverse transcription, can serve as a polycistronic mRNA for translation of genes I through V and perhaps also gene VI. Expression of all the genes on the polycistronic mRNA is trans activated by the gene VI protein. Reporter gene expression appears most efficient when its start codon is in close proximity to the stop codon of the preceding gene, as for the native genes of caulimoviruses. We propose that the gene VI product enables expression of the polycistronic mRNA by promoting reinitiation of ribosomes to give translational coupling of individual genes.

The six major conserved genes of figwort mosaic virus (FMV), a caulimovirus, appear in tandem array on an RNA transcript that spans the entire viral genome. Gene VI, the only cistron that appears as a separate subgenomic RNA, has been reported to transactivate the expression of downstream genes of the full-length transcript. This transcript has a long 5′-leader of about 600 nucleotides followed by a small nonconserved region (gene VII), a smaller intergenic region (57 nucleotides), and the major conserved genes in a closely spaced array. In our present experiments we have constructed expression units containing the promoter for the full-length transcript followed by the 5′ leader region, gene VII, and a reporter gene. These have been tested for expression with and without gene VI as a separate plasmid by electroporation into plant protoplasts. A series of these expression units containing truncated versions of the 5′ leader region placed upstream of a reporter gene (CAT) showed that gene VI transactivation occurred only when gene VII sequences were present in cis between the leader region and the reporter gene. In addition, a more complete version of the FMV genome containing the reporter gene further downstream (in viral gene IV) showed CAT expression only when gene VII sequences were present in an upstream position. A similar construct failed to express CAT activity when gene VII was absent.

A naturally occurring deletion mutant is observed in plants infected with figwort mosaic virus (FMV), a caulimovirus. The encapsidated mutant genome is formed spontaneously in association with two different strains of FMV in four host plant species. The mutant also appears when cloned wild-type viral DNA is used as the inoculum. The deletion mutant alone is not infectious and it appears unable to replicate after its formation, even in the presence of wild-type virus. The gene for chloramphenicol acetyltransferase was inserted at different positions in the deletion mutant genome, and subsequent transient assays showed that gene expression of the mutant occurs despite the deletion. Sequence analyses of the mutant genome revealed a deletion of a 1237-bp segment encompassing a major portion of the coat protein gene and the 5′ end of the downstream reverse transcriptase gene. This deletion is associated with consensus signals for RNA splicing including the conserved 5′ and 3′ splice sites plus surrounding sequences, putative branch point(s) for lariat formation, and an extremely high adenosine content (41 %) of the removed fragment. This suggests that splicing of the FMV full-length transcript has occurred prior to reverse transcription and this accounts for the presence and accumulation of encapsidated DNAs with the same deletion.

This Update reviews how virus-encoded suppressors of RNA silencing are currently being used in certain areas of plant research and biotechnology. Keeping within the scope of this special Focus issue, an emphasis is placed on the use of gene vectors for expression of suppressors in plants. Toward the end of this review, a discussion is presented on how the choice of vector system may influence the functionality of suppressors and the occurrence of side effects. The purpose of this brief review is to illustrate, for a general audience of biologists, how individual properties of viral-encoded suppressors might affect their performance in plants when expressed by heterologous vectors.

This unit describes principles and protocols for expressing a gene of interest in plant cells using gene vectors that are derived from an infectious full-length cDNA plasmid of the tomato bushy stunt virus (TBSV) genomic RNA, and from defective interfering RNAs (DIs). The TBSV gene vector system permits convenient cloning, allows modification and abundant expression of the gene of interest, and facilitates biosecure containment of the gene vectors. These vectors can be employed for functional genomics studies and for analyzing the biochemical properties and subcellular distribution of expressed RNAs and/or their cognate proteins. As with other plant virus gene vectors, recombination and deletion of the gene of interest during virus multiplication limits the application of the TBSV gene vectors to the inoculated cells or leaves.

Since its discovery in the late 1980s, the status of the Tombusvirus-encoded p19 protein (P19) changed from being thought obsolete to its identification a decade later as an important viral pathogenicity factor. The recent finding that P19 suppresses RNA interference (RNAi) by appropriating short interfering RNAs led to its widespread use as an RNAi-probing tool in various plant and animal models. Here, I discuss how our knowledge of p19 has developed over the years, with emphasis on the relevance of understanding its biological roles during Tombusvirus infection of plants.

This study on Tomato bushy stunt virus identified and defined three previously unknown regulatory sequences involved in RNA accumulation that are located within the 3′-proximal nested movement protein genes p22 and p19 . The first is a 16-nucleotide (nt) element termed III-A that is positioned at the very 3′ end of p22 and is essential for RNA accumulation. Approximately 300 nt upstream of III-A resides an ∼80-nt inhibitory element (IE) that is obstructive to replication only in the absence of a third regulatory element of ∼30 nt (SUR-III) that is positioned immediately upstream of III-A. Inspection of the nucleotide sequences predicted that III-A and SUR-III can form looped hairpins. A comparison of different tombusviruses showed, in each case, conservation for potential base pairing between the two predicted hairpin-loops. Insertion of a spacer adjacent to the predicted hairpins had no or a minimal effect on RNA accumulation, whereas an insertion in the putative III-A loop abolished genomic RNA multiplication. We conclude that the sequences composing the predicted III-A and SUR-III hairpin-loops are crucial for optimal RNA accumulation and that the inhibitory effect of IE surfaces when the alleged interaction between SUR-III and III-A is disturbed.

Zebra complex (ZC) disease on potatoes is associated with Candidatus Liberibacter solanacearum (CLs), an α-proteobacterium that resides in the plant phloem and is transmitted by the potato psyllid Bactericera cockerelli (Šulc). The name ZC originates from the brown striping in fried chips of infected tubers, but the whole plants also exhibit a variety of morphological features and symptoms for which the physiological or molecular basis are not understood. We determined that compared to healthy plants, stems of ZC-plants accumulate starch and more than three-fold total protein, including gene expression regulatory factors (e.g. cyclophilin) and tuber storage proteins (e.g., patatins), indicating that ZC-affected stems are reprogrammed to exhibit tuber-like physiological properties. Furthermore, the total phenolic content in ZC potato stems was elevated two-fold, and amounts of polyphenol oxidase enzyme were also high, both serving to explain the ZC-hallmark rapid brown discoloration of air-exposed damaged tissue. Newly developed quantitative and/or conventional PCR demonstrated that the percentage of psyllids in laboratory colonies containing detectable levels of CLs and its titer could fluctuate over time with effects on colony prolificacy, but presumed reproduction-associated primary endosymbiont levels remained stable. Potato plants exposed in the laboratory to psyllid populations with relatively low-CLs content survived while exposure of plants to high-CLs psyllids rapidly culminated in a lethal collapse. In conclusion, we identified plant physiological biomarkers associated with the presence of ZC and/or CLs in the vegetative potato plant tissue and determined that the titer of CLs in the psyllid population directly affects the rate of disease development in plants.

Experimental evidence for a molecular function for gene VI of the caulimoviruses is presented. Based on experiments with the figwort mosaic virus (FMV), it appears that gene VI has a role in the posttranscriptional expression of the closely packed genes (VII and I-V), which appear on the larger, full-length RNA transcript of this virus. Gene VI with its flanking 5'/3' expression signals included as a separate plasmid during electroporation of DNA into protoplasts of Nicotiana edwardsonii shows an unusual type of transactivation of a chloramphenicol acetyltransferase (CAT) gene fused at its 5' end to a small open reading frame (gene VII) of the long 5' leader of the full-length RNA transcript of the FMV genome. The level of activity of the CAT gene is increased up to 20-fold over the activity of control plasmids when gene VI is included in the electroporation mixture. Mutagenesis of the coding portions of gene VI of pGS1 RVI, a transactivating plasmid used in the electroporation experiments, demonstrated that it was probably the polypeptide product of gene VI that was responsible for the transactivating effect. Experiments with various portions of the 5' leader of the large, full-length RNA of FMV showed that the coding region of gene VII is necessary for the transactivation event. Clones of cauliflower mosaic virus (CaMV) or FMV with intact gene VI were found to reciprocally transactivate gene VII-CAT fusions (FMV) or gene I-CAT fusions (CaMV) located downstream of the 5' leader sequences of either viral genome.

Tomato bushy stunt virus (TBSV) is an RNA plant virus encoding a protein of approximately 19 kDa (P19) that is involved in various activities important for pathogenicity, including virus transport and suppression of gene silencing. In this study, we provide evidence in vivo and in vitro that P19 specifically interacts with itself to predominantly form dimers, and with a novel host protein, Hin19. Hin19 has a high degree of similarity with a class of RNA-binding proteins of which many are involved in RNA processing. The binding of P19 to itself and to Hin19 both depend on a structurally important central region of P19 that was previously shown critical for its biological function in plants. Our findings provide evidence for a model in which virus spread through suppression of defense-related gene silencing involves the formation of a complex that includes P19 dimers and a newly identified host RNA-binding protein.

The nucleotide sequence of an infectious clone of figwort mosaic virus (FMV) was determined using the dideoxynucleotide chain termination method. The double-stranded DNA genome (7743 base pairs) contained eight open reading frames (ORFs), seven of which corresponded approximately in size and location to the ORFs found in the genome of cauliflower mosaic virus (CaMV) and carnation etched ring virus (CERV). ORFs I and V of FMV demonstrated the highest degrees of nucleotide and amino acid sequence homology with the equivalent coding regions of CaMV and CERV. Regions II, III and IV showed somewhat less homology with the analogous regions of CaMV and CERV, and ORF VI showed homology with the corresponding gene of CaMV and CERV in only a short segment near the middle of the putative gene product. A 16 nucleotide sequence, complementary to the 3' terminus of methionine initiator tRNA (tRNAimet) and presumed to be the primer binding site for initiation of reverse transcription to produce minus strand DNA, was found in the FMV genome near the discontinuity in the minus strand. Sequences near the three interruptions in the plus strand of FMV DNA bear strong resemblance to similarly located sequences of 3 other caulimoviruses and are inferred to be initiation sites for second strand DNA synthesis. Additional conserved sequences in the small and large intergenic regions are pointed out including a highly conserved 35 bp sequence that occurs in the latter region.