Herman B. Scholthof

The Tombusvirus -encoded P19: from irrelevance to elegance

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.

Identification of an ARGONAUTE for Antiviral RNA Silencing in Nicotiana benthamiana

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.

RNAi-associated ssRNA-specific ribonucleases in Tombusvirus P19 mutant-infected plants and evidence for a discrete siRNA-containing effector complex

Tomato bushy stunt virus (TBSV) and other tombusviruses encode a p19 protein (P19), which is a suppressor of RNAi. Wild-type TBSV or p19-defective mutants initially show a similar infection course in Nicotiana benthamiana, but the absence of an active P19 results in viral RNA degradation followed by recovery from infection. P19 homodimers sequester 21-nt virus-derived duplex siRNAs, and it is thought that this prevents the programming of an antiviral RNA-induced silencing complex to avoid viral RNA degradation. Here we report on chromatographic fractionation (gel filtration, ion exchange, and hydroxyapatite) of extracts from healthy or infected Nicotiana benthamiana plants in combination with in vitro assays for ribonuclease activity and detection of TBSV-derived siRNAs. Only extracts of plants infected with p19 mutants provided a source of sequence-nonspecific but ssRNA-targeted in vitro ribonuclease activity that coeluted with components of a wide molecular weight range. In addition, we isolated a discrete ≈500-kDa protein complex that contained ≈21-nt TBSV-derived siRNAs and that exhibited ribonuclease activity that was TBSV sequence-preferential, ssRNA-specific, divalent cation-dependent, and insensitive to a ribonuclease inhibitor. We believe that this study provides biochemical evidence for a virus–host system that infection in the absence of a fully active RNAi suppressor induces ssRNA-specific ribonuclease activity, including that conferred by a RNA-induced silencing complex, which is likely the cause for the recovery of plants from infection.

Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene

Nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (TBSV) failed to elicit effective virus resistance when inoculated with wildtype virus. Subsequently, R1 and R2 progeny from 13 transgenic lines were inoculated with a TBSV mutant containing a defective coat protein gene. Mild symptoms typical of those elicited in nontransformed plants infected with the TBSV mutant initially appeared. However, within 2 to 4 weeks, up to 20% of the transgenic plants sporadically began to develop the lethal syndrome characteristic of wild-type virus infections. RNA hybridization and immunoblot analyses of these plants and nontransformed N. benthamiana inoculated with virus from the transgenic lines indicated that wild-type virus had been regenerated by a double recombination event between the defective virus and the coat protein transgene. Similar results were obtained with a TBSV deletion mutant containing a nucleotide sequence marker, and with a chimeric cucumber necrosis virus (CNV) containing the defective TBSV coat protein gene. In both cases, purified virions contained wild-type TBSV RNA or CNV chimeric RNA derived by recombination with the transgenic coat protein mRNA. These results thus demonstrate that recombinant tombus-viruses can arise frequently from viral genes expressed in transgenic plants.

Tobacco Mosaic Virus: Pioneering Research for a Century

One century ago, M.W. Beijerinck contended that the filterable agent of tobacco mosaic disease was neither a bacterium nor any corpuscular body, but rather that it was a contagium vivum fluidum ([Beijerinck, 1898][1]). Beijerincks contribution followed A. Mayers path-breaking work on tobacco

Plant responses against invasive nucleic acids: RNA silencing and its suppression by plant viral pathogens

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.

Biological Relevance of a Stable Biochemical Interaction between the Tombusvirus-Encoded P19 and Short Interfering RNAs

ABSTRACT The Tomato bushy stunt virus (TBSV)-encoded p19 protein (P19) is widely used as a robust tool to suppress RNA interference (RNAi) in various model organisms. P19 dimers appropriate 21-nucleotide (nt) duplex short interfering RNAs (siRNAs) generated by Dicer presumably to prevent programming of the RNA-induced silencing complex (RISC). In the context of virus infection, this model predicts that P19 mutants compromised for siRNA binding cannot prevent RISC-mediated degradation of TBSV RNA and thus reduce viral pathogenicity. To test this, we used P19/43 (R→W), which is less pathogenic than wild-type P19 (wtP19), and P19/75-78 (RR→GG), with pathogenicity properties (i.e., viral spread and symptom induction) comparable to those of a P19-null mutant. We demonstrate that P19/43 still suppresses RNAi-mediated viral RNA degradation in infected Nicotiana benthamiana, while P19/75-78 is unable to prevent this clearance of viral RNA, leading to an irreversible recovery phenotype. Gel filtration and immunoprecipitation assays show that at the onset of the infection, wtP19, P19/43, and P19/75-78 readily accumulate, and they form dimers. The wtP19 is stably associated with duplex ∼21-nt TBSV siRNAs, while P19/75-78 does not bind these molecules, and the electrostatic interaction of P19/43 with siRNAs is perturbed for ∼21-nt duplexes but not for longer siRNAs. This is the first clear demonstration of a direct correlation between a novel structurally orchestrated siRNA binding of an RNAi suppressor and its roles in viral pathogenesis. The findings should be particularly valuable for the RNAi field in general because the P19 mutants enable precise determination of siRNA appropriation effects.

A Novel Plant Homeodomain Protein Interacts in a Functionally Relevant Manner with a Virus Movement Protein

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.

Control of Plant Virus Diseases by Pathogen-Derived Resistance in Transgenic Plants.

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

Recombination with Host Transgenes and Effects on Virus Evolution: An Overview and Opinion

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.