T. Jack Morris

Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1

Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis.

The Coat Protein of Turnip Crinkle Virus Suppresses Posttranscriptional Gene Silencing at an Early Initiation Step

ABSTRACT Posttranscriptional gene silencing (PTGS), or RNA silencing, is a sequence-specific RNA degradation process that targets foreign RNA, including viral and transposon RNA for destruction. Several RNA plant viruses have been shown to encode suppressors of PTGS in order to survive this host defense. We report here that the coat protein (CP) of Turnip crinkle virus (TCV) strongly suppresses PTGS. The Agrobacterium infiltration system was used to demonstrate that TCV CP suppressed the local PTGS as strongly as several previously reported virus-coded suppressors and that the action of TCV CP eliminated the small interfering RNAs associated with PTGS. We have also shown that the TCV CP must be present at the time of silencing initiation to be an effective suppressor. TCV CP was able to suppress PTGS induced by sense, antisense, and double-stranded RNAs, and it prevented systemic silencing. These data suggest that TCV CP functions to suppress RNA silencing at an early initiation step, likely by interfering the function of the Dicer-like RNase in plants.

HRT gene function requires interaction between a NAC protein and viral capsid protein to confer resistance to turnip crinkle virus.

An Arabidopsis protein was found to interact specifically with the capsid protein (CP) of turnip crinkle virus (TCV) through yeast two-hybrid screening. This protein, designated TIP (for TCV-interacting protein), was found to be a member of the recently recognized NAC family of proteins. NAC proteins have been implicated in the regulation of development of plant embryos and flowers. TIP alone was able to activate expression of reporter genes in yeast if fused to a DNA binding domain, suggesting that it may be a transcriptional activator. The TIP binding region in the TCV CP has been mapped to the N-terminal 25 amino acids. Site-directed mutagenesis within this region revealed that loss of the TIP–CP interaction in the yeast two-hybrid assay correlated with loss of the ability of TCV to induce the hypersensitive response and resistance in the TCV-resistant Arabidopsis ecotype Dijon (Di-0 and its inbred line Di-17). These data suggest that TIP is an essential component in the TCV resistance response pathway.

RDR6 Has a Broad-Spectrum but Temperature-Dependent Antiviral Defense Role in Nicotiana benthamiana

ABSTRACT SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.

Suppressors of RNA silencing encoded by plant viruses and their role in viral infections

RNA silencing as a robust host defense mechanism against plant viruses is generally countered by virus‐encoded silencing suppressors. This strategy is now increasingly recognized to be used by animal viruses as well. We present here an overview of the common features shared by some of the better studied plant viral silencing suppressors. We then briefly describe the characteristics of the few reported animal viral suppressors, notably their extraordinary ability of cross‐kingdom suppression. We next discuss the basis for biased protection of viral RNA and subviral parasites by silencing suppressors, the link between movement and silencing suppression, the influence of temperature on the outcome of viral infection and the effect of viral silencing suppressors on the microRNA pathway.

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.

Cap-Independent Translational Enhancement of Turnip Crinkle Virus Genomic and Subgenomic RNAs

ABSTRACT The presence of translational control elements and cap structures has not been carefully investigated for members of theCarmovirus genus, a group of small icosahedral plant viruses with positive-sense RNA genomes. In this study, we examined both the 5′ and 3′ untranslated regions (UTRs) of the turnip crinkle carmovirus (TCV) genomic RNA (4 kb) as well as the 5′ UTR of the coat protein subgenomic RNA (1.45 kb) for their roles in translational regulation. All three UTRs enhanced translation of the firefly luciferase reporter gene to different extents. Optimal translational efficiency was achieved when mRNAs contained both 5′ and 3′ UTRs. The synergistic effect due to the 5′-3′ cooperation was at least fourfold greater than the sum of the contributions of the individual UTRs. The observed translational enhancement of TCV mRNAs occurred in a cap-independent manner, a result consistent with the demonstration, using a cap-specific antibody, that the 5′ end of the TCV genomic RNA was uncapped. Finally, the translational enhancement activity within the 5′ UTR of 1.45-kb subgenomic RNA was shown to be important for the translation of coat protein in protoplasts and for virulent infection in Arabidopsis plants.

The Capsid Protein of Turnip Crinkle Virus Overcomes Two Separate Defense Barriers To Facilitate Systemic Movement of the Virus in Arabidopsis

ABSTRACT The capsid protein (CP) of Turnip crinkle virus (TCV) is a multifunctional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long-distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV CP and evaluated the interdependence of these functions. A series of TCV mutants containing alterations in the CP coding region were generated. These alterations range from single-amino-acid substitutions and domain truncations to knockouts of CP translation. The latter category also contained two constructs in which the CP coding region was replaced by either the cDNA of a silencing suppressor of a different virus or that of green fluorescent protein. These mutants were used to infect Arabidopsis plants with diminished antiviral silencing capability (dcl2 dcl3 dcl4 plants). There was a strong correlation between the ability of mutants to reach systemic leaves and the silencing suppressor activity of mutant CP. Virus particles were not essential for entry of the viral genome into vascular bundles in the inoculated leaves in the absence of antiviral silencing. However, virus particles were necessary for egress of the viral genome from the vasculature of systemic leaves. Our experiments demonstrate that TCV CP not only allows the viral genome to access the systemic movement channel through silencing suppression but also ensures its smooth egress by way of assembled virus particles. These results illustrate that efficient long-distance movement of TCV requires both functions afforded by the CP.

Wheat streak mosaic virus Lacking Helper Component-Proteinase Is Competent to Produce Disease Synergism in Double Infections with Maize chlorotic mottle virus

ABSTRACT The tritimovirus Wheat streak mosaic virus (WSMV) and the machlomovirus Maize chlorotic mottle virus (MCMV) each cause systemic chlorosis in infected maize plants. Infection of maize with both viruses produces corn lethal necrosis disease (CLND). Here, we report that complete deletion of the WSMV helper component-proteinase (HC-Pro) coding region had no effect on induction of CLND symptoms following coinoculation of maize with WSMV and MCMV. We further demonstrated that elevation of virus titers in double infections, relative to single infections, also was independent of WSMV HC-Pro. Thus, unlike potyvirus HC-Pro, WSMV HC-Pro was dispensable for disease synergism. Because disease synergism involving potyviruses requires HC-Pro-mediated suppression of posttranscriptional gene silencing (PTGS), we hypothesized that WSMV HC-Pro may not be a suppressor of PTGS. Indeed, WSMV HC-Pro did not suppress PTGS of a green fluorescent protein (GFP) transgene in an Agrobacterium-mediated coinfiltration assay in which potyvirus HC-Pro acted as a strong suppressor. Furthermore, coinfiltration with potyvirus HC-Pro, but not WSMV HC-Pro, resulted in elevated levels of the GFP target mRNA under conditions which trigger PTGS. Collectively, these results revealed significant differences in HC-Pro function among divergent genera of the family Potyviridae and suggest that the tritimovirus WSMV utilizes a gene other than HC-Pro to suppress PTGS and mediate synergistic interactions with unrelated viruses.

Robust RNAi-Based Resistance to Mixed Infection of Three Viruses in Soybean Plants Expressing Separate Short Hairpins from a Single Transgene

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.