A tomato bushy stunt virus-based vector for simultaneous editing and sensing to survey the host antiviral RNA silencing machinery.

A tomato bushy stunt virus (TBSV)-derived vector system was applied for the delivery of CRISPR/Cas9 gene editing materials, to facilitate rapid, transient assays of host-virus interactions involved in the RNA silencing pathway. Toward this, single guide RNAs designed to target key components of the virus-induced host RNA silencing pathway (AGO2, DCL2, HEN1) were inserted into TBSV-based GFP-expressing viral vectors TBSV-GFP (TG) and its P19 defective mutant TGΔP19. This produced rapid, efficient, and specific gene editing in planta. Targeting AGO2, DCL2, or HEN1 partially rescued the lack of GFP accumulation otherwise associated with TGΔP19. Since the rescue phenotypes are normally only observed in the presence of the P19 silencing suppressor, the results support that the DCL2, HEN1, and AGO2 proteins are involved in anti-TBSV RNA silencing. Additionally, we show that knockdown of the RNA silencing machinery increases cargo expression from a nonviral binary Cas9 vector. The TBSV-based gene editing technology described in this study can be adapted for transient heterologous expression, rapid gene function screens, and molecular interaction studies in many plant species considering the wide host range of TBSV. In summary, we demonstrate that a plant virus can be used to establish gene editing while simultaneously serving as an accumulation sensor for successful targeting of its homologous antiviral silencing machinery components.

Practicing virology: making and knowing a mid-twentieth century experiment with Tobacco mosaic virus.

Tobacco mosaic virus (TMV) has served as a model organism for pathbreaking work in plant pathology, virology, biochemistry and applied genetics for more than a century. We were intrigued by a photograph published in Phytopathology in 1934 showing that Tabasco pepper plants responded to TMV infection with localized necrotic lesions, followed by abscission of the inoculated leaves. This dramatic outcome of a biological response to infection observed by Francis O. Holmes, a virologist at the Rockefeller Institute for Medical Research, was used to score plants for resistance to TMV infection. Our objective was to gain a better understanding of early to mid-twentieth century ideas of genetic resistance to viruses in crop plants. We investigated Holmes' observation as a practical exercise in reworking an experiment, having been inspired by Pamela Smith's innovative Making and Knowing Project. We had a great deal of difficulty replicating Holmes' experiment, finding that biological materials and experimental customs change over time, in ways that ideas do not. Using complementary tools plus careful study and interpretation of the original text and figures, we were able to rework, yet only partially replicate, this experiment. Reading peer-reviewed manuscripts that cited Holmes' 1934 report provided an additional level of insight into the interpretation and replication of this work in the decades that followed. From this, we touch on how experimental reworking can inform our strategies to address the reproducibility "crisis" in twenty-first century science.

Expression of Separate Proteins in the Same Plant Leaves and Cells Using Two Independent Virus-Based Gene Vectors.

Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies) it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV) referred to as TG, and Tobacco mosaic virus (TMV) annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.

Tobacco rattle virus (TRV)-Mediated Silencing of Nicotiana benthamiana ARGONAUTES (NbAGOs) Reveals New Antiviral Candidates and Dominant Effects of TRV-NbAGO1.

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.

Transgenic down-regulation of ARGONAUTE2 expression in Nicotiana benthamiana interferes with several layers of antiviral defenses.

The present study aimed to analyze the contribution of Nicotiana benthamiana ARGONAUTE2 (NbAGO2) to its antiviral response against different viruses. For this purpose, dsRNA hairpin technology was used to reduce NbAGO2 expression in transgenic plants as verified with RT-PCR. This reduction was specific because the expression of other NbAGOs was not affected, and did not cause obvious developmental defects under normal growth conditions. Inoculation of transgenic plants with an otherwise silencing-sensitive GFP-expressing Tomato bushy stunt virus (TBSV) variant resulted in high GFP accumulation because antiviral silencing was compromised. These transgenic plants also exhibited accelerated spread and/or enhanced susceptibility and symptoms for TBSV mutants defective for P19 or coat protein expression, other tombusviruses, Tobacco mosaic virus, and Potato virus X; but not noticeably for Foxtail mosaic virus. These findings support the notion that NbAGO2 in N. benthamiana can contribute to antiviral defense at different levels.

Tombusvirus-based vector systems to permit over-expression of genes or that serve as sensors of antiviral RNA silencing in plants.

A next generation Tomato bushy stunt virus (TBSV) coat protein gene replacement vector system is described that can be applied by either RNA inoculation or through agroinfiltration. A vector expressing GFP rapidly yields high levels of transient gene expression in inoculated leaves of various plant species, as illustrated for Nicotiana benthamiana, cowpea, tomato, pepper, and lettuce. A start-codon mutation to down-regulate the dose of the P19 silencing suppressor reduces GFP accumulation, whereas mutations that result in undetectable levels of P19 trigger rapid silencing of GFP. Compared to existing virus vectors the TBSV system has a unique combination of a very broad host range, rapid and high levels of replication and gene expression, and the ability to regulate its suppressor. These features are attractive for quick transient assays in numerous plant species for over-expression of genes of interest, or as a sensor to monitor the efficacy of antiviral RNA silencing.