Plant Genome Editing and the Relevance of Off-Target Changes.

Site-directed nucleases (SDNs) used for targeted genome editing are powerful new tools to introduce precise genetic changes into plants. Like traditional approaches, such as conventional crossing and induced mutagenesis, genome editing aims to improve crop yield and nutrition. Next-generation sequencing studies demonstrate that across their genomes, populations of crop species typically carry millions of single nucleotide polymorphisms and many copy number and structural variants. Spontaneous mutations occur at rates of ∼10-8 to 10-9 per site per generation, while variation induced by chemical treatment or ionizing radiation results in higher mutation rates. In the context of SDNs, an off-target change or edit is an unintended, nonspecific mutation occurring at a site with sequence similarity to the targeted edit region. SDN-mediated off-target changes can contribute to a small number of additional genetic variants compared to those that occur naturally in breeding populations or are introduced by induced-mutagenesis methods. Recent studies show that using computational algorithms to design genome editing reagents can mitigate off-target edits in plants. Finally, crops are subject to strong selection to eliminate off-type plants through well-established multigenerational breeding, selection, and commercial variety development practices. Within this context, off-target edits in crops present no new safety concerns compared to other breeding practices. The current generation of genome editing technologies is already proving useful to develop new plant varieties with consumer and farmer benefits. Genome editing will likely undergo improved editing specificity along with new developments in SDN delivery and increasing genomic characterization, further improving reagent design and application.

The Impact of "Coat Protein-Mediated Virus Resistance" in Applied Plant Pathology and Basic Research.

Worldwide, plant viruses cause serious reductions in marketable crop yield and in some cases even plant death. In most cases, the most effective way to control virus diseases is through genetically controlled resistance. However, developing virus-resistant (VR) crops through traditional breeding can take many years, and in some cases is not even possible. Because of this, the demonstration of the first VR transgenic plants in 1985 generated much attention. This seminal report served as an inflection point for research in both basic and applied plant pathology, the results of which have dramatically changed both basic research and in a few cases, commercial crop production. The typical review article on this topic has focused on only basic or only applied research results stemming from this seminal discovery. This can make it difficult for the reader to appreciate the full impact of research on transgenic virus resistance, and the contributions from fundamental research that led to translational applications of this technology. In this review, we take a global view of this topic highlighting the significant changes to both basic and applied plant pathology research and commercial food production that have accumulated in the last 30 plus years. We present these milestones in the historical context of some of the scientific, economic, and environmental drivers for developing specific VR crops. The intent of this review is to provide a single document that adequately records the significant accomplishments of researchers in both basic and applied plant pathology research on this topic and how they relate to each other. We hope this review therefore serves as both an instructional tool for students new to the topic, as well as a source of conversation and discussion for how the technology of engineered virus resistance could be applied in the future.

A historical overview of RNAi in plants.

RNA interference, or RNAi, is arguably one of the most significant discoveries in biology in the last several decades. First recognized in plants (where it was called post-transcriptional gene silencing, PTGS) RNAi is a gene down-regulation mechanism since demonstrated to exist in all eukaryotes. In RNAi, small RNAs (of about 21-24 nucleotides) function to guide specific effector proteins (members of the Argonaute protein family) to a target nucleotide sequence by complementary base pairing. The effector protein complex then down-regulates the expression of the targeted RNA or DNA. Small RNA-directed gene regulation systems were independently discovered (and named) in plants, fungi, worms, flies, and mammalian cells. Collectively, PTGS, RNA silencing, and co-suppression (in plants); quelling (in fungi and algae); and RNAi (in Caenorhabditis elegans, Drosophila, and mammalian cells) are all examples of small RNA-based gene regulation systems. From the very beginning, plant research has had a major impact on our understanding of RNAi. The purpose of this chapter is to provide an historical perspective and overview on the discovery, characterization, and applications of RNAi in plants.

RNAi effects on actin mRNAs in Homalodisca vitripennis cells.

The xylem feeding leafhopper Homalodisaca vitripennis (H. vitripennis) is an unusually robust and efficient vector of Xylella fastidiosa, a Gram-negative bacterium which causes several very important plant diseases. Here we investigated RNA interference (RNAi) to target actin, a key component of insect cells and whole bodies, in H. vitripennis cells. RNAi effectors were delivered via lipid based transfection and real-time RT-PCR, RNA hybridization, and microscopic analyses were employed to verify RNAi effects. When actin dsRNAs were used, a 10-fold decrease in the target H. vitripennis actin mRNA level was seen in cells. Altered phenotypic effects also were evident in transfected cells, as were small interfering RNAs, hallmarks of RNAi. The use of H. vitripennis cells and RNAi offers new opportunities to research hemipterans, the most important insect vectors of plant pathogens.

Agroinoculation of the Crinivirus, Lettuce infectious yellows virus, for systemic plant infection.

Lettuce infectious yellows virus (LIYV) is phloem-limited, non-mechanically transmissible, and is transmitted to plants only by Bemisia tabaci. Here, we developed agroinoculation to deliver LIYV to plants thereby obviating the need for B. tabaci. Agroinfiltration of RNA 1 containing a green fluorescent protein gene into Nicotiana benthamiana leaves resulted in subliminal infections, as judged by green fluorescence. Agroinfiltration of LIYV wild-type RNA 1 and 2 constructs resulted in systemic infections in N. benthamiana plants and typical LIYV symptoms. In addition, partially purified LIYV virions from agroinoculated N. benthamiana plants were successfully acquired via membrane-feeding and transmitted to lettuce plants by B. tabaci. Agroinoculation coupled with targeted mutagenesis technologies will greatly enhance LIYV reverse genetics studies to characterize LIYV gene functions in planta for processes such as virus replication, recombination, trafficking, symptom elicitation and virus-vector interactions.

Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression.

The effects of six different plant viral suppressors of gene silencing were compared using an automated image collection and analysis system developed for continual monitoring of GFP expression. Suppressors were introduced into lima bean cotyledonary tissues either as 3'-GFP translational fusions or as co-introductions with the GFP gene on a separate plasmid. The resultant transient expression profiles for each suppressor depended on whether the suppressor was introduced as a fusion or co-introduced on separate plasmids. As co-introductions, the silencing suppressors HCPro (from Tobacco etch virus), p19 (from Tomato bushy stunt virus), gammab (from Barley stripe mosaic virus) and p21 (from Beet yellows virus) led to an almost twofold increase in initial GFP expression levels, followed by a rapid decline. In contrast, fusions of HCPro, p19, and gammab to the 3'-end of GFP resulted in slightly lower but more prolonged GFP expression. Compared with the co-introductions, all GFP::Suppressor translational fusions gave reduced GFP fluorescence levels, suggesting interference of the fusion partner with GFP fluorescence. Regardless of the configuration, introductions of the silencing suppressors AL2 (from Tomato golden mosaic virus) and 126-kDa protein (from Tobacco mosaic virus) resulted in very low GFP fluorescence. This is the first report that directly compares the effects of a large number of viral suppressors of silencing on transient transgene expression using both translational fusions and co-introductions.

Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing.

Using particle bombardment, a DNA expression vector containing the green fluorescent protein (GFP) reporter gene was introduced into plant cells. Expression of the GFP gene was transient; resulting in peak GFP Expression about 24 h post introduction and a rapid decline thereafter. This well known decline in gene expression has previously been attributed to pre-integrative DNA events that involved the loss of introduced DNA or cell death. Here, we show that post-transcriptional gene silencing (PTGS) is also involved. Introduction of a GFP expression vector alone resulted in a rapid decline in transient expression after 30 h. However, if GFP was expressed as a translational fusion to the RNA silencing suppressor protein HCPro from tobacco etch potyvirus, transgene expression was extended to well over 100 h. Mutant analyses of HCPro showed that a functional HCPro protein was required for this extension of transient expression. Various deletion and translational fusion analyses confirmed that the C-terminal region of the protein was important for suppressor activity and the entire protein was required for optimal suppression of host silencing. The transient nature of gene expression during particle bombardment appears to result from induction of PTGS, which can be mitigated by the presence of a suppressor of silencing. The use of RNA silencing suppressor proteins may make particle bombardment-mediated transient expression assays more useful for evaluating factors that effect gene expression.

High-efficiency protein expression in plants from agroinfection-compatible Tobacco mosaic virus expression vectors.

BACKGROUND: Plants are increasingly being examined as alternative recombinant protein expression systems. Recombinant protein expression levels in plants from Tobacco mosaic virus (TMV)-based vectors are much higher than those possible from plant promoters. However the common TMV expression vectors are costly, and at times technically challenging, to work with. Therefore it was a goal to develop TMV expression vectors that express high levels of recombinant protein and are easier, more reliable, and more cost-effective to use. RESULTS: We have constructed a Cauliflower mosaic virus (CaMV) 35S promoter-driven TMV expression vector that can be delivered as a T-DNA to plant cells by Agrobacterium tumefaciens. Co-introduction (by agroinfiltration) of this T-DNA along with a 35S promoter driven gene for the RNA silencing suppressor P19, from Tomato bushy stunt virus (TBSV) resulted in essentially complete infection of the infiltrated plant tissue with the TMV vector by 4 days post infiltration (DPI). The TMV vector produced between 600 and 1200 micrograms of recombinant protein per gram of infiltrated tissue by 6 DPI. Similar levels of recombinant protein were detected in systemically infected plant tissue 10-14 DPI. These expression levels were 10 to 25 times higher than the most efficient 35S promoter driven transient expression systems described to date. CONCLUSION: These modifications to the TMV-based expression vector system have made TMV vectors an easier, more reliable and more cost-effective way to produce recombinant proteins in plants. These improvements should facilitate the production of recombinant proteins in plants for both research and product development purposes. The vector should be especially useful in high-throughput experiments.

Functional modulation of the geminivirus AL2 transcription factor and silencing suppressor by self-interaction.

The DNA genomes of geminiviruses have a limited coding capacity that is compensated for by the production of small multifunctional proteins. The AL2 protein encoded by members of the genus Begomovirus (e.g., Tomato golden mosaic virus) is a transcriptional activator, a silencing suppressor, and a suppressor of a basal defense. The related L2 protein of Beet curly top virus (genus Curtovirus) shares the pathogenicity functions of AL2 but lacks transcriptional activation activity. It is known that AL2 and L2 can suppress local silencing by interacting with adenosine kinase (ADK) and can suppress basal defense by interacting with SNF1 kinase. However, how the activities of these viral proteins are regulated remains an unanswered question. Here, we provide some answers by demonstrating that AL2, but not L2, interacts with itself. The zinc finger-like motif (CCHC) is required but is not sufficient for AL2 self-interaction. Alanine substitutions for the invariant cysteine residues that comprise the motif abolish self-interaction or cause aberrant subnuclear localization but do not abolish interaction with ADK and SNF1. Using bimolecular fluorescence complementation, we show that AL2:AL2 complexes accumulate primarily in the nucleus, whereas AL2:ADK and L2:ADK complexes accumulate mainly in the cytoplasm. Further, the cysteine residue mutations impair the ability of AL2 to activate the coat protein promoter but do not affect local silencing suppression. Thus, AL2 self-interaction correlates with nuclear localization and efficient activation of transcription, whereas AL2 and L2 monomers can suppress local silencing by interacting with ADK in the cytoplasm.

TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector.

Transient expression is a rapid, useful approach for producing proteins of interest in plants. Tobacco mosaic virus (TMV)-based transient expression vectors can express very high levels of foreign proteins in plants. However, TMV vectors are, in general, not efficiently delivered to plant cells by agroinfection. It was determined that agroinfection was very efficient with a 35S promoter-driven TMV replicon that lacked the TMV coat protein gene sequence. This coat protein deletion vector had several useful features as a transient expression system, including improved ease of use, higher protein expression rates, and improved biocontainment. Using this TMV expression vector, some foreign proteins were expressed at levels of 3 to 5 mg/g fresh weight of plant tissue. It is proposed that this new transient expression vector will be a useful tool for expressing recombinant proteins in plants for either research or production purposes.

Assembly of trans-encapsidated recombinant viral vectors engineered from Tobacco mosaic virus and Semliki Forest virus and their evaluation as immunogens.

RNA virus vectors are attractive vaccine delivery agents capable of directing high-level gene expression without integration into host cell DNA. However, delivery of non-encapsidated RNA viral vectors into animal cells is relatively inefficient. By introducing the tobacco mosaic virus (TMV) origin of assembly into the RNA genome of Semliki Forest virus (SFV), we generated an SFV expression vector that could be efficiently packaged (trans-encapsidated) in vitro by purified TMV coat protein (CP). Using cellular assays, pseudovirus disassembly, RNA replication and reporter gene expression were demonstrated. We also evaluated the immune response to trans-encapsidated recombinant SFV carrying a model antigen gene (beta-galactosidase) in C57/B6 mice. Relative to RNA alone, vector encapsidation significantly improved the humoral and cellular immune responses. Furthermore, reassembly with recombinant TMV CPs permitted the display of peptide epitopes on the capsid surface as either genetic fusions or through chemical conjugation, to complement the immunoreactivity of the encapsidated RNA genetic payload. The SFV vector/TMV CP system described provides an alternative nucleic acid delivery mechanism that is safe, easy to manufacture in vitro and that also facilitates the generation of unique nucleic acid/protein antigen compositions.

Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications.

Display of peptides or proteins in an ordered, repetitive array, such as on the surface of a virus-like particle, is known to induce an enhanced immune response relative to vaccination with the "free" protein antigen. The coat protein of Tobacco mosaic virus (TMV) can accommodate short peptide insertions into the primary sequence, but the display of larger protein moieties as genetic fusions to the capsid protein has not been possible. We employed a randomized library approach to introduce a reactive lysine at the externally located amino terminus of the coat protein, which facilitated biotinylation of the capsid. To characterize display of heterologous proteins on the virion surface, we bound a model antigen (green fluorescent protein (GFP)-streptavidin (SA), expressed and purified from plants) to the biotinylated TMV particles, creating a GFP-SA decorated virus particle. A GFP-SA tetramer loading of 26% was obtained, corresponding to approximately 2200 GFP moieties displayed per intact virion. We evaluated the immunogenicity of GFP decorated virions in both mice and guinea pigs and found augmented humoral IgG titers in both species, relative to unbound GFP-SA tetramer. Next, we fused an N-terminal fragment of the Canine oral papillomavirus L2 protein to streptavidin. With TMV display, the L2 protein fragment was significantly more immunogenic than uncoupled antigen when tested in mice. By demonstrating the presentation of whole proteins, this study expands the utility of TMV as a vaccine scaffold beyond that which is possible by genetic manipulation.

Plant pathology and RNAi: a brief history.

This article describes the discovery of RNA-activated sequence-specific RNA degradation, a phenomenon now referred to as RNA silencing or RNA interference (RNAi). From 1992 to 1996, a series of articles were published on virus resistant transgenic plants expressing either translatable or nontranslatable versions of the coat protein gene of Tobacco etch virus (TEV). Certain transgenic plant lines were resistant to TEV but not to closely related viruses. In these plants a surprising correlation was observed: Transgenic plant lines with the highest degree of TEV resistance had actively transcribed transgenes but low steady-state levels of transgene RNA. Molecular analysis of these transgenic plants demonstrated the existence of a cellular-based, sequence-specific, posttranscriptional RNA-degradation system that was programmed by the transgene-encoded RNA sequence. This RNA-degradation activity specifically targeted both the transgene RNA and TEV (viral) RNA for degradation and was the first description of RNA-mediated gene silencing.

Making an ally from an enemy: plant virology and the new agriculture.

Historically, the study of plant viruses has contributed greatly to the elucidation of eukaryotic biology. Recently, concurrent with the development of viruses into expression vectors, the biotechnology industry has developed an increasing number of disease therapies utilizing recombinant proteins. Plant virus vectors are viewed as a viable option for recombinant protein production. Employing pathogens in the process of creating added value to agriculture is, in effect, making an ally from an enemy. This review discusses the development and use of viruses as expression vectors, with special emphasis on (+) strand RNA virus systems. Further, the use of virus expression vectors in large-scale agricultural settings to produce recombinant proteins is described, and the technical challenges that need to be addressed by agriculturists and molecular virologists to fully realize the potential of this latest evolution of plant science are outlined.

Epigenetic modification of plants with systemic RNA viruses.

Knowledge of gene function is critical to the development of new plant traits for improved agricultural and industrial applications. Viral expression vectors offer a rapid and proven method to provide epigenetic expression of foreign sequences throughout infected plants. Expression of these sequences from viral vectors can lead to gain- or loss-of-function phenotypes, allowing gene function to be determined by phenotypic or biochemical effects in the infected plant. Tobacco mosaic virus and barley stripe mosaic virus expression vectors have been developed to express foreign gene sequences in dicotyledonous and monocotyledonous hosts, respectively. Large-scale application of both viral vector systems for gene function discovery in Nicotiana and barley hosts resulted in high infection rates and produced distinctive visual phenotypes in approximately 5% of transfected plants. Novel genes expressing potential herbicide target proteins in addition to genes promoting stem elongation, leaf development and apical dominance were identified in the large-scale screening. This report illustrates the adaptability of viral vectors for gene function discovery in higher plants.