Systemic delivery of engineered compact AsCas12f by a positive-strand RNA virus vector enables highly efficient targeted mutagenesis in plants.

Because virus vectors can spread systemically autonomously, they are powerful vehicles with which to deliver genome-editing tools into plant cells. Indeed, a vector based on a positive-strand RNA virus, potato virus X (PVX), harboring SpCas9 and its single guide RNA (sgRNA), achieved targeted mutagenesis in inoculated leaves of . However, the large size of the gene makes it unstable in the PVX vector, hampering the introduction of mutations in systemic leaves.

Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana through Viral Delivery of SpCas9 and sgRNA.

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in Nicotiana benthamiana PHYTOENE DESATURASE.

Inhibition of Cell-Free Translation and Replication of Tobacco Mosaic Virus RNA by Exogenously Added 5'-Proximal Fragments of the Genomic RNA.

Replication proteins of tobacco mosaic virus (TMV), a positive-sense RNA virus, co-translationally bind to a 5'-proximal ~70-nucleotide (nt) region of the genomic RNA, referred to as the nuclease-resistant (NR) region for replication template selection. Therefore, disruption of the interaction between the viral replication proteins and viral genomic RNA is expected to inhibit the replication of TMV. In this study, we demonstrate that the addition of small RNA fragments (18-33 nts in length) derived from different regions within the NR region inhibit the binding of TMV replication proteins to viral RNA and TMV RNA replication in a cell-free system.

Tomato brown rugose fruit virus resistance generated by quadruple knockout of homologs of TOBAMOVIRUS MULTIPLICATION1 in tomato.

Tomato brown rugose fruit virus (ToBRFV) is an emerging virus of the genus Tobamovirus. ToBRFV overcomes the tobamovirus resistance gene Tm-22 and is rapidly spreading worldwide. Genetic resources for ToBRFV resistance are urgently needed.

Potato Virus X Vector-Mediated DNA-Free Genome Editing in Plants.

Genome editing technology is important for plant science and crop breeding. Genome-edited plants prepared using general CRISPR-Cas9 methods usually contain foreign DNA, which is problematic for the production of genome-edited transgene-free plants for vegetative propagation or highly heterozygous hybrid cultivars. Here, we describe a method for highly efficient targeted mutagenesis in Nicotiana benthamiana through the expression of Cas9 and single-guide (sg)RNA using a potato virus X (PVX) vector.

Soybean antiviral immunity conferred by dsRNase targets the viral replication complex.

Eukaryotic positive-strand RNA viruses replicate their genomes in membranous compartments formed in a host cell, which sequesters the dsRNA replication intermediate from antiviral immune surveillance. Here, we find that soybean has developed a way to overcome this sequestration. We report the positional cloning of the broad-spectrum soybean mosaic virus resistance gene Rsv4, which encodes an RNase H family protein with dsRNA-degrading activity.

A Cell-Free Replication System for Positive-Strand RNA Viruses for Identification and Characterization of Plant Resistance Gene Products.

Plant cells have lytic vacuoles, which contain ribonucleases and proteinases. The vacuoles are fragile and easily collapsed upon homogenization of plant tissues or cells. Thus, with a few exceptions, plant cell extracts are contaminated by vacuole-derived lytic enzymes and unsuitable for biochemical analyses.

Purification and functional characterization of tomato mosaic virus 130K protein expressed in silkworm pupae using a baculovirus vector.

Tomato mosaic virus (ToMV; genus, Tobamovirus) is a member of the alpha-like virus superfamily of positive-strand RNA viruses, which includes many plant and animal viruses of agronomical and clinical importance. The genomes of alpha-like viruses encode replication-associated proteins that contain methyltransferase, helicase and/or polymerase domains. The three-dimensional structure of the helicase domain fragment of ToMV has been determined, but the structures of the other domains of alpha-like virus replication proteins are not available.

[Virus resistance genes in plants].

Plants defend themselves from virus infection by RNA silencing and resistance (R) gene-mediated mechanisms. Many dominant R genes encode nucleotide-binding site and leucine-rich repeat (NB-LRR)-containing proteins. NB-LRR proteins are also encoded by R genes against bacteria or fungi, suggesting a similar mechanism underlies defense systems to diverse pathogens.

A Tomato Spotted Wilt Virus S RNA-based Replicon System in Yeast.

Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus of the order Bunyavirales, family Tospoviridae, genus Orthotospovirus. TSWV infects a broad range of plant species, causing serious economic losses. Despite its agronomic importance, molecular biological understanding of TSWV has been limited, partly due to the lack of a reverse genetics system, which would enable genetic manipulation of the virus.

A removable virus vector suitable for plant genome editing.

Plant genome editing is achieved by the expression of sequence-specific nucleases (SSNs). RNA virus vector-mediated expression of SSNs is a promising approach for transgene integration-free targeted mutagenesis in plants. However, the removal of virus vectors from infected plants is challenging because no antiviral drugs are available against plant viruses.

A Split Staphylococcus aureus Cas9 as a Compact Genome-Editing Tool in Plants.

Split-protein methods-where a protein is split into two inactive fragments that must re-assemble to form an active protein-can be used to regulate the activity of a given protein and reduce the size of gene transcription units. Here, we show that a Staphylococcus aureus Cas9 (SaCas9) can be split, and that split-SaCas9 expressed from Agrobacterium can induce targeted mutagenesis in Nicotiana benthamiana. Since SaCas9 is smaller than the more commonly used Cas9 derived from Streptococcus pyogenes, the split-SaCas9 provides the smallest tool yet for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) plant genome editing.

Replication of Tobamovirus RNA.

Tobacco mosaic virus and other tobamoviruses have served as models for studying the mechanisms of viral RNA replication. In tobamoviruses, genomic RNA replication occurs via several steps: (a) synthesis of viral replication proteins by translation of the genomic RNA; (b) translation-coupled binding of the replication proteins to a 5'-terminal region of the genomic RNA; (c) recruitment of the genomic RNA by replication proteins onto membranes and formation of a complex with host proteins TOM1 and ARL8; (d) synthesis of complementary (negative-strand) RNA in the complex; and (e) synthesis of progeny genomic RNA. This article reviews current knowledge on tobamovirus RNA replication, particularly regarding how the genomic RNA is specifically selected as a replication template and how the replication proteins are activated.

Viruses roll the dice: the stochastic behavior of viral genome molecules accelerates viral adaptation at the cell and tissue levels.

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1-20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus.

Functions of the 5'- and 3'-untranslated regions of tobamovirus RNA.

The tobamovirus genome is a 5'-m(7)G-capped RNA that carries a tRNA-like structure at its 3'-terminus. The genomic RNA serves as the template for both translation and negative-strand RNA synthesis. The 5'- and 3'-untranslated regions (UTRs) of the genomic RNA contain elements that enhance translation, and the 3'-UTR also contains the elements necessary for the initiation of negative-strand RNA synthesis.

Mechanisms of tomato mosaic virus RNA replication and its inhibition by the host resistance factor Tm-1.

In the plant immune system, sensor proteins encoded by dominant resistance genes activate a defense response upon pathogen infection. The tomato mosaic virus (ToMV) resistance gene Tm-1 is exceptional in that it inhibits ToMV multiplication without inducing a defense response. Several lines of evidence had suggested that Tm-1 encodes a direct inhibitor of ToMV RNA replication.

Possible involvement of eEF1A in Tomato spotted wilt virus RNA synthesis.

Tomato spotted wilt virus (TSWV) is a negative-strand RNA virus in the family Bunyaviridae and propagates in both insects and plants. Although TSWV can infect a wide range of plant species, host factors involved in viral RNA synthesis of TSWV in plants have not been characterized. In this report, we demonstrate that the cell-free extract derived from one of the host plants can activate mRNA transcriptional activity of TSWV.

Structural basis for the recognition-evasion arms race between Tomato mosaic virus and the resistance gene Tm-1.

The tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homology to functionally characterized proteins. Tm-1 binds ToMV replication proteins and thereby inhibits replication complex formation. ToMV mutants that overcome this resistance have amino acid substitutions in the helicase domain of the replication proteins (ToMV-Hel).

Replication protein of tobacco mosaic virus cotranslationally binds the 5' untranslated region of genomic RNA to enable viral replication.

Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes.

[Template selection by replication protein of tobacco mosaic virus].

Replication proteins of eukaryotic positive-strand RNA viruses specifically recognize the genomic RNA as replication template, recruit them to the surfaces of intracellular membranes, and form replication complexes. We recently revealed that tobacco mosaic virus (TMV) replication protein cotranslationally binds 5' untranslated region (UTR) of the genomic RNA, and that a full-length replication protein cannot posttranslationally bind TMV RNA in trans. This result provides a mechanistic explanation for the previously reported property of TMV replication protein that it selects replication template preferentially in cis.

Crystallization and preliminary X-ray crystallographic analysis of the inhibitory domain of the tomato mosaic virus resistance protein Tm-1.

Tm-1, an inhibitor protein of Tomato mosaic virus RNA replication, contains two conserved domains: an uncharacterized domain at its N-terminus and a TIM-barrel-like domain at its C-terminus. The N-terminal domain of Tm-1 has an inhibitory activity and its three-dimensional structure has not been determined. Here, the crystallization and preliminary X-ray diffraction of the N-terminal domain of Tm-1 are reported.

Functional characterization of the mutations in Pepper mild mottle virus overcoming tomato tm-1-mediated resistance.

In tomato plants, Pepper mild mottle virus (PMMoV) cannot replicate because the tm-1 protein inhibits RNA replication. The resistance of tomato plants to PMMoV remains durable both in the field and under laboratory conditions. In this study, we constructed several mutant PMMoVs and analysed their abilities to replicate in tomato protoplasts and plants.

The resistance protein Tm-1 inhibits formation of a Tomato mosaic virus replication protein-host membrane protein complex.

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition.

Expression, purification, and functional characterization of an N-terminal fragment of the tomato mosaic virus resistance protein Tm-1.

Tm-1, the protein product of Tm-1, a semidominant resistance gene of tomato, inhibits tomato mosaic virus (ToMV) replication by binding to ToMV replication proteins. Previous studies suggested the importance of the Tm-1 N-terminal region for its inhibitory activity; however, it has not been determined if the N-terminal region is sufficient for inhibition. Furthermore, the three-dimensional structure of Tm-1 has not been determined.