RNA virus-mediated gene editing for tomato trait breeding.

Virus-induced genome editing (VIGE) leverages viral vectors to deliver CRISPR-Cas components into plants for robust and flexible trait engineering. We describe here a VIGE approach applying an RNA viral vector based on potato virus X (PVX) for genome editing of tomato, a mayor horticultural crop. Viral delivery of single-guide RNA into Cas9-expressing lines resulted in efficient somatic editing with indel frequencies up to 58%.

Methyltransferase-like (METTL) homologues participate in antiviral responses.

Methyltransferase (MTase) enzymes catalyze the addition of a methyl group to a variety of biological substrates. MTase-like (METTL) proteins are Class I MTases whose enzymatic activities contribute to the epigenetic and epitranscriptomic regulation of multiple cellular processes. N-adenosine methylation (mA) is a common chemical modification of eukaryotic and viral RNA whose abundance is jointly regulated by MTases and METTLs, demethylases, and mA binding proteins.

Assembly of plant virus agroinfectious clones using biological material or DNA synthesis.

Infectious clone technology is universally applied for biological characterization and engineering of viruses. This protocol describes procedures that implement synthetic biology advances for streamlined assembly of virus infectious clones. Here, I detail homology-based cloning using biological material, as well as SynViP assembly using type IIS restriction enzymes and chemically synthesized DNA fragments.

AlkB RNA demethylase homologues and N -methyladenosine are involved in Potyvirus infection.

Proteins of the alkylation B (AlkB) superfamily show RNA demethylase activity removing methyl adducts from N -methyladenosine (m A). m A is a reversible epigenetic mark of RNA that regulates human virus replication but has unclear roles in plant virus infection. We focused on Potyvirus-the largest genus of plant RNA viruses-and report here the identification of AlkB domains within P1 of endive necrotic mosaic virus (ENMV) and an additional virus of a putative novel species within Potyvirus.

Proteome expansion in the Potyviridae evolutionary radiation.

Potyviridae, the largest family of known RNA viruses (realm Riboviria), belongs to the picorna-like supergroup and has important agricultural and ecological impacts. Potyvirid genomes are translated into polyproteins, which are in turn hydrolyzed to release mature products. Recent sequencing efforts revealed an unprecedented number of potyvirids with a rich variability in gene content and genomic layouts.

Maf/ham1-like pyrophosphatases of non-canonical nucleotides are host-specific partners of viral RNA-dependent RNA polymerases.

Cassava brown streak disease (CBSD), dubbed the "Ebola of plants", is a serious threat to food security in Africa caused by two viruses of the family Potyviridae: cassava brown streak virus (CBSV) and Ugandan (U)CBSV. Intriguingly, U/CBSV, along with another member of this family and one secoviridae, are the only known RNA viruses encoding a protein of the Maf/ham1-like family, a group of widespread pyrophosphatase of non-canonical nucleotides (ITPase) expressed by all living organisms. Despite the socio-economic impact of CDSD, the relevance and role of this atypical viral factor has not been yet established.

Oligonucleotide abundance biases aid design of a type IIS synthetic genomics framework with plant virome capacity.

Synthetic genomics-driven dematerialization of genetic resources facilitates flexible hypothesis testing and rapid product development. Biological sequences have compositional biases, which, I reasoned, could be exploited for engineering of enhanced synthetic genomics systems. In proof-of-concept assays reported herein, the abundance of random oligonucleotides in viral genomic components was analyzed and used for the rational design of a synthetic genomics framework with plant virome capacity (SynViP).

Abscisic Acid Connects Phytohormone Signaling with RNA Metabolic Pathways and Promotes an Antiviral Response that Is Evaded by a Self-Controlled RNA Virus.

A complex network of cellular receptors, RNA targeting pathways, and small-molecule signaling provides robust plant immunity and tolerance to viruses. To maximize their fitness, viruses must evolve control mechanisms to balance host immune evasion and plant-damaging effects. The genus Potyvirus comprises plant viruses characterized by RNA genomes that encode large polyproteins led by the P1 protease.

Home-made enzymatic premix and Illumina sequencing allow for one-step Gibson assembly and verification of virus infectious clones.

An unprecedented number of viruses have been discovered by leveraging advances in high-throughput sequencing. Infectious clone technology is a universal approach that facilitates the study of biology and role in disease of viruses. In recent years homology-based cloning methods such as Gibson assembly have been used to generate virus infectious clones.

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses.

Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses.

Streamlined generation of plant virus infectious clones using the pLX mini binary vectors.

Recent metagenomic surveys have provided unprecedented amounts of data that have revolutionized our understanding of virus evolution and diversity. Infectious clones are powerful tools to aid the biological characterization of viruses. We recently described the pLX vectors, a set of mini binary T-DNA vectors (∼3 kb) that includes strong bacterial terminators and a minimal replicon from the broad-host-range plasmid pBBR1, which replicate autonomously in both Escherichia coli and Agrobacterium.

Plant Viral Proteases: Beyond the Role of Peptide Cutters.

Almost half of known plant viral species rely on proteolytic cleavages as key co- and post-translational modifications throughout their infection cycle. Most of these viruses encode their own endopeptidases, proteases with high substrate specificity that internally cleave large polyprotein precursors for the release of functional sub-units. Processing of the polyprotein, however, is not an all-or-nothing process in which endopeptidases act as simple peptide cutters.

Truncation of a P1 leader proteinase facilitates potyvirus replication in a non-permissive host.

The Potyviridae family is a major group of plant viruses that includes c. 200 species, most of which have narrow host ranges. The potyvirid P1 leader proteinase self-cleaves from the remainder of the viral polyprotein and shows large sequence variability linked to host adaptation.

The Potyviridae P1a leader protease contributes to host range specificity.

The P1a protein of the ipomovirus Cucumber vein yellowing virus is one of the self-cleavage serine proteases present in Potyviridae family members. P1a is located at the N-terminal end of the viral polyprotein, and is closely related to potyviral P1 protease. For its proteolytic activity, P1a requires a still unknown host factor; this might be linked to involvement in host specificity.

Rapid fluorescent reporter quantification by leaf disc analysis and its application in plant-virus studies.

Background: Fluorescent proteins are extraordinary tools for biology studies due to their versatility; they are used extensively to improve comprehension of plant-microbe interactions. The viral infection process can easily be tracked and imaged in a plant with fluorescent protein-tagged viruses. In plants, fluorescent protein genes are among the most commonly used reporters in transient RNA silencing and heterologous protein expression assays.

The hypervariable amino-terminus of P1 protease modulates potyviral replication and host defense responses.

The replication of many RNA viruses involves the translation of polyproteins, whose processing by endopeptidases is a critical step for the release of functional subunits. P1 is the first protease encoded in plant potyvirus genomes; once activated by an as-yet-unknown host factor, it acts in cis on its own C-terminal end, hydrolyzing the P1-HCPro junction. Earlier research suggests that P1 cooperates with HCPro to inhibit host RNA silencing defenses.