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. Here, using an infectious cDNA clone and reverse genetics, we demonstrate that UCBSV requires the ITPase activity for infectivity in cassava, but not in the model plant Nicotiana benthamiana. HPLC-MS/MS experiments showed that, quite likely, this host-specific constraint is due to an unexpected high concentration of non-canonical nucleotides in cassava. Finally, protein analyses and experimental evolution of mutant viruses indicated that keeping a fraction of the yielded UCBSV ITPase covalently bound to the viral RNA-dependent RNA polymerase (RdRP) optimizes viral fitness, and this seems to be a feature shared by the other members of the Potyviridae family expressing Maf/ham1-like proteins. All in all, our work (i) reveals that the over-accumulation of non-canonical nucleotides in the host might have a key role in antiviral defense, and (ii) provides the first example of an RdRP-ITPase partnership, reinforcing the idea that RNA viruses are incredibly versatile at adaptation to different host setups.

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. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T-DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus-based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next-generation virus-based tools will be indispensable landmarks in the synthetic biology roadmap to better crops.

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. Using Plum pox virus as a model, we show that although P1 does not have a major direct role in RNA silencing suppression, it can indeed modulate HCPro function by its self-cleavage activity. To study P1 protease regulation, we used bioinformatic analysis and in vitro activity experiments to map the core C-terminal catalytic domain. We present evidence that the hypervariable region that precedes the protease domain is predicted as intrinsically disordered, and that it behaves as a negative regulator of P1 proteolytic activity in in vitro cleavage assays. In viral infections, removal of the P1 protease antagonistic regulator is associated with greater symptom severity, induction of salicylate-dependent pathogenesis-related proteins, and reduced viral loads. We suggest that fine modulation of a viral protease activity has evolved to keep viral amplification below host-detrimental levels, and thus to maintain higher long-term replicative capacity.

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%. By proof-of-concept VIGE of PHYTOENE DESATURASE (PDS) and plant regeneration from edited somatic tissue, we recovered loss-of-function pds mutant progeny displaying an albino phenotype. VIGE of STAYGREEN 1 (SGR1), a gene involved in fruit color variation, generated sgr1 mutant lines with recolored red-brown fruits and high chlorophyll levels. The obtained editing events were heritable, overall confirming the successful breeding of fruit color. Altogether, our VIGE approach offers great potential for accelerated functional genomics of tomato variation, as well as for precision breeding of novel tomato traits.

Heritable CRISPR-Cas9 editing of plant genomes using RNA virus vectors.

Viral vectors hold enormous potential for genome editing in plants as transient delivery vehicles of CRISPR-Cas components. Here, we describe a protocol to assemble plant viral vectors for single-guide RNA (sgRNA) delivery. The obtained viral constructs are based on compact T-DNA binary vectors of the pLX series and are delivered into Cas9-expressing plants through agroinoculation. This approach allows rapidly assessing sgRNA design for plant genome targeting, as well as the recovery of progeny with heritable mutations at targeted loci. For complete details on the use and execution of this protocol, please refer to Uranga et al. (2021)1 and Aragonés et al. (2022).2.

Methyltransferase-like (METTL) homologues participate in Nicotiana benthamiana 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. N6-adenosine methylation (m6A) is a common chemical modification of eukaryotic and viral RNA whose abundance is jointly regulated by MTases and METTLs, demethylases, and m6A binding proteins. m6A affects various cellular processes including RNA degradation, post-transcriptional processing, and antiviral immunity. Here, we used Nicotiana benthamiana and plum pox virus (PPV), an RNA virus of the Potyviridae family, to investigated the roles of MTases in plant-virus interaction. RNA sequencing analysis identified MTase transcripts that are differentially expressed during PPV infection; among these, accumulation of a METTL gene was significantly downregulated. Two N. benthamiana METTL transcripts (NbMETTL1 and NbMETTL2) were cloned and further characterized. Sequence and structural analyses of the two encoded proteins identified a conserved S-adenosyl methionine (SAM) binding domain, showing they are SAM-dependent MTases phylogenetically related to human METTL16 and Arabidopsis thaliana FIONA1. Overexpression of NbMETTL1 and NbMETTL2 caused a decrease of PPV accumulation. In sum, our results indicate that METTL homologues participate in plant antiviral responses.

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. The assembled virus clones are based on compact T-DNA binary vectors of the pLX series and are delivered to host plants by Agrobacterium-mediated inoculation. For complete details on the use and execution of this protocol, please refer to Pasin et al. (2017, 2018) and Pasin (2021).

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. Here, we review the heterogeneity of non-core modules that expand the structural and functional diversity of the potyvirid proteomes. We provide a family-wide classification of P1 proteinases into the functional Types A and B, and discuss pretty interesting sweet potato potyviral ORF (PISPO), putative zinc fingers, and alkylation B (AlkB)-non-core modules found within P1 cistrons. The atypical inosine triphosphate pyrophosphatase (ITPase/HAM1), as well as the pseudo tobacco mosaic virus-like coat protein (TMV-like CP) are discussed alongside homologs of unrelated virus taxa. Family-wide abundance of the multitasking helper component proteinase (HC-pro) is revised. Functional connections between non-core modules are highlighted to support host niche adaptation and immune evasion as main drivers of the Potyviridae evolutionary radiation. Potential biotechnological and synthetic biology applications of potyvirid leader proteinases and non-core modules are finally explored.

Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors.

Viral vectors provide a quick and effective way to express exogenous sequences in eukaryotic cells and to engineer eukaryotic genomes through the delivery of CRISPR/Cas components. Here, we present JoinTRV, an improved vector system based on tobacco rattle virus (TRV) that simplifies gene silencing and genome editing logistics. Our system consists of two mini T-DNA vectors from which TRV RNA1 (pLX-TRV1) and an engineered version of TRV RNA2 (pLX-TRV2) are expressed. The two vectors have compatible origins that allow their cotransformation and maintenance into a single Agrobacterium cell, as well as their simultaneous delivery to plants by a one-Agrobacterium/two-vector approach. The JoinTRV vectors are substantially smaller than those of any known TRV vector system, and pLX-TRV2 can be easily customized to express desired sequences by one-step digestion-ligation and homology-based cloning. The system was successfully used in Nicotiana benthamiana for launching TRV infection, for recombinant protein production, as well as for robust virus-induced gene silencing (VIGS) of endogenous transcripts using bacterial suspensions at low optical densities. JoinTRV-mediated delivery of single-guide RNAs in a Cas9 transgenic host allowed somatic cell editing efficiencies of ≈90%; editing events were heritable and >50% of the progeny seedlings showed mutations at the targeted loci.

AlkB RNA demethylase homologues and N6 -methyladenosine are involved in Potyvirus infection.

Proteins of the alkylation B (AlkB) superfamily show RNA demethylase activity removing methyl adducts from N6 -methyladenosine (m6 A). m6 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. We show that Nicotiana benthamiana m6 A levels are reduced by infection of plum pox virus (PPV) and potato virus Y (PVY). The two potyviruses lack AlkB and the results suggest a general involvement of RNA methylation in potyvirus infection and evolution. Methylated RNA immunoprecipitation sequencing of virus-infected samples showed that m6 A peaks are enriched in plant transcript 3' untranslated regions and in discrete internal and 3' terminal regions of PPV and PVY genomes. Down-regulation of N. benthamiana AlkB homologues of the plant-specific ALKBH9 clade caused a significant decrease in PPV and PVY accumulation. In summary, our study provides evolutionary and experimental evidence that supports the m6 A implication and the proviral roles of AlkB homologues in Potyvirus infection.

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). Type IIS endonucleases with low abundance in the plant virome, as well as Golden Gate and No See'm principles were combined with DNA chemical synthesis for seamless viral clone assembly by one-step digestion-ligation. The framework described does not require subcloning steps, is insensitive to insert terminal sequences, and was used with linear and circular DNA molecules. Based on a digital template, DNA fragments were chemically synthesized and assembled by one-step cloning to yield a scar-free infectious clone of a plant virus suitable for Agrobacterium-mediated delivery. SynViP allowed rescue of a genuine virus without biological material, and has the potential to greatly accelerate biological characterization and engineering of plant viruses as well as derived biotechnological tools. Finally, computational identification of compositional biases in biological sequences might become a common standard to aid scalable biosystems design and engineering.

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. We detail herein the preparation of home-made cloning materials for Gibson assembly. The home-made materials were used in one-step generation of the infectious cDNA clone of a plant RNA virus into a T-DNA binary vector. The clone was verified by a single Illumina reaction and a de novo read assembly approach that required no primer walking, custom primers or reference sequences. Clone infectivity was finally confirmed by Agrobacterium-mediated delivery to host plants. We anticipate that the convenient home-made materials, one-step cloning and Illumina verification strategies described herein will accelerate characterization of viruses and their role in disease development.

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. A P1 autoinhibitory domain controls polyprotein processing, the release of a downstream functional RNA-silencing suppressor, and viral replication. Here, we show that P1Pro, a plum pox virus clone that lacks the P1 autoinhibitory domain, triggers complex reprogramming of the host transcriptome and high levels of abscisic acid (ABA) accumulation. A meta-analysis highlighted ABA connections with host pathways known to control RNA stability, turnover, maturation, and translation. Transcriptomic changes triggered by P1Pro infection or ABA showed similarities in host RNA abundance and diversity. Genetic and hormone treatment assays showed that ABA promotes plant resistance to potyviral infection. Finally, quantitative mathematical modeling of viral replication in the presence of defense pathways supported self-control of polyprotein processing kinetics as a viral mechanism that attenuates the magnitude of the host antiviral response. Overall, our findings indicate that ABA is an active player in plant antiviral immunity, which is nonetheless evaded by a self-controlled RNA virus.

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. On the contrary, spatial-temporal modulation of these polyprotein cleavage events is crucial for a successful viral infection. In this way, the processing of the polyprotein coordinates viral replication, assembly and movement, and has significant impact on pathogen fitness and virulence. In this mini-review, we give an overview of plant viral proteases emphasizing their importance during viral infections and the varied functionalities that result from their proteolytic activities.

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. In this study, a workflow that encompassed pLX binary vectors, overlap-based assembly strategies, and sequencing-by-synthesis verification steps is described and applied for the streamlined generation of infectious clones suitable for Agrobacterium-mediated delivery. The pLX-based vectors herein assembled include the first infectious clone of Wasabi mottle virus, a crucifer-infecting tobamovirus, as well as binary vectors of positive-single-stranded RNA and single- and double-stranded DNA viruses from the Potyviridae, Geminiviridae and Caulimoviridae families, respectively. Finally, the clones generated were used to agro-inoculate the model plant Arabidopsis thaliana and infections were confirmed by a multiplex RT-PCR assay. This workflow facilitated the rapid generation of infectious clones which, together with agro-infection scalability, would allow the pursuit of systematic insights into virus biology and physiology of plant infections and the design of novel biotechnological applications.

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. P1 proteins can be classified as Type A or Type B on the basis, amongst other things, of their dependence or not on a host factor to develop their protease activity. In this work, we studied Type A proteases from the Potyviridae family, characterizing their host factor requirements. Our in vitro cleavage analyses of potyvirid P1 proteases showed that the N-terminal domain is relevant for host factor interaction and suggested that the C-terminal domain is also involved. In the absence of plant factors, the N-terminal end of Plum pox virus P1 antagonizes protease self-processing. We performed extended deletion mutagenesis analysis to define the N-terminal antagonistic domain of P1. In viral infections, removal of the P1 protease antagonistic domain led to a gain-of-function phenotype, strongly increasing local infection in a non-permissive host. Altogether, our results shed new insights into the adaptation and evolution of potyvirids.

Multiple T-DNA Delivery to Plants Using Novel Mini Binary Vectors with Compatible Replication Origins.

Improved plants are necessary to meet human needs. Agrobacterium-mediated transformation is the most common method used to rewire plant capabilities. For plant gene delivery, DNA constructs are assembled into binary T-DNA vectors that rely on broad host range origins for bacterial replication. Here we present pLX vectors, a set of mini binary T-DNA plasmids suitable for Type IIS restriction endonuclease- and overlap-based assembly methods. pLX vectors include replicons from compatible broad host range plasmids. Simultaneous usage of pBBR1- and RK2-based pLX vectors in a two-plasmid/one-Agrobacterium strain strategy allowed multigene delivery to plants. Adoption of pLX vectors will facilitate routine plant transformations and targeted mutagenesis, as well as complex part and circuit characterization.

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. Here we built a series of constructs and chimeric viruses to help elucidate the role of P1a cleavage in host range definition. We demonstrate that host-dependent separation of P1a from the remainder of the polyprotein is essential for suppressing RNA silencing defenses and for efficient viral infection. These findings support the role of viral proteases as important determinants in host adaptation.

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. Fluorescence intensity is used to quantify fluorescent protein accumulation by image analysis or spectroscopy of protein extracts; however, these methods might not be suitable for medium- to large-scale comparisons. RESULTS: We report that laser scanners, used routinely in proteomic studies, are suitable for quantitative imaging of plant leaves that express different fluorescent protein pairs. We developed a microtiter plate fluorescence spectroscopy method for direct quantitative comparison of fluorescent protein accumulation in intact leaf discs. We used this technique to measure a fluorescent reporter in a transient RNA silencing suppression assay, and also to monitor early amplification dynamics of a fluorescent protein-labeled potyvirus. CONCLUSIONS: Laser scanners allow dual-color fluorescence imaging of leaf samples, which might not be acquired in standard stereomicroscope devices. Fluorescence microtiter plate analysis of intact leaf discs can be used for rapid, accurate quantitative comparison of fluorescent protein accumulation.