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

Pasin, Fabio

doi:10.1016/j.xpro.2022.101716

Cited by 5 publications

(10 citation statements)

References 78 publications

(120 reference statements)

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“… Note: If your sample arcs when electroporating, dialyze or purify the assembly reaction to remove salt excess. Alternatives: Heat shock of chemically-competent E. coli cells for high-efficiency transformation 23 may be used. Incubate the cells at 37°C, shaking vigorously at 200–250 rpm (1 h).…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Alternatives: Heat shock of chemically-competent E. coli cells for high-efficiency transformation 23 may be used.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Both viral systems are based on compact T-DNA binary vectors of the pLX series, 20 which have been successfully used for starting RNA and DNA virus infections by Agrobacterium -mediated inoculation (agroinoculation). 21 , 22 , 23 Recombinant viral replicons with sgRNA constructs are assembled and delivered into Cas9-expressing plants via agroinoculation. Systemic viral infection results in germline genome editing and recovery of edited progeny ( Figure 1 ).…”

Section: Before You Beginmentioning

confidence: 99%

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

et al. 2023

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Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…Alternatives: Heat shock of chemically-competent E. coli cells for high-efficiency transformation 23 may be used.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Section: Before You Beginmentioning

confidence: 99%

See 1 more Smart Citation

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

et al. 2023

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“…Alternative: Heat shock of chemically-competent E. coli cells for high-efficiency transformation 23 may be used.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

“…pLX-TRV1 provides the replicase function, whereas pLX-TRV2 includes an engineered TRV RNA2 sequence with a heterologous sub-genomic promoter of pea early browning virus (PEBV) to drive insert expression (Figure 2). Both viral systems are based on compact T-DNA binary vectors of the pLX series 20 , which have been successfully used for starting RNA and DNA virus infections by Agrobacterium-mediated inoculation (agroinoculation) [21][22][23] . Recombinant viral replicons with sgRNA constructs are assembled and delivered into Cas9-expressing plants via agroinoculation.…”

Section: Before You Beginmentioning

confidence: 99%

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

Uranga

Aragonés

Daròs

et al. 2022

Preprint

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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.

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Methyltransferase-like (METTL) homologues participate in Nicotiana benthamiana antiviral responses

Yue

Sun

et al. 2023

Plant Signaling & Behavior

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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 6 -adenosine methylation (m 6 A) is a common chemical modification of eukaryotic and viral RNA whose abundance is jointly regulated by MTases and METTLs, demethylases, and m 6 A binding proteins. m 6 A 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.

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

Cited by 5 publications

References 78 publications

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

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

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

Methyltransferase-like (METTL) homologues participate in Nicotiana benthamiana antiviral responses

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