Robust Survival-Based RNA Interference of Gene Families Using in Tandem Silencing of Adenine Phosphoribosyltransferase

Orr, Robert G.; Foley, Stephen J; Sherman, Catherine A.; Abreu, Isidro; Galotto, Giulia; Liu, Boyuan; González‐Guerrero, Manuel; Vidali, Luis

doi:10.1104/pp.20.00865

Cited by 8 publications

(4 citation statements)

References 65 publications

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“…In order to expand the toolbox for gene function analysis in P. patens, we explored the possibility of editing specific bases of the genome through CBE and ABE in this model plant. For this purpose, we used the APT gene that we and other groups had previously used as a reporter of gene modification or modulation in P. patens (Trouiller et al, 2006;Holá et al, 2013;Orr et al, 2020). We demonstrate here for the first time that the CRISPR-Cas9 deaminase systems CBE and ABE are very efficient tools for base editing, including multiplex editing, in P. patens.…”

Section: Introductionmentioning

confidence: 90%

“…For this purpose, we used the APT gene that we and other groups had previously used as a reporter of gene modification or modulation in P . patens (Trouiller et al ., 2006; Holá et al ., 2013; Orr et al ., 2020). We demonstrate here for the first time that the CRISPR‐Cas9 deaminase systems CBE and ABE are very efficient tools for base editing, including multiplex editing, in P .…”

Section: Introductionmentioning

confidence: 99%

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A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

et al. 2021

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CRISPR-Cas9 has proven to be highly valuable for genome editing in plants, including the model plant Physcomitrium patens. However, the fact that most of the editing events produced using the native Cas9 nuclease correspond to small insertions and deletions is a limitation. CRISPR-Cas9 base editors enable targeted mutation of single nucleotides in eukaryotic genomes and therefore overcome this limitation. Here, we report two programmable baseediting systems to induce precise cytosine or adenine conversions in P. patens. Using cytosine or adenine base editors, site-specific single-base mutations can be achieved with an efficiency up to 55%, without off-target mutations. Using the APT gene as a reporter of editing, we could show that both base editors can be used in simplex or multiplex, allowing for the production of protein variants with multiple amino-acid changes. Finally, we set up a co-editing selection system, named selecting modification of APRT to report gene targeting (SMART), allowing up to 90% efficiency site-specific base editing in P. patens. These two base editors will facilitate gene functional analysis in P. patens, allowing for sitespecific editing of a given base through single sgRNA base editing or for in planta evolution of a given gene through the production of randomly mutagenised variants using multiple sgRNA base editing.

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Section: Introductionmentioning

confidence: 90%

Section: Introductionmentioning

confidence: 99%

A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

et al. 2021

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“…Additionally, the Rab‐E11i targeting sequence was specific to the Rab‐E clade, as it showed 54% and 63% identity to representative Rabs of the A and D clades, respectively. To investigate our rab‐E plants without the need for a specialised RNAi reporter line we used our recently developed RNAi system that exploited positive selection of an endogenous marker to enable robust, high‐throughput phenotyping (Orr et al ., 2020b). To characterise the growth phenotype of the mutant moss plants, we quantified two morphological parameters of the plant, area and solidity (Vidali et al ., 2010).…”

Section: Resultsmentioning

confidence: 99%

“…The loss of Rab‐E phenotype was elucidated using a recently developed RNAi method that allowed us to use the Δrab‐E14/15 line without the need for reporter insertion (Orr et al ., 2020b). To maximise silencing efficacy, our target was 401 bp of the Rab‐E11 coding sequence (CDS) (RabE11i), which shares 88% and 90% identity with Rab‐E12 and Rab‐E13, respectively.…”

Section: Methodsmentioning

confidence: 99%

Rab‐E and its interaction with myosin XI are essential for polarised cell growth

et al. 2020

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The fundamental process of polarised exocytosis requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. In plants, few mechanistic details are known about how molecular motors, such as myosin XI, associate with their secretory cargo to support the ubiquitous processes of polarised growth and cell division. Live-cell imaging coupled with targeted gene knockouts and a high-throughput RNAi assay enabled the first characterisation of the loss of Rab-E function. Yeast two-hybrid and subsequent in silico structural prediction uncovered a specific interaction between Rab-E and myosin XI that is conserved between P. patens and A. thaliana. Rab-E co-localises with myosin XI at sites of active exocytosis, and at the growing tip both proteins are spatiotemporally coupled. Rab-E is required for normal plant growth in P. patens and the rab-E and myosin XI phenotypes are rescued by A. thaliana's Rab-E1c and myosin XI-K/E, respectively. Both PpMyoXI and AtMyoXI-K interact with PpRabE14, and the interaction is specifically mediated by PpMyoXI residue V1422. This interaction is required for polarised growth. Our results suggest that the interaction of Rab-E and myosin XI is a conserved feature of polarised growth in plants.

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When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems

Trudeau,

Berbee

2024

Fungal Associations

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Robust Survival-Based RNA Interference of Gene Families Using in Tandem Silencing of Adenine Phosphoribosyltransferase

Abstract: Generation of double-stranded RNA targeting the adenine phosphoribosyltransferase gene in tandem with a target gene enables positive selection of strongly silencing plants.

Cited by 8 publications

References 65 publications

A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

A blueprint for gene function analysis through Base Editing in the model plant Physcomitrium (Physcomitrella) patens

Rab‐E and its interaction with myosin XI are essential for polarised cell growth

When Plants and Animals First Met Fungi: Insights from the Evolution of Host Immune Systems

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