Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava

Bull, Simon E.; Owiti, Judith; Niklaus, Michael; Beeching, John R.; Gruissem, Wilhelm; Vanderschuren, Hervé

doi:10.1038/nprot.2009.208

Cited by 103 publications

(126 citation statements)

References 23 publications

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“…Crop improvement efforts have aimed at attaining CBB resistance; however, these efforts have been largely unsuccessful, most likely due to genetic diversity among Xam strains (15). Introduction of R proteins into cassava through genetic engineering or breeding is possible (16,17); however, both processes are time consuming and laborious, highlighting the importance of first identifying the most promising R proteins. Here we report full-genome sequencing, PAMP, and effector identification from 65 geographically and temporally diverse Xam strains.…”

Section: P-eif2αmentioning

confidence: 99%

High-throughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance

Bart

Cohn

Kassen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

129

128

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Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis ( Xam ), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Here we present a widely applicable strategy for elucidating the virulence components of a pathogen population. We report Illumina-based draft genomes for 65 Xam strains and deduce the phylogenetic relatedness of Xam across the areas where cassava is grown. Using an extensive database of effector proteins from animal and plant pathogens, we identify the effector repertoire for each sequenced strain and use a comparative sequence analysis to deduce the least polymorphic of the conserved effectors. These highly conserved effectors have been maintained over 11 countries, three continents, and 70 y of evolution and as such represent ideal targets for developing resistance strategies.

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Section: P-eif2αmentioning

confidence: 99%

High-throughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance

Bart

Cohn

Kassen

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

129

128

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“…Cassava storage roots of the model cv 60444 that is routinely used for transformation (Bull et al, 2009) were harvested in the greenhouse and sliced. Root slices were collected for characterization of the root proteome at harvest (0 h).…”

Section: Properties Of the Samples Used For Analysismentioning

confidence: 99%

“…The cDNA encoding cytosolic At-GPX2 (AT2G31570.1) was amplified by RT-PCR from Arabidopsis total RNA using primers 59-ATGGCGGAT-GAATCTCCAAAGTC-39 and 59-TTAAGAAGAGGCCTGTCCCAAC-39 and cloned downstream of the patatin promoter to generate the PAT-GPX transformation vector. Transgenic cassava plants were generated according to an optimized transformation procedure (Bull et al, 2009). Transgenic plant analysis and characterization were performed according to procedures described previously ).…”

Section: Plant Transformationmentioning

confidence: 99%

Large-Scale Proteomics of the Cassava Storage Root and Identification of a Target Gene to Reduce Postharvest Deterioration

et al. 2014

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ORCID IDs: 0000-0003-2102-9737 (H.V.); 0000-0002-1904-9440 (K.B.) Cassava (Manihot esculenta) is the most important root crop in the tropics, but rapid postharvest physiological deterioration (PPD) of the root is a major constraint to commercial cassava production. We established a reliable method for image-based PPD symptom quantification and used label-free quantitative proteomics to generate an extensive cassava root and PPD proteome. Over 2600 unique proteins were identified in the cassava root, and nearly 300 proteins showed significant abundance regulation during PPD. We identified protein abundance modulation in pathways associated with oxidative stress, phenylpropanoid biosynthesis (including scopoletin), the glutathione cycle, fatty acid a-oxidation, folate transformation, and the sulfate reduction II pathway. Increasing protein abundances and enzymatic activities of glutathione-associated enzymes, including glutathione reductases, glutaredoxins, and glutathione S-transferases, indicated a key role for ascorbate/glutathione cycles. Based on combined proteomics data, enzymatic activities, and lipid peroxidation assays, we identified glutathione peroxidase as a candidate for reducing PPD. Transgenic cassava overexpressing a cytosolic glutathione peroxidase in storage roots showed delayed PPD and reduced lipid peroxidation as well as decreased H 2 O 2 accumulation. Quantitative proteomics data from ethene and phenylpropanoid pathways indicate additional gene candidates to further delay PPD. Cassava root proteomics data are available at www.pep2pro.ethz.ch for easy access and comparison with other proteomics data.

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“…We generated cassava transgenic lines expressing the Cas9 protein together with sgRNA1 (referred to as Cas9+sgRNA1 lines) as well as control lines expressing only the Cas9 protein (referred to as Cas9 lines) using an established Agrobacterium tumefaciens-mediated transformation protocol 19 . Twenty transgenic cassava lines were characterized for T-DNA copy number (Supplementary Fig.…”

Section: Plant Transformation and Growth Conditionsmentioning

confidence: 99%

CRISPR-Cas9 interference in cassava linked to the evolution of editing-resistant geminiviruses

Mehta

Sturchler

Hirsch‐Hoffmann

et al. 2018

Preprint

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We used CRISPR-Cas9 in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family of plant-pathogenic DNA viruses. We found that between 33 and 48% of edited virus genomes evolved a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. Our study highlights the potential for virus escape from this technology. Care should be taken to design CRISPR-Cas9 experiments that minimize the risk of virus escape.The bacterial CRISPR-Cas9 (Clustered, regularly interspaced short palindromic repeats-CRISPR associated 9) gene editing system can be used to engineer resistance to DNA viruses through direct cleavage of the virus genome. Unlike conventional gene-editing using CRISPRCas9, engineering virus resistance requires constitutive and permanent expression of the ribonucleoprotein complex in the host. For example, CRISPR/Cas9 system has been used to engineer immunity to latent HIV-1 proviruses, Hepatitis B viruses, Herpes simplex virus and the Human papillomavirus in mammalian cell lines 1 . CRISPR-Cas9 has also been used in the model plants Arabidopsis thaliana and Nicotiana benthamiana to engineer resistance to ssDNA geminiviruses [2][3][4] . However, the degree to which using CRISPR-Cas9 to engineer virus-resistance results in the evolution of resistant viruses is unknown. One concern (which has previously been highlighted 5 ) might be that planting transgenic, virus-resistant CRISPR-Cas9 plants in the field will impose a selection pressure on viruses, while simultaneously providing viruses with a mechanism (via Cas9-induced mutations) to escape resistance. We applied CRISPR-Cas9 to engineer resistance to geminiviruses in cassava and investigated the impact of engineering resistance on geminivirus evolution. We failed to engineer geminivirus resistance, but, we found that use of CRISPR-Cas9 led to emergence of a novel, conserved mutant virus that cannot be cleaved again. We urge caution in the application of CRISPR-Cas9 for virus resistance in plants, both in glasshouse and field settings, to prevent the evolution of novel viruses.Cassava is a tropical staple food crop consumed by more than a billion people. Cassava production in Africa and South Asia can be decimated by cassava mosaic geminiviruses 6 . Biotechnology has proven effective for engineering cassava mosaic virus resistance by using plant endogenous RNA interference (RNAi) pathways to limit the expression of virus genes 7 . However, the fact that plant viruses have developed effective suppressors of RNAi 8 , as well as the limited success of RNAi-mediated geminivirus resistance suggest that newer methods of engineering resistance are needed. Using orthogonal systems like CRISPR-Cas9 to which plant viruses are unlikely to have developed suppressing mechanisms hence appears particularly attractive.. CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a...

show abstract

Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava

Cited by 103 publications

References 23 publications

High-throughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance

High-throughput genomic sequencing of cassava bacterial blight strains identifies conserved effectors to target for durable resistance

Large-Scale Proteomics of the Cassava Storage Root and Identification of a Target Gene to Reduce Postharvest Deterioration

CRISPR-Cas9 interference in cassava linked to the evolution of editing-resistant geminiviruses

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