Abstract
Biotic diseases cause substantial agricultural losses annually, spurring research into plant pathogens and strategies to mitigate them. Nicotiana benthamiana is a commonly used model plant for studying plant–pathogen interactions because it is host to numerous plant pathogens and because many research tools are available for this species. The clustered regularly interspaced short palindromic repeats (CRISPR) system is one of several powerful tools available for targeted gene editing, a crucial strategy for analyzing gene function. Here, we demonstrate the use of various CRISPR-associated (Cas) proteins for gene editing of N. benthamiana protoplasts, including Staphylococcus aureus Cas9 (SaCas9), Streptococcus pyogenes Cas9 (SpCas9), Francisella novicida Cas12a (FnCas12a), and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID). We successfully mutated Phytoene Desaturase (PDS) and Ethylene Receptor 1 (ETR1) and the disease-associated genes RNA-Dependent RNA Polymerase 6 (RDR6), and Suppressor of Gene Silencing 3 (SGS3), and confirmed that the mutated alleles were transmitted to progeny. sgs3 mutants showed the expected phenotype, including absence of trans-acting siRNA3 (TAS3) siRNA and abundant expression of the GFP reporter. Progeny of both sgs3 and rdr6 null mutants were sterile. Our analysis of the phenotypes of the regenerated progeny indicated that except for the predicted phenotypes, they grew normally, with no unexpected traits. These results confirmed the utility of gene editing followed by protoplast regeneration in N. benthamiana. We also developed a method for in vitro flowering and seed production in N. benthamiana, allowing the regenerants to produce progeny in vitro without environmental constraints.
Highlights
Nicotiana benthamiana is a host to many plant pathogens, especially viruses, and is widely used to study plant–pathogen interactions (Goodin et al, 2008)
We placed protoplasts isolated from the leaves of in vitro-grown shoots (Figure 1a) in 1N0.3K liquid medium, incubated them in the dark for 1 month (Figure 1b), and transferred them to fresh 1N0.3K medium and incubated them in the dark for another month (Figure 1c)
Unlike in our previous method described for tobacco (Lin et al, 2018), we incubated N. benthamiana calluses directly in liquid 1 mg/L 6-benzylaminopurine (1B) medium in light without embedding (Figure 1d)
Summary
Nicotiana benthamiana is a host to many plant pathogens, especially viruses, and is widely used to study plant–pathogen interactions (Goodin et al, 2008). Many tools for functional genomics are available for this species, including viral vectors, RNA interference (RNAi), ethyl methanesulfonate mutagenesis, agroinfiltration, protoplast transfection, and Agrobacterium-mediated stable transformation. These tools are useful for research in genomics, biochemistry, metabolomics, cell biology, and pathology, as well as other topics in agriculture (Derevnina et al, 2019). Notwithstanding its many advantages, the fact that N. benthamiana is allotetraploid, with a very large genome (3.1 Gb) (Bombarely et al, 2012), makes it difficult to edit the genome of this plant and to obtain mutants for plant biological and gene functional studies. The use of CRISPR-Cas has greatly accelerated plant research and crop breeding in recent years (Li et al, 2013; Nekrasov et al, 2013; Shan et al, 2013; Li and Xia, 2020; Yue et al, 2020)
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