Genome Editing and Protoplast Regeneration to Study Plant–Pathogen Interactions in the Model Plant Nicotiana benthamiana

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.

Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration

“…The protoplast regeneration protocol (Figure 1) used in the present study was modified from previously published protocols. 24,25,29 A key step to our approach is the observation that the phase of the cell cycle largely governs the choice of pathway used for DNA repair: NHEJ is the major DNA repair pathway during the G1, S, and G2 phases, whereas HDR occurs only during the late S and G2 phases 31,32 . Cell cycle synchronization is an effective strategy for enhancing TI efficiency in human embryonic kidney 293T cells.…”

“…These protoplasts were cultured and regenerated (Figure 1C). 24,25,29 The rooted plants were incubated in the growth chamber and genotyped (Figure 1D). These regenerated plants grew normally and produced seeds.…”

“…Nicotiana benthamiana and rapid cycling Brassica oleracea (RCBO) plants were propagated in ½ -strength Murashige and Skoog (½ MS) medium supplemented with 30 g/L sucrose and 1% agar under a 12-h/12-h light/dark cycle at 25°C. Protoplast isolation was performed according to Lin et al 24 and Hsu et al 29 except for the digestion solution [½ MS medium supplemented with 1 mg/L 1-naphthaleneacetic acid (NAA), 0.3 mg/L kinetin, 30 g/L sucrose, 0.4 M mannitol (1N0.3K), 1% cellulose, 0.5% Macerozyme] and digestion time (3 days) in N. benthamiana. The protoplasts were co-transfected with RNP and 50 μg synthetic single-stranded oligonucleotide DNA (ssODN; Genomics, Taipei, Taiwan) according to Woo et al 22 Transfected protoplasts were incubated in a 5-cm-diameter Petri dish containing liquid callus medium (1N0.3K) for 3 weeks.…”

“…19 Like the transcription activatorlike effector nuclease genome editing system 21 , CRISPR reagents can be delivered into protoplasts via polyethylene glycol (PEG)-Ca 2+ -mediated transfection of ribonucleoprotein (RNP) or plasmid DNA, and targeted mutations can be achieved. [22][23][24][25][26][27][28][29] The mutated protoplasts can then be regenerated into plants without chimerism and the mutated alleles are passed onto the progeny. 22,24,25 Here, we describe a simple, high-efficiency TI method based on the protoplast strategy for genome editing in plants using Ca9-gRNA and non-modified synthetic single-stranded oligonucleotide DNA (ssODN) DDs that does not require expensive equipment.…”

DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration

“…The CRISPR-Cas system uses Agrobacterium tumefaciens -mediated stable transformation to deliver DNA encoding Cas protein and single guide RNA (sgRNA) into the nuclei of tomato cells. As an alternative approach, CRISPR ribonucleoprotein (RNP) or plasmids harboring the Cas and sgRNA sequences can be introduced directly into protoplasts using transient transfection, allowing recombinant DNA-free plants to be regenerated to circumvent concerns about genetically modified organisms (GMOs) ( Woo et al, 2015 ; Andersson et al, 2018 ; Lin et al, 2018 ; Hsu et al, 2019 , 2021a, 2021b; De Bruyn et al, 2020 ; Yu et al, 2021 ). This protocol is important for use with hybrids or plants with a long juvenile period and for vegetative propagation because the transgenes from stable transformation (selection markers and CRISPR reagent genes) cannot be removed from these crops by crossing.…”