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.

Keywords: FnCas12a; RDR6; SGS3; SaCas9; SpCas9; nCas9-Target-AID.

Figure 1

Protoplast regeneration in N. benthamiana. The gray background indicates that the explants were incubated in the dark. All liquid media were supplemented with 0.4 M mannitol. The abbreviated names of the media are shown in the top left corner of the images. 1N0.3K: 1/2 MS basal medium supplemented with 1 mg/L NAA and 0.3 mg/L kinetin. 1B: ½ MS basal medium supplemented with 1 mg/L BA and 20 g/L sucrose. All solid media were solidified with 7 g/L Phytagel. 2B: 1/2 MS basal medium supplemented with 2 mg/L BA. HB1: 3 g/L Hyponex No. 1 (N:P:K = 7:6:19), 2 g/L tryptone, 20 g/L sucrose, and 1 g/L activated charcoal. (a) Transfected mesophyll protoplasts. Bar = 200 μm. (b) Microcallus formation after 1 month of incubation in 1N0.3K. Bar = 1 cm. (c) Microcallus amplification. (d) Microcalli transferred to 1B medium and incubated in the light. Bar = 1 cm. (e) Callus amplification in 2B. Bar = 1 cm. (f) Shoot formation in 2B. Bar = 1 cm. (g) Shoots incubated in HB1 for root formation. (h) In vitro seed formation. Bar = 5 mm. (i) Plantlet incubated in a growth chamber. Bar = 1 cm.

Figure 2

Phenotypes of seed-derived seedlings and seedlings regenerated from protoplasts.

Figure 3

Target mutagenesis efficiencies. Mu, mutants. Gray: sgRNA mismatch. *: mutation. There were 10 regenerated plants (Total) analysis in each transfection. The mutants were confirmed by Sanger sequencing. % = No. of mutants/total no. of regenerated plants analyzed) X 100.

Figure 4

Mutants regenerated from CRISPR-Cas-edited Nicotiana benthamiana protoplasts. (A) Genotypes of F₁ N. benthamiana sgs3 mutants used for small RNA analysis. NbSGS3-1: Niben101Scf03392Ctg069; NbSGS3-2: Niben101Scf05468Ctg070. Red -: deleted nucleotide. Letter in gray: inserted nucleotide. Letter in green: edited nucleotide. (B) Five-week-old N. benthamiana plants subjected to Agrobacterium-mediated transient infiltration with 1 OD Agrobacterium cultures (OD₆₀₀ = 1) harboring binary vector with Green fluorescence protein (GFP) driven by the cauliflower mosaic virus 35S promoter. Leaves were harvested 3 d after infiltration. GFP and tubulin levels were analyzed by immunoblot analysis. (C) RNA gel blot analysis of the progeny of the Nbsgs mutants. WT, wild type; rdri, RNAi line of NbRDR6; D, 3-bp deletion. T insertion. (D) The progeny of Nbsgs3-14. Uppercase letters: in frame; lowercase letters: out of frame. E: base editing. a: A insertion. d: 5-bp deletion. Bar = 5 mm.

Figure 5

In vitro flowering and seed production in N. benthamiana. (a) In vitro flower. an: anther. Bar = 5 mm. (b) Mature fruit. Bar = 0.5 mm. (c) Open capsule. Bar = 0.5 mm. (d) Seedlings derived from in vitro seeds. Bar = 1 cm. (e) Bottle lip sealed with our newly developed sealing material. Bar = 1 cm. (f) Subculture in HB1 medium. Bar = 1 cm.