Virus-induced systemic and heritable gene editing in pepper (Capsicum annuum L.)

Abstract

Genome editing using the CRISPR/Cas system enables rapid and efficient plant breeding by directly introducing desired traits into elite lines within a short time frame. However, challenges associated with conventional Agrobacterium tumefaciens-mediated transformation and regeneration have limited gene editing in pepper (Capsicum annuum L.). In this study, we applied and optimized a virus-induced gene editing (VIGE) system to overcome these limitations. We inoculated transgenic pepper seedlings already expressing Cas9 with vectors based on tobacco rattle virus 2 (TRV2) expressing single guide RNAs (sgRNAs) targeting Phytoene desaturase (PDS); shoots regenerated from inoculated cotyledons displayed photobleaching phenotypes. To promote sgRNA mobility and maintain its integrity, we modified the pTRV2-sgRNA vector by incorporating a self-cleaving hammerhead ribozyme (HH) sequence to produce an intact sgRNA fused to part of the mobile RNA of FLOWERING LOCUS T. Additionally, we tested alternative mobile elements, such as tRNA^Ile and tRNA^Met. Furthermore, we cultivated plants at the low temperature of 20°C following TRV inoculation to increase TRV persistence and spread. These optimizations, including vector modifications and cultivation conditions, resulted in a systemic editing efficiency of 36.3%, as evidenced by systemic leaves showing photobleaching phenotypes. We determined that 8.5% of progeny from plants inoculated with the pTRV-HH-CaPDS-sgRNA-FT construct were mutated at the CaPDS locus. In addition, we used our VIGE system to successfully edit FASCICULATE, producing mutants whose inflorescences showed a fasciculate phenotype. Direct inoculation with a TRV-based vector expressing a mobile sgRNA to bypass tissue culture, therefore, offers an effective tool for molecular studies and breeding in pepper.

Keywords: CRISPR/Cas9; Capsicum annuum; FASCICULATE (FA); Phytoene desaturase (PDS); heritable gene editing; tobacco rattle virus (TRV); virus‐induced gene editing (VIGE).

Figure 1

Generation of CaPDS‐edited pepper plants via TRV‐mediated gene editing and regeneration. (a) Diagram of the pTRV2‐sgRNA construct targeting CaPDS. 35S, cauliflower mosaic virus 35S promoter; CP, coat protein; LB, left border; nosT, nopaline synthase terminator; RB, right border; RZ, terminating ribozyme; U6, U6‐26 promoter. (b) Position of the CRISPR/Cas9 target site in the CaPDS locus. The CaPDS‐sgRNA target site is located in the second exon of CaPDS. The restriction enzyme site for BstXI, used for mutation detection, is underlined within the sgRNA target site. The protospacer adjacent motif (PAM) is shown in blue. (c) Mutation detection in leaves infiltrated with the pTRV‐U6‐CaPDS‐sgRNA construct. Cut, wild‐type sample treated with the restriction enzyme; Uncut, undigested wild‐type sample. (d) Regeneration of mutants of CaPDS via tissue culture. Scale bars, 1 cm. (e) Table summarizing the regeneration rate and mutant rate of regenerated shoots from infiltrated leaves. (f) Photobleaching phenotypes of E₁ progenies derived from the pds 1‐1 mutant. Scale bars, 1 cm. (g) Sequence of the sgRNA target site for E₁ progenies derived from each E₀ plant.

Figure 2

Improvement of systemic editing through modification of the pTRV2‐sgRNA vector and cultivation at low temperature. (a) Diagrams of the pTRV2‐CaPDS‐sgRNA constructs modified with the PEBV promoter, hammerhead ribozyme (HH), and/or FLOWERING LOCUS T (FT) sequences. LB, left border; 35S, cauliflower mosaic virus 35S promoter; CP, coat protein; PEBV, PEBV subgenomic promoter; RZ, terminating ribozyme; nosT, nopaline synthase terminator; RB, right border. (b–d) Mutation frequencies (b), photobleaching phenotypes (c), and sgRNA expression levels (d) in systemic leaves from plants grown at different temperatures after plant infiltration with various pTRV2‐CaPDS‐sgRNA constructs. (e) Agarose gel analysis of mutations in CaPDS in flowers from plants cultivated at 20°C following infiltration with pTRV‐HH‐CaPDS‐sgRNA‐FT. Uncut, undigested wild‐type sample. For (b), values represent means ± standard errors (SE) from six replicates. For (d), values represent means ± standard deviations (SD) from 3 to 6 replicates.

Figure 3

Comparison of editing efficiency with different mobile elements incorporated into the pTRV constructs. (a) Diagrams of the pTRV2‐CaPDS‐sgRNA constructs carrying different mobile elements. 35S, cauliflower mosaic virus 35S promoter; CP, coat protein; FT, FLOWERING LOCUS T; HH, hammerhead ribozyme; LB, left border; nosT, nopaline synthase terminator; PEBV, PEBV subgenomic promoter; RB, right border; RZ, terminating ribozyme; tRNA‐Ile, tRNA isoleucine; tRNA‐Met, tRNA methionine. (b) Photobleaching phenotypes of plants infiltrated with the pTRV2‐CaPDS‐sgRNA construct carrying the indicated mobile element. (c) Mutation frequencies in the uppermost systemic leaves of plants infiltrated with each pTRV2‐CaPDS‐sgRNA construct. (d) sgRNA expression levels in the uppermost systemic leaves of plants infiltrated with each pTRV2‐CaPDS‐sgRNA construct. PC, inoculated leaves with pTRV2‐HH‐CaPDS‐sgRNA‐FT (positive control); NC, non‐inoculated leaves (negative control). For (c, d), values represent means ± standard errors (SE) from 4 to 6 replicates. Statistical significance was determined using a two‐tailed t‐test in Microsoft Excel: NS, not significant; *P < 0.05.

Figure 4

FASCICULATE editing via TRV‐mediated gene editing and regeneration in pepper. (a) Diagrams of the pTRV2‐sgRNA constructs targeting FA for editing in Capsicum annuum. 35S, cauliflower mosaic virus 35S promoter; CP, coat protein; FT, FLOWERING LOCUS T; HH, hammerhead ribozyme; LB, left border; nosT, nopaline synthase terminator; PEBV, PEBV subgenomic promoter; RB, right border; RZ, terminating ribozyme; tRNA‐Ile, tRNA isoleucine; tRNA‐Met, tRNA methionine. (b) Positions of the CRISPR/Cas9 target sites in the CaFA locus. The different sgRNA target sites are located in different FA exons. The pTRV2‐CaFA‐sgRNA1‐2 construct includes multiplexed targets (site 1 and site 2), and pTRV2‐CaFA‐sgRNA3 and pTRV2‐CaFA‐sgRNA4 target sites 3 and 4, respectively. The upper arrows indicate conserved residues associated with substrate binding. The restriction enzyme site for BstNI, used to detect mutations in target sites 3 and 4, is underlined in the sgRNA target sites. The PAMs are highlighted in blue. (c, d) Sequences in the FA locus in the wild type (WT) and the mutants (c), and the mutant rate of plants infiltrated with and regenerated from each pTRV‐CaFA‐sgRNA construct. (e) Fasciculate phenotypes of the biallelic mutant plants, fa1‐2.01 and fa1‐2.02, in E₀ plants regenerated from plants infiltrated with the pTRV‐CaFA‐sgRNA1‐2 construct. Scale bar, 10 cm.

Figure 5

Transgenerational editing through an optimized TRV‐mediated gene editing system. (a) Photobleaching phenotypes in the pedicel and pericarp of fruits from infiltrated plants after infiltration of pTRV‐HH‐CaPDS‐sgRNA‐FT and cultivation at 20°C. Scale bars, 1 cm. (b, c) Photobleaching phenotype (b) and sequences of FA in the wild type (WT) and fa mutant plants (c) of E₁ plants derived from plants infiltrated with pTRV‐HH‐CaPDS‐sgRNA‐FT.

Figure 6

Transgenerational TRV‐mediated FA editing in pepper. (a) Fasciculate phenotype branches from TRV‐CaFA‐sgRNA1‐2‐infiltrated plants. (b) Mutation detection in the systemic leaves of fasciculate phenotype branches and non‐edited branch. (c, d) Mutation detection (c) and mutant sequences (d) of the E₁ seedlings from a TRV‐CaFA‐sgRNA1‐2‐infiltrated plant.

Figure 7

Diagram of the TRV‐mediated gene editing system in pepper. The pTRV2‐sgRNA vector with hammerhead ribozyme (HH) and FLOWERING LOCUS T (FT) sequences, along with pTRV1 and P19 constructs, is infiltrated into Cas9‐overexpressing pepper plants via Agrobacterium. Edited plants can be obtained through two methods: tissue culture‐mediated gene editing (GE) or tissue culture‐free transgenerational GE. In the latter method, an intact sgRNA produced via cleavage mediated by a HH spreads through systemic infection by the TRV‐based vector, whereas FT promotes its mobility to the shoot apical meristem, facilitating systemic editing and the generation of edited plants.