![Gene editing in lateral shoots of TRSV vector–inoculated plants. (A) A near completely photobleached lateral shoot of a plant inoculated with TRSV-SpCas9-NbPDS. (B) The rate of lateral shoots bearing partially photobleached leaves. Bar graphs and error bars represent the mean values and standard deviations, respectively. Statistical significance was determined using Brown-Forsythe and Welch ANOVA tests with Dunnett T3 multiple comparison test. ns, *, **, *** and **** indicate P > 5.00 × 10−2, P = 1.10 × 10−2, 2.14 × 10−2, 8.60 × 10−4 and 2.03 × 10−3, respectively. Sample numbers are 8, 15, 8 and 7 for TRSV-SpCas9-NbPDS, ALSV-NbRDR6 + TRSV-SpCas9-NbPDS, TRSV-SpCas9-NbPDS-16K and ALSV-NbRDR6 + TRSV-SpCas9-NbPDS-16K, respectively. (C) Heritability of the introduced mutations for progenies from lateral shoots containing highly gene-edited tissues. Mutation frequencies of each allele and nucleotide sequences of the target sites with mutations are shown in Supplementary Table S5–S8. Phenotypes (D) and genotypes (E) of the progenies derived from a fruit harvested from a lateral shoot with highly photobleached leaves of a plant inoculated with ALSV-NbRDR6 and TRSV-SpCas9-NbPDS-16K with possible seed transmission of the virus (F). The PAM sequence is underlined, and a red letter and hyphen indicate a nucleotide insertion and deletion, respectively, in (E). TRSV (upper) and ALSV (middle) RNAs in the progenies were detected by RT-PCR in (F). Lane numbers correspond to the progeny numbers in (E). The endogenous N. benthamiana Actin gene (lower) was used as an internal control. ‘Uninoc’ and ‘inoc’ indicate an uninoculated (negative control) and inoculated (positive control) N. benthamiana plant, respectively. (G) Amplicon sequencing analysis of NbPDSa and NbPDSb in leaves from the primary and lateral shoots of a plant inoculated with ALSV-NbRDR6 and TRSV-SpCas9-NbPDS-16K. The unedited sequences are shown first in blue, followed by edited sequences in other colors in the order of read counts. Numbers indicate the position of nodes where the lateral branches or leaves arose. Zero, minus and plus indicate the node of an inoculated leaf, lower nodes and upper nodes, respectively. A biological replicate is shown in Supplementary Fig. S6C. (H) Nucleotide sequences around the target sites of NbPDSa and NbPDSb in a leaf from a lateral shoot that arose from the third lower node from the TRSV-inoculated leaf of the plant inoculated with ALSV-NbRDR6 and TRSV-SpCas9-NbPDS-16K [‘−3’ in (G)]. The 20-nt sgRNA target sequence and the GGG PAM sequence are shown in blue and brown, respectively. A red letter and hyphen indicate a nucleotide insertion and deletion, respectively. Numbers of nucleotide insertion (+) and deletion (−) are denoted. WT indicates the sequence with no mutations.](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/pcp/65/11/10.1093_pcp_pcae100/1/pcae100f3.jpeg?Expires=1748903376&Signature=VTMGZnhna1y~GbgLeFGTLIl2aM986iOlHZGPS4IQVtp6fOltPTdJ775HMECcisuC~DGnY2uhYWrySh1YmnxzfAwltP6Vd~ydRSavJQM010jhZrfaA2tUo2~Y~-oSZTpMNBpJIlwXRnyMOYcA3TBswoDZH2Ao3izk5Io2ck6Z5hzgYjXpDMaA8zjUf3vpH-M3k09ViNT2AAAxF17Sh52y~gOzGq9~t2cB79xLwuhgqdSzKgEJxeva7g11bBo78HqleVLN61IGelQ1EOTCl7hXWBLynQXPS698q5WZ4zlfSCJ0RmbOubiwJXYUF1l322KiH72w3Hp1h96F1g2OlN5RHQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Gene editing in lateral shoots of TRSV vector–inoculated plants. (A) A near completely photobleached lateral shoot of a plant inoculated with TRSV-SpCas9-NbPDS. (B) The rate of lateral shoots bearing partially photobleached leaves. Bar graphs and error bars represent the mean values and standard deviations, respectively. Statistical significance was determined using Brown-Forsythe and Welch ANOVA tests with Dunnett T3 multiple comparison test. ns, *, **, *** and **** indicate P > 5.00 × 10−2, P = 1.10 × 10−2, 2.14 × 10−2, 8.60 × 10−4 and 2.03 × 10−3, respectively. Sample numbers are 8, 15, 8 and 7 for TRSV-SpCas9-NbPDS, ALSV-NbRDR6 + TRSV-SpCas9-NbPDS, TRSV-SpCas9-NbPDS-16K and ALSV-NbRDR6 + TRSV-SpCas9-NbPDS-16K, respectively. (C) Heritability of the introduced mutations for progenies from lateral shoots containing highly gene-edited tissues. Mutation frequencies of each allele and nucleotide sequences of the target sites with mutations are shown in Supplementary Table S5–S8. Phenotypes (D) and genotypes (E) of the progenies derived from a fruit harvested from a lateral shoot with highly photobleached leaves of a plant inoculated with ALSV-NbRDR6 and TRSV-SpCas9-NbPDS-16K with possible seed transmission of the virus (F). The PAM sequence is underlined, and a red letter and hyphen indicate a nucleotide insertion and deletion, respectively, in (E). TRSV (upper) and ALSV (middle) RNAs in the progenies were detected by RT-PCR in (F). Lane numbers correspond to the progeny numbers in (E). The endogenous N. benthamiana Actin gene (lower) was used as an internal control. ‘Uninoc’ and ‘inoc’ indicate an uninoculated (negative control) and inoculated (positive control) N. benthamiana plant, respectively. (G) Amplicon sequencing analysis of NbPDSa and NbPDSb in leaves from the primary and lateral shoots of a plant inoculated with ALSV-NbRDR6 and TRSV-SpCas9-NbPDS-16K. The unedited sequences are shown first in blue, followed by edited sequences in other colors in the order of read counts. Numbers indicate the position of nodes where the lateral branches or leaves arose. Zero, minus and plus indicate the node of an inoculated leaf, lower nodes and upper nodes, respectively. A biological replicate is shown in Supplementary Fig. S6C. (H) Nucleotide sequences around the target sites of NbPDSa and NbPDSb in a leaf from a lateral shoot that arose from the third lower node from the TRSV-inoculated leaf of the plant inoculated with ALSV-NbRDR6 and TRSV-SpCas9-NbPDS-16K [‘−3’ in (G)]. The 20-nt sgRNA target sequence and the GGG PAM sequence are shown in blue and brown, respectively. A red letter and hyphen indicate a nucleotide insertion and deletion, respectively. Numbers of nucleotide insertion (+) and deletion (−) are denoted. WT indicates the sequence with no mutations.
