Fig 1.
Somatic embryogenesis and plant regeneration process.
(A) pro-embryogenic masses transformed with the CRISPR-dCas12a expression vectors, (B) individualization and selection of somatic embryos on hygromycin-containing medium, (C) edited plant regeneration, (D) edited plant elongation and rooting, (E) acclimatization of edited plants, (F) transcript levels of SlPAL2 in single parental epigenetically edited tomato plants (with three technical replicates of each PCR reaction). Data represents mean ± SD. Statistical analysis was performed by using Welch’s one-way ANOVA followed by Dunnett’s multiple comparisons test. Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****; the absence of significance bars indicate that no significant (ns) differences were detected.
Fig 2.
Transcript levels of SlPAL2 in edited tomato plants in non-infected plants (A, upper row) and infected plants (B, lower row) at different time points (days post-infection, dpi). Foliage leaves from 3-weeks-old plants were taken at 24 h before Cmm (t0), 1 dpi (t1), 3 dpi (t2), and 60 dpi (t4). Data were normalized to the SlLSM7 reference gene (based on the 2^(-ΔΔCt) method; [38]). Data represent mean ± SD from three independent clones (n = 3) with three technical replicates per clone (except for the WT line, for which a single representative dataset with three technical replicates is shown). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****; the absence of significance bars indicate that no significant (ns) differences were detected.
Fig 3.
Colony forming units and lesion development in tomato plants after inoculation with CmmAcR42.
(A) Colony Forming Unit (CFU) count in leaves taken 9 days after Cmm infection. (B) Disease severity as percentage of leaf damage in tomato leaves 9 days after infection. (C) Disease severity as percentage of leaf damage in tomato leaves 120 days after infection. For (A) a Tukey’s test was performed at α=0.01, with n = 3 biological replicates, each with three technical replicates (data represents mean ± SEM); for (B) and (C), statistical analysis was performed from three biological replicates (n = 3), using Welch’s one-way ANOVA followed by Dunnett’s multiple comparisons test. Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****; the absence of significance bars indicates that no significant (ns) differences were detected (data represents mean ± SD). (D) Phenotypes of non-infected and infected tomato leaves 120 days after infection. (E) Cmm symptomatology in upper, middle and lower stem regions from longitudinal sections from distinct infected tomato plants (reference image taken from INRAE, [44]). (F) Cmm symptomatology in upper, middle and lower stem from transversal sections from distinct infected tomato plants (reference image taken from INRAE, [44]).
Fig 4.
Agronomic characteristics in edited tomato plants.
Plant height at different time points (days post-germination): (A) non-infected plants, (B) plants infected with Cmm 10 − 8 CFU mL − 1. Number of fruits: (C) non-infected plants, (D) plants infected with Cmm 10 − 8 CFU mL − 1. Fruit weight: (E) non-infected plants, (F) plants infected with Cmm 10 − 8 CFU mL − 1. For A-B, statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test. Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****; data represents mean ± SD. For C-F, statistical analysis was performed using Welch’s one-way ANOVA followed by Dunnett’s multiple comparisons test, based on six biological replicates (n = 6) (except for the WT line, for which a single representative dataset with three technical replicates is shown). Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****; data represents mean ± SEM. The absence of significance bars indicates that no significant (ns) differences were detected.
Fig 5.
Quantification of lignin contents.
Quantification of lignin contents in leaf tissue to assess the effect of PAL2 gene activation on lignin deposition 120 days post-infection. (A) Non-infected plants and (B) plants infected with Cmm 10 − 8 CFU mL − 1. Statistical analysis was performed using Welch’s one-way ANOVA followed by Dunnett’s multiple comparisons test, based on three biological replicates (n = 3) each with three technical replicates. Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****; data represents mean ± SEM. The absence of significance bars indicates that no significant (ns) differences were detected.
Fig 6.
Transcript levels of SlPAL2 in edited tomato plants in non-infected plants (A, upper row) and infected plants (B, lower row) at different time points (days post-infection, dpi). Foliage leaves from 3-weeks-old plants were taken at 24 h before Cmm (t0), 1 dpi (t1), 3 dpi (t2), and 120 dpi (t4). Data were normalized to the SlLSM7 reference gene (based on the 2^(-ΔΔCt) method; [38]). Data represent mean ± SD from three independent clones with three technical replicates per clone (except for the WT line, for which a single representative dataset with three technical replicates is shown). Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. Data represents mean ± SEM. Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****. The absence of significance bars indicates that no significant (ns) differences were detected.
Fig 7.
Colony forming units and lesion development in tomato plants.
(A) Colony Forming Unit (CFU) count in leaves taken 9 days after Cmm infection. (B) Disease severity as percentage of leaf damage in tomato leaves 9 days after infection. (C) Disease severity as percentage of leaf damage in tomato leaves 120 days after infection. For (A), a Tukey’s test was performed at α=0.01, with n = 3 biological replicates, each with three technical replicates (except for the WT line, for which n = 9 biological replicates with three technical replicates is shown); data represent mean ± SD. For (B) and (C), statistical analysis was performed from three biological replicates, using Welch’s one-way ANOVA followed by Dunnett’s multiple comparisons test. Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****; the absence of significance bars indicates that no significant (ns) differences were detected. (D) Phenotypes of non-infected and infected tomato leaves 120 days after infection. (E) Cmm symptomatology in upper, middle and lower stem regions from longitudinal sections from distinct infected tomato plants (reference image taken from INRAE [44]). (F) Cmm symptomatology in upper, middle and lower stem from transversal sections from distinct infected tomato plants (reference image taken from INRAE [44]).
Fig 8.
Histone methylation profile in tomato plants.
Chromatin Immunoprecipitation (ChIP) assays to analyze the histone H3 lysine-4 trimethylation mark (H3K4me3) at the SlPAL2 first exonic region. (A) Schematic representation of the SlPAL2 gene targeted in the ChIP assay. The purple rectangle denotes the promoter region, red rectangles indicate exonic regions, purple lines show the crRNA binding sites, pink lines represent the regions amplified by PCR using two distinct sets of primers, and the bent arrow marks the transcription start site (TSS). (B) ChIP assays conducted on 21-day-old plants, 24 hours before infection with Clavibacter michiganensis subsp. michiganensis (Cmm). (C) ChIP assays from control plants that were not exposed to the pathogen (24-days-old plants). (D) ChIP assays performed 72 hours after Cmm infection. Data are mean ± SEM. Each ChIP experiment was independently conducted in duplicate (two plants per line as technical replicates) from two biological replicates. ChIP-qPCR data were normalized to input samples (1% starting chromatin), according to the Percent Input method (% Input = 2((Cq(IN)-Log2(DF))-Cq(IP)) * 100; [40]).
Fig 9.
Lignin contents in edited tomato plants.
Quantification of lignin contents in leaf tissue to assess the effect of PAL2 gene activation on lignin deposition. (A) Non-infected plants (upper row) and (B) infected plants (lower row) at different time points: t1 (1-day post-infection, dpi), t2 (3 dpi), t3 (12 dpi) and t4 (120 dpi). Statistical analysis was performed using Welch’s one-way ANOVA followed by Dunnett’s multiple comparisons test, based on three biological replicates (n = 3), each with three technical replicates (for t3 and t4). Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****. Data represents mean ± SEM. The absence of significance bars indicates that no significant (ns) differences were detected.
Fig 10.
Agronomic characteristics in edited tomato plants.
Plant height at different time points (days post-germination): (A) non-infected plants, (B) plants infected with Cmm. Number of fruits: (C) non-infected plants, (D) plants infected with Cmm. Fruit weight: (E) non-infected plants, (F) plants infected with Cmm. For A-B, statistical analysis was performed using two-way ANOVA followed by Tukey’s multiple comparisons test (n = 3 biological replicates). Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****); data represents mean ± SD. For C-F, statistical analysis was performed using Welch’s one-way ANOVA followed by Dunnett’s multiple comparisons test (n = 3 biological replicates, each with three technical replicates, except for the WT line for which n = 3 biological replicates). Statistically significant differences are indicated as follows: p < 0.005, * ; p < 0.01, **, p < 0.001, ***; p < 0.0001, ****. The absence of significance bars indicates that no significant (ns) differences were detected.