Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L

Abstract

Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T₁ and T₂ progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

Keywords: CRISPR/Cas 9; eggplant; gene editing; knock-out; polyphenol oxydase.

Figure 1

(A) Relative position and organization of PPO1-9 genes on chromosome 8 of Solanum melongena. All eggplant PPOs except SmelPPO10 are located on chromosome 8. (B) Phylogenetic analysis of PPO proteins. The neighbor-joining trees was constructed by aligning the PPO protein sequences contained in Data Sheet 1. Clade A (orange) and B (yellow) proteins. The number at each node represents the bootstrap percentage value from 1,000 replicates. Smel, Solanum melongena; Sl, Solanum lycopersicum; Stu, Solanum tuberosum.

Figure 2

Transcriptional levels of 10 PPO-encoding genes in the Black Beauty variety 30 min after fruit cutting. The values are expressed as relative mRNA abundance at 30 min after cutting compared to time 0 (just after cutting). Eggplant actin and elongation factor genes were used as the reference genes. Data are means of three biological replicates ± SE. Different letters associated with the set of means indicate a significant difference based on Tukey b test (P ≤ 0.05).

Figure 3

(A) Alignment of SmelPPO4, SmelPPO5 SmelPPO6 with selected gRNA. (B) Genotyping of targeted gene mutations induced by CRISPR/Cas9 in the T₀ generation. Quantification of Illumina reads edited at the target locus in T₀_3 and T₀_4. For each line, the percentage of reads carrying mutated (blue) as well as not mutated (red) target sequence is reported together with the pattern and frequency of targeted gene mutations.

Figure 4

Genotyping of targeted gene mutations induced by CRISPR/Cas9 in the T₁ and T₂ generations. (A) Mutagenesis frequencies for all three targeted loci in T₁ and T₂ progenies. (B) Zygosity of targeted gene mutations in T₁ and T₂ populations.

Figure 5

Genotyping of targeted gene mutations induced by CRISPR/Cas9 in the T₂_4_10_1 plant. The dashed lines represent nucleotide deletions. The reported number represents the frequency and the number of reads carrying mutated (edited) target sequence.

Figure 6

Phenotypical and biochemical changes associated with postcut browning. (A) Cut fruits of Wild Type, T₁_4_8, T₁_4_9 and T₁_4_10 showing post-cut browning 30 min after cutting. (B) Polyphenoloxidase (PPO) activity in fruits of Wild Type, T₁_4_8, T₁_4_9 and T₁_4_10. Data are means of eight biological replicates ± SD. Asterisks indicate a significant difference based on Tukey's HSD test (P ≤ 0.05).