CRISPR/Cas9-Mediated SlNPR1 mutagenesis reduces tomato plant drought tolerance

Abstract

Background: NPR1, nonexpressor of pathogenesis-related gene 1, is a master regulator involved in plant defense response to pathogens, and its regulatory mechanism in the defense pathway has been relatively clear. However, information about the function of NPR1 in plant response to abiotic stress is still limited. Tomato is the fourth most economically crop worldwide and also one of the best-characterized model plants employed in genetic studies. Because of the lack of a stable tomato NPR1 (SlNPR1) mutant, little is known about the function of SlNPR1 in tomato response to biotic and abiotic stresses.

Results: Here we isolated SlNPR1 from tomato 'Ailsa Craig' and generated slnpr1 mutants using the CRISPR/Cas9 system. Analysis of the cis-acting elements indicated that SlNPR1 might be involved in tomato plant response to drought stress. Expression pattern analysis showed that SlNPR1 was expressed in all plant tissues, and it was strongly induced by drought stress. Thus, we investigated the function of SlNPR1 in tomato-plant drought tolerance. Results showed that slnpr1 mutants exhibited reduced drought tolerance with increased stomatal aperture, higher electrolytic leakage, malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) levels, and lower activity levels of antioxidant enzymes, compared to wild type (WT) plants. The reduced drought tolerance of slnpr1 mutants was further reflected by the down-regulated expression of drought related key genes, including SlGST, SlDHN, and SlDREB.

Conclusions: Collectively, the data suggest that SlNPR1 is involved in regulating tomato plant drought response. These results aid in further understanding the molecular basis underlying SlNPR1 mediation of tomato drought sensitivity.

Keywords: CRISPR/Cas9; Drought; ROS; SlNPR1; Stomatal closure; Tomato plant.

Fig. 1

Phylogenetic, gene structure, and domain analyses of SlNPR1. (a) Phylogenetic tree of 35 plant NPR1 homologous proteins identified from nine plant species (MEGA 5.0; Neighbour-Joining (NJ) method; bootstrap of 1000). (b) Exon/intron structure and (c) domain organization of NPR proteins identified from tomato and Arabidopsis thaliana. The domains and motifs are drawn to scale. Among them, the unmarked pink areas don’t code any known domain.

Fig. 2

CRISPR/Cas9-mediated genome editing. (a) Schematic illustration of the two target sites in SlNPR1 genomic sequence. Target 1 and target 2 sequences are shown in capital letters and the protospacer adjacent motif (PAM) sequence is marked in red. (b) Schematic diagram of pYLCRISPR/Cas9-SlNPR1 vector. HPT, hygromycin B phosphotransferase; Ubi, maize ubiquitin promoter; NLS, nuclear localization sequence; Tnos, gene terminator; AtU3d, Arabidopsis thaliana U3d promoter; AtU3b, A. thaliana U3b promoter. (c) CRISPR/Cas9-mediated efficient edit and variant genotypes of two target sequences in T0 plants.

Fig. 3

Expression patterns and phenotype under drought stress. (a) Expression patterns of SlNPR1 in WT plants within 3 days after PEG treatment. (b) Relative expression of SlNPR1 in different tissues of WT plants. The error bars indicate the standard deviations of three biological replicates. Asterisks indicate significant differences as determined by Student’s t-test (*, P < 0.05; **, P < 0.01). (c) Phenotype of slnpr1 mutants and WT plants under drought stress. Photographs were taken 6 days after stopping watering.

Fig. 4

Stomatal aperture of slnpr1 mutants and wild type (WT) plants under drought stress. Stomatal condition in leaves of (a) WT plants and (b) slnpr1 mutants after 3 days’ drought stress. (c) Stomatal length, (d) stomatal width, and (e) stomatal aperture after 3-day drought stress. The error bars indicate the standard deviations of three biological replicates. Asterisks indicate significant differences as determined by Student’s t-test (*, P < 0.05; **, P < 0.01).

Fig. 5

Effects of CRISPR/Cas9-mediated mutations on (a) electrolytic leakage, (b) hydrogen peroxide (H₂O₂), and (c) malondialdehyde (MDA) content after drought stress. The error bars indicate the standard deviations of three biological replicates. Asterisks indicate significant differences as determined by Student’s t-test (*, P < 0.05; **, P < 0.01).

Fig. 6

Effects of CRISPR/Cas9-mediated mutations on activities of (a) superoxide dismutase (SOD), (b) ascorbate peroxidase (APX), (c) peroxidase (POD), and (d) catalase (CAT) after drought stress. The error bars indicate the standard deviations of three biological replicates. Asterisks indicate significant differences as determined by Student’s t-test (*, P < 0.05; **, P < 0.01).

Fig. 7

Effects of CRISPR/Cas9-mediated mutants on the relative expression of (a) SlGST (GenBank ID: XM_004246333), (b) SlDHN (GenBank ID: NM_001329436), and (c) SlDREB (GenBank ID: XM_004241698) after drought stress. The β-Actin (GenBank ID: NM_001308447) was used as the reference gene. The error bars indicate the standard deviations of three biological replicates. Asterisks indicate significant differences as determined by Student’s t-test (*, P < 0.05; **, P < 0.01).