Fig. 1: The identification and successful knockout of SPX gene.

a Graphical motif representation showing conserved amino acid residues of the SPX domain. b Alignment of SPX conserved sites with land plants. Identical residues are yellow-shaded while chemically similar ones are green-shaded. Included in the alignment are the six amino acid sequences from P. tricornutum, one from Oryza sativa Japonica Group (OsSPX2, XP_015614909.1), and one from Arabidopsis thaliana (AtSPX, NP_567674.1). c Schematic diagram showing the N-terminal SPX domain (yellow) and the effective sgRNA-targeting site (sgRNA3, arrow). The hatched area with number 210 indicates the location of the conserved domain shown in a, b. The red horizontal arrows indicate primers designed for RT-qPCR assay of SPX expression (results shown in g). F1 and R1 are located near the mutation target site whereas F2 and R2 are located far downstream from the mutation target site. d Target region of SPX gene amplified with specific primers, with the red arrowhead showing a deletion mutant band. e Sequence alignment of representative mutant clones with the wild type (WT). The sgRNA sequences, PAM sequences, and inserted sequences are labeled in green, red, and yellow, respectively. Clones of sequence 1-4 (mSPX1-4) and sequence 15-3 (mSPX15-3) were selected for downstream analyses. f Summary of on-target rate (targeting efficiency) in SPX knockout. NHEJ non-homologous end joining. g The expression of P stress-induced SPX-related genes in P. tricornutum mSPX1-4 and mSPX15-3 under P+ condition. Two SPX mutants (mSPX1-4 and mSPX15-3) were selected for comparisons with WT on the third day of inoculation into P+ growth medium (n = 3). Error bars, SD. Statistically significant changes in gene expression (*) in mSPX relative to WT is based on one-way ANOVA test (p < 0.05).