Amino acid alignments of PAM16 homologues and mutations identified in Mppam16 mutants.

(A) Trimmed amino acid alignment of PAM16 homologues. Protein sequences similar to AtPAM16 (At3G59280) from 11 species were identified using the BlastP algorithm to search their proteomes [37]. The sequences were aligned via the L-INS-i strategy in MAFFT [76] and manually trimmed. The predicted signal peptides are marked in red and magnified in (B). (C) Mppam16 mutants were generated via CRISPR-Cas9 mutagenesis to target the MpPAM16 gene. Wild type spores (Tak-1 x Tak-2) were transformed with Agrobacterium tumefaciens strains carrying the vector containing the Cas9 gene and an sgRNA targeting MpPAM16. Positive transformants were genotyped using Sanger sequencing to determine the mutations induced by the CRISPR-Cas9 complex. Predicted protein sequences were determined based on the mutations in each line. Nucleotide or protein sequences were aligned via the L-INS-i strategy using MAFFT version 7. The top row is the reference MpPAM16 nucleotide sequence or the reference MpPAM16 protein sequence (MpTak v6.1).

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