Physcomitrella

To identify the presence of shared single copy genes in non-seed plants, we used the PlantTribes database ([63 (link)]; http://fgp.huck.psu.edu/tribe.html) to identify shared single copy genes in Selaginella, Physcomitrella, Arabidopsis, Populus, Vitis and Oryza. In addition, the presence of genes in the Physcomitrella, Selaginella and Chlamydomonas genomes that are shared single copy genes in Arabidopsis, Populus, Vitis, and Oryza was also identified. The PlantTribes database identifies clusters of genes from sequenced genomes through TRIBEMCL clustering of BLASTP searches against various combinations of plant genomes [63 (link),93 (link),94 (link)]. The presence of shared single copy genes in other non-seed plant lineages were detected by using top hits to TBLASTX of all shared single copy Arabidopsis, Populus, Vitis and Oryza protein sequences against the Plant Transcript Assemblies ([73 (link)]; http://plantta.tigr.org/).

The pAct‐Cas9 plasmid used in this study contains a Cas9 expression cassette containing the rice actin 1 promoter and a codon‐optimized version of Cas9 from Streptococcus pyogenes fused to a SV40 nuclear localization (Mali et al., 2013). The pAct‐Cas9 plasmid was constructed as follows: the hCas9 plasmid (plasmid#41815 from AddGene) was digested by NcoI and PmeI and the hCas9 gene was ligated to the pCOR104‐CaMVter plasmid (Proust et al., 2011) previously digested by NcoI and SmaI.
Two sgRNA expression cassettes were designed, each containing a U6 promoter from P. patens, the 5′‐G‐N₍₁₉₎‐3′ sequences targeting PpAPT and the tracrRNA scaffold (Mali et al., 2013; Figures 1 and S1). P. patens genomic sequence for the U6 gene (coordinates 5050300–5050958 on chromosome 1) was identified by Basic Local Alignment Search Tool (http://www.phytozome.net/physcomitrella_er.php) using the Arabidopsis U6‐26 snRNA sequence (X52528; Li et al., 2007) as query. U6 promoter sequence coordinates used for gRNA expression are 5050300–5050621 on chromosome 1. For the design of CRISPR‐Cas targets in the PpAPT gene, both strands of the P. patens adenine phosphoribosyltransferase gene (PpAPT, Phytozome # Pp3c8_16590) were searched using the CRISPOR, free software (http://tefor.net/crispor/crispor.cgi), for sequences of the form 5′‐G‐N(18 or 19)NGG‐3′ with respect to the U6 promoter and Cas9 specificity conditions. Two target loci were selected, one in exon 5 (sgRNA#1) and one in exon 3 (sgRNA#2) of the PpAPT gene (Figure 1). The sgRNA1 and sgRNA2 cassettes were synthesized as gBlocks^® by IDT (www.idtdna.com), PCR‐amplified and introduced into pCR^®II‐TOPO^® TA‐cloning vectors (www.lifetechnologies.com) to give the plasmids psgRNA#1 and psgRNA#2.
Two donor DNA cassettes were used for gene targeting experiments. The PpAPT‐KO4 knockout cassette used for gene targeting experiments bears a 715‐bp 5′ targeting fragment (coordinate 772–1486 on Pp3c8_16590 in Phytozome) and a 702‐bp 3′ targeting fragment (coordinate 1487–2188 on Pp3c8_16590 in Phytozome) of the PpAPT gene, flanking a pAct :: hygroR cassette from the pActHygR plasmid. The pActHygR carries a HPH gene for resistance to hygromycin (Bilang et al., 1991) in fusion with the rice actin 1 promoter from pCOR104 (McElroy et al., 1991) and before a NOS terminator. The 5′ and 3′ sequences of the PpAPT gene present in the PpAPT‐KO4 cassette are flanking the predicted CRISPR‐mediated DSB for target#1 sequence (coordinate 1468–1487 on Pp3c8_16590 in Phytozome). The PpAPT‐KO7 knockout cassette bears a 743‐bp 5′ targeting fragment (coordinate 156–898 on Pp3c8_16590 in Phytozome) and a 778‐bp 3′ targeting fragment (coordinate 917–1694 on Pp3c8_16590 in Phytozome) of the PpAPT gene, flanking a 35S :: neoR cassette from pBNRF for resistance to G418 (Schaefer et al., 2010) cloned in a pCR^®II‐TOPO^® TA‐cloning vector. The 5′ and 3′ sequences of the PpAPT gene present in the PpAPT‐KO7 cassette are flanking the predicted CRISPR‐mediated DSB for the target#2 sequence (coordinate 890–909 on Pp3c8_16590 in Phytozome).

To investigate the evolutionary relationships between LHT genes in various plant species, the LHT protein sequences of Arabidopsis thaliana, Oryza sativa, Physcomitrella patens, Selaginella moellendorffii, Medicago truncatula, Solanum lycopersicum and Sorghum bicolor were used to construct an unrooted phylogenetic tree. Multiple-sequence alignment was performed using Clustal X (1.83) software, and the tree was constructed according to the maximum-likelihood (ML) method with the p-distance substitution model in MEGA X software. We used 1000 replicates in a bootstrap analysis to determine a support value for each branch.

In this study, we performed name searches with keywords (e.g., Lysine/Histidine transporter) to obtain annotated candidates of AtLHT and OsLHT family members in the Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/) (accessed on 25 June 2022) and RAP-DB (http://rapdb.dna.affrc.go.jp/) (accessed on 20 June 2022), respectively. As 10 AtLHT (AtLHT1–10) and 6 OsLHT (OsLHT1-6) transporters have been previously identified, we used the gene nomenclature for Arabidopsis LHTs proposed by Rentsch et al. [4 (link)] and that for rice LHTs proposed by Zhao et al. [5 (link)]. LHT sequences were selected from Physcomitrella patens, Selaginella moellendorffii, Medicago truncatula, Solanum lycopersicum and Sorghum bicolor protein sequences via BLAST searches for Arabidopsis LHTs on the Phytozome v13.1 website (http://www.phytozome.net/) (accessed on 30 June 2022). Information on rice LHT genes, including open reading frame (ORF) lengths, locations and numbers of amino acids were obtained from RAP-DB. The physicochemical parameters of each OsLHT protein were found using ExPASy (http://web.expasy.org/protparam/) (accessed on 20 August 2022). The subcellular localization of the OsLHT proteins was predicted using WoLF PSORT (http://wolfpsort.org) (accessed on 9 September 2022) with the amino acid sequences.

Azide-modified amino acids and AF488-Alkyne were purchased from Jena Bioscience (Jena, Germany). Atto-514-Alkyne was purchased from ATTO-TEC (Siegen, Germany) and the Click-iT cell reaction buffer kit from Invitrogen (Darmstadt, Germany).
To visualize PGN in Physcomitrella patens, protonema cells were incubated in BCD medium with ADA (0.25 mM) for 22 h. The cells were washed once with BCD medium and incubated in BCD medium mixed with a Click-iT cell reaction cocktail (1:1) containing Atto-514 (1–5 µM) for 2 h. Cells were washed once with the medium prior to imaging with a confocal microscope.
For click chemistry reactions of A. thaliana and N. benthamiana, adult plants leaves were infiltrated with 1/2 MS medium containing 0.25 mM azide-modified D-amino acids or 0.125 mM azide modified L-amino acids and incubated for 24–48 h. Infiltrated leaves were either cut off (A. thaliana) or partially cut out (N. benthamiana) and incubated in ½ MS medium mixed with Click-iT cell reaction cocktail (1:1) containing Atto-514 or AF488 (1–5 µM) for 1.5–2 h. The leaves were washed once with the medium prior to imaging. In the experiments with A. thaliana seedlings, all incubation and washing steps were performed as a whole.