Evolutionary Analysis of Plant Receptor-Like Cytoplasmic Kinases

For protein alignment of AthRLCK XI and representative RLKs in Figure 1, we aligned members of RLCK XI from A. thaliana together with well-studied RLK AthCLV1 from LRR-RLK XI-1 subfamily, AthBIK1 from RLCK VIIa-2 subfamily, and the distant RLK homolog HsaIRAK1 from humans. For Supplementary Figure 1, we used the RLK dataset from Dievart et al. (2020) (link), which included grouped subfamilies of RLKs from the iTAK database of protein kinases (http://itak.feilab.net/cgi-bin/itak/index.cgi). To identify suitable RLK representatives, each RLK subfamily was aligned with AthRLCK XI-2. The sequence with the highest pairwise comparison score with AthRLCK XI-2 was chosen to represent their subfamily. AthRLCK XI-2 was selected because it has the highest average pairwise similarity score among all AthRLCK XI members. Each RLK representative selected for alignment contained all eleven conserved protein kinase subdomains. RLK subfamilies whose members lacked one or more conserved protein kinase subdomains were excluded from the alignment. To determine the evolutionary conservation of the RLCK XI subfamily in plants, full RLCK XI protein sequences were aligned using the L-INS-i strategy from MAFFT version 7 (Katoh and Standley, 2013 (link)). The resulting alignment was used to construct a phylogenetic tree using the maximum likelihood method from IQ-TREE (Nguyen et al., 2015 (link)). The Phylogenetic tree was illustrated using iTOL (https://itol.embl.de) (Letunic and Bork, 2021 (link)).