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As we usually find out with clones, they just do not work as good. We also investigated the transmembrane topology of all identified LjPINs using the TMHMM tool [32]. We found conserved transmembrane helices in both C- and N-termini of the seven LjPINs and a hydrophilic loop between them (Figure S2), which is consistent with the typical structure of PIN-formed family proteins [31].

There was an assumption that one of the PIN proteins—LjPIN6—is incorrectly assigned in the Kazusa database [27] with two different accession numbers: Lj0g3v0178829 (LjPIN6a, probable N-terminus) and Lj1g3v0264160.1 (LjPIN6b, possible C-terminus) [27]. Our quantitative polymerase chain reaction (qPCR) analysis revealed that their expression level in L. japonicus root nodules appeared to be identical (Figure S3). Thus, to examine whether LjPIN6b is situated on the same chromosome, downstream to LjPIN6a, and whether both sequences could constitute one gene, we designed a pair of primers, from which the forward one was anchored to the end fragment of LjPIN6a, while the reverse one to the beginning of the LjPIN6b coding sequence. The performed RT-PCR analysis proved that LjPIN6a and LjPIN6b, in fact, constituted one sequence since they were jointly transcribed (Figure S4). Nevertheless, the complete amplicon was longer than expected, thus the PCR product was sequenced, and the full-length LjPIN6 sequence is provided (Figure S5A). The transmembrane domain prediction using the complete, assembled sequence of LjPIN6 was repeated. This allowed us to identify transmembrane domains, analogous to those from A. truncatula PIN6 orthologs, at both N- and C-termini.

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