Truncated forms of PvPGIP1 and 2 consisting of only LRR5 to LRR8 retains similar level of interaction with AnPG2 and BcPG1 compared to the full length PvPGIP2.
The modular structure of leucine-rich repeat proteins (Figure 1) leads to the hypothesis that a fraction of PGIP may be sufficient to efficiently inhibit PG activity. Binding of PGs to truncated versions of PvPGIP2 were tested using the Y2H system. PvPGIPs lacking the N-terminus (Figure 3a) or C-terminus (Figure 3b) exhibited similar activity levels to full-length PvPGIP2, displaying comparable growth rates and final OD. This indicates that the N- and C-terminus likely do not harbor any residues integral to PG recognition. To narrow down which LRRs to truncate, we noted that a previous docking simulation of BcPG1 and PvPGIP2 found the optimal docking area of PvPGIP2 was between Val-152 and His-291, and played a vital role in the interaction with PGs. (Sicilia et al., 2005). Thus, PvPGIP2 was truncated to retain only LRR5 to LRR8 (tPvPGIP2_5-8, Figure 1c), which flanks the optimal docking area. tPvPGIP2_5-8 exhibited a comparable growth curve to full length PvPGIP2 in the Y2H assay, with only slight decreases in AnPG2 and BcPG1 interactions (Figure 3c). Further truncation of LRR5 from tPvPGIP2_5-8 to make tPvPGIP2_6-8 resulted in an almost complete loss of inhibitory activity to all the PGs tested (Figure 3d). Likewise, further truncation of tPvPGIP2_6-8 to tPvPGIP2_6-7 (Figure 3e) also exhibited no inhibitory activity to the tested PGs. Truncation of LRR8 from tPvPGIP2_5-8 to make tPvPGIP2_5-7 lead to reduced interactions with AnPG2 and BcPG1 compared to tPvPGIP2_5-8 (Figure 3f). Other truncations that contained only LRR4 to LRR5 (Figure 3g) and LRR4 to LRR6 (Figure 3h) resulted in a complete loss of interaction with PGs as well. These results imply that tPvPGIP2_5-8 is likely the smallest truncation that retains similar level of inhibitory activity towards AnPG2 and BcPGs, compared with full length PvPGIP2. Taken together, our results indicate that the regions outside of LRR5 to LRR8 in PvPGIP2 are not essential for the interaction with PGs, and the specific function of these parts of PGIPs remain to be clearly elucidated.
We then measured the interactions of both tPvPGIP1_5-8 and tPvPGIP2_5-8 with different PGs. The interactions of both the truncated PGIPs with different PGs well mirrored that of the full length PGIPs (Figure S2). The growth curves for yeast harboring AnPG2 and BcPG1 had the highest stationary phase among all the yeast strains and were comparable to the positive control. This again shows that the properly truncated PGIPs (here, tPvPGIP2_5-8 and tPvPGIP1_5-8) are able to interact with PGs in a similar capacity to their full-length counterparts (Figure S2).