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).