Both full length and truncated PvPGIP2 can efficiently inhibit the growth of A. niger when applied exogenously.
To validate the inhibitory activity of PGIPs to PG, and the potential of using PGIPs exogenously to inhibit fungal growth, we constructed yeast strains that can secret PGIPs and monitored fungal growth on pectin in the presence of the PGIP-secreting yeast strains. The Ost1 signal sequence, a well-documented secretion tag (Fitzgerald & Glick, 2014), was fused to PvPGIP1 and PvPGIP2. Since the corresponding clone of PvPGIP2 (the OST1-PvPGIP2-HA construct) was not successful, we used PvPGIP1 construct instead to validate the utilization ofOST1 signal sequence. The presence of PvPGIP1 in both the cell lysate and medium was verified through western blot (Figure 4a). Interestingly, a faint band also detected in the medium of yeast harboring PvPGIP1 without the Ost1 signal sequence, which implies that PvPGIP1 can be natively secreted out of the cell. This is consistent with its defense function reported previously (Kalunke et al., 2015).
With the secretion of PvPGIP1 aided by the Ost1 signal sequence confirmed, PvPGIP1 and PvPGIP2-secreting yeast strains without the HA tag were constructed and evaluated in an in vitro assay to evaluate whether PGIPs can inhibit the growth of fungi in the presence of pectin. A. niger or B. cinerea was inoculated on agar plate amended with 0.5% citrus pectin (weight/volume) in the presence of PGIP-secreting yeast (roughly 1.2 x 106 cells), the antifungal agent natamycin at a concentration of 100 mg/L (positive control), or wild-type yeast strains (negative control). The growth ofA. niger and B. cinerea were estimated with OD readings at 630nm (Langvad, 1999). Both the full length PvPGIP2 and tPvPGIP2_5-8 efficiently delayed the growth of both fungi (Figure 4b and 4c). Meanwhile, when PvPGIP2 or tPvPGIP2 were expressed from high copy plasmids, lower OD readings at 630nm were detected than when expressed from low copy plasmids, which shows that the inhibitory activity of PGIP is likely dose dependent. To further confirm the activities of PGIPs, the PGIP- secreting yeast was spread onto the entire plate and 2µL ofA. niger (5 x 105 conidia/mL) was spotted into four locations. The growth of fungi was also monitored over seven days and both full length PvPGIP2 and tPvPGIP2_5-8 delayed sporulation ofA. niger by at least one day (Figure S3). Similarly, PvPGIP2 or tPvPGIP2_5-8 expressed from high copy plasmids was more effective in inhibiting the growth of A. niger , compared to the ones expressed from low copy plasmids (Figure S3). These results suggest that PvPGIP2 and tPvPGIP2, when applied exogenously, can inhibit A. niger andB. cinerea utilization of pectin and thus impede fungal growth when pectin is present as one carbon source, though with a lower efficiency than the antifungal agent natamycin. The consistency between the Y2H assay and fungal-pectin assay also demonstrates that Y2H can be utilized as a functional monitoring or screening method for PGIP-PG interactions.