Polygalacturonase-inhibiting proteins (PGIPs), extracellular proteins that specifically inhibit fungal endopolygalacturonases (PGs), play a critical role in plant protection by favouring the accumulation of oligogalacturonides (OGs), which are elicitors of plant defence responses. The genes encoding PGIP2 of P. vulgaris and the variant PGIP2.Q224K were subjected to error prone PCR (epPCR) to generate mutated inhibitors with novel and improved recognition capabilities. Using a Pichia pastoris expression library and a high-throughput screening method, two mutated PvPGIP2.Q224K derived inhibitors active against the PG produced by the phytopathogenic fungus F. phyllophilum (FpPG) were isolated. Both variants were better inhibitors than PGIP2.Q224K and were characterized by the replacement of the lysine in position 224, supporting the view that the absence of this positively charged amino acid at position 224 is a primary requirement for gaining the inhibition capability against FpPG.
ARTIFICIAL EVOLUTION CORRECTS A REPULSIVE AMINO ACID IN POLYGALACTURONASE INHIBITING PROTEINS (PGIPs)
CAPRARI, Claudio
2011-01-01
Abstract
Polygalacturonase-inhibiting proteins (PGIPs), extracellular proteins that specifically inhibit fungal endopolygalacturonases (PGs), play a critical role in plant protection by favouring the accumulation of oligogalacturonides (OGs), which are elicitors of plant defence responses. The genes encoding PGIP2 of P. vulgaris and the variant PGIP2.Q224K were subjected to error prone PCR (epPCR) to generate mutated inhibitors with novel and improved recognition capabilities. Using a Pichia pastoris expression library and a high-throughput screening method, two mutated PvPGIP2.Q224K derived inhibitors active against the PG produced by the phytopathogenic fungus F. phyllophilum (FpPG) were isolated. Both variants were better inhibitors than PGIP2.Q224K and were characterized by the replacement of the lysine in position 224, supporting the view that the absence of this positively charged amino acid at position 224 is a primary requirement for gaining the inhibition capability against FpPG.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
