IN VITRO EVOLUTION HIGHLIGHTS THE RESIDUE 224 OF PGIPS (POLYGALACTURONASE-INHIBITING PROTEINS) AS A PRIMARY SPOT OF VARIABILITY FOR IMPROVEMENT OF INHIBITION CAPABILITY

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular proteins that specifically inhibit fungal endopolygalacturonases produced by fungi during the infection process. PGIPs play an important role in plant protection by favouring the accumulation of oligogalacturonides (OGs), which are elicitors of plant defence responses. We have subjected the gene encoding PGIP2 of P. vulgaris to error prone PCR (epPCR) with the aim of obtaining mutated inhibitors with novel and improved recognition capabilities. By using an expression library in Pichia pastoris and a high-throughput screening method, two mutated PvPGIP2 active against PG produced by the phytopathogenic fungus F. phyllophilum (FpPG) were isolated. Both mutants were affected by a mutation in the residue 224 indicating that this is a primary spot of variability of the protein. Both mutants were better inhibitors of FpPG