Direct evidence for a new mode of plant defense against insects via a novel polygalacturonase-inhibiting protein expression strategy

Plant cell wall-associated polygalacturonase-inhibiting proteins (PGIPs) are widely distributed in the plant kingdom. They play a crucial role in plant defense against phytopathogens by inhibiting microbial polygalacturonases (PGs). PGs hydrolyze the cell wall polysaccharide pectin and are among the first enzymes to be secreted during plant infection. Recent studies demonstrated that herbivorous insects express their own PG multi-gene families, raising the question whether PGIPs also inhibit insect PGs and protect plants from herbivores. Preliminary evidence suggested that PGIPs may negatively influence larval growth of the leaf beetlePhaedon cochleariae(Coleoptera: Chrysomelidae) and identified BrPGIP3 from Chinese cabbage (Brassica rapassp.pekinensis) as a candidate. PGIPs are predominantly studiedin plantabecause their heterologous expression in microbial systems is problematic and instability and aggregation of recombinant PGIPs has complicatedin vitroinhibition assays. To minimize aggregate formation, we heterologously expressed BrPGIP3 fused to a glycosylphosphatidylinositol (GPI) membrane anchor, immobilizing it on the extracellular surface of insect cells. We demonstrated that BrPGIP3_GPI inhibited severalP. cochleariaePGsin vitro, providing the first direct evidence of an interaction between a plant PGIP and an animal PG. Thus, plant PGIPs not only confer resistance against phytopathogens, but may also aid in defense against herbivorous beetles.