Abstract
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 beetle Phaedon cochleariae (Coleoptera: Chrysomelidae) and identified BrPGIP3 from Chinese cabbage (Brassica rapa ssp. pekinensis) as a candidate. PGIPs are predominantly studied in planta because their heterologous expression in microbial systems is problematic and instability and aggregation of recombinant PGIPs has complicated in vitro inhibition 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 several P. cochleariae PGs in 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.
Highlights
Plant cells are encased by cell walls of crosslinked polysaccharides that provide protection and structural integrity and contribute to cell–cell adhesion and signal transduction [1, 2]
BrPGIP3 is unstable as a soluble protein (Fig. 1A)
The detection of bands of higher molecular weight for BrPGIP3_GPI but not BrPGIP3 indicated that aggregation was reduced to the point that protein precipitates were small enough to run in the gel matrix of the SDS-PAGE (Fig. 2)
Summary
Plant cells are encased by cell walls of crosslinked polysaccharides that provide protection and structural integrity and contribute to cell–cell adhesion and signal transduction [1, 2]. Plants defend themselves by secreting PG-inhibiting proteins (PGIPs) into their cell wall to counteract microbial PGs [14,15,16]. Thereby, we aim to elucidate if the PGIP plant defense system targets microbial PGs and influences the pectin digestion of an herbivorous beetle. In 2001, De Lorenzo et al [14] even went so far as to rate yeast or other fungal expression systems as “unsuitable for PGIP expression,” as no protein could be detected despite high levels of transcripts. It took 10 years until they found a way to express enough protein to solve the X-ray structure of PvPGIP2 from the bean Phaseolus vulgaris in a complex with the fungal FpPG from Fusarium phyllophilum [52]. Which properties enable the expression and increased stability compared with other PGIPs in vitro is unknown
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