1.8.62
SPECIFIC RECOGNITION OF FUNGAL POLYGALACTURONASES IS DETERMINED BY AMINO ACID VARIATIONS IN THE SOLVENT EXPOSED SURFACE OF THE POLYGALACTURONASE-INHIBITING PROTEIN (PGIP)

F LECKIE, C CAPODICASA, B MATTEI, A DEVOTO, L NUSS, G SALVI, G DE LORENZO and F CERVONE

Dipartimento di Biologia Vegetale, Universita di Roma 'La Sapienza', Piazzale Aldo Moro 5, 00185 Roma, Italy

Background and objectives
A major goal in plant pathology is to understand the molecular basis of pathogen perception. Polygalacturonase (PG) which degrades the homogalacturonan of the plant cell wall and the PG-inhibiting protein (PGIP) offer a simple and elegant system to investigate molecular recognition at the level of the plant cell wall which could play a part in plant defence. PGIP is a leucine-rich repeat (LRR) protein structurally related to several resistance gene products (R) cloned in plants. Unlike PGIP, no information is as yet available on the molecular basis of the interaction between products of the R genes and microbial molecules. PGIPs can specifically interact with a range of different PGs and are differentially induced in resistant or susceptible interactions and in response to treatment with elicitors [1].

Materials and methods
A bean CDNA library was screened to yield two different pgip clones. A heterologous expression system based on the potato virus X (PVX) successfully expressed PGIPs in Nicotiana benthamiana. Site-directed mutagenesis allowed the generation of a range of mutant proteins which were expressed and purified from N. benthamiana. Surface plasmon resonance measurements were used to evaluate the kinetic parameters of the PG-PGIP interactions.

Results and conclusions
Two CDNA clones were isolated from a bean CDNA library and the gene products were separately expressed and purified. Inhibitory assays with polygalacturonases purified from Aspergillus niger and Fusarium moniliforme have shown that PGIP-2 is able to interact with and inhibit the PG from both fungi, but PGIP-1 is not able to interact with or inhibit the PG of F. moniliforme [2]. The difference between the two proteins resides in only eight isolated amino acids located in the part of the protein that is predicted to form the solvent exposed surface (-sheet/-turn) and therefore is likely to be involved in the interaction with the ligand.

A series of pgip genes mutant for each of the eight amino acids was generated. A loss-of-function approach was adopted and the amino acids in the pgip-2 gene were mutated to the corresponding amino acid in the pgip-1 gene to create a series of eight mutant proteins. All the mutant proteins were able to bind and inhibit to some degree the PG of F. moniliforme showing that more than one amino acid is responsible for the interaction. The purified mutant proteins were individually immobilized on a sensor chip and subjected to surface plasmon resonance analysis using the PG of A. niger or F. moniliforme as analytes passed over the surface. Chimaeric PGIP proteins where the PGIP-1 N-terminal portion was substituted into PGIP-2 and vice versa have also been analysed. The results obtained show that three amino acids are critical for the specific interaction of PGIP-2 with the PG of F. moniliforme

References
1. Devoto A., Clark AJ, Nuss L et al., 1997. Planta 202, 284-292.
2. Desiderio A, Aracri B, Leckie F et al., 1997. Molecular Plant-Microbe Interactions 10, 852-860.