Theor Appl Genet (2009) 118:1371–1379 DOI 10.1007/s00122-009-0987-4 123 ORIGINAL PAPER The bean polygalacturonase-inhibiting protein 2 (PvPGIP2) is highly conserved in common bean (Phaseolus vulgaris L.) germplasm and related species Anna Farina · Valentina Rocchi · Michela Janni · Stefano Benedettelli · Giulia De Lorenzo · Renato D’Ovidio Received: 28 October 2008 / Accepted: 31 January 2009 / Published online: 24 February 2009 © Springer-Verlag 2009 Abstract Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant protein inhibitors of endo-polygalac- turonases (PGs) that belong to the leucine-rich repeat (LRR) protein family. In bean, PGIP is encoded by a small gene family of four members among which Pvpgip2 encodes the most wide-spectrum and eYcient inhibitor of fungal PGs. In order to evaluate the sequence polymorphism of Pvpgip2 and its functional signiWcance, we have analyzed a number of wild and cultivated bean (P. vulgaris) accessions of Andean and Mesoamerican origin, and some genotypes from the related species P. coccineus, P. acutifolius, and P. lunatus. Our analyses indicate that the protein encoded by Pvpgip2 is highly conserved in the bean germplasm. The few detected polymorphic sites correspond to synonymous substitutions and only two wild genotypes contain a Pvpgip2 with a single non-synonymous replacement. Sequence com- parison showed a slightly larger variation in the related bean species P. coccineus, P. acutifolius, and P. lunatus and con- Wrmed the known phylogenetic relationships with P. vulga- ris. The majority of the replacements were within the xxLxLxx region of the leucine rich repeat (LRR) domain and none of them aVected residues contributing to structural features. The variant PGIPs were expressed in Nicotiana benthamiana using PVX as vector and their inhibitory activ- ity compared to that of PvPPGIP2. All the variants were able to fully inhibit the four fungal PGs tested with minor diVerences. Taken together these results support the hypoth- esis that the overall sequence conservation of PGIP2 and minor variation at speciWc sites is necessary for high-aYnity recognition of diVerent fungal PGs. Introduction Pathogens produce a number of molecules to overcome the host barriers. Among these, cell wall degrading enzymes (CWDE) play an important role in several host–pathogen interactions. To counteract these arrays of enzymes and hamper the invasion process, plants produce protein inhibi- tors among which the polygalacturonase-inhibiting proteins (PGIPs) have been shown to play an important role in limit- ing host tissue colonization by fungal pathogens (Powell et al. 2000; Ferrari et al. 2003; Manfredini et al. 2006; Aguero et al. 2005; Joubert et al. 2006; Janni et al. 2008). Polygalacturonase-inhibiting proteins are extracellular plant inhibitors of fungal and insect endo-polygalacturon- ases (PGs) that belong to the superfamily of leucine-rich repeat proteins (LRRs) of the extracytoplasmic type (Jones and Jones 1997). They contain 9–10 imperfect LRRs of 24 amino acid each that are organized to form two  sheets, one of which—sheet B1—occupies the concave inner side of the molecule and contains residues crucial for PG Communicated by D. Lightfoot. Electronic supplementary material The online version of this article (doi:10.1007/s00122-009-0987-4) contains supplementary material, which is available to authorized users. A. Farina · V. Rocchi · M. Janni · R. D’Ovidio (&) Dipartimento di Agrobiologia e Agrochimica, Università della Tuscia, 01100 Viterbo, Italy e-mail: dovidio@unitus.it S. Benedettelli Dipartimento Scienze Agronomiche e Gestione del Territorio, Università di Firenze, 50144 Florence, Italy G. De Lorenzo Dipartimento di Biologia Vegetale, Università di Roma ‘La Sapienza’, 00185 Rome, Italy