Two members of the pgip gene family (pgip-1 and pgip-2) of Phaseolus vulgaris L. were expressed separately in Nicotiana benthamiana and the ligand specificity of their products was analysed by surface plasmon resonance (SPR). Polygalacturonase-inhibiting protein-1 (PGIP-1) was unable to interact with PG from Fusarium moniliforme and interacted with PG from Aspergillus niger; PGIP-2 interacted with both PGs. Only eight amino acid variations distinguish the two proteins: five of them are confined within the β-sheet/β-turn structure and two of them are contiguous to this region. By site-directed mutagenesis, each of the variant amino acids of PGIP-2 was replaced with the corresponding amino acid of PGIP-1, in a loss-of-function approach. The mutated PGIP-2s were expressed individually in N. benthamiana, purified and subjected to SPR analysis. Each single mutation caused a decrease in affinity for PG from F. moniliforme; residue Q253 made a major contribution, and its replacement with a lysine led to a dramatic reduction in the binding energy of the complex. Conversely, in a gain-of-function approach, amino acid K253 of PGIP-1 was mutated into the corresponding amino acid of PGIP-2, a glutamine. With this single mutation, PGIP-1 acquired the ability to interact with F. moniliforme PG.

Cited By :160Export Date: 29 September 2015CODEN: EMJODCorrespondence Address: Cervone, F.; Dipartimento di Biologia Vegetale, Universita di Roma ’La Sapienza’, Piazzale Aldo Moro 5, 00185 Roma, Italy; email: cervone@axrma.uniromal.itChemicals/CAS: glutamine, 56-85-9, 6899-04-3; leucine, 61-90-5, 7005-03-0; lysine, 56-87-1, 6899-06-5, 70-54-2; polygalacturonase, 9023-92-1, 9032-75-1References: Anderson, P.A., Lawrence, G.J., Morrish, B.C., Alyffe, M.A., Finnegan, J., Ellis, J.G., Inactivation of the flax rust resistance gene M associated with loss of a repeated unit within the leucine-rich repeat coding region (1997) Plant Cell, 9, pp. 641-651;Baulcombe, D., Chapman, S., Santa Cruz, S., Jellyfish green fluorescent protein as a reporter for virus infections (1995) Plant J., 7, pp. 1045-1053; Bent, A.F., Plant disease resistance genes: Function meets structure (1996) Plant Cell, 8, pp. 1757-1771; Bent, A.F., Kunkel, B.N., Dahlbeck, D., Brown, K.L., Schmidt, R., Giraudat, J., Leung, J., Staskawicz, B.J., KPS2 of Arabidopsis thaliana: A leucine-rich repeat class of plant disease resistance genes (1994) Science, 265, pp. 1856-1860; Botella, M.A., Parker, J.E., Frost, L.N., Bittner-Eddy, P.D., Beynon, J.L., Daniels, M.J., Holub, E.B., Jones, J.D.G., Three genes of the Arabidopsis RPP1 complex resistance locus recognize distinct Peronospora parasitica avirulence determinants (1998) Plant Cell, 10, pp. 1847-1860; Brünger, A.T., Kuriyan, J., Karplus, M., X-PLOR version 3.1: A system for X-ray and NMR (1987) Science, 235, p. 458; Buchanan, S.G.S., Gay, N.J., Structural and functional diversity in the leucine-rich repeat family of proteins (1996) Prog. Biophys. Mol. Biol., 65, pp. 1-44; Caprari, C., Richter, A., Bergmann, C., Lo Cicero, S., Salvi, G., Cervone, F., De Lorenzo, G., Cloning and characterization of the gene encoding the endopolygalacturonase of Fusarium moniliforme (1993) Mycol. Res., 97, pp. 497-505; Caprari, C., Mattei, B., Basile, M.L., Salvi, G., Crescenzi, V., De Lorenzo, G., Cervone, F., Mutagenesis of endopolygalacturonase from Fusarium moniliforme: Histidine residue 234 is critical for enzymatic and macerating activities and not for binding to polygalacturonase-inhibiting protein (PGIP) (1996) Mol. Plant Microbe Interact., 9, pp. 617-624; Cervone, F., De Lorenzo, G., Degrà, L., Salvi, G., Bergami, M., Purification and characterization of a polygalacturonase-inhibiting protein from Phaseolus vulgaris L (1987) Plant Physiol., 85, pp. 631-637; Cervone, F., Hahn, M.G., De Lorenzo, G., Darvill, A., Albersheim, P., Host-pathogen interactions. XXXIII. A plant protein converts a fungal pathogenesis factor into an elicitor of plant defense responses (1989) Plant Physiol., 90, pp. 542-548; Cervone, F., Castoria, R., Leckie, F., De Lorenzo, G., Perception of fungal elicitors and signal transduction (1997) Signal Transduction in Plants, pp. 153-177. , Aducci, P. (ed.), Birkäuser Verlag, Basel, Switzerland; Clark, S.E., Williams, R.W., Meyerowitz, E.M., The CLAVATAI gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis (1997) Cell, 89, pp. 575-585; Dayhoff, M.O., Eck, R.V., Park, C.M., A model of evolutionary change in proteins (1972) Atlas of Protein Sequence and Structure, 5, pp. 89-99. , Dayhoff, M.O. (ed.), National Biomedical Research Foundation, Georgetown University Medical Center, Washington, DC; De Lorenzo, G., Cervone, F., Polygalacturonase-inhibiting proteins (PGIPs): Their role in specificity and defense against pathogenic fungi (1997) Plant-Microbe Interactions, 3, pp. 76-93. , Stacey, G. and Keen, N.T. (eds), Chapman & Hall, New York, NY; Desiderio, A.E.A., Polygalacturonase-inhibiting proteins (PGIPs) with different specificities are expressed in Phaseolus vulgaris (1997) Mol. Plant Microbe Interact., 10, pp. 852-860; Devoto, A., Leckie, F., Lupotto, E., Cervone, F., De Lorenzo, G., The promoter of a gene encoding pgip (PolyGalacturonase-Inhibiting Protein) of Phaseolus vulgaris L. is activated by wounding but not by elicitors or pathogen infection (1998) Planta, 205, pp. 165-174; Dixon, M.S., Hatzixanthis, K., Jones, D.A., Harrison, K., Jones, J.D.G., The tomato Cf-5 disease resistance gene and six homologs show pronounced allelic variation in leucine-rich repeat copy number (1998) Plant Cell, 10, pp. 1915-1925; Favaron, F., D’Ovidio, R., Porceddu, E., Alghisi, P., Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein (1994) Planta, 195, pp. 80-87; Frediani, M., Cremonini, R., Salvi, G., Caprari, C., Desiderio, A., D’Ovidio, R., Cervone, F., De Lorenzo, G., Cytological localization of the pgip genes in the embryo suspensor cells of Phaseolus vulgaris L (1993) Theor. Appl. Genet., 87, pp. 369-373; Grant, M.R., Godiard, L., Straube, E., Ashfield, T., Lewald, J., Sattler, A., Innes, R.W., Dangl, J.L., Structure of the Arabidopsis KPM1 gene enabling dual specificity disease resistance (1995) Science, 269, pp. 843-846; Granzow, R., Reed, R., Interactions in the fourth dimension (1992) Biotechnology, 10, pp. 390-393; Hammond-Kosack, K.E., Jones, J.D.G., Plant disease resistance genes (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol., 48, pp. 575-607; Johnston, D.J., Ramanathan, V., Williamson, B., A protein from immature raspberry fruits which inhibits endopolygalacturonases from Botrytis cinerea and other micro-organisms (1993) J. Exp. Bot., 44, pp. 971-976; Kajava, A.V., Structural diversity of leucine-rich repeat proteins (1998) J. Mol. Biol., 277, pp. 519-527; Kester, H.C.M., Visser, J., Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger (1990) Biotechnol. Appl. Biochem., 12, pp. 150-160; Kobe, B., Deisenhofer, J., Crystal structure of porcine ribonuclease inhibitor, a protein with leucine-rich repeats (1993) Nature, 366, pp. 751-756; Kobe, B., Deisenhofer, J., The leucine-rich repeat: A versatile binding motif (1994) Trends Biochem. Sci., 19, pp. 415-421; Kobe, B., Deisenhofer, J., A structural basis of the interactions between leucine-rich repeats and protein ligands (1995) Nature, 374, pp. 183-186; Kobe, B., Deisenhofer, J., Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A (1996) J. Mol. Biol., 264, pp. 1028-1043; Kresse, H., Liszio, C., Schönherr, E., Fisher, L.W., Critical role of glutamate in a central leucine-rich repeat of decorin for interaction with type I collagen (1997) J. Biol. Chem., 272, pp. 18404-18410; Li, J.M., Chory, J., A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction (1997) Cell, 90, pp. 929-938; McDowell, J.M., Dhandaydham, M., Long, T.A., Aarts, M.G.M., Goff, S., Holub, E.B., Dangl, J.L., Intragenic recombination and diversifying selection contribute to the evolution of downy mildew resistance at the RPP8 locus of Arabidopsis (1998) Plant Cell, 10, pp. 1861-1874; Meyers, B.C., Chin, D.B., Shen, K.A., Sivaramakrishnan, S., Lavelle, D.O., Zhang, Z., Michelmore, R.W., The major resistance gene cluster in lettuce is highly duplicated and spans several megabases (1998) Plant Cell, 10, pp. 1817-1832; Meyers, B.C., Shen, K.A., Rohani, P., Gaut, B.S., Michelmore, R.W., Receptor-like genes in the major resistance locus of lettuce are subject to divergent selection (1998) Plant Cell, 10, pp. 1833-1846; Katagiri, F., Yu, G.-L., Ausubel, F.M., The A.thaliana disease resistance gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats (1994) Cell, 78, pp. 1089-1099; Papageorgiou, A.C., Shapiro, R., Acharya, K.R., Molecular recognition of human angiogenin by placental ribonuclease inhibitor - An X-ray crystallographic study at 2.0 Å resolution (1997) EMBO J., 16, pp. 5162-5177; Parniske, M., Hammond-Kosack, K.E., Golstein, C., Thomas, C.M., Jones, D.A., Harrison, K., Wulff, B.B.H., Jones, J.D.G., Novel disease resistance specificities result from sequence exchange between tandemly repeated genes at the Cf-4/9 locus of tomato (1997) Cell, 91, pp. 821-832; Puett, D., Bhowmick, N., Fernandez, L.M., Huang, J., Wu, C., Narayan, P., hCG-receptor binding and transmembrane signaling (1996) Mol. Cell. Endocrinol., 125, pp. 55-64; Ronald, P.C., The molecular basis of disease resistance in rice (1997) Plant Mol. Biol., 35, pp. 179-186; Sambrook, J., Fritsch, E.F., Maniatis, T., (1989) Molecular Cloning: A Laboratory Manual, , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Schuster, S.C., Swanson, R.Y., Alex, L.A., Bourret, R.B., Simon, M.I., Assembly and function of a quaternary signal transduction complex monitored by surface plasmon resonance (1993) Nature, 365, pp. 343-347; Stotz, H.U., Powell, A.L.T., Damon, S.E., Greve, L.C., Bennett, A.B., Labavitch, J.M., Molecular characterization of a polygalacturonase inhibitor from Pyrus communis L. cv Bartlett (1993) Plant Physiol., 102, pp. 133-138; Stotz, H.U., Contos, J.J.A., Powell, A.L.T., Bennett, A.B., Labavitch, J.M., Structure and expression of an inhibitor of fungal polygalacturonases from tomato (1994) Plant Mol. Biol., 25, pp. 607-617; Thomas, C.M., Jones, D.A., Parniske, M., Harrison, K., Balint-Kurti, P.J., Hatzixanthis, K., Jones, J.D.G., Characterization of the tomato Cf-4 gene for resistance to Cladosporium fulvum identities sequences that determine recognitional specificity in Cf-4 and Cf-9 (1997) Plant Cell, 9, pp. 2209-2224; Thomas, D., Rozell, T.G., Liu, X.B., Segaloff, D.L., Mutational analyses of the extracellular domain of the full-length lutropin/ choriogonadotropin receptor suggest leucine-rich repeats 1-6 are involved in hormone binding (1996) Mol. Endocrinol., 10, pp. 760-768; Torii, K.U., Mitsukawa, N., Oosumi, T., Matsuura, Y., Yokoyama, R., Whittier, R.F., Komeda, Y., The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats (1996) Plant Cell, 8, pp. 735-746; Toubart, P., Desiderio, A., Salvi, G., Cervone, F., Daroda, L., De Lorenzo, G., Bergmann, C., Albersheim, P., Cloning and characterization of the gene encoding the endopolygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L (1992) Plant J., 2, pp. 367-373; Wang, G.L., Song, W.Y., Ruan, D.L., Sideris, S., Ronald, P.C., The cloned gene, Xa21, confers resistance to multiple Xanthomonas oryzae pv oryzae isolates in transgenic plants (1996) Mol. Plant Microbe Interact., 9, pp. 850-855; Warren, R.F., Henk, A., Mowery, P., Holub, E., Innes, R.W., A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes (1998) Plant Cell, 10, pp. 1439-1452