RESUMEN
A detailed analysis of the secondary structure has been carried out on the polygalacturonase-inhibiting protein (PGIP) from Phaseolus vulgaris, a leucine-rich repeat (LRR) protein present in the cell wall of many plants. Far-UV CD and infrared spectroscopies coupled to constrained secondary structure prediction methods indicated the presence of 12 alpha- and 12 beta-segments, thus allowing a schematic representation of three domains of the protein, namely, the central LRR region and the two cysteine-rich flanking domains. Peptides from endoproteinase-degraded PGIP were analyzed by mass spectrometry, and four disulfide bonds were identified. Mass spectrometric analysis in combination with glycosidase treatments revealed two N-linked oligosaccharides located on Asn 64 and Asn 141. The main structure resembled the typical complex plant N-glycan consisting of a core pentasaccharide beta1,2-xylosylated, carrying an alpha1,3-fucose linked to the innermost N-acetylglucosamine and one outer arm N-acetylglucosamine residue. The schematic representation of PGIP structural domains is discussed in the framework of the structure and function of LRR proteins.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Leucina/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Poligalacturonasa/antagonistas & inhibidores , Procesamiento Proteico-Postraduccional , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Dicroismo Circular , Disulfuros/química , Fabaceae , Glicosilación , Datos de Secuencia Molecular , Plantas Medicinales , Estructura Secundaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
A cDNA clone encoding a leucine-rich repeat (LRR) receptor-like protein kinase (LRPKm1) of Malus x domestica cv. Florina has been isolated using as a heterologous probe a cloned gene encoding a polygalacturonase-inhibiting protein (PGIP) of Phaseolus vulgaris L. A genomic clone containing the 5'-regulatory region and a 5' portion of the open reading frame of the LRPKm1 gene has also been isolated. An open reading frame of 2997 nt (999 amino acids) was present in the cDNA clone, encoding a receptor-like protein comprising a 21 amino acid signal peptide for secretion, a leucine zipper, 23 LRRs, a putative membrane-spanning region and a serine/threonine protein kinase domain. LRPKm1 shows homology to the A. thaliana receptor-like protein kinase RLK5 and, to a minor extent, to PGIP. The LRPKm1 region from +5 to +600 exhibits an alternative reading frame that encodes a product corresponding to a proline-rich protein fragment homologous to several hydroxyproline-rich proteins. Southern blot analysis showed that LRPKm1 belongs to a multigene family and that there is length polymorphism of the hybridizing restriction fragments among different M. x domestica cultivars. Northern blot analysis was carried out on mRNA extracted from infected leaves of either cv. Florina (resistant to Venturia inaequalis) or cv. Golden Delicious (susceptible to V. inaequalis), and from tissues treated with salicylic acid. A 3500 bp transcript hybridizing at high stringency with the LRPKm1 cDNA accumulated in response to infection or salicylic acid treatment. Transcript accumulation was more intense in the incompatible interaction than in the compatible one. The possible involvement of this receptor-like protein kinase in resistance of apple to phytopathogenic fungi is discussed.
Asunto(s)
Antifúngicos/farmacología , Frutas/genética , Enfermedades de las Plantas/microbiología , Proteínas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Ácido Salicílico/farmacología , Secuencia de Aminoácidos , Ascomicetos/crecimiento & desarrollo , Secuencia de Bases , Northern Blotting , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , ADN de Plantas/química , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Frutas/enzimología , Frutas/microbiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas/genética , Proteínas Repetidas Ricas en Leucina , Datos de Secuencia Molecular , Familia de Multigenes/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinasas , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción Genética/efectos de los fármacosRESUMEN
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 beta-sheet/beta-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.
Asunto(s)
Proteínas de Plantas/química , Poligalacturonasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Secuencia de Bases , ADN Complementario/genética , Fabaceae/genética , Fusarium/enzimología , Biblioteca de Genes , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/genética , Plantas Medicinales , Plantas Tóxicas , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Secuencias Repetitivas de Aminoácido , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Nicotiana/genéticaRESUMEN
Polygalacturonase-inhibiting proteins (PGIPs), leucine-rich repeat (LRR) proteins evolutionarily related to several plant resistance genes, bind to and regulate the action of fungal endopolygalacturonases. In Phaseolus vulgaris L., PGIPs are encoded by a gene family comprising at least five members. As a start for a systematic analysis of the regulation of the pgip family, we have analysed the ability of the promoter of the bean gene pgip-1 to direct expression of beta-glucuronidase (GUS) in transfected tobacco protoplasts, microbombarded bean and tobacco leaves, and transgenic tobacco plants. In protoplasts, the pgip-1 gene region from nucleotide (nt) -2004 to nt +27 directed a level of expression that was as high as that directed by the cauliflower mosaic virus (CaMV) 35S promoter and could not be further induced by elicitor treatment; alteration of the region immediately following the TATAA sequence at nt -29 abolished expression. Upon stable integration into tobacco plants of the pgip-1 promoter-GUS construct, as well as of a -394 deletion, expression was detected for both constructs mainly in the stigma and, to a lesser extent, in the anthers and in the conductive vascular tissue. The promoter responded to wounding but not to oligogalacturonides, fungal glucan, salicylic acid, cryptogein, or pathogen infection. This expression pattern does not mirror that of the whole pgip gene family.
Asunto(s)
Inhibidores Enzimáticos , Fabaceae/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Proteínas de Plantas/genética , Plantas Medicinales , Poligalacturonasa/antagonistas & inhibidores , Regiones Promotoras Genéticas , Fusión Artificial Génica , Fabaceae/microbiología , Glucuronidasa/genética , Phytophthora/fisiología , Plantas Modificadas Genéticamente , Plantas Tóxicas , Pseudomonas/fisiología , NicotianaRESUMEN
The filamentous fungus Cochliobolus carbonum produces endo-alpha 1,4-polygalacturonase (endoPG), exo-alpha 1,4-polygalacturonase (exoPG), and pectin methylesterase when grown in culture on pectin. Residual activity in a pgn1 mutant (lacking endoPG) was due to exoPG activity, and the responsible protein has now been purified. After chemical deglycosylation, the molecular mass of the purified protein decreased from greater than 60 to 45 kDa. The gene that encodes exoPG, PGX1, was isolated with PCR primers based on peptide sequences from the protein. The product of PGX1, Pgx1p, has a predicted molecular mass of 48 kDa, 12 potential N-glycosylation sites, and 61% amino acid identity to an exoPG from the saprophytic fungus Aspergillus tubingensis. Strains of C. carbonum mutated in PGX1 were constructed by targeted gene disruption and by gene replacement. Growth of pgx1 mutant strains on pectin was reduced by ca. 20%, and they were still pathogenic on maize. A double pgn1/pgx1 mutant strain was constructed by crossing. The double mutant grew as well as the pgx1 single mutant on pectin and was still pathogenic despite having less than 1% of total wild-type PG activity. Double mutants retained a small amount of PG activity with the same cation-exchange retention time as Pgn1p and also pectin methylesterase and a PG activity associated with the mycelium. Continued growth of the pgn1/pgx1 mutant on pectin could be due to one or more of these residual activities.
Asunto(s)
Ascomicetos/enzimología , Ascomicetos/genética , Mutación , Poligalacturonasa/genética , Secuencia de Aminoácidos , Ascomicetos/crecimiento & desarrollo , Secuencia de Bases , Clonación Molecular , ADN de Hongos/genética , Expresión Génica , Marcación de Gen , Genes Fúngicos , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/aislamiento & purificación , Datos de Secuencia Molecular , Pectinas/metabolismo , Fenotipo , Poligalacturonasa/aislamiento & purificación , Mapeo Restrictivo , Virulencia/genética , Zea mays/microbiologíaRESUMEN
The pgip-1 gene of Phaseolus vulgaris, encoding a polygalacturonase-inhibiting protein (PGIP), PGIP-1 (P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Bergmann, A. G. Darvill, and P. Albersheim, Plant J. 2:367-373, 1992), was expressed under control of the cauliflower mosaic virus 35S promoter in tomato plants via Agrobacterium tumefaciens-mediated transformation. Transgenic tomato plants with different expression levels of PGIP-1 were used in infection experiments with the pathogenic fungi Fusarium oxysporum f. sp. lycopersici, Botrytis cinerea, and Alternaria solani. No evident enhanced resistance, compared with the resistance of untransformed plants, was observed. The pgip-1 gene was also transiently expressed in Nicotiana benthamiana with potato virus X (PVX) as a vector. PGIP-1 purified from transgenic tomatoes and PGIP-1 in crude protein extracts of PVX-infected N. benthamiana plants were tested with several fungal polygalacturonases (PGs). PGIP-1 from both plant sources exhibited a specificity different from that of PGIP purified from P. vulgaris (bulk bean PGIP). Notably, PGIP-1 was unable to interact with a homogeneous PG from Fusarium moniliforme, as determined by surface plasmon resonance analysis, while the bulk bean PGIP interacted with and inhibited this enzyme. Moreover, PGIP-1 expressed in tomato and N. benthamiana had only a limited capacity to inhibit crude PG preparations from F. oxysporum f. sp. lycopersici, B. cinerea, and A. solani. Differential affinity chromatography was used to separate PGIP proteins present in P. vulgaris extracts. A PGIP-A with specificity similar to that of PGIP-1 was separated from a PGIP-B able to interact with both Aspergillus niger and F. moniliforme PGs. Our data show that PGIPs with different specificities are expressed in P. vulgaris and that the high-level expression of one member (pgip-1) of the PGIP gene family in transgenic plants is not sufficient to confer general, enhanced resistance to fungi.
Asunto(s)
Fabaceae/genética , Proteínas de Plantas/genética , Plantas Medicinales , Inhibidores Enzimáticos , Fabaceae/microbiología , Solanum lycopersicum/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Especificidad por SustratoRESUMEN
The sequence encoding the endopolygalacturonase (PG) of Fusarium moniliforme was cloned into the E. coli/yeast shuttle vector Yepsec1 for secretion in yeast. The recombinant plasmid (pCC6) was used to transform Saccharomyces cerevisiae strain S150-2B; transformed yeast cells were able to secrete PG activity into the culture medium. The enzyme (wtY-PG) was purified, characterized, and shown to possess biochemical properties similar to those of the PG purified from F. moniliforme. The wtY-PG was able to macerate potato medullary tissue disks and was inhibited by the polygalacturonase-inhibiting protein (PGIP) purified from Phaseolus vulgaris. The sequence encoding PG in pCC6 was subjected to site-directed mutagenesis. Three residues in a region highly conserved in all the sequences known to encode PGs were separately mutated: His 234 was mutated into Lys (H 234-->K), and Ser 237 and Ser 240 into Gly (S 237-->G and S 240-->G). Each of the mutated sequences was used to transform S. cerevisiae and the mutated enzymes were purified and characterized. Replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence that a His residue is critical for enzyme activity. Replacement of either Ser 237 or Ser 240 with Gly reduced the enzymatic activity to 48% and 6%, respectively, of the wtY-PG. When applied to potato medullary tissue, F. moniliforme PG and wtY-PG caused comparable maceration, while the variant PGs exhibited a limited (S 234-->G and S 240-->G) or null (H 234-->K) macerating activity. The interaction between the variant enzymes and the P. vulgaris PGIP was investigated using a biosensor based on surface plasmon resonance (BIAlite). The three variant enzymes were still able to interact and bind to PGIP with association constants comparable to that of the wild type enzyme.
Asunto(s)
Fusarium/enzimología , Histidina , Poligalacturonasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia Conservada , Cartilla de ADN , Inhibidores Enzimáticos/metabolismo , Fabaceae , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/metabolismo , Plantas Medicinales , Poligalacturonasa/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiaeRESUMEN
Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.
Asunto(s)
Fabaceae/metabolismo , Enfermedades de las Plantas , Proteínas de Plantas/metabolismo , Plantas Medicinales , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Fabaceae/microbiología , Fabaceae/ultraestructura , Microscopía Electrónica , Hongos Mitospóricos/ultraestructura , Datos de Secuencia Molecular , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/genética , ARN Mensajero/biosíntesis , Salicilatos/farmacología , Ácido SalicílicoAsunto(s)
Oligosacáridos/metabolismo , Fenómenos Fisiológicos de las Plantas , Pared Celular/metabolismo , Glucano Endo-1,3-beta-D-Glucosidasa/metabolismo , Interacciones Huésped-Parásitos , Phytophthora/fisiología , Desarrollo de la Planta , Extractos Vegetales/biosíntesis , Proteínas de Plantas/metabolismo , Plantas/microbiología , Poligalacturonasa/antagonistas & inhibidores , Sesquiterpenos , Terpenos , FitoalexinasRESUMEN
Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein purified from hypocotyls of true bean (Phaseolus vulgaris L.). PGIP inhibits fungal endopolygalacturonases and is considered to be an important factor for plant resistance to phytopathogenic fungi (Albersheim and Anderson, 1971; Cervone et al., 1987). The amino acid sequences of the N-terminus and one internal tryptic peptide of the PGIP purified from P. vulgaris cv. Pinto were used to design redundant oligonucleotides that were successfully utilized as primers in a polymerase chain reaction (PCR) with total DNA of P. vulgaris as a template. A DNA band of 758 bp (a specific PCR amplification product of part of the gene coding for PGIP) was isolated and cloned. By using the 758-bp DNA as a hybridization probe, a lambda clone containing the PGIP gene was isolated from a genomic library of P. vulgaris cv. Saxa. The coding and immediate flanking regions of the PGIP gene, contained on a subcloned 3.3 kb SalI-SalI DNA fragment, were sequenced. A single, continuous ORF of 1026 nt (342 amino acids) was present in the genomic clone. The nucleotide and deduced amino acid sequences of the PGIP gene showed no significant similarity with any known databank sequence. Northern blotting analysis of poly(A)+ RNAs, isolated from various tissues of bean seedlings or from suspension-cultured bean cells, were also performed using the cloned PCR-generated DNA as a probe. A 1.2 kb transcript was detected in suspension-cultured cells and, to a lesser extent, in leaves, hypocotyls, and flowers.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Genes de Plantas , Proteínas de Plantas/genética , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN/genética , Sondas de ADN , Fabaceae/genética , Datos de Secuencia Molecular , Plantas Medicinales , Poligalacturonasa/antagonistas & inhibidoresRESUMEN
A pectolytic enzyme from culture filtrates of Rhizoctonia fragariae was purified approx. 29-fold. The enzyme exhibited maximal depolymerizing activity on Na-polypectate rather than pectin at pH 5.0 and was inhibited at different extent by divalent cations Mg++, Mn++, Ca++. The analysis by paper chromatography of the products of enzyme hydrolysis suggested that the enzyme attacks the substrate by a random mechanism. The absorption spectrum of the chromogen formed by the hydrolysis products and thiobarbituric acid suggested that the enzyme is a hydrolytic enzyme. On the basis of these results the enzyme is classifiable as endo-polygalacturonase (poly alpha-1,4-galacturonide glycanohydrolase E.C. 3.2.1.15).