RESUMEN
The plant pathogen Botrytis cinerea can infect undamaged plant tissue directly by penetration of the cuticle. This penetration has been suggested to be enzyme-mediated, and an important role for cutinase in the infection process has been proposed. In this study the expression of the cutinase encoding gene cutA of B. cinerea was analyzed using a cutA promoter-GUS reporter gene fusion. Transformants containing the fusion construct were examined for GUS expression on gerbera flowers and tomato fruits. High GUS activity was detected from the onset of conidial germination and during penetration into epidermal cells, indicating that cutA is expressed during the early stages of infection. To determine the biological relevance of cutinase A for successful penetration, cutinase A-deficient mutants were constructed by gene disruption. Pathogenicity of two transformants lacking a functional cutA gene was studied on gerbera flowers and tomato fruits. Their ability to penetrate and cause symptoms was unaltered compared to the wild-type strain. These results exclude an important role for cutinase A during direct penetration of host tissue by B. cinerea.
Asunto(s)
Hidrolasas de Éster Carboxílico/biosíntesis , Hongos Mitospóricos/enzimología , Plantas/microbiología , Solanum lycopersicum/microbiología , Secuencia de Bases , Southern Blotting , Cartilla de ADN , ADN de Hongos/análisis , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Glucuronidasa/biosíntesis , Hongos Mitospóricos/genética , Hongos Mitospóricos/patogenicidad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Especificidad de la EspecieRESUMEN
Cutinase of Botrytis cinerea has been suggested to play an important role in penetration of host tissues. A protein fraction with cutin hydrolyzing activity was purified from culture filtrates of B. cinerea induced for cutinase activity. An 18-kDa protein in this fraction was identified as cutinase and the corresponding gene cutA was cloned. The gene is present in a single copy in the genome of B. cinerea strain SAS56 and its predicted amino acid sequence shows significant homology (31 to 35% identity) to other fungal cutinases. RNA blot analysis showed that cutA mRNA is induced in vitro by the cutin monomer 16-hydroxyhexadecanoic acid and repressed by glucose. The expression of cutA during infection of tomato leaves is low during early phases of infection, but high when the fungus has colonized the leaf and starts to sporulate.
Asunto(s)
Hidrolasas de Éster Carboxílico/biosíntesis , Hidrolasas de Éster Carboxílico/química , Genes Fúngicos , Hongos Mitospóricos/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Cromatografía en Gel , Clonación Molecular , Biblioteca Genómica , Hongos Mitospóricos/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Mapeo Restrictivo , Homología de Secuencia de AminoácidoRESUMEN
Colonies of Botrytis cinerea were obtained from spore samplers placed inside and outside a glasshouse with a rose crop. Pure cultures were made from five colonies collected on one sampling date every month throughout the year. These isolates were tested for germination on water agar and for pathogenicity on gerbera and rose petals. The germination rate of the conidia on water agar varied between 60 and 99%. Pathogenicity of the isolates on gerbera and rose flowers ranged from 14 to 166% relative to reference isolate Bc16 and varied among isolates collected on the same day as much as among isolates collected in different months. The pathogenicity of the isolates on rose flowers was overall higher than on gerbera flowers. Random amplified polymorphic DNA (RAPD) analysis was performed on 30 selected isolates with different relative pathogenicity, collected both inside and outside the glasshouse. Almost all of the isolates were genetically different. No correlation was found among pathogenicity, sampling time, sampling place, and RAPD patterns. Results are further evidence for the statement that B. cinerea inoculum in glasshouses continuously originates from many different sources in their vicinity.
RESUMEN
Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated from Cladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joosten et al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatible C. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.
Asunto(s)
Quitinasas/genética , Cladosporium/fisiología , Plantas/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN/genética , ADN/aislamiento & purificación , Genes de Plantas , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plantas/genética , Plantas/microbiología , Homología de Secuencia de AminoácidoRESUMEN
Tomato leaves infected by the fungal pathogen Cladosporium fulvum contain several types of intracellular and extracellular pathogenesis-related (PR) proteins. Previously, we reported the purification and serological characterization of five extracellular PR proteins: P2, P4, P6, a chitinase and a beta-1,3-glucanase [22, 23]. Here we describe the purification of a basic intracellular 33 kDa beta-1,3-glucanase and the isolation and characterization of cDNA clones encoding the two extracellular P14 isomers P4 and P6, the extracellular acidic beta-1,3-glucanase and a basic 35 kDa beta-1,3-glucanase, different from the purified 33 kDa protein. Southern blot analysis demonstrated that tomato PR proteins are not encoded by large gene families, as is the case in tobacco. The number of genes corresponding to each protein was estimated to vary between one and three. A northern blot analysis indicated that the mRNAs for the extracellular PR proteins (P4, P6 and acidic beta-1,3-glucanase) accumulate to similar levels in compatible and incompatible tomato-C. fulvum interactions, although the maximum level of expression is reached much faster in the incompatible interaction. On the other hand, the mRNA for the basic 35 kDa beta-1,3-glucanase is induced rapidly to high levels in both interactions, but declines in time to background levels only in the incompatible interaction. The relevance of this difference in relation to plant defence is discussed.
Asunto(s)
Cladosporium/genética , Enfermedades de las Plantas , Proteínas de Plantas/genética , Plantas/metabolismo , ARN Mensajero/biosíntesis , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cladosporium/patogenicidad , Clonación Molecular , Glucano 1,3-beta-Glucosidasa , Datos de Secuencia Molecular , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Homología de Secuencia de Aminoácido , Transcripción Genética , Virulencia , beta-Glucosidasa/genética , beta-Glucosidasa/aislamiento & purificación , beta-Glucosidasa/metabolismoRESUMEN
The immunoreactivity of a panel of monoclonal antibodies raised to tomato spotted wilt virus (TSWV) was examined in enzyme-linked immunosorbent assays (ELISA) and dot immunobinding assays (DIBA) procedures. MAbs 6.12.15 and 2.9 were specific for the nucleocapsid protein of TSWV. The sensitivity of the two immunoassays was compared with that of a dot-blot hybridization technique using riboprobes (RNA transcripts) to TSWV M RNA. Using deproteinized plant extracts or purified virus preparations, as little as 1 pg RNA could be detected. Although an ELISA using MAb 6.12.15, a DIBA procedure using MAb 3.22.6 and the dot-blot hybridization, detected several TSWV isolates in different host species equally well, the ELISA was most precise and most suitable for routine diagnosis in the field.