RESUMEN
Identification of enzymes that are expressed during host colonization and characterization of their biochemical properties are prerequisite to understanding their role in the pathogen-host interaction. Nine alpha-1,2-mannosidase homologs were identified in the analysis of the Magnaporthe oryzae genome. Endoplasmic reticulum localized alpha-1,2-mannosidases play an important role in protein glycosylation. However, several members of the alpha-1,2-mannosidase gene family are predicted to be secreted. The biological role of such extracellular enzymes in host colonization has not been defined. Here, we characterized a secreted alpha-1,2-mannosidase of M. oryzae, MGG_00994.6, and found that the mature polypeptide is a glycoprotein capable of hydrolyzing alpha-1,2 linked mannobiose. The gene is expressed during growth in vitro and during colonization on rice plants, however, deletion of the gene did not affect pathogenicity. Five other members of the alpha-1,2-mannosidase of M. oryzae were expressed with a pattern similar to MGG_00994.6, suggesting the potential for functional redundancy. These results form the basis for additional studies on the role of this gene family in the rice blast fungus and its interaction with rice.
Asunto(s)
Magnaporthe/genética , alfa-Manosidasa/análisis , alfa-Manosidasa/metabolismo , Algoritmos , Secuencia de Aminoácidos , Análisis por Conglomerados , Bases de Datos Genéticas , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Vectores Genéticos , Genoma Fúngico , Glicosilación , Histidina/metabolismo , Interacciones Huésped-Patógeno , Magnaporthe/crecimiento & desarrollo , Magnaporthe/patogenicidad , Datos de Secuencia Molecular , Peso Molecular , Oryza/microbiología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Plásmidos/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , alfa-Manosidasa/química , alfa-Manosidasa/genéticaRESUMEN
Microbial pathogens secrete an array of cell wall-degrading enzymes to break down the structure of the host cell wall to facilitate colonization of the host tissue. To better understand their role in the pathogenesis, a putative endoglucanase from Magnaporthe grisea was characterized in this paper. SignalP-3.0 analysis indicates that the protein encoded by gene MGG_02532.5 in M. grisea (named MgEGL1 for M. grisea endoglucanase 1) contains a secretory signal peptide. Multiple alignment shows that MgEGL1 has high level of homology to endoglucanases (EC 3.1.1.4) from Aspergillus nidulans and Trichoderma reesei. The three proteins share a conserved catalytic domain, but only the one from T. reesei contains a cellulose binding module. MgEGL1 was constitutively expressed with the highest level in mycelia and the lowest in conidia. Interestingly, the MgEGL1 RNA could be alternatively processed when cultured in vitro and after infection of rice. Expression analysis confirmed that the MgEGL1 is a secreted protein. Its endoglucanase activity was assayed by Congo red plates, and further confirmed by the dinitrosalicylic acid method. The finding in this paper will provide the basis for further determination of the biochemical properties of the endoglucanase protein and its relevant function in fungal pathogenesis.