RESUMEN
Reactive oxygen species (ROS) are produced in conserved cellular processes either as by-products of the cellular respiration in mitochondria, or purposefully for defense mechanisms, signaling cascades or cell homeostasis. ROS have two diametrically opposed attributes due to their highly damaging potential for DNA, lipids and other molecules and due to their indispensability for signaling and developmental processes. In filamentous fungi, the role of ROS in growth and development has been studied in detail, but these analyses were often hampered by the lack of reliable and specific techniques to monitor different activities of ROS in living cells. Here, we present a new method for live cell imaging of ROS in filamentous fungi. We demonstrate that by use of a mixture of two fluorescent dyes it is possible to monitor H2O2 and superoxide specifically and simultaneously in distinct cellular structures during various hyphal differentiation processes. In addition, the method allows for reliable fluorometric quantification of ROS. We demonstrate that this can be used to characterize different mutants with respect to their ROS production/scavenging potential.
Asunto(s)
Botrytis/metabolismo , Imagen Óptica/métodos , Especies Reactivas de Oxígeno/análisis , Colorantes Fluorescentes , Fluorometría/métodos , Peróxido de Hidrógeno/metabolismo , Hifa/metabolismo , Cebollas/metabolismo , Cebollas/microbiología , Epidermis de la Planta/metabolismo , Epidermis de la Planta/microbiología , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismoRESUMEN
The putative Claviceps purpurea homologue of the Saccharomyces cerevisiae stretch-activated calcium ion channel Mid1 was investigated for its role in vegetative growth, differentiation and pathogenicity on rye (Secale cereale). Gene replacement mutants of Cl. purpurea mid1 were not affected in polar growth and branching in axenic culture but showed a significantly reduced growth rate. The growth defect could not be complemented by Ca(2+) supplementation, in contrast to mid1 mutants in yeast, but the altered sensitivity of the mutants to changes in external and internal Ca(2+) concentrations indicates some role of Mid1 in Ca(2+) homeostasis. The major effect of mid1 deletion, however, was the complete loss of virulence: infected rye plants showed no disease symptoms at all. Detailed analyses of in vitro-infected rye ovaries demonstrated that the Deltamid1 mutants had multiple apical branches and were unable to infect the host tissue, suggesting that Mid1 is essential for generating the necessary mechanical force for penetration. This is believed to be the first report of an essential role for a Mid1 homologue in the virulence of a plant-pathogenic fungus.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Claviceps/genética , Claviceps/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Calcio/metabolismo , Pared Celular/metabolismo , Claviceps/crecimiento & desarrollo , Claviceps/patogenicidad , ADN de Hongos/genética , Eliminación de Gen , Genes Fúngicos , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Presión Osmótica , Enfermedades de las Plantas/microbiología , Secale/microbiología , Homología de Secuencia de Aminoácido , Estrés Fisiológico , Virulencia/genética , Virulencia/fisiologíaRESUMEN
The ergot diseases of grasses, caused by members of the genus Claviceps, have had a severe impact on human history and agriculture, causing devastating epidemics. However, ergot alkaloids, the toxic components of Claviceps sclerotia, have been used intensively (and misused) as pharmaceutical drugs, and efficient biotechnological processes have been developed for their in vitro production. Molecular genetics has provided detailed insight into the genetic basis of ergot alkaloid biosynthesis and opened up perspectives for the design of new alkaloids and the improvement of production strains; it has also revealed the refined infection strategy of this biotrophic pathogen, opening up the way for better control. Nevertheless, Claviceps remains an important pathogen worldwide, and a source for potential new drugs for central nervous system diseases.