Microtubule based cytoskeleton for septin ring assembly during appressorium formation by the rice blast fungus

Abstract

The rice blast fungus, Magnaporthe oryzae, causes the major destructive disease on worldwide rice production and understanding its infectious process must be a priority to develop new control strategies. The purpose of this work is to investigate the appressorium formation in germ tube of M. oryzae conidia and the involvement of the microtubule based cytoskeleton for septin ring assembly. For that purpose, the experiment was developed at the Department of Plant Pathology at the University of Arkansas and used conidia of the mutant ∆Sep5-GFP of M. oryzae in seven treatments. The conidia were collected into chamber slides, allocated into seven wells with 2000 conidia/mL concentration. Right after, the fungicide benomyl (5 μg/mL) was applied within a 2 hours difference, each, over a 10-hour period (0, 2, 4, 6, 8 and 10 hours), plus control with 0,5% of dimethyl sulfoxide (DMSO), an organosulfur compound. The chamber slides were kept overnight in an incubator, adjusted to 24oC and 12:12 light cycles, after the last benomyl application. On the following day, conidia were visualized on the live-cell imaging microcope with magnification of 200x and five frames of each treatment were taken. Data sets were analyzed using linear regression to examine the relationships between fungicide exposure time and apressoria formation. In order to the confirm or not the septin ring assembly in the appressoria, epifluorescence imaging were also taking from the 0 hours, 10 hours and DMSO treatment with magnification of 1000x. The results achieved demonstrated that germination and appressorial formation were lower in conidia at initials exposure times of the fungicide. Furthermore, as benomyl action interrupts mitosis and so depolymerizes microtubules, the septin ring assembly was only visualized in the DMSO treatment.