Abstract
Rice blast is one of the most serious diseases affecting rice yield which is caused by Magnaporthe oryzae, a model organism for studies on plant pathogenic fungi. Lipids stored in M. oryzae cells have been shown to be crucial for the development of appressorium turgor and the ability of the pathogen to cause infection. Nile red staining is a common method to study lipid dynamics in phytopathogenic fungi. However, the disadvantages of this dye include its wide spectrum, poor water solubility, and susceptibility to quenching. Boron dipyrromethene (BODIPY) is a new type of fluorescent dye that has a different emission wavelength to that of Nile red as well as many desirable spectral and chemical properties. In this study, we used BODIPY to stain the lipids in M. oryzae cells to seek a possible substitute to Nile red in the study of lipid dynamics in plant pathogenic fungi. Our data showed that through simple and routine procedures, BODIPY was able to distinctly label lipids in the cells of mycelia and conidia. The positions of lipids labeled by BODIPY were essentially identical to those labeled by Nile red, but with more clear fluorescence labelling, lower background, and higher specificity. The use of BODIPY to stain germinating M. oryzae conidia allowed the lipid dynamics to be clearly tracked during this process. We also achieved double and multiple fluorescent staining conidia by combining BODIPY with the red fluorescent protein mCherry and other fluorescent dyes, such as Calcofluor white and DAPI, in conidia, mycelia, and sexual structures of M. oryzae. These results indicate that BODIPY is an ideal fluorescent dye for staining fungal lipids and provide a method for the study of the lipid dynamics and lipid metabolism in plant pathogenic fungi.
Highlights
Rice blast is one of the most serious diseases on rice and occurs widely throughout the world, causing average reductions in rice yields of 10–30% annually and even total loss of yields in severe cases [1]
The mycelia and conidia of strain Guy11 that were collected from a culture incubated in the dark for six days were stained for 10 min with 20 μL of the Boron dipyrromethene (BODIPY) staining solution
The distribution of green fluorescent spots was rather uniform, while that observed in conidial cells was primarily in the periphery, which is consistent with the distribution of lipid droplets in M. oryzae cells
Summary
Rice blast is one of the most serious diseases on rice and occurs widely throughout the world, causing average reductions in rice yields of 10–30% annually and even total loss of yields in severe cases [1]. The causative pathogen of rice blast is a heterothallic ascomycete, referred to as Magnaporthe oryzae in the perfect stage and Pyricularia grisea in the anamorph stage [2,3]. Molecules 2018, 23, 1594 system, it has become an important model organism for studying the interactions between plant pathogenic fungi and their hosts [5,6]. M. oryzae produces specific infection structures called appressoria. A high concentration of glycerol accumulates in appressoria that produces high turgor pressure and results in high mechanical pressure, facilitating the invasion and dissemination of the pathogen in the host. If the glycerol in the appressorium is inadequate, the osmotic pressure is too low for the pathogen to penetrate the host epidermis and cannot cause an infection [5]. The glycerol in appressoria is converted from material stored in the fungal conidia [8]. Lipids are one of the primary substances stored in the conidia of
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