DTAF: an efficient probe to study cyanobacterial-plant interaction using confocal laser scanning microscopy (CLSM).
A variety of microscopic techniques have been utilized to study cyanobacterial associations with plant roots, but confocal laser scanning microscopy (CLSM) is the least used due to the unavailability of a suitable fluorescent dye. Commonly used lectins have problems with their binding ability with root cells and their visualization under CLSM. DTAF (5-(4,6-dichlorotriazinyl) aminofluorescein) is a fluorescent dye that has been widely used for staining various biological samples for fluorescent microscopy. It reacts with polysaccharides and peptides at ordinary conditions. The possible application and efficiency of DTAF for CLSM studies were examined in various aspects of cyanobacterial-plant interactions. Seedlings of Pisum sativum, Vigna rediata and Triticum aestivum were co-cultivated and stained with DTAF as a fluorochrome. Extracellular and intracellular interactions of cyanobacteria and the plant root surface were observed by CLSM. Results were compared with staining by other commonly used lectins. Advantages of the use of DTAF over other stains are its penetration into root tissues and binding with polysaccharides, mainly the cellulose. The staining was smooth, which clearly showed minute details on the cell of surface and root hairs with higher resolution. The emission wavelength for DTAF is 517 nm, which is highly advantageous as cyanobacteria have auto-fluorescence at 665 nm, and both can be simultaneously used in CLSM by visualizing in different channels. This worked efficiently with all three plants used and with filamentous and unicellular cyanobacterial strains. Cyanobacterial presence was not only clearly observed on the root surface, but also inside the root tissue and epidermal cells. The easy protocol and absence of tissue processing make DTAF a useful probe for studies of cyanobacterial associations with plant roots by CLSM.
Ahmed
Mehboob
M
Department of Microbiology and Molecular Genetics, Quaid-e-Azam Campus, University of the Punjab, Lahore, 54590, Pakistan.
Stal
Lucas J
LJ
Hasnain
Shahida
S
D000458:Cyanobacteria / Q000254:growth & development*; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted; D018613:Microscopy, Confocal; D018517:Plant Roots / Q000382:microbiology*; D010944:Plants / Q000382:microbiology; D013194:Staining and Labeling
D005452:Fluoresceins; D005456:Fluorescent Dyes; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein
Folia Microbiol (Praha). 2009;54(1):43-51
19330544
Int Microbiol. 2007 Jun;10(2):91-6
17661286
Curr Opin Plant Biol. 2006 Dec;9(6):621-30
17011813
FEMS Microbiol Rev. 2009 Sep;33(5):917-41
19453747
Methods Enzymol. 1999;310:145-52
10547788
Microb Ecol. 2009 May;57(4):649-56
18982381