Characterization of Root Hair Branching Factor in White Clover/Rhizobium Trifolii Symbiosis

Abstract

In white clover Rhizobium infections that lead to nodule formation occur frequently in root hairs that are developmentally mature at the time of inoculation. These mature root hairs become susceptible to infections only after they develop lateral branches (Bhuvaneswari, T.V. this volume). Cell free filtrates of R. trifolii cultured in the presence of white clover plants induced branching in mature root hairs. A bioassay for branching was developed to test the fractions purified from the filtrate. Each fraction was tested on a minimum of 14 plants growing in 1.5 ml chambers (7 plants/chamber) made up of a microscope slide and a 22 × 60 mm cover slip. Roots were scored after 14 h on a scale of 0–4 (0 is no root hairs branched and 4 is almost all root hairs branched). Control roots exposed to water averaged a score of 0.5. Freeze dried cell free filtrates were sequentially extracted with 95%, 60% ethanol and water. The 60% ethanol soluble fraction which was biologically most active was concentrated by evaporation at reduced pressure, freeze dried and applied to a DEAE Sephadex column (imidazole/HCl 0.05M pH 7.5). Active unbound components were pooled, dialysed (1200 dalton cut off), freeze dried and applied to a Biogel P4 column. Three active fractions were collected. Further separation of these fractions by HPLC with a silica column yielded eight active fractions ranging from 1200–10,000 daltons in molecular weight. All these fractions contained neutral sugars and their biological activity was stable to autoclaving above pH 6.0.