Structural Requirements for Uptake and Bioactivity of Rhizobium Chitolipooligosaccharides in Legume Roots as Revealed by Synthetic Analogs and Fluorescent Probes

Abstract

We study the structure / function relationships of chitolipooligosaccharides (CLOSs) made by authentic wild type R. leguminosarum bv. trifolii ANU843 and their role in symbiotic development with white clover. Previously, we found that ANU843 accumulates a large (22+) family of CLOS glycolipids primarily in its membranes, and this structural diversity is unaltered in the ANU297nodE::Tn5 mutant derivative [from M. Djordjevic], despite its change in legume host range (Philip-Hollingsworth et al. 1995). Nevertheless, (sub)nano-molar concentrations of ANU843 membrane CLOSs fully elicit symbiosis-related responses in white clover roots, including Had and Ccd leading to nodule primordia, modulation of growth dynamics, localized disruption of crystalline wall architecture, and development of new infection sites in root hairs, and elevated chitinase activity in root exudate (Dazzo et al. 1996; Orgambide et al. 1996). Here, we prepared fluorescent-tagged NBD derivatives and synthetic analogs of ANU843 CLOSs to define their structural requirements for uptake and mitogenicity in living root cells, using laser scanning confocal microscopy and axenic seedling bioassays. We found that (i) NBD-CLOS conjugates are still Had+ and Ccd+, and are rapidly internalized within cell membranes and nuclei of living clover root hairs and cortical cells; (ii) the structural requirements for uptake and biological activity of CLOSs are directly correlated; and (iii) the smallest CLOS analog still internalized and mitogenic on both clover and alfalfa is a N-fattyacylglucosamine, without an essential requirement for oligomerization or functionalization of the polar head group or unsaturation of the fatty acyl tail (Philip-Hollingsworth et al. 1997).KeywordsRoot HairRoot ExudateCortical CellEssential RequirementGrowth DynamicThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.