Modulation of development, growth dynamics, wall crystallinity, and infection sites in white clover root hairs by membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii

Abstract

We used bright-field, time-lapse video, cross-polarized, phase-contrast, and fluorescence microscopies to examine the influence of isolated chitolipooligosaccharides (CLOSs) from wild-type Rhizobium leguminosarum bv. trifolii on development of white clover root hairs, and the role of these bioactive glycolipids in primary host infection. CLOS action caused a threefold increase in the differentiation of root epidermal cells into root hairs. At maturity, root hairs were significantly longer because of an extended period of active elongation without a change in the elongation rate itself. Time-series image analysis showed that the morphological basis of CLOS-induced root hair deformation is a redirection of tip growth displaced from the medial axis as previously predicted. Further studies showed several newly described infection-related root hair responses to CLOSs, including the localized disruption of the normal crystallinity in cell wall architecture and the induction of new infection sites. The application of CLOS also enabled a NodC ؊ mutant of R. leguminosarum bv. trifolii to progress further in the infection process by inducing bright refractile spot modifications of the deformed root hair walls. However, CLOSs did not rescue the ability of the NodC ؊ mutant to induce marked curlings or infection threads within root hairs. These results indicate that CLOS Nod factors elicit several host responses that modulate the growth dynamics and symbiont infectibility of white clover root hairs but that CLOSs alone are not sufficient to permit successful entry of the bacteria into root hairs during primary host infection in the Rhizobium-clover symbiosis.

Rhizobium cellulase CelC2 is essential for primary symbiotic infection of legume host roots

“…Third, the substrate specificity of CelC2 cellulase for noncrystalline cellulose significantly restricts its in vivo site of wall-penetrating erosive action to the highly localized root-hair infection sites lacking crystalline wall architecture. This finding provides an explanation of the remarkable infection event that rhizobia typically only erode and penetrate one side of the root-hair wall despite being sandwiched by both sides when cradled in the overlap of the markedly curled shepherd's crook (10,15,29,30,35,36). Development of these localized infection sites is strategically modulated by the action of the rhizobial chitolipooligosaccharide Nod factors that disrupt the crystallization of the root-hair wall architecture and activate symbiotic root-hair infection (15).…”

“…This finding provides an explanation of the remarkable infection event that rhizobia typically only erode and penetrate one side of the root-hair wall despite being sandwiched by both sides when cradled in the overlap of the markedly curled shepherd's crook (10,15,29,30,35,36). Development of these localized infection sites is strategically modulated by the action of the rhizobial chitolipooligosaccharide Nod factors that disrupt the crystallization of the root-hair wall architecture and activate symbiotic root-hair infection (15). Fourth, the diameter of the hole eroded completely through the root-hair wall by CelC2 cellulase action is precisely within the same in situ size range that wild type R. leguminosarum bv trifolii cells use to traverse the clover root-hair wall during primary host infection (10,30,31).…”

“…That morphological feature commonly precedes root-hair penetration and infection-thread formation (15). As is also the case for root hairs responsive to Nod factors (15), a subset of root hairs is particularly susceptible to deformation after inoculation with either the wild type or the CelC2 Ϫ mutant derivative strain (Fig.…”

“…Previous studies (10) showed that the DEAE fraction containing the partially purified cellulase CelC2 isozyme from wild-type ANU843 was capable of degrading the cell wall at the apex of the root hair tip (the ''Hot'' phenotype) in vivo when incubated with intact white clover host-seedling roots, and this activity was not found in the other DEAE fraction containing the cellulase C1 isozyme (10). The symbiotic relevance of this enzyme bioactivity was revealed by studies indicating that (i) its highly localized site of cell wall degradation matched the restricted distribution of the isotropic noncrystalline wall architecture at root hair tips of axenic white clover seedlings that typically undergo marked curling and infection (10), (ii) the discrete hole through the plant wall that is ultimately produced there has a diameter precisely within the 2-to 3-m range typically found at primary infection sites in deformed root hairs (10,30,31), (iii) it was more extensive in walls of root hairs that had been grown in the presence of wild-type chitolipooligosaccharide Nod factors that increase root hair infectibility (10), and (iv) it was less extensive when clover seedlings were grown with sufficient nitrate supply to inhibit primary infection (10,32) or with root hair walls isolated from the nonhost legume, alfalfa (15).…”

“…trifolii can completely erode through the white clover root hair wall at a highly localized site on the isotropic, noncrystalline apex of its root-hair tip [expression of the Hot (Hole on the tip) phenotype], and can more extensively degrade isolated clover root-hair walls as a substrate if they were grown in the presence (rather than the absence) of purified chitolipooligosaccharide Nod factors from clover rhizobia (10). Other studies showed that these Nod factors from clover rhizobia evoked a localized disruption in the normal crystallization of the host cell wall architecture in growing root hairs (15). Considered collectively, the results implicate a complimentary role of Rhizobium cellulase and Nod factors in promoting root-hair infectibility at strategic sites during primaryhost infection.…”