Jacques Vasse

Alfalfa nodulation in the absence of Rhizobium

SummaryCertain alfalfa plants can develop non-nitrogen fixing structures on their root systems when grown in testtubes under strictly axenic conditions. We demonstrate that these structures possess all the histological features characteristic of indeterminate nodules and that their formation is inhibited by combined nitrogen. The nodule morphogenesis-related gene ENOD2 is expressed in these nodules whereas leghemoglobin transcripts cannot be detected. The capacity to Nodulate in the Absence of Rhizobium (NAR) is maintained during clonal propagation of these alfalfa plants. Our results show that Rhizobium is not absolutely required for nodule morphogenesis and suggest that plant genetic determinants are involved in the NAR phenomenon.

Ralstonia solanacearum produces Hrp-dependent pili that are required for PopA secretion but not for attachment of bacteria to plant cells

As in many other Gram‐negative plant pathogenic bacteria, the Ralstonia solanacearum hrp genes are involved in the production of a type III secretion apparatus that allows the translocation of PopA protein to the external medium. Here, we show that hrp genes are also involved in the biogenesis of pili that are mainly composed of the HrpY protein. These pili are produced at one pole of the bacterium and are also released into the external medium where they can form very long straight bundles. An hrpY mutant is defective in pilus production, impaired in interactions with plants and in secretion of the PopA protein but not in attachment to plant cells.

Saprophytic intracellular rhizobia in alfalfa nodules.

In indeterminate alfalfa nodules, the establishment of the senescent zone IV, in which both symbionts undergo simultaneous degeneration, has been considered, until now, as the end point of the symbiotic interaction. However, we now describe an additional zone, zone V, proximal to the senescent zone IV and present in alfalfa nodules more than 6 weeks old. In zone V, a new round of bacterial release occurs from remaining infection threads, leading to the reinvasion of plant cells that have completely senesced. These intracellular rhizobia are rod shaped and do not display the ultrastructural differentiation features of bacteroids observed in the more distal zones of the nodule. Interestingly, we have found that oxygen is available in zone V at a concentration compatible with both bacterial development and nitrogen fixation gene expression in newly released rhizobia. However, this expression is not correlated with acetylene reduction. Moreover, the pattern of nifH expression in this zone, as well as new data relating to expression in zone II, strongly suggest that nifH transcription in the nodule is under the control of a negative regulator in addition to oxygen. Our results support the conclusion that zone V is an ecological niche where intracellular rhizobia take advantage of the interaction for their exclusive benefit and live as parallel saprophytic partners. The demonstration of such an advantage for rhizobia in nodules was the missing evidence that Rhizobium-legume interactions are indeed symbiotic and, in particular, suggests that benefits to the two partners are associated with different developmental stages within the nodule.

Interactions of rhizobia with rice and wheat

Recently, evidence has been obtained that naturally occurring rhizobia, isolated from the nodules of non-legume Parasponia species and from some tropical legumes, are able to enter the roots of rice, wheat and maize at emerging lateral roots by crack entry. We have now investigated whether Azorhizobium caulinodans strain ORS571, which induces root and stem nodules on the tropical legume Sesbania rostrata as a result of crack entry invasion of emerging lateral roots, might also enter rice and wheat by a similar route. Following inoculation with ORS571 carrying a lacZ reporter gene, azorhizobia were observed microscopically within the cracks associated with emerging lateral roots of rice and wheat. A high proportion of inoculated rice and wheat plants had colonized lateral root cracks. The flavanone naringenin at 10 and 10 M stimulated significantly the colonization of lateral root cracks and also intercellular colonization of wheat roots. Naringenin does not appear to be acting as a carbon source and may act as a signal molecule for intercellular colonization of rice and wheat by ORS571 by a mechanism which is nod gene-independent, unlike nodule formation in Sesbania rostrata. The opportunity now arises to compare and to contrast the ability of Azorhizobium caulinodans with that of other rhizobia, such as Parasponia rhizobia, to intercellularly colonize the roots of non-legume crops.

Specific Flavonoids Promote Intercellular Root Colonization of Arabidopsis thaliana by Azorhizobium caulinodans ORS571

The ability of Azorhizobium caulinodans ORS571 and other diazotrophic bacteria to internally colonize roots of Arabidopsis thaliana has been studied. Strains tagged with lacZ or gusA reporter genes were used, and the principal colonization sites were found to be the points of emergence of lateral roots, lateral root cracks (LRCs). High frequencies of colonization were found; 63 to 100% of plants were colonized by ORS571, and 100% of plants were colonized by Herbaspirillum seropedicae. After LRCs were colonized, bacteria moved into intercellular spaces between the cortical and endodermal cell layers. Specific flavonoids, naringenin and daidzein, at 5 x 10(-5) M, significantly promoted colonization by ORS571. By using a nodC and a nodD mutant of ORS571, it was shown that neither Nod factors nor NodD are involved in colonization or flavonoid stimulation of colonization. Flavonoids did not appear to be stimulating LRC colonization by their activity as nutritional factors. LRC and intercellular colonization by H. seropedicae was stimulated by naringenin and daidzein at the same concentration that stimulated colonization by ORS571.

The hrpB and hrpG Regulatory Genes of Ralstonia solanacearum Are Required for Different Stages of the Tomato Root Infection Process

hrp genes, encoding type III secretion machinery, have been shown to be key determinants for pathogenicity in the vascular phytopathogenic bacterium Ralstonia solanacearum GMI1000. Here, we show phenotypes of R. solanacearum mutant strains disrupted in the prhJ, hrpG, or hrpB regulatory genes with respect to root infection and vascular colonization in tomato plants. Tests of bacterial colonization and enumeration in tomato plants, together with microscopic observations of tomato root sections, revealed that these strains display different phenotypes in planta. The phenotype of a prhJ mutant resembles that of the wild-type strain. An hrpB mutant shows reduced infection, colonization, and multiplication ability in planta, and induces a defense reaction similar to a vascular hypersensitive response at one protoxylem pole of invaded plants. In contrast, the hrpG mutant exhibited a wild-type level of infection at secondary root axils, but the ability of the infecting bacteria to penetrate into the vascular cylinder was significantly impaired. This indicates that bacterial multiplication at root infection sites and transit through the endodermis constitute critical stages in the infection process, in which hrpB and hrpG genes are involved. Moreover, our results suggest that the hrpG gene might control, in addition to hrp genes, other functions required for vascular colonization.

TheRhizobium-legume symbiosis Two methods to discriminate between nodules and other root-derived structures

SummaryTwo methods have been developed in order to discriminate between lateral roots, nodules and root-derived structures which exhibit both root and nodule histological features and which can develop on legumes inoculated with certainRhizobium mutants. The first method, known as the “clearing method”, allows the observation by light microscopy of cleared undissected root-structures. The second, known as the “slicing method”, is a complementary technique which provides a greater degree of structural information concerning such structures. The two methods have proved invaluable in defining unequivocally the nature of the interaction between a rhizobial strain and a legume host.

The Rhizobium - legume symbiosis: observation of root infection by bright-field microscopy after staining with methylene blue

Staining of infected legume roots with 0.01% methylene blue facilitated the observation of the initial steps of the Rhizobium—legume symbiosis. It allowed particularly the visualization by bright-field microscopy of infection threads in the root hairs and the root cortex of the host plant.

Detection and visualization of the major acidic exopolysaccharide of Ralstonia solanacearum and its role in tomato root infection and vascular colonization

Exopolysaccharides play an important role in the pathogenicity of Ralstonia solanacearum. We compared in vitro and in planta exopolysaccharide production of the pathogenic strain AW1 with that of three related mutant strains impaired in both their exopolysaccharide production and aggressiveness on tomato. The distinction between the two hexosamine-rich exopolysaccharides, namely the N-acetyl-glucosaminorhamnan and the major N-acetyl-galactosamine-containing acidic polymer was emphasized. The major acidic polymer was identified specifically by electron microscopy using glutaraldehyde/ruthenium red/uranyl acetate staining, by immunofluorescence using specific monoclonal antibodies and correlated to an appropriate biochemical analysis. The two mutant strains AW1-1 and AW-19A were totally devoid of any production of the major exopolysaccharide in vitro or in planta whatever the technique used. Infection and vascular colonization of tomato roots by the pathogenic strain were also compared to those of the mutant strains by light microscopy. Pathogenicity on tomato was assessed by root infection without any artificial injury. Light microscopy showed that the two mutant strains AW1-1 and AW-19A were poorly infective and unable to invade xylem vessels, while they induced defence mechanisms in root tissues and appeared aggregated or degenerated within cortical infection pockets. These two mutant strains were non-pathogenic or weakly aggressive, respectively. In contrast, the pathogenic strain AW1 and the hypoaggressive AW1-41 strains, which produce large amounts of the major acidic exopolysaccharide in planta, were both infective and invasive, and tomato root tissues exhibited only limited defence reactions. Thus, the major acidic exopolysaccharide produced by Ralstonia solanacearum is involved in root infection and vascular colonization, though its precise role is still unknown.

Xylem Colonization by an HrcV¯ Mutant of Ralstonia solanacearum Is a Key Factor for the Efficient Biological Control of Tomato Bacterial Wilt

Microscopic studies of the colonization of the vascular tissues of tomato by an HrcV¯ (formerly HrpO¯) mutant strain of Ralstonia solanacearum were carried out after either root inoculation of the mutant strain alone or delayed challenge inoculation by a pathogenic strain. The use of two different marker genes, lacZ and uidA, introduced into either mutant or wild-type strains, respectively, permitted histological observation for the presence of both strains simultaneously. In roots, both strains could be found together in infected root tips and in lateral root emergence sites (lateral root cracks), but these bacterial strains subsequently invaded separate xylem vessels in the root system. At the hypocotyl level, a novel staining procedure, in conjunction with bacterial isolation and counting, showed three vascular colonization patterns: exclusive colonization by each of the competitors or simultaneous presence of each strain in separate xylem vessels. The relative frequencies of these patterns depended up...