Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection

Abstract

During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) The morphological steps involved in the nodulation of leguminous plants by Rhizobium spp. have been clearly described. In general, the initial step involves a characteristic curling of the root hair, followed by an invagination of the root hair cell wall. This invagination develops into an infection thread which grows through and between root hair cells until it finally enters meristematic cells. At this point, bacteria that have grown along the infection thread are budded off and surrounded by a membrane (the peribacteroid membrane) of plant origin (14,15,18).Previously, we identified a series of transposon-(Tn5) induced mutants of R. leguminosarum (7) which were affected in their ability to nodulate Pisum sativum (peas). The mutations were all located within a 10-kilobase-pair (kb) region of DNA which had been shown to contain nodulation and host range genes (6,8). Five different classes of mutants were identified on the basis of the phenotypes of the mutant strains and on the locations of the TnS inserts (7). These observations, taken together with DNA sequence data from this part of the nod region (16; C. A. Shearman, L. Rossen, A. W. B. Johnston, and J. A. Downie, submitted for publication), allowed six different nod genes (called nodA, nodB, nodC, nodD, nodE, and nodF) to be identified (Fig. 1) required for root hair curling, and mutations within these genes blocked nodulation; nodD mutant were Nod-but did induce a delayed root hair curling and deformation response; nodE and class V mutations (e.g., nodE68::TnS) caused a delay in nodulation and induced root hair curlingdeformation (7).Only the nodABC genes are essential for root hair curling and deformation because a strain cured of its symbiotic plasmid but containing the subcloned nodABC genes could induce root hair curling and deformation (16) Table 1; media and general growth conditions were those described by Beringer (2). For inoculation of plants, Rhizobium cultures were grown for 2 days on agar slants containing 10 ml of medium, the bacteria were suspended in 4 ml of water, and 0.1 ml was added to the plant roots (this corresponds to about 5 x 107 bacteria). Bacteria were isolated from surface-sterilized nodules as described previously (3).Growth of plants and analysis of nodulation. Peas (variety Wisconsin Perfection) were surface sterilized and grown in 200 ml of sterile agar plant medium as described previously (3). V. hirsuta seeds were treated with concentrated H2SO4 for 20 min, rinsed three times in sterile water, and then left 552

Characterization of the cycHJKL genes involved in cytochrome c biogenesis and symbiotic nitrogen fixation in Rhizobium leguminosarum

“…Tn5 mutagenesis and isolation of respiration-deficient mutants were done as described previously (37) by screening for mutants with the NADI cytochrome oxidase test (17), staining Tn5-mutagenized colonies on TY medium, and screening for colonies that did not stain blue. Plants were grown and inoculated with R. leguminosarum strains as described previously (13).…”

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

“…In addition, several dicarboxylic acids also inhibit the expression of nod genes in B. japonicum and may therefore be responsible, at least in part, for the observed reduction in nod expression in the bacteroids (442,544). The negative regulation of nod genes seems to be important, since artificial constitutive expression of these genes led to a Fix Ϫ phenotype (270).…”

Regulation of nodulation gene expression by NodD in rhizobia

“…viciae and apparently is caused by ineffective binding of NodD in bacteroids to nod boxes, because of either a conformational change of the protein or its presence in another complex (47). Since high-level constitutive expression of the inducible nod genes in bacteroids results in Fix-nodules (3,24), the expression of these genes is undesirable in bacteroids. Moreover, the transcription of nodD is reduced in bacteroids (46,49).…”