Suppression of nodulation gene expression in bacteroids of Rhizobium leguminosarum biovar viciae

Abstract

The expression of nod genes of Rhizobium leguminosarum bv. viciae in nodules of Pisum sativum was investigated at both the translational and transcriptional levels. By using immunoblots, it was found that the levels of NodA, NodI, NodE, and NodO proteins were reduced at least 14-fold in bacteroids compared with cultured cells, whereas NodD protein was reduced only 3-fold. Northern (RNA) blot hybridization, RNase protection assays, and in situ RNA hybridization together showed that, except for the nodD transcript, none of the other nod gene transcripts were present in bacteroids. The amount of nodD transcript in bacteroids was reduced only two-to threefold compared with that in cultured cells. Identical results were found with a Rhizobium strain harboring multicopies of nodD and with a strain containing a NodD protein (NodD604) which is activated independently of flavonoids. Furthermore, it was found that mature pea nodules contain inhibitors of induced nod gene transcription but that NodD604 was insensitive to these compounds. In situ RNA hybridization on sections from P. sativum and Vicia hirsuta nodules showed that transcription of inducible nod genes is switched off before the bacteria differentiate into bacteroids. This is unlikely to be due to limiting amounts of NodD, the absence of inducing compounds, or the presence of anti-inducers. The observed switch off of transcription during the development of symbiosis is a general phenomenon and is apparently caused by a yet unknown negative regulation mechanism.Bacteria of the genus Rhizobium are able to establish a symbiosis with leguminous plants, resulting in formation of root nodules in which the bacteria, in an altered form designated as bacteroids, reduce atmospheric nitrogen to ammonia. Successful nodulation is a host-specific process in the sense that Pisum and Vicia species are host plants for Rhizobium leguminosarum biovar (bv.) viciae, alfalfa is a host for R. meliloti, and Trifolium sp. is a host for R. leguminosarum bv. trifolii.Bacterial nod (for nodulation) genes localized on a Sym (for symbiosis) plasmid code for proteins involved in early steps in nodulation. The nodD gene is the only constitutively transcribed nod gene in free-living cells. In R. leguminosarum bv. viciae and R. leguminosarum bv. trifolii, nodD is present as a single copy whereas in R. meliloti four allelic forms, designated nodDi, nodD2, nodD3, and syrM, have been identified. The NodD protein binds specifically to nod boxes (18,19,22,28), conserved DNA sequences in the upstream untranslated region of other nod genes (11,40,46,49), and induces transcription of the other nod genes, provided that NodD protein is activated by an inducer of plant origin. These inducers have been identified as flavones and flavanones (17,34,37,65), while isoflavones and coumarins act as anti-inducers for these species (13,17 early steps of nodulation, as reflected by the Nod-phenotype of nodABC mutants and the strongly reduced nodulation of nodFEL mutants. The products of these genes function in root...

Infection and Invasion of Roots by Symbiotic, Nitrogen-Fixing Rhizobia during Nodulation of Temperate Legumes

“…That the LYK3 and LYK4 knockdown mutants are altered in infection thread initiation and extension may also indicate that Nod on May 12, 2018 by guest http://mmbr.asm.org/ factor production is important for processes beyond root hair deformation (101). Finally, work showing that nod genes are expressed by bacteria in tissues of the nodule that contain actively growing infection threads supports the idea that infection thread propagation probably requires Nod factor-dependent targeting of material to infection thread tips in these cells as well (143,144).…”

The Medicago truncatula Lysine Motif-Receptor-Like Kinase Gene Family Includes NFP and New Nodule-Expressed Genes

“…The occurrence of Nod factor recognition in the infection zone of M. truncatula nodules is corroborated by the expression pattern of LYK3 (Limpens et al, 2005). Also, in nodules, NFP expression coincides with the localization of rhizobial nod gene transcripts and Nod factors, whereas the decline in NFP expression toward the nitrogen-fixing zone coincides with the decline of nod gene transcripts, as well as with organelle redistribution and cytoskeleton reorganization (Sharma and Signer, 1990;Schlaman et al, 1991;Timmers et al, 1998).…”

Rhizobium nodM and nodN genes are common nod genes: nodM encodes functions for efficiency of nod signal production and bacteroid maturation

“…The peribacteroid membranes (which are of plant origin) of the mutant bacteroids looked normal. In light of recent reports that nod genes are switched off after bacteroid development (25,28) it is unlikely that nodM would affect symbiotic nitrogen fixation in the fully developed nitrogenfixing nodule tissue. Our finding that the delayed-nodulation phenotype and the changes in nodule ultrastructure observed with nodM mutants on M. sativa could be suppressed by GlcN or related amino sugars again did not reveal differences between the functions of gimS and nodM.…”

“…After evaporation of the solvent, the different fractions were repurified on the same column by elution with 20% acetonitrile-H20 isocratic for 5 min followed by a 20-min gradient to 60% acetonitrile-H20 without the addition of ammonium acetate. Fractions absorbing at 205 nm were lyophilized, taken up in 60% methanol-H20, and analyzed by fast atom bombardment mass spectrometry (FAB-MS) as described elsewhere (26 (29) by using 1-ml cultures in minimal medium (26) containing 25 ,uCi of [1-14C]acetate (Amersham; specific activity, 55 mCi/mmol). The supernatants of cultures with OD6.…”