T. Bisseling

Root Hair Deformation Activity of Nodulation Factors and Their Fate on Vicia sativa

We used a semiquantitative root hair deformation assay for Vicia sativa (vetch) to study the activity of Rhizobium leguminosarum bv viciae nodulation (Nod) factors. Five to 10 min of Nod factor-root interaction appears to be sufficient to induce root hair deformation. The first deformation is visible within 1 h, and after 3 h about 80% of the root hairs in a small susceptible zone of the root are deformed. This zone encompasses root hairs that have almost reached their maximal size. The Nod factor accumulates preferentially to epidermal cells of the young part of the root, but is not restricted to the susceptible zone. In the interaction with roots, the glucosamine backbone of Nod factors is shortened, presumably by chitinases. NodRlv-IV(C18:4,Ac) is more stable than NodRlv-V(C18:4,Ac). No correlation was found between Nod factor degradation and susceptibility. Degradation occurs both in the susceptible zone and in the mature zone. Moreover, degradation is not affected by NH4NO3 and is similar in vetch and in the nonhost alfalfa (Medicago sativa).

Rhizobium Lipooligosaccharides Rescue a Carrot Somatic Embryo Mutant

At a nonpermissive temperature, somatic embryos of the temperature-sensitive (ts) carrot cell mutant ts11 only proceed beyond the globular embryo stage in the presence of medium conditioned by wild-type embryos. The causative component in the conditioned medium has previously been identified as a 32-kD acidic endochitinase. In search of a function for this enzyme in plant embryogenesis, several compounds that contain oligomers of N-acetylglucosamine were tested for their ability to promote ts11 embryo formation. Of these compounds, only the Rhizobium lipooligosaccharides or nodulation (Nod) factors were found to be effective in rescuing the formation of ts11 embryos. These results suggest that N-acetylglucosamine-containing lipooligosaccharides from bacterial origin can mimic the effect of the carrot endochitinase. This endochitinase may therefore be involved in the generation of plant analogs of the Rhizobium Nod factors.

Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation.

Rhizobia induce the formation of root nodules on the roots of leguminous plants. In temperate legumes, nodule organogenesis starts with the induction of cell divisions in regions of the root inner cortex opposite protoxylem poles, resulting in the formation of nodule primordia. It has been postulated that the susceptibility of these inner cortical cells to Rhizobium nodulation (Nod) factors is conferred by an arrest at a specific stage of the cell cycle. Concomitantly with the formation of nodule primordia, cytoplasmic rearrangement occurs in the outer cortex. Radially aligned cytoplasmic strands form bridges, and these have been called preinfection threads. It has been proposed that the cytoplasmic bridges are related to phragmosomes. By studying the in situ expression of the cell cycle genes cyc2, H4, and cdc2 in pea and alfalfa root cortical cells after inoculation with Rhizobium or purified Nod factors, we show that the susceptibility of inner cortical cells to Rhizobium is not conferred by an arrest at the G2 phase and that the majority of the dividing cells are arrested at the G0/G1 phase. Furthermore, the outer cortical cells forming a preinfection thread enter the cell cycle although they do not divide.

Modification of Phytohormone Response by a Peptide Encoded by ENOD40 of Legumes and a Nonlegume

The gene ENOD40 is expressed during early stages of legume nodule development. A homolog was isolated from tobacco, which, as does ENOD40 from legumes, encodes an oligopeptide of about 10 amino acids. In tobacco protoplasts, these peptides change the response to auxin at concentrations as low as 10−12 to 10−16 M. The peptides encoded by ENOD40 appear to act as plant growth regulators. Sequence alignment of full ENDO40 gene sequences from soybean, pea, alfalfa, and tocacco plants.

Isolation of total, poly(A) and polysomal RNA from plant tissue.

Most plant material contains relativly high levels of RNase activity that is normally located in the vacuoles. During the RNA extraction procedure RNA should be protected against these endogenous RNases. In this chapter we describe four procedures for the isolation of RNA. In all procedures, a high pH of the extraction buffer and a chelating agent (EDTA or EGTA) are used to prevent RNA degradation. In addition, during the isolation of total RNA (a) detergent(s) (SDS or LiDS, Na-deoxycholate and Nonidet P40) are used. In one procedure, the pulverized plant material is directly thawed in a mixture of phenol and extraction buffer for immediate denaturation of RNase. a) The ‘extraction of total RNA’ is the most simple and convenient method of the four and yields RNA that is directly suitable for in vitro translation and Northern blot analysis [1,2]. After isolation of poly(A) RNA it can be used for cDNA synthesis [2]. This RNA isolation procedure was originally developed for the extraction of RNA from fungi [3] that are notorious for their high RNase activities.

Sym2 of Pea Is Involved in a Nodulation Factor-Perception Mechanism That Controls the Infection Process in the Epidermis

In pea (Pisum sativum) up to 50 nodulation mutants are known, several of which are affected in the early steps of the symbiotic interaction with Rhizobium sp. bacteria. Here we describe the role of the sym2 gene in nodulation (Nod) factor perception. Our experiments show that the sym2A allele from the wild pea variety Afghanistan confers an arrest in infection-thread growth if the Rhizobium leguminosarum bv viciae strain does not produce Nod factors with a NodX-mediated acetylation at their reducing end. Since the induction of the early nodulin gene ENOD12 in the epidermis and the formation of a nodule primordium in the inner cortex were not affected, we conclude that more than one Nod factor-perception mechanism is active. Furthermore, we show that sym2A-mediated control of infection-thread growth was affected by the bacterial nodulation gene nodO.

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH4NO3 on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteroids in the nodule tissue were studied. The addition of NH4NO3 decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [35S]sulfate into the components I and II of nitrogenase was similarly not affected. The addition of NH4NO3 decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.

Expression of plant genes during the development of pea root nodules

The expression of plant genes involved in the pea‐Rhizobium symbiosis was studied by analysing mRNA from root nodules. The RNA was translated in vitro and the translation products were separated by two‐dimensional gel electrophoresis. The results show differential expression of nodulin genes during root nodule development. One gene encoding N‐40′ is expressed at a significant level 5 days before the leghemoglobin genes. Most other nodulin genes are expressed more or less concomitantly with the leghemoglobin genes whereas the N‐21 mRNA is only present late during the development. In the development of ineffective root nodules induced by infection with different nod+fix− mutants of R. leguminosarum all nodulin genes are expressed except for the N‐21 gene. The results suggest that neither bacteroid development, heme excretion nor nitrogen fixation are essential for the induction of nodulin gene expression in the host plant. Further, it appears that the amount of leghemoglobin in ineffective nodules is regulated at a post‐transcriptional level.

Nod factor-induced expression of leghemoglobin to study the mechanism of NH4NO3 inhibition on root hair deformation

Nod factors secreted by Rhizobium leguminosarum by, viciae induce root hair deformation, the formation of nodule primordia, and the expression of early nodulin genes in Vicia sativa (vetch). Root hair deformation is induced within 3 h in a small, susceptible zone (+/-2 mm) of the root. NH4NO3, known to be a potent blocker of nodule formation, inhibits root hair deformation, initial cortical cell divisions, and infection thread formation. To test whether NH4NO3 affects the formation of a component of the Nod factor perception-transduction system, we studied Nod factor-induced gene expression. The differential display technique was used to search for marker genes, which are induced within 1 to 3 h after Nod factor application. Surprisingly, one of the isolated cDNA clones was identified as a leghemoglobin gene (VsLb1), which is induced in vetch roots within 1 h after Nod factor application. By using the drug brefeldin A, it was then shown that VsLb1 activation does not require root hair deformation. The pVsLb1 clone was used as a marker to show that in vetch plants grown in the presence of NH4NO3, Nod factor perception and transduction leading to gene expression are unaffected.

Developmental aspects of Rhizobium-legume symbiosis.

In the plant kingdom a great variety of pathogenic, saprophytic and symbiotic interactions between plants and microorganisms occur, and several of these interactions have been the subject of intensive research. One of the best studied interactions is the symbiosis of Rhizobium, Bradyrhizobium or Azorhizobium bacteria with legume plants, which results in the formation of root nodules, in which bacteria are able to fix atmospheric nitrogen into ammonia. This process of symbiotic N2 fixation is the major naturally occurring mechanism by which nitrogen is reduced and the ecological and agricultural importance of this process has been an important incentive to study this plant-microbe relation. The process of symbiotic N2 fixation has been discussed in several recent reviews [33].