Nod factors activate both heterotrimeric and monomeric G-proteins in <Emphasis Type="Italic">Vigna unguiculata</Emphasis> (L.) Walp

Nod factors are lipo-chito-oligosaccharides secreted by rhizobia that initiate many responses in the root hairs of the legume hosts, culminating in deformed hairs. The heterotrimeric G-protein agonists mastoparan, Mas7, melittin, compound 48/80 and cholera toxin provoke root hair deformation, whereas the heterotrimeric G-protein antagonist pertussis toxin inhibits mastoparan and Nod factor NodNGR[S]- (from Rhizobiumsp. NGR234) induced root hair deformation. Another heterotrimeric G-protein antagonist, isotetrandrine, only inhibited root hair deformation provoked by mastoparan and melittin. These results support the notion that G-proteins are implicated in Nod factor signalling. To study the role of G-proteins at a biochemical level, we examined the GTP-binding profiles of root microsomal membrane fractions isolated from the nodulation competent zone of Vigna unguiculata(L.) Walp. GTP competitively bound to the microsomal membrane fractions labelled with [(35)S]GTPgammaS, yielding a two-site displacement curve with displacement constants ( K(i)) of 0.58 micro M and 0.16 mM. Competition with either ATP or GDP revealed a one-site displacement curve with K(i) of 4.4 and 29 micro M, respectively, whereas ADP and UTP were ineffective competitors. The GTP-binding profiles of microsomal membrane fractions isolated from roots pretreated with either NodNGR[S] or the four-sugar, N- N'- N"- N'"-tetracetylchitotetraose (TACT) backbone of Nod factors were significantly altered compared with control microsomal fractions. To identify candidate proteins, membrane proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose. GTP overlay experiments revealed that membrane fractions isolated from roots pretreated with NodNGR[S] or TACT contained two proteins (28 kDa and 25 kDa) with a higher affinity for GTPgammaS than control membrane fractions. Western analysis demonstrated that membranes from the pretreated roots contained more of another protein (~55 kDa) recognised by Galpha(common) antisera. These results provide pharmacological and biochemical evidence supporting the contention that G-proteins are involved in Nod factor signalling and, importantly, implicate monomeric G-proteins in this process.     

Role of the Differentiation of Root Epidermal Cells in Nod Factor (from Rhizobium meliloti)-Induced Root-Hair Depolarization of Medicago sativa

The stage of differentiation of epidermal cells and the development of root hairs was found to be important for the induction of depolarization in root hairs of Medicago sativa by Nod factor [NodRm-IV(S)] isolated from the bacterium Rhizobium meliloti. The electrical membrane response was concentration dependent, having its major effect (amplitude of the depolarization and number of root hairs that responded) at 10-8 and 10-7 M Nod factor. This response was correlated with a morphological effect of Nod factor in the root-hair-deformation bioassay at similar concentrations. The effect of Nod factor on depolarization and root-hair deformation showed specificity with respect to the structure, since unsulfated Nod molecules were inactive, as was the synthetic N,N',N",N"'- tetraacetylchitotetraose. The Nod factor that is O-acetylated at the nonreducing sugar was as efficient in root-hair deformation and membrane depolarization as the sulfated Nod factor.     

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo-oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis-specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR-factors to signal-production-deficient mutants of the broad-host-range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 ⁻⁷ M and 10⁻⁶ M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non-host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild-type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.     

Pharmacological evidence for activation of phospholipid and small GTP binding protein signalling cascades by Nod factors.

The effects of lipo-chitin oligosaccharide Nod factors (NodNGR[S] from Rhizobium sp. NGR234) on root hair deformation in Vigna unguiculata (L.) Walp. were studied using pharmacological agents to mimic and/or inhibit their action. It was hypothesised that the rearrangement of the cytoskeleton seen during Nod factor induced root hair deformation is modulated by protein kinase C, monomeric G proteins of the Rho superfamily and the location and amount of phosphatidylinositol 3-phosphates (PI3Ps). This hypothesis is supported by the following observations. The protein kinase C activators, 12-deoxyphorbol 13-acetate (DPA) and diacylglycerol kinase inhibitor 1, stimulated root hair deformation to a level similar to that seen with Nod factors or mastoparan, whereas the inhibitor Gö 6976 inhibited root hair deformations induced by NodNGR[S], mastoparan, DPA and diacylglycerol kinase inhibitor 1. The Ras antagonists mevastatin and sulindac sulphide, and the Rho antagonist exoenzyme C3 toxin from Clostridium botulinum all inhibited Nod factor stimulated root hair deformation. Pasteurella multocida toxin activates Rho and stimulated root hair deformation, this stimulation was inhibited by both neomycin and exoenzyme C3 toxin. The PI3 kinase inhibitors, wortmannin and LY-294002 attenuated Nod factor induced root hair deformation. These studies were complemented with actin immunoprecipitations of root hair enriched microsomal membrane preparations from V. unguiculata which pulled down small GTP binding proteins. Root hair deformation is an important early stage in the formation of nitrogen fixing nodules and this study highlights that these processes may depend on signalling cascades involving phospholipids and small GTP binding proteins.     

Identical accumulation and immobilization of sulfated and nonsulfated Nod factors in host and nonhost root hair cell walls

Nod factors are signaling molecules secreted by Rhizobium bacteria. These lipo-chitooligosaccharides (LCOs) are required for symbiosis with legumes and can elicit specific responses at subnanomolar concentrations on a compatible host. How plants perceive LCOs is unclear. In this study, using fluorescent Nod factor analogs, we investigated whether sulfated and nonsulfated Nod factors were bound and perceived differently by Medicago truncatula and Vicia sativa root hairs. The bioactivity of three novel sulfated fluorescent LCOs was tested in a root hair deformation assay on M. truncatula, showing bioactivity down to 0.1 to 1 nM. Fluorescence microscopy of plasmolyzed M. truncatula root hairs shows that sulfated fluorescent Nod factors accumulate in the cell wall of root hairs, whereas they are absent from the plasma membrane when applied at 10 nM. When the fluorescent Nod factor distribution in medium surrounding a root was studied, a sharp decrease in fluorescence close to the root hairs was observed, visualizing the remarkable capacity of root hairs to absorb Nod factors from the medium. Fluorescence correlation microscopy was used to study in detail the mobilities of sulfated and nonsulfated fluorescent Nod factors which are biologically active on M. truncatula and V. sativa, respectively. Remarkably, no difference between sulfated and nonsulfated Nod factors was observed: both hardly diffuse and strongly accumulate in root hair cell walls of both M. truncatula and V. sativa. The implications for the mode of Nod factor perception are discussed.     

Nod factor-induced phosphatidic acid and diacylglycerol pyrophosphate formation: a role for phospholipase C and D in root hair deformation

Rhizobium-secreted nodulation factors are lipochitooligosaccharides that trigger the initiation of nodule formation on host legume roots. The first visible effect is root hair deformation, but the perception and signalling mechanisms that lead to this response are still unclear. When we treated Vicia sativa seedlings with mastoparan root hairs deformed, suggesting that G proteins are involved. To investigate whether mastoparan and Nod factor activate lipid signalling pathways initiated by phospholipase C (PLC) and D (PLD), seedlings were radiolabelled with [(32)P]orthophosphate prior to treatment. Mastoparan stimulated increases in phosphatidic acid (PA) and diacylglycerol pyrophosphate, indicative of PLD or PLC activity in combination with diacylglycerol kinase (DGK) and PA kinase. Treatment with Nod factor had similar effects, although less pronounced. The inactive mastoparan analogue Mas17 had no effect. The increase in PA was partially caused by the activation of PLD that was monitored by its in vivo transphosphatidylation activity. The application of primary butyl alcohols, inhibitors of PLD activity, blocked root hair deformation. Using different labelling strategies, evidence was provided for the activation of DGK. Since the PLC antagonist neomycin inhibited root hair deformation and the formation of PA, we propose that PLC activation produced diacylglycerol (DAG), which was subsequently converted to PA by DGK. The roles of PLC and PLD in Nod factor signalling are discussed.     

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 sp. strain NGR234 NodZ protein is a fucosyltransferase.

Rhizobium sp. strain NGR234 produces a large family of lipochitooligosaccharide Nod factors carrying specific substituents. Among them are 3-O- (or 4-O-) and 6-O-carbamoyl groups, an N-methyl group, and a 2-O-methylfucose residue which may bear either 3-O-sulfate or 4-O-acetyl substitutions. Investigations on the genetic control of host specificity revealed a number of loci which directly affect Nod factor structure. Here we show that insertion and frameshift mutations in the nodZ gene abolish fucosylation of Nod factors. In vitro assays using GDP-L-fucose as the fucose donor show that fucosyltransferase activity is associated with the nodZ gene product (NodZ). NodZ is located in the soluble protein fraction of NGR234 cells. Together with extra copies of the nodD1 gene, the nodZ gene and its associated nod box were introduced into ANU265, which is NGR234 cured of the symbiotic plasmid. Crude extracts of this transconjugant possess fucosyltransferase activity. Fusion of a His6 tag to the NodZ protein expressed in Escherichia coli yielded a protein able to fucosylate both nonfucosylated NodNGR factors and oligomers of chitin. NodZ is inactive on monomeric N-acetyl-D-glucosamine and on desulfated Rhizobium meliloti Nod factors. Kinetic analyses showed that the NodZ protein is more active on oligomers of chitin than on nonfucosylated NodNGR factors. Pentameric chitin is the preferred substrate. These data suggest that fucosylation occurs before acylation of the Nod factors.     

NolL of Rhizobium sp. Strain NGR234 Is Required for O-Acetyltransferase Activity

Following (iso)flavonoid induction, nodulation genes of the symbiotic nitrogen-fixing bacterium Rhizobium sp. strain NGR234 elaborate a large family of lipooligosaccharidic Nod factors (NodNGR factors). When secreted into the rhizosphere of compatible legumes, these signal molecules initiate root hair deformation and nodule development. The nonreducing glucosamine residue of NodNGR factors are N acylated, N methylated, and mono- or biscarbamoylated, while position C-6 of the reducing extremity is fucosylated. This fucose residue is normally 2-O methylated and either sulfated or acetylated. Here we present an analysis of all acetylated NodNGR factors, which clearly shows that the acetate group may occupy position C-3 or C-4 of the fucose moiety. Disruption of the flavonoid-inducible nolL gene, which is preceded by a nod box, results in the synthesis of NodNGR factors that lack the 3-O- or 4-O-acetate groups. Interestingly, the nodulation capacity of the mutant NGRΩnolL is not impaired, whereas introduction of the nod box::nolL construct into the related strain Rhizobium fredii USDA257 extends the host range of this bacterium to Calopogonium caeruleum, Leucaena leucocephala, and Lotus halophilus. Nod factors produced by a USDA257(pnolL) transconjugant were also acetylated. The nod box::nolL construct was also introduced into ANU265 (NGR234 cured of its symbiotic plasmid), along with extra copies of the nodD1 gene. When permeabilized, these cells possessed acetyltransferase activity, although crude extracts did not.     

GTPγS antagonizes the mastoparan-induced in vitro activity of PIP2-phospholipase C from symbiotic root nodules of Phaseolus vulgaris

Phospholipase C (PLC) has been suggested to have a role in signal perception by Nod factors (NFs) in legume root hair cells. For instance, mastoparan, a well-described agonist of heterotrimeric G protein, induces nodulin expression after NFs treatment or Rhizobium inoculation. Furthermore, it has been recently demonstrated that mastoparan also mimics calcium oscillations induced by NFs, suggesting that PLC could play a key role during the nodulation process. In this study, we elucidate a biochemical relationship between PLC and heterotrimeric G proteins during NFs signaling in legumes. In particular, the effect of NFs on in vitro PLC activity from nodule membrane fractions in the presence of guanosine 5'-[γ-thio]triphosphate (GTPγS) and mastoparan was assayed. Our results indicate that for phosphatidylinositol 4,5 bisphosphate (PIP₂)-PLC, there is a specific activity of 20–27 nmol mg⁻¹ min⁻¹ in membrane fractions of nodules 18–20 days after inoculation with Rhizobium tropici . Interestingly, in the presence of 5 μ M mastoparan, PIP₂-PLC activity was almost double the basal level. In contrast, PIP₂-PLC activity was downregulated by 1–10 μ M GTPγS. Also, PLC activity was decreased by up to 64% in the presence of increasing concentrations of NFs (10⁻⁸ to 10⁻⁵  M ). NFs are critical signaling molecules in rhizobia/legume symbiosis that can activate many of the plant's early responses during nodule development. Calcium spiking, kinases, PLC activity and possibly G proteins appear to be components downstream of the NFs perception pathway. Our results suggest the occurrence of a dual signaling pathway that could involve both G proteins and PLC in Phaseolus vulgaris during the development of root nodules.     

Nod signal-induced plasma membrane potential changes in alfalfa root hairs are differentially sensitive to structural modifications of the lipochitooligosaccharide

Lipochitooligosaccharide Nod signals are important determinants of host specificity in the Rhizobium -legume symbiosis. The most rapid response of plant cells to the R. meliloti Nod signal NodRm-IV(C16:2,S) reported so far is the depolarization of the plasma membrane potential in alfalfa root hairs. In order to investigate whether this response may be part of a specific signal transduction cascade involved in the nodulation process, its specificity was studied with respect to host-specific modifications of the lipochitooligosaccharide. Five different Nod factors displaying different degrees of activity in inducing root hair deformation or cortical cell divisions on alfalfa were tested. The ability of the Nod factors to elicit plasma membrane depolarization correlated well with their activity in the bioassays. Removal of the sulfate group (NodRm-IV(C16:2)) led to inactivation of the Nod factor. An increase in the length of the chitooligosaccharide backbone (NodRm-V(C16:2,S)) or saturation of the acyl chain (NodRm-IV(C16:0,S)) resulted in severely reduced activity. In contrast, the O -acetyl group at the non-reducing terminus in NodRm-IV(Ac,C16:2,S), which confers substantially higher activity in long-term bioassays, did not enhance plasma membrane depolarization significantly in comparison with the non- O -acetylated factor. Thus, the rapid plasma membrane response is differentially sensitive to various structural motifs of the lipochitooligosaccharide. These data suggest that the different substituents modifying the basic Nod factor structure may have distinct functions, not all of them contributing to the interaction with a putative receptor in root hair cells. However, the overall specificity of the membrane depolarization for the cognate Nod factors raises the possibility that it is involved in a Nod signal transduction pathway.     

NodS is an S-adenosyl-l-methionine-dependent methyltransferase that methylates chitooligosaccharides deacetylated at the non-reducing end

In response to phenolic compounds exuded by the host plant, symbiotic Rhizobium bacteria produce signal molecules (Nod factors), consisting of lipochitooligosaccharides with strain-specific substitutions. In Azorhizobium caulinodans strain ORS571 these modifications are an O -arabinosyl group, an O -carbamoyl group, and an N -methyl group. Several lines of evidence indicate that the nodS gene located in the nodABCSUIJ operon is implicated in the methylation of Nod factors. Previously we have shown that NodS is an S -adenosyl- l -methionine (SAM)-binding protein, essential for the l -[³H-methyl]-methionine labelling of ORS571 Nod factors in vivo . Here, we present an in vitro assay showing that NodS from either A. caulinodans or Rhizobium species NGR234 methylates end-deacetylated chitooligosaccharides, using [³H-methyl]-SAM as a methyl donor. The enzymatic and SAM-binding activity were correlated with the nodS gene and localized within the soluble protein fraction. The A. caulinodans nodS gene was expressed in Escherichia coli and a glutathione- S -transferase—NodS fusion protein purified. This protein bound SAM and could methylate end-deacetylated chitooligosaccharides, but could not fully methylate acetylated chitooligosaccharides or unmethylated lipo-chitooligosaccharides. These data implicate that the methylation step in the biosynthesis pathway of ORS571 Nod factors occurs after deacetylation and prior to acylation of the chitooligosaccharides.     

Early nodulin gene expression during Nod factor-induced processes in Vicia sativa

Rhizobium leguminosarum bv. viciae -secreted Nod factors are able to induce root hair deformation, the formation of nodule primordia and the expression of early nodulin genes in Vicia sativa (vetch). To obtain more insight into the mode of action of Nod factors the expression of early nodulin genes was followed during Nod factor-induced root hair deformation and nodule primordium formation. The results of these studies suggested that the expression of VsENOD5 and VsENOD12 is not required for root hair deformation. In the Nod factor-induced primordia both VsENOD12 and VsENOD40 are expressed in a spatially controlled manner similar to that found in Rhizobium -induced nodule primordia. In contrast, VsENOD5 expression has never been observed in Nod factor-induced primordia, showing that the induction of VsENOD5 and VsENOD12 expression are not coupled. VsENOD5 expression is induced in the root epidermis by Nod factors and in Rhizobium -induced nodule primordia only in cells infected by the bacteria, suggesting that the Nod factor does not reach the inner cortical cells.     

Nod factors modulate the concentration of cytosolic free calcium differently in growing and non-growing root hairs of Medicago sativa L.

Using Ca²⁺-selective microelectrodes, the concentration of free calcium ([Ca²⁺]) in the cytosol has been measured in root hair cells of Medicago sativa L. in the presence of nodulation (Nod) factors. Growing root hairs of M. sativa displayed a steep apical [Ca²⁺] gradient, i.e. 604–967 nM in the tip compared with 95–235 nM in the basal region. When tested within the first 5 to 10 μm of the tip, addition of NodRm-IV(C16:2,S) decreased the cytosolic [Ca²⁺], whereas an increase was observed when tested behind the tip. Overall, this led to a partial dissipation of the [Ca²⁺] gradient. The Ca²⁺ response was specific: it was equally well observed in the presence of NodRm-IV(Ac,C16:2,S), reduced with NodRm-IV(C16:0,S), but not with chitotetraose, the nonactive glucosamine backbone. In contrast to growing root hairs, non-growing root hairs without a tip-to-base cytosolic [Ca²⁺] gradient responded to NodRm-IV(C16:2,S) with an increase in cytosolic [Ca²⁺] at the tip as well as at the root hair base. We suggest that the response to Nod factors depends on the stage of development of the root hairs, and that changes in cytosolic [Ca²⁺] may play different roles in Nod-factor signaling: changes of cytosolic [Ca²⁺] in the apical part of the root hair may be related to root hair deformation, while the increase in [Ca²⁺] behind the tip may be essential for the amplification of the Nod signal, for its propagation and transduction to trigger downstream events. Received: 5 January 1999 / Accepted: 14 April 1999     

Calcium oscillations and Nod signal transduction in Rhizobium-legume symbiosis

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting a number of key developmental responses in the roots of legume hosts. One of the earliest responses of root hairs to Nod factors is the induction of sharp oscillations of cytoplasmic calcium ion concentration ("calcium spiking"). This response was first characterised in Medicago sativa and Nod factors were found to be unable to induce calcium spiking in a nodulation-defective mutant of M. sativa. The fact that this mutant lacked any morphological response to Nod factors raised the question of whether calcium spiking could be part of a Nod factor-induced signal transduction pathway leading to nodulation. More recently, calcium spiking has been described in a model legume, Medicago truncatula, and in pea. When nodulation-defective mutants were tested for the induction of calcium spiking in response to Nod factors, three loci of pea and two of M. truncatula were found to be necessary for Nod factor-induced calcium spiking. These loci are also known to be necessary for Nod factor-induction of symbiotic responses such as root hair deformation, nodulin gene expression and cortical cell division. These results therefore constitute strong genetic evidence for the role of calcium spiking in Nod factor transduction. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.     

Early interactions between Alnus glutinosa and Frankia strain ArI3. Production and specificity of root hair deformation factor(s)

Actinomycetes from the genus Frankia are able to form symbiotic associations with more than 200 different species of woody angiosperms, so called actinorhizal plants. Many actinorhizal plants are infected via deformed root hairs. Factor(s) eliciting root hair deformation in actinorhizal symbioses have been found to be released into the culture medium, but the factor(s) has (have) not yet been characterized. In the present work, we describe the constitutive production of factor(s) by Frankia strain ArI3 causing root hair deformation on Alnus glutinosa . Deformation was detected after 4–5 h of incubation with both Frankia cultures and their cell-free culture filtrates. When culture filtrate was used, deformation was concentration dependent. A contact time of 2 min between culture filtrate and host roots was sufficient to induce subsequent root hair deformation. No root hair deformation on A. glutinosa could be detected with purified Nod factors from Rhizobium meliloti or R. leguminosarum biovar viciae . No correlation was found between Frankia strains belonging to different host specificity groups and their ability to deform root hairs on A. glutinosa. However, strains not able to deform root hairs on A. glutinosa were also unable to nodulate.     

Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.     

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

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.     

The mode of action of cell wall-degrading enzymes and their interference with Nod factor signalling in <Emphasis Type="Italic">Medicago sativa</Emphasis> root hairs

Medicago sativa L. (alfalfa) root hairs respond to Nod factors [NodRm-IV(C16:2,S)] in a host-specific manner with depolarization and rapid ion fluxes. Protoplasts prepared from these cells using the cell wall-digesting enzymes pectolyase and cellulase do not, or to a rather small extent, respond to Nod factors. In an effort to understand this activity loss we analyzed the mode of action of both enzymes with respect to their effects on the root hairs as well as their interference with the Nod factor response. (i) In the presence of the enzymes, Nod factor at saturating concentrations neither depolarized the plasma membrane of root hairs nor caused ion fluxes. Even after removal of the enzymes, Nod factor responses were strongly refractory. (ii) After a lag-phase of 12-18 s, pectolyase depolarized the plasma membrane, alkalized the external space, acidified the cytosol and increased the cytosolic Ca(2+) activity. (iii) Cellulase, without a lag-phase, depolarized the plasma membrane, acidified the cytosol, but only marginally increased the cytosolic Ca(2+) activity. Unlike pectolyase, the cellulase response was only weakly refractory to a second addition. (iv) Neither enzyme increased the membrane conductance, but pectolyase inhibited the H(+)-pump. (v) Pectolyase shows all the signs of an elicitor, while cellulase yields a mixed response. (vi) Denatured enzymes yielded strong effects similar to those of untreated enzymes. We conclude that the effects shown do not originate from enzymatic activity, but from interactions of the proteins with cell wall or plasma membrane constituents. It is further concluded that these enzymes (pectolyase more so than cellulase) trigger defense-related signal pathways, which makes protoplasts prepared with such enzymes unsuitable for studies of symbiotic or defense-related signalling.