Tris(methylthio)methane produced by Mortierella hyalina affects sulfur homeostasis in Arabidopsis.

Microbial volatiles are important factors in symbiotic interactions with plants. Mortierella hyalina is a beneficial root-colonizing fungus with a garlic-like smell, and promotes growth of Arabidopsis seedlings. GC-MS analysis of the M. hyalina headspace and NMR analysis of the extracted essential oil identified the sulfur-containing volatile tris(methylthio)methane (TMTM) as the major compound. Incorporation of the sulfur from the fungal volatile into plant metabolism was shown by 34S labeling experiments. Under sulfur deficiency, TMTM down-regulated sulfur deficiency-responsive genes, prevented glucosinolate (GSL) and glutathione (GSH) diminishment, and sustained plant growth. However, excess TMTM led to accumulation of GSH and GSL and reduced plant growth. Since TMTM is not directly incorporated into cysteine, we propose that the volatile from M. hyalina influences the plant sulfur metabolism by interfering with the GSH metabolism, and alleviates sulfur imbalances under sulfur stress.

PRR2, a pseudo-response regulator, promotes salicylic acid and camalexin accumulation during plant immunity.

Calcium signalling mediated by Calmodulin (CaM) and calmodulin-like (CML) proteins is critical to plant immunity. CaM and CML regulate a wide range of target proteins and cellular responses. While many CaM-binding proteins have been identified, few have been characterized for their specific role in plant immunity. Here, we report new data on the biological function of a CML-interacting partner, PRR2 (PSEUDO-RESPONSE REGULATOR 2), a plant specific transcription factor. Until now, the physiological relevance of PRR2 remained largely unknown. Using a reverse genetic strategy in A. thaliana, we identified PRR2 as a positive regulator of plant immunity. We propose that PRR2 contributes to salicylic acid (SA)-dependent responses when challenged with the phytopathogenic bacterium Pseudomonas syringae. PRR2 is transcriptionally upregulated by SA and P. syringae, enhances SA biosynthesis and SA signalling responses; e.g. in response to P. syringae, PRR2 induces the production of SA and the accumulation of the defence-related protein PR1. Moreover, PRR2 overexpressing lines exhibit an enhanced production of camalexin, a phytoalexin that confers enhanced resistance against pathogens. Together, these data reveal the importance of PRR2 in plant immune responses against P. syringae and suggest a novel function for this particular plant specific transcription factor in plant physiology.

Isolation of a French bean (Phaseolus vulgaris L.) homolog to the beta-glucan elicitor-binding protein of soybean (Glycine max L.).

A high-affinity membrane-bound beta-glucan elicitor-binding protein has been purified from microsomal preparations of French bean (Phaseolus vulgaris L.) roots. A 5900-fold purification was achieved by affinity chromatography of functionally solubilized membrane proteins. The beta-glucan-binding protein had an apparent molecular mass of 78 kDa when subjected to SDS-PAGE. Western blot analysis showed specific crossreactivity of this French bean protein with an antiserum raised against a synthetic peptide representing an internal 15 amino acid fragment of the beta-glucan-binding protein from soybean. Northern blot analysis with a cDNA probe of the soybean beta-glucan-binding protein gene revealed a crosshybridizing transcript of 2.4 kb in French bean. These results indicate that the beta-glucan-binding proteins of French bean and soybean are conserved homologs involved in beta-glucan elicitor recognition.

Functional reconstitution of beta-glucan elicitor-binding activity upon incorporation into lipid vesicles.

In temperature-induced Triton X-114 phase separation experiments the beta-glucan elicitor-binding site from soybean (Glycine max L.) root membranes was identified as (a) hydrophobic membrane protein(s). The Zwittergent 3-12-solubilized beta-glucan-binding proteins were incorporated into lipid vesicles by the detergent-dilution procedure. Reconstituted binding proteins were functional in that binding of the hepta-beta-glucoside ligand was saturable, reversible and of high affinity (K(d)=6-7 nM). Competition studies using beta-glucans with different degrees of polymerization (DP 7-15; DP 15-25) showed effective displacement of the radioligand from the binding site whereas beta-glucan fragments with DP <7 were ineffective. The total amount of reconstituted binding activity was dependent on the acyl chain length of the phospholipids used for the reconstitution with a preference for decanoic (C10) and dodecanoic (C12) chains. Restored ligand binding was maximally 37% as compared to the former detergent-solubilized binding activity. The presence of a lipid environment stabilized the purified beta-glucan-binding proteins.

One-step purification of the beta-glucan elicitor-binding protein from soybean (Glycine max L.) roots and characterization of an anti-peptide antiserum.

A low abundance beta-glucan elicitor-binding protein from soybean was isolated by a rapid, simple and one-step purification method yielding about 9000-fold enrichment. The affinity-based purification technique was more efficient than a procedure that uses conventional methods and preserved the binding activity to a much larger extent. The final preparation consisted of one major protein with an apparent molecular mass of about 75 kDa. Electrophoretic analyses of the purified and photoaffinity-labeled binding protein showed that the native protein was an oligomer with apparent molecular mass of about 240 kDa. A polyclonal anti-peptide antiserum was raised against a synthetic 15-mer internal oligopeptide sequence derived from the 75-kDa protein. The antiserum recognized the purified binding protein in immunoblotting experiments and precipitated the affinity-labeled protein from a crude extract of the membrane fraction.

Induction of H(2)O(2) synthesis by beta-glucan elicitors in soybean is independent of cytosolic calcium transients.

Soybean cell suspension cultures have been used to investigate the role of the elevation of the cytosolic Ca(2+) concentration in beta-glucan elicitors-induced defence responses, such as H(2)O(2) and phytoalexin production. The intracellular Ca(2+) concentration was monitored in transgenic cells expressing the Ca(2+)-sensing aequorin. Two lines of evidence showed that a transient increase of the cytosolic Ca(2+) concentration is not necessarily involved in the induction of H(2)O(2) generation: (i) a Bradyrhizobium japonicum cyclic beta-glucan induced the H(2)O(2) burst without increasing the cytosolic Ca(2+) concentration; (ii) two ion channel blockers (anthracene-9-carboxylate, A9C; 5-nitro-2-(3-phenylpropylamino)-benzoate, NPPB) could not prevent a Phytophthora soja beta-glucan elicitor-induced H(2)O(2) synthesis but did prevent a cytosolic Ca(2+) concentration increase. Moreover, A9C and NPPB inhibited P. sojae beta-glucan-elicited defence-related gene inductions as well as the inducible accumulation of phytoalexins, suggesting that the P. sojae beta-glucan-induced transient cytosolic Ca(2+) increase is not necessary for the elicitation of H(2)O(2) production but is very likely required for phytoalexin synthesis.

Bradyrhizobium japonicum mutants defective in cyclic beta-glucan synthesis show enhanced sensitivity to plant defense responses.

Susceptibility of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum to inducible plant defense metabolites such as phytoalexin and H2O2, was investigated. On the wild-type strain USDA 110 the soybean phytoalexin, glyceollin, showed bacteriostatic activity. Viable bacteria isolated from intact nodules were adapted to glyceollin. H2O2 in physiological concentrations did not affect wild-type bacteria. B. japonicum mutants defective in the biosynthesis of cyclic beta-(1-->3)-(1-->6)-glucans showed higher susceptibility to both phytoalexin and H2O2.

Wound and insect-induced jasmonate accumulation in carnivorous Drosera capensis: two sides of the same coin.

Carnivorous sundew plants catch and digest insect prey for their own nutrition. The sundew species Drosera capensis shows a pronounced leaf bending reaction upon prey capture in order to form an 'outer stomach'. This formation is triggered by jasmonates, phytohormones typically involved in defence reactions against herbivory and wounding. Whether jasmonates still have this function in D. capensis in addition to mediating the leaf bending reaction was investigated here. Wounded, insect prey-fed and insect-derived oral secretion-treated leaves of D. capensis were analysed for jasmonates (jasmonic acid, JA; jasmonic acid-isoleucine conjugate, JA-Ile) using LC-MS/MS. Prey-induced jasmonate accumulation in D. capensis leaves was persistent, and showed high levels of JA and JA-Ile (575 and 55.7 pmol · g · FW(-1) , respectively), whereas wounding induced a transient increase of JA (maximum 500 pmol · g · FW(-1) ) and only low (3.1 pmol · g · FW(-1) ) accumulation of JA-Ile. Herbivory, mimicked with a combined treatment of wounding plus oral secretion (W+OS) obtained from Spodoptera littoralis larvae induced both JA (4000 pmol · g · FW(-1) ) and JA-Ile (25 pmol · g · FW(-1) ) accumulation, with kinetics similar to prey treatment. Only prey and W+OS, but not wounding alone or OS, induced leaf bending. The results indicate that both mechanical and chemical stimuli trigger JA and JA-Ile synthesis. Differences in kinetics and induced jasmonate levels suggest different sensing and signalling events upon injury and insect-dependent challenge. Thus, in Drosera, jasmonates are still part of the response to wounding. Jasmonates are also employed in insect-induced reactions, including responses to herbivory and carnivory.

Methyljasmonate and α-linolenic acid are potent inducers of tendril coiling.

A coiling-inducing factor was isolated from tendrils of Bryonia dioica Jacq. and identified by infrared, (1)H-, (13)C-nuclear magnetic resonance and mass spectrometry as α-linolenic acid. When applied to detached tendrils, exogenous α-linolenic acid, but not linoleic acid or oleic acid, induced tendril coiling. Further investigations showed that metabolites of α-linolenic acid, jasmonic acid and, even more so, methyljasmonate, are highly effective inducers of tendril coiling in B. dioica. Methyljasmonate was most active when administered by air and, in atmospheric concentrations as low as 40-80 nM, induced a full free-coiling response with kinetics similar to mechanical stimulation. Even atmospheric levels as low as 4-5 nM methyljasmonate were still found to be significantly active. Methyljasmonate could be one of the endogenous chemical signals produced in mechanically stimulated parts of a tendril and, being highly volatile, act as a diffusible gaseous mediator spreading through the intracellular spaces to trigger free coiling of tendrils.

Cloning and expression of an Arabidopsis nitrilase which can convert indole-3-acetonitrile to the plant hormone, indole-3-acetic acid.

From an Arabidopsis thaliana cDNA expression library, a cDNA clone was isolated, characterized and sequenced which, at the amino acid level, resembled the Klebsiella ozaenae bromoxynil nitrilase encoded by the bxn gene. The cDNA contained a long open reading frame, starting from two possible neighbouring ATG codons and capable of encoding 340 or 346 amino acids with calculated molecular masses of 37526 Da or 38176 Da, respectively. The sequence similarity between the deduced polypeptides from the Arabidopsis cDNA and bxn was clustered in three domains, one at the C-terminus, one in the center and one near the N-terminus of the two proteins, suggesting important functional elements in these parts of the proteins. The cDNA was cloned into different vectors under the control of the lacZ promotor and was functionally expressed by induction with isopropyl-beta-D-thiogalactoside. Using a combination of high-performance liquid chromatography, monoclonal-antibody based enzyme-linked immunosorbent assay and mass spectroscopy, it was shown that the isolated cDNA clone encodes an enzymatically active nitrilase which is able to convert indole-3-acetonitrile to the plant growth hormone, indole-3-acetic-acid.

The hepta-beta-glucoside elicitor-binding proteins from legumes represent a putative receptor family.

The ability of legumes to recognize and respond to beta-glucan elicitors by synthesizing phytoalexins is consistent with the existence of a membrane-bound beta-glucan-binding site. Related proteins of approximately 75 kDa and the corresponding mRNAs were detected in various species of legumes which respond to beta-glucans. The cDNAs for the beta-glucan-binding proteins of bean and soybean were cloned. The deduced 75-kDa proteins are predominantly hydrophilic and constitute a unique class of glucan-binding proteins with no currently recognizable functional domains. Heterologous expression of the soybean beta-glucan-binding protein in tomato cells resulted in the generation of a high-affinity binding site for the elicitor-active hepta-beta-glucoside conjugate (Kd = 4.5 nM). Ligand competition experiments with the recombinant binding sites demonstrated similar ligand specificities when compared with soybean. In both soybean and transgenic tomato, membrane-bound, active forms of the glucan-binding proteins coexist with immunologically detectable, soluble but inactive forms of the proteins. Reconstitution of a soluble protein fraction into lipid vesicles regained beta-glucoside-binding activity but with lower affinity (Kd = 130 nM). We conclude that the beta-glucan elicitor receptors of legumes are composed of the 75 kDa glucan-binding proteins as the critical components for ligand-recognition, and of an as yet unknown membrane anchor constituting the plasma membrane-associated receptor complex.

Further studies of the role of cyclic beta-glucans in symbiosis. An NdvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1-->3)-beta-glucosyl.

The cyclic beta-(1-->3),beta-(1-->6)-D-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize beta-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic beta-glucan lacking beta-(1-->6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1-->3)-beta-D-glucosyl, a cyclic beta-(1-->3)-linked decasaccharide in which one of the residues is substituted in the 6 position with beta-laminaribiose. Cyclodecakis-(1-->3)-beta-D-glucosyl did not suppress the fungal beta-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic beta-(1-->3),beta-(1-->6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic beta-glucans may function as suppressors of a host defense response.