The habitat of the nitrogen-fixing endophyte Azoarcus sp. strain BH72 is grass roots grown under waterlogged conditions that produce, under these conditions, ethanol. Strain BH72 is well equipped to metabolize ethanol, with eight alcohol dehydrogenases (ADHs), of which ExaA2 and ExaA3 are the most relevant ones. exaA2 and exaA3 cluster and are surrounded by genes encoding two-component regulatory systems (TCSs) termed ExaS-ExaR and ElmS-GacA. Functional genomic analyses revealed that i) expression of the corresponding genes was induced by ethanol, ii) the genes were also expressed in the rhizoplane or even inside of rice roots, iii) both TCSs were indispensable for growth on ethanol, and iv) they were important for competitiveness during rice root colonization. Both TCSs form a hierarchically organized ethanol-responsive signal transduction cascade with ExaS-ExaR as the highest level, essential for effective expression of the ethanol oxidation system based on ExaA2. Transcript and expression levels of exaA3 increased in tcs deletion mutants, suggesting no direct influence of both TCSs on its ethanol-induced expression. In conclusion, this underscores the importance of ethanol for the endophytic lifestyle of Azoarcus sp. strain BH72 and indicates a tight regulation of the ethanol oxidation system during root colonization.

Azoarcus sp. strain BH72 is an endophytic betaproteobacterium able to colonize rice roots without induction of visible disease symptoms. BH72 possesses one polar flagellum. The genome harbors three copies of putative fliC genes, generally encoding the major structural protein flagellin. It is not clear whether, in endophytic interactions, flagella mediate endophytic competence or act as MAMPs (microbe-asscociated molecular patterns) inducing plant defense responses. Therefore, possible functions of the three FliC proteins were investigated. Only fliC3 was found to be highly expressed in pure culture and in association with rice roots and to be required for bacterial motility, suggesting that it encodes the major flagellin. Endophytic colonization of rice roots was significantly reduced in the in-frame deletion mutant, while the establishment of microcolonies on the root surface was not affected. Moreover, an elicitation of defense responses related to FliC3 was not observed. In conclusion, our data support the hypothesis that FliC3 does not play a major role as a MAMP but is required for endophytic colonization in the Azoarcus-rice interaction, most likely for spreading inside the plant.

The endophytic bacterium Azoarcus sp. strain BH72 is capable of colonizing the interior of rice roots, where it finds suitable physicochemical properties for multiplying and fixing nitrogen. Because these properties are poorly understood, a microtiter-plate-based screening of a transcriptional gfp (green fluorescent protein) fusion library of Azoarcus sp. grown under different conditions was performed. Monitoring of the GFP activity allowed the identification of a gene highly expressed in medium supplemented with ethanol. Sequence analysis revealed that this gene encodes a pyrrolo-quinoline quinone-dependent alcohol dehydrogenase (ADH). Inspection of the complete genome sequence of the Azoarcus sp. strain BH72 identified seven additional genes encoding putative ADH, indicating that BH72 is well equipped to survive in different environmental conditions offering various alcohols as carbon source. Analyses of these eight putative ADH showed that expression of three was induced by ethanol, of which two were also expressed inside rice roots. The fact that waterlogged plants such as rice accumulate ethanol suggests that ethanol occurs in sufficiently high concentration within the root to induce expression of bacterial ADH. Disruption of these two ADH evoked a reduced competitiveness to the wild type in colonizing rice roots internally. Thus, it is likely that ethanol is an important carbon source for the endophytic life of Azoarcus sp.

Interactions between legumes and rhizobia are controlled by the sequential exchange of symbiotic signals. Two different techniques, 2D-PAGE electrophoresis and differential display were used to study the effects of rhizobial signals on legume development. Application of variously substituted lipo-oligo-saccharidic Nod-factors to roots of Vigna unguiculata resulted in changes in the phosphorylation patterns of microsomal proteins. Reliable amino-acid sequences were obtained for one Nod-factor enhanced protein which was highly homologous to the 57-kDa subunit from Arabidopsis thaliana vacuolar membrane H(+)-ATPase. Immuno-blotting techniques demonstrated that Nod-factors cause rapid and massive increases of this enzyme in treated roots, suggesting that H(+)-ATPases play symbiotic roles. Concomitantly, we used differential display (DD) techniques on mRNA isolated from root-hairs to analyse early root responses to NGR234. Significant matches of several DD clones to known sequences were found. Clone D2.62 was homologous to a multitude of receptor kinases including S receptor-like kinases of A. thaliana and clone D4.1 showed similarities to Lotus japonicus phosphatidylinositol transfer-like protein III and late nodulin 16. Independent confirmatory analyses of these differentially expressed clones indicated expression at very low levels.

The FEBS JournalVolume 274, Issue s1 p. 6-17 Free Access Symposia Lectures First published: 16 July 2007 https://doi.org/10.1111/j.0014-2956.2007.05866_1.xCitations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume274, Issues1July 2007Pages 6-17 RelatedInformation

Nature Biotechnology 24, 1385–1391 (2006); published online 22 October 2006; corrected after print 26 March 2007 In the version of this article initially published, the gene name nosRZDYFLX on p. 1388, left column, line 5, is incorrect; the correct name is nosZRDFYL. The error has been corrected in the PDF version of the article.

Nature Biotechnology 24, 1385–1391 (2006); published online 22 October 2006; corrected after print 26 March 2007 In the version of this article initially published, the gene name nosRZDYFLX on p. 1388, left column, line 5, is incorrect; the correct name is nosZRDFYL. The error has been corrected in the PDF version of the article.

Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agrobiotechnological interest because it supplies biologically fixed nitrogen to its host and colonizes plants in remarkably high numbers without eliciting disease symptoms. The complete genome sequence is 4,376,040-bp long and contains 3,992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. Coding sequences involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant-associated bacteria. Strain BH72 appears to be 'disarmed' compared to plant pathogens, having only a few enzymes that degrade plant cell walls; it lacks type III and IV secretion systems, related toxins and an N-acyl homoserine lactones–based communication system. The genome contains remarkably few mobile elements, indicating a low rate of recent gene transfer that is presumably due to adaptation to a stable, low-stress microenvironment.

Flagellin is the bulk protein secreted by Bradyrhizobium japonicum. For easier identification of minor protein fractions, the flagellin genes bll6865 and bll6866 were deleted. Extracellular proteins of the corresponding mutant were purified and separated by 2D gel electrophoresis. Several of the protein spots were detectable only after addition of genistein to the growth medium—genistein is an isoflavone secreted by soybean that activates the expression of genes encoding a type III secretion system. These secreted proteins were not present in supernatants of mutants in which conserved genes of the type III secretion system or the regulatory gene ttsI, which is essential for activation of the type III secretion system, are deleted. Out of 22 genistein-inducible protein spots 8 different proteins could be identified by mass spectrometry. One of the proteins, Blr1752, has similarity to NopP of Rhizobium sp. strain NGR234 that is known to be secreted. Another protein is Blr1656 (GunA2) that was shown previously to have endoglucanase activity. Three proteins have similarity to subunits of the flagellar apparatus. Some proteins appeared in several separate spots indicating posttranslational modification. A conserved tts box motif was found in the putative promoter region of six genes encoding secreted proteins.

Im Gegensatz zu online Job- oder Automobilbörsen leidet der internetbasierte Immobilienmarkt unter einem hohen Maß an Fragmentierung. Trotz der einsetzenden Konsolidierung konnte aufgrund von marktimmanenten Problemen bisher keine der existierenden Börsen eine kritische Masse erreichen. Vor diesem Hintergrund und in Anbetracht der Tatsache, dass sich die Erosion von Kleinanzeigen aus den Printmedien hin ins Internet fortsetzen wird, wurde eine Case Study durchgeführt, die das Design und die Erfolgsfaktoren von internetbasierten Immobilienbörsen analysiert.

Sequencing the symbiotic region of Bradyrhizobium japonicum revealed a gene cluster (tts) encoding a type III secretion system (TTSS) that is similar to those found in Mesorhizobium loti MAFF303099 and Rhizobium strain NGR234. In addition to genes that are likely to encode structural core components of the TTSS, the cluster contains several open reading frames that are found exclusively in rhizobia or that are specific to B. japonicum. Depending on the host, mutations within this cluster affected nodulation capacity to different extents. One of the genes likely encodes a transcriptional activator (TtsI) of the two-component regulatory family. Upstream of ttsI, a nod box promoter was identified. Expression of ttsI could be induced by genistein. This induction depended on the transcriptional activator protein NodW as well as the nodD1nodD2nolA gene region. TtsI was found to be involved in transcriptional regulation of the tts gene cluster. Sequence comparison revealed a conserved tts box element within putative promoter regions of several genes. Here, we propose a model of the regulatory cascade leading to the induction of the tts gene cluster.

Three cDNAs (ext3, ext127, and ext26), originally isolated by differential screening from a root-hair cDNA library of Vigna unguiculata, were found to encode extensin-like cell wall proteins. Transcripts homologous to these cDNAs were only detected in root hairs where mRNA levels decreased 1 day after inoculation with rhizobia. This coincided with the onset of root-hair deformation, the first morphological step in the Rhizobium-legume interaction. Decreases in transcript levels following inoculation with wild-type Rhizobium sp. NGR234 were more pronounced than with NGR delta nodABC, a mutant deficient in Nod-factor production. Inoculation with a rhizobial strain carrying a mutation in a gene encoding a transcriptional activator for nod genes (NGR delta nodD1) did not repress mRNA levels, indicating that a second nodulation signal may be present that is nodD dependent. Application of purified NodNGR factors only affected transcript levels of ext3. The genomic locus of the gene homologous to ext26 (Ext26G) was cloned. In the 5' flanking region, several potential TATA boxes and CAP signals were identified. Part of the promoter region shares homology with the Pisum sativum seed lectin promoter and the Nicotiana tabacum nitrate reductase promoter region. Nonetheless, the function of these homologous regions in gene regulation is unknown.

Chalcone synthase (CHS) of Vigna unguiculata is encoded by a gene family that is abundantly transcribed in leaves and nodules. Inoculation with Rhizobium sp. NGR234, which nodulates V. unguiculata, or with NGR delta nodABC, a mutant deficient in Nod factor production, induced rapid accumulation of CHS mRNAs in roots and root hairs. As both Nod+ and Nod- bacteria provoke responses, induction of CHS gene expression may involve symbiotic or defense responses. Four days after inoculation with the wild-type Rhizobium sp., the transcript levels increased in roots but decreased in root hairs. Use of a region unique to the 5' end of a specific CHS gene (VuCHS1) showed that increases of transcript levels in root hairs 24 h after inoculation with both rhizobia were specific to this gene. Transcripts of this gene in roots were only detectable 4 days after treatment with NGR234. It is possible therefore that accumulation of VuCHS1 follows the infection pathway of rhizobia entering legume roots. Purified Nod factors induced accumulation of transcripts, showing that they might be part of the signal transduction pathway leading to CHS expression.

Temporal and spatial observations on the attachment of Nod-factors were made by biotinylating the reducing terminus of the lipo-chitooligosaccharides of the broad host-range Rhizobium sp. NGR234 with the fluorescent reagent 2-amino-(6-amidobiotinyl)pyridine. Complex formation between the biotinylated Nod-factors and fluorescent streptavidin allowed localisation of the binding-site. At concentrations of > 10-7 M, these fluorescently tagged moleculs bound rapidly and asymmetrically (≅ 1 min) to nodulation competent root-hairs but they did not bind to root-hairs of the non-host Arabidopsis thaliana. Early cellular events within the root-hairs were studied by loading root-segments with the calcium indicators Fura-2 and Fluo-3. Fluorescence ratio imaging showed that addition of NodNGR factors provoked almost immediate, plateau-like increases in intra-cellular free calcium {[Ca2+]i} in root-hairs and epidermal cells. Confocal laser scanning microscopy (CLSM) revealed that calcium accumulation was concentrated at the tips and the sides of the responsive root-hairs. A gene encoding a lipid transfer-like protein (LPT2) was isolated from a Vigna unguiculata root-hair cDNA bank. Levels of the LTP2-transcript increased in root-hairs 24 h after treatment with NGR234, or its Nod-factors. The LTP2-gene was cloned into the pMalTM expression vector and LTP2 purified by affinity chromatography. It was unable to transfer phospholipids between liposomes and mitochondria. Anti-sense analysis in which the LTP2 coding region was cloned between the 35S promoter and terminator sequences reduced nodulation when transformed into V. unguiculata.

The incidence of abnormal flower development in some clonal variants of tissue-culture derived oil palm is high. This is attributed to hormonal changes, genetic changes or both but conclusive evidence is lacking. Hence, an attempt was made to isolate a homeotic gene involved in the control of flower morphogenesis. The deficiens gene which has been characterised only in dicotyledenous plants, was identified based on the preliminary results obtained. The possible role of this gene and a likely mechanism has been suggested to explain the occurrence of mantled inflorescences in tissue-culture derived oil palm.

A cDNA library was constructed from RNA of Vigna unguiculata root hairs harvested 1 day and 4 days after inoculation with Rhizobium sp. NGR234. A heterologous probe was used to identify a cDNA clone, the predicted 99-amino-acid sequence of which shares homology with a nonspecific lipid transfer protein (LTP) of Hordeum vulgare. Other characteristics, including an estimated molecular weight of 10.4 kD, an isoelectric point of 8.6, and a signal peptide with a hydrophobic region at the amino-terminal end, are shared by most LTPs. A transcript of 630 nt was found in all tissues tested, except nodules. Levels of mRNA increased in root hairs 24 hr after treatment with Rhizobium sp. NGR234, with different hormones, or with Nod factors. Amounts of transcripts were dependent on the concentration of Nod factors. Accumulation of transcripts during nodule development correlated with root hair deformation, the first visible step in the Rhizobium-legume symbiosis.