Gary J. Loake

Chemotaxis of Rhizobium leguminosarum biovar phaseoli towards Flavonoid Inducers of the Symbiotic Nodulation Genes

Summary: Chemotaxis of Rhizobium leguminosarum biovar phaseoli RP8002 towards a range of carbohydrates, phenolic compounds and flavonoids was assayed. Xylose (peak response 10−4 M), sucrose (peak response 10−6 M) and raffinose (peak response 10−5 M) were strong chemoattractants amongst the carbohydrates, whilst glucose, fructose, galactose and maltose produced little or no detectable response. Of the monocyclic phenolic compounds, vanillyl alcohol, p-hydroxybenzoic acid (both peak responses 10−6 M) and 3,4-dihydroxybenzoic acid (peak response 10−4 M) all evoked strong chemotactic responses. Amongst the nod-inducing flavonoids, apigenin and luteolin were both strong chemoattractants (peaks at 10−5 M) while naringenin produced a very low response. Competition experiments suggest that apigenin and luteolin are recognized by a common receptor, but that there exists a separate receptor for luteolin alone. The inhibitors of nod-induction, umbelliferone and acetosyringone, both produced strong chemotactic responses, with peaks at 10−3 M and 10−2 M respectively. This evidence is indicative of a role for chemotaxis towards nod-inducing flavonoids in the initiation of root nodule formation by rhizobia, and also suggests that chemotaxis may influence the host range of the interaction.

virA and virG are the Ti‐plasmid functions required for chemotaxis of Agrobacterium tumefaciens towards acetosyringone

Octopine and nopaline Ti‐plasmids confer upon Agrobacterium tumefaciens C58C1 the ability to respond chemotactically to the vir‐inducing phenolic wound exudate, acetosyringone. A. tumefaciens C58C1 containing Ti‐plasmids with Tn5 insertions in virB, C., D or E exhibited marked chemotaxis towards acetosyringone. However, Ti‐plasmids with mutations in virA or VirG were unable to confer the responsive phenotype. Of the cosmid clones pVK219 (virAB) pVK221 (virBGC) pVK225 (virGCDE) and pVK257 (virABGC) mobilized to cured A. tumefaciens C58C1, only pVK257 bestowed acetosyringone chemotaxis. virA and virG are thus required for chemotaxis of A. tumefaciens towards acetosyringone. This suggests a multifunctional role for virA and virG: at low concentrations of acetosyringone they mediate chemotaxis and at higher concentrations they effect vir‐induction.

Attraction of Agrobacterium tumefaciens C58C1 towards Sugars Involves a Highly Sensitive Chemotaxis System

Summary: Motility of Agrobacterium tumefaciens C58C1 consisted of long straight runs, with relatively few tumbles. Speeds of up to 60 μm s−1 and runs of up to 500 μm were recorded. The propulsive mechanism appeared to resemble that of Rhizobium. Chemotaxis towards carbohydrates resolved four groups of sugars: chemoattractants with peaks at 10−6 M (sucrose, glucose and fructose); 10−5 M (maltose, lactulose and galactose); 10−4 M (raffinose, stachyose and arabinose); and weak or non-attractants (palatinose, lactose, cellobiose and xylose). In descending order, the magnitude of the responses was as follows: sucrose ≫ maltose > lactulose > glucose > galactose/fructose > stachyose/arabinose/raffinose. The amino acids valine and arginine were good chemoattractants with peaks at 10−3 M, but no significant attraction was observed with alanine, cysteine, methionine or glycine. These results are indicative of a highly sensitive chemotaxis system towards sugars in A. tumefaciens C58C1, and suggest a role for this process in the ecology of the organism.

Agrobacterium tumefaciens possesses a fourth flagellin gene located in a large gene cluster concerned with flagellar structure, assembly and motility

The authors have identified a fourth flagellin gene in a 21850 bp region of the Agrobacterium tumefaciens C58C1 chromosome containing at least 20 genes concerned with flagellar structure, assembly and function. Three flagellin genes, flaA, flaB and flaC, orientated rightward, are positioned in a tandem array at the right end, with the fourth, substantially larger gene, flaD, in the opposite orientation, at the left end. Between these lie four apparent operons, two transcribed in each direction (motA, flhB leftward; flgF, flgB rightward) from a divergent position approx 7.5 kb from the left end. This unifies the previously published motA, flgB and flaABC sequences into a single region, also containing the homologues of flhB, flgF and fliI. Site-specific mutagenesis of fliI results in a non-flagellate phenotype, while a Tn5-induced flhB mutant possesses abnormal flagella. Mutagenesis and protein profiling demonstrate that all four flagellins contribute to flagellar structure: FlaA is the major protein, FlaB and FlaC are present in lesser amounts, and FlaD is a minor component. FlaA has anomolous electrophoretic mobility, possibly due to glycosylation.

Transcriptional Regulation of Phytoalexin Biosynthetic Genes

In legumes, isoflavonoid derivatives function as antimicrobial phytoalexins, whereas phytoalexins of solanaceous species are of terpenoid origin. The phenylpropanoid and isoprenoid pathways leading to these phytoalexins are involved in the synthesis of a wide range of secondary metabolites with important functions in plant growth, development and responses to the environment. Elicitation of phytoalexin biosynthesis involves transcriptional activation of the genes encoding enzymes of general phenylpropanoid/terpenoid biosynthesis, and of the genes for the specific branch pathways leading to antimicrobial compounds. In order to understand the molecular controls determining the developmental and environmental regulation of the general and specific enzymes of phytoalexin synthesis, we are studying the promoter regions of three elicitor inducible genes, chalcone synthase (chs, isoflavonoid pathway, general), isoflavone reductase (ifr,isoflavonoid pathway, specific) and 3-hydroxy-3-methylglutaryl CoA reductase (hmgr,terpenoid pathway, general). We describe cis-elements and trans-factors involved in the expression of these genes in relation to their tissue specific expression and response to biotic stress. Two elements, the G-box and H-box, located within 50 bp of the TATA box, are important for regulation of expression of chs and probably hmgr, but are not present in the alfalfa ifr promoter.

Molecular Biology of Chemotaxis in Agrobacterium

Our interest in Chemotaxis stems from the concept that unless bacteria are present in huge numbers, and evenly distributed throughout the soil, they must arrive from somewhere to interact with a plant. Agrobaeterium tumefaeiens thus possesses a multifunctional system capable of triggering either Chemotaxis or gene-induction depending upon the ligand concentration, as part of an intricate mechanism for sensing and guiding A. tumefaeiens towards wounded cells.