Marcelle Holsters

Characterization of different plaque-forming and defective temperate phages in Agrobacterium strains

Four Agrobacterium tumefaciens temperate phages (PB2A, PB6(omega), PV-1(LV-1) and PS8), were shown to have the same genome size. Moreover hybridization experiments by the heteroduplex method and electron microscopy showed a 100% homology between these four phage genomes. Indications for lysogeny were found by direct means for the Agrobacterum timefaciens strain 396, Agrobacterium radiobacter strain 8149 and Agrobacterium species 0362 and by the electron microscope negative staining technique for the A.tumefaciens strains b6-806,b6-6,b6s3,b2as,cv-1,4452,11156,11158,396, and 925; for A.radiobacter strains tr-1 and 8149, the latter being bi-lysogenic, and for the A. species 0362. These isolated phage particles, most of which appear to be defective, could be grouped into different classes. No particles could be detected in the lysates of A. tumefaciens RV3, A. radiobacter strains 4718 and S1005, and A. species 0363. Further characterization by genome size was carried out for the defective temperate phages PB6-806, P4452,P8149 and P0362. No evidence for homology between PB6-806 and PB6 omega could be found. The defective phages PB6-806 and P4452 showed the same morphology but a different genome size, whereas the two phages P0362 and P8149 had a very different morphology and genome size.

CLE Peptides Control Medicago truncatula Nodulation Locally and Systemically

The CLAVATA3/embryo-surrounding region (CLE) peptides control the fine balance between proliferation and differentiation in plant development. We studied the role of CLE peptides during indeterminate nodule development and identified 25 MtCLE peptide genes in the Medicago truncatula genome, of which two genes, MtCLE12 and MtCLE13, had nodulation-related expression patterns that were linked to proliferation and differentiation. MtCLE13 expression was up-regulated early in nodule development. A high-to-low expression gradient radiated from the inner toward the outer cortical cell layers in a region defining the incipient nodule. At later stages, MtCLE12 and MtCLE13 were expressed in differentiating nodules and in the apical part of mature, elongated nodules. Functional analysis revealed a putative role for MtCLE12 and MtCLE13 in autoregulation of nodulation, a mechanism that controls the number of nodules and involves systemic signals mediated by a leucine-rich repeat receptor-like kinase, SUNN, which is active in the shoot. When MtCLE12 and MtCLE13 were ectopically expressed in transgenic roots, nodulation was abolished at the level of the nodulation factor signal transduction, and this inhibition involved long-distance signaling. In addition, composite plants with roots ectopically expressing MtCLE12 or MtCLE13 had elongated petioles. This systemic effect was not observed in transgenic roots ectopically expressing MtCLE12 and MtCLE13 in a sunn-1 mutant background, although nodulation was still strongly reduced. These results suggest multiple roles for CLE signaling in nodulation.

Reactive oxygen species and ethylene play a positive role in lateral root base nodulation of a semiaquatic legume

Lateral root base nodulation on the tropical, semiaquatic legume Sesbania rostrata results from two coordinated, Nod factor-dependent processes: formation of intercellular infection pockets and induction of cell division. Infection pocket formation is associated with cell death and production of hydrogen peroxide. Pharmacological experiments showed that ethylene and reactive oxygen species mediate Nod factor responses and are required for nodule initiation, whereby induction of division and infection could not be uncoupled. Application of purified Nod factors triggered cell division, and both Nod factors and ethylene induced cavities and cell death features in the root cortex. Thus, in S. rostrata, ethylene and reactive oxygen species act downstream from the Nod factors in pathways that lead to formation of infection pockets and initiation of nodule primordia.

Aging in Legume Symbiosis. A Molecular View on Nodule Senescence in Medicago truncatula

Rhizobia reside as symbiosomes in the infected cells of legume nodules to fix atmospheric nitrogen. The symbiotic relation is strictly controlled, lasts for some time, but eventually leads to nodule senescence. We present a comprehensive transcriptomics study to understand the onset of nodule senescence in the legume Medicago truncatula. Distinct developmental stages with characteristic gene expression were delineated during which the two symbiotic partners were degraded consecutively, marking the switch in nodule tissue status from carbon sink to general nutrient source. Cluster analysis discriminated an early expression group that harbored regulatory genes that might be primary tools to interfere with pod filling-related or stress-induced nodule senescence, ultimately causing prolonged nitrogen fixation. Interestingly, the transcriptomes of nodule and leaf senescence had a high degree of overlap, arguing for the recruitment of similar pathways.

Identification of Rhodococcus fascians cytokinins and their modus operandi to reshape the plant

Decades ago, the importance of cytokinins (CKs) during Rhodococcus fascians pathology had been acknowledged, and an isopentenyltransferase gene had been characterized in the fas operon of the linear virulence plasmid, but hitherto, no specific CK(s) could be associated with virulence. We show that the CK receptors AHK3 and AHK4 of Arabidopsis thaliana are essential for symptom development, and that the CK perception machinery is induced upon infection, underlining its central role in the symptomatology. Three classical CKs [isopentenyladenine, trans-zeatin, and cis-zeatin (cZ)] and their 2-methylthio (2MeS)-derivatives were identified by CK profiling of both the pathogenic R. fascians strain D188 and its nonpathogenic derivative D188–5. However, the much higher CK levels in strain D188 suggest that the linear plasmid is responsible for the virulence-associated production. All R. fascians CKs were recognized by AHK3 and AHK4, and, although they individually provoked typical CK responses in several bioassays, the mixture of bacterial CKs exhibited clear synergistic effects. The cis- and 2MeS-derivatives were poor substrates of the apoplastic CK oxidase/dehydrogenase enzymes and the latter were not cytotoxic at high concentrations. Consequently, the accumulating 2MeScZ (and cZ) in infected Arabidopsis tissue contribute to the continuous stimulation of tissue proliferation. Based on these results, we postulate that the R. fascians pathology is based on the local and persistent secretion of an array of CKs.

Decaprenylphosphoryl Arabinofuranose, the Donor of the d-Arabinofuranosyl Residues of Mycobacterial Arabinan, Is Formed via a Two-Step Epimerization of Decaprenylphosphoryl Ribose

The major cell wall polysaccharide of mycobacteria is a branched-chain arabinogalactan in which arabinan chains are attached to the 5 carbon of some of the 6-linked galactofuranose residues; these arabinan chains are composed exclusively of D-arabinofuranose (Araf) residues. The immediate precursor of the polymerized Araf is decaprenylphosphoryl-D-Araf, which is derived from 5-phosphoribose 1-diphosphate (pRpp) in an undefined manner. On the basis of time course, feedback, and chemical reduction experiment results we propose that decaprenylphosphoryl-Araf is synthesized by the following sequence of events. (i) pRpp is transferred to a decaprenyl-phosphate molecule to form decaprenylphosphoryl-beta-D-5-phosphoribose. (ii) Decaprenylphosphoryl-beta-D-5-phosphoribose is dephosphorylated to form decaprenylphosphoryl-beta-D-ribose. (iii) The hydroxyl group at the 2 position of the ribose is oxidized and is likely to form decaprenylphosphoryl-2-keto-beta-D-erythro-pentofuranose. (iv) Decaprenylphosphoryl-2-keto-beta-D-erythro-pentofuranose is reduced to form decaprenylphosphoryl-beta-D-Araf. Thus, the epimerization of the ribosyl to an arabinosyl residue occurs at the lipid-linked level; this is the first report of an epimerase that utilizes a lipid-linked sugar as a substrate. On the basis of similarity to proteins implicated in the arabinosylation of the Azorhizobium caulidans nodulation factor, two genes were cloned from the Mycobacterium tuberculosis genome and expressed in a heterologous host, and the protein was purified. Together, these proteins (Rv3790 and Rv3791) are able to catalyze the epimerization, although neither protein individually is sufficient to support the activity.

The Gram-positive side of plant-microbe interactions

Plant growth and development are significantly influenced by the presence and activity of microorganisms. To date, the best-studied plant-interacting microbes are Gram-negative bacteria, but many representatives of both the high and low G+C Gram-positives have excellent biocontrol, plant growth-promoting and bioremediation activities. Moreover, actinorhizal symbioses largely contribute to the global biological nitrogen fixation and many Gram-positive bacteria promote other types of symbioses in tripartite interactions. Finally, several prominent and devastating phytopathogens are Gram-positive. We summarize the present knowledge of the beneficial and detrimental interactions of Gram-positive bacteria with plants to underline the importance of this particular group of bacteria.

MOLECULAR MECHANISMS OF NOD FACTOR DIVERSITY

The rhizobia–legume symbiosis is highly specific. Major host specificity determinants are the bacterial Nod factor signals that trigger the nodulation programme in a compatible host. Nod factors are lipo‐chitooligosaccharides (LCOs) varying in the oligosaccharide chain length, the nature of the fatty acids and substitutions on the oligosaccharide. The nod genotype of rhizobia, which forms the genetic basis for this structural variety, includes a set of nodulation genes encoding the enzymes that synthesize LCOs. Allelic and non‐allelic variation in these genes ensures the synthesis of different LCO structures by the different rhizobia. The nod genotypes co‐evolved with host plant divergence in contrast to the rhizobia, which followed a different evolution. Horizontal gene transfer probably played an important role during evolution of symbiosis. The nod genotypes are particularly well equipped for horizontal gene transfer because of their location on transmissible plasmids and/or on ‘symbiosis islands’, which are symbiotic regions associated with movable elements.

Srchi13, a Novel Early Nodulin from Sesbania rostrata, Is Related to Acidic Class III Chitinases

On the tropical legume Sesbania rostrata, stem-borne nodules develop after inoculation of adventitious root primordia with the microsymbiont Azorhizobium caulinodans. A cDNA clone, Srchi13, with homology to acidic class III chitinase genes, corresponds to an early nodulin gene with transiently induced expression during nodule ontogeny. Srchi13 transcripts accumulated strongly 2 days after inoculation, decreased from day 7 onward, and disappeared in mature nodules. Induction was dependent on Nod factor–producing bacteria. Srchi13 was expressed around infection pockets, in infection centra, around the developing nodule and its vascular bundles, and in uninfected cells of the central tissue. The specific and transient transcript accumulation together with the lipochitooligosaccharide degradation activity of the recombinant protein hint at a role of Srchi13 in normal nodule ontogeny by limiting the action of Nod factors.

Roles for Azorhizobial Nod Factors and Surface Polysaccharides in Intercellular Invasion and Nodule Penetration, Respectively

In the symbiotic interaction between Azorhizobium caulinodans and Sesbania rostrata root and stem-borne nodules are formed. The bacteria enter the host via intercellular spaces at lateral or adventitious root bases and form infection pockets in outer cortical layers. Infection threads guide the bacteria to nodule primordia where plant cells are invaded. To identify bacterial functions that are required for this infection process, two mutants defective in nodulation were studied; one produced no Nod factors (nodA mutant), the other had altered surface polysaccharides (SPS) and induced the formation of pseudo-nodules. Bacteria were visualized with the help of a nodA-uidA reporter fusion that was functional during nodule development and in bacteroids. In contrast to the SPS mutant, nodA mutants were unable to colonize outer cortical regions. In mixed inoculations with both mutants, functional nodules were formed, the central tissue of which was occupied by the nodA mutant. These observations suggest that SPS...