Joanna Kopcińska

Expression of genes encoding PR10 class pathogenesis-related proteins is inhibited in yellow lupine root nodules

Abstract Pathogenesis-related proteins of the PR10 class have been found in many plant species, are induced under various stress conditions and act as common allergens. Here we demonstrate the presence of two PR10 proteins in yellow lupine (Lupinus luteus L. cv. Ventus). Both 17 kDa proteins, referred to as LlPR10.1A and LlPR10.1B, are composed of 156 amino acids, and have 76% parities identity (91% similarity). Identity to homologues from other plants ranges from 25 to 67% (46–82% similarity). Patterns of their expression in lupine organs and tissues were investigated using Western blotting and immunocytochemistry. Both proteins are constitutively expressed in roots, but expression is significantly decreased in young and mature root nodules (9–26 days post infection (dpi)), but not in senescent nodules (36 dpi). Immunocytochemical staining localised the proteins in the parenchymatous tissues of the root and senescent nodule, primarily in the cortex. The PR10 proteins were not detected in nodule bacteroid tissue. Expression in aerial parts of the plant is generally lower and only one of the proteins, LlPR10.1B, is expressed constitutively in the stem, leaf and petiole, while the other, LlPR10.1A, is only present in the stem and is induced in senescent leaves.

Molecular Characterization of pssCDE Genes of Rhizobium leguminosarum bv. trifolii strain TA1: pssD Mutant Is Affected in Exopolysaccharide Synthesis and Endocytosis of Bacteria

We have identified the three genes pssCDE in Rhizobium leguminosarum bv. trifolii TA1. Even though they were almost identical to earlier identified pssCDE genes of R. leguminosarum, they differed in gene lengths and gene overlaps. The predicted gene products of pssCDE genes shared significant homology to prokaryotic glycosyl transferases involved in exopolysaccharide synthesis. The Tn5 insertion in pssD created the nonmucoid mutant that induced non-nitrogen-fixing nodules. The microscopic analysis of the nodules, induced on Trifolium pratense by the pssD133 mutant, showed abnormally enlarged infection threads densely packed with bacteria, which were released from the infection threads in an unusual way. The symbiosomes were observed very rarely and the nodule remained almost empty. Symbiotic phenotype of the pssD133 suggested a correlation between this mutation and defective endocytosis of bacteria into nodule cells.

Mutation in the pssB-pssA intergenic region of Rhizobium leguminosarum bv. trifolii affects the surface polysaccharides synthesis and nitrogen fixation ability

Summary A Rhizobium leguminosarum bv. trifolii Tn 5 transposon mutant deficient in exopolysaccharide biosynthesis was found to form non-nitrogen fixing nodules on clover. Root nodules induced by the mutant contained aberrant infection threads and few bacteroids. Sequence analysis of the transposon insertion site localized the mutation in the pssB-pssA intergenic region affecting the exopolysaccharide biosynthesis. The mutant also showed decreased sensitivity to SDS and deoxycholate and displayed a changed lipopolysaccharide (LPS) banding pattern compared to the wild-type strain TA1. The alteration in the O-polysaccharide part of LPS was confirmed by Western immunoblotting with polyclonal antibodies. LPS preparations of strain TA1 and the mutant strain only reacted with their homologous sera. The common epitopes in LPS from bacteroids and free-living rhizobia were revealed by immunogold assay. The results of this study indicate that the pssB-pssA region of R. leguminosarum bv. trifolii is important for the polysaccharide synthesis.

Review article: The meristem in indeterminate root nodules of Faboideae

In this review, the anatomy of indeterminate legume root nodule is briefly summarized. Next, the indeterminate nodule meristem activity, organization and cell ultrastructure are described in species with a distinct nodule meristem zonation. Finally, the putative primary endogenous factors controlling nodule meristem maintenance are discussed in the context of the well-studied root apical meristem (RAM) of Arabidopsis thaliana.

Infection of clover by plant growth promoting Pseudomonas fluorescens strain 267 and Rhizobium leguminosarum bv. trifolii studied by mTn5-gusA.

Plant growth promoting Pseudomonas fluorescens strain 267, isolated from soil, produced pseudobactin A, 7-sulfonic acid derivatives of pseudobactin A and several B group vitamins. In coinoculation with Rhizobium leguminosarum bv. trifolii strain 24.1, strain 267 promoted clover growth and enhanced symbiotic nitrogen fixation under controlled conditions. To better understand the beneficial effect of P. fluorescens 267 on clover inoculated with rhizobia, the colonization of clover roots by mTn5-gusA marked bacteria was studied in single and mixed infections under controlled conditions. Histochemical assays combined with light and electron microscopy showed that P. fluorescens 267.4 (i) efficiently colonized clover root surface; (ii) was heterogeneously distributed along the roots without the preference to defined root zone; (iii) formed microcolonies on the surface of clover root epidermis; (iv) penetrated the first layer of the primary root cortex parenchyma and (v) colonized endophytically the inner root tissues of clover.

Genetic characterization of the Pss region and the role of PssS in exopolysaccharide production and symbiosis of Rhizobium leguminosarum bv. trifolii with clover

Background and aimsIn the symbiotic bacterium Rhizobium leguminosarum bv. trifolii, a majority of proteins involved in exopolysaccharide (EPS) synthesis are encoded by genes located in a large polysaccharide synthesis cluster (Pss). The aim of this study was genetic characterization of the Pss region in the Rt24.2 strain in the context of EPS production and symbiosis with red clover (Trifolium pratense).MethodsThe expression of genes located in the Pss cluster was determined using constructed pss-lacZ transcriptional fusions. The role of transcriptional regulator RosR in pss transcription was confirmed using a rosR mutant and the Rt24.2(pBR1) strain carrying multiple rosR copies. An EPS-deficient mutant, Rt770 was obtained using a random mutagenesis and mTn5SSgusA40 transposon. Symbiotic properties of the Rt770 strain in interaction with clover were characterized in inoculation experiments. Infection of host roots and nodule occupancy by this mutant were investigated using both light and electron microscopy.ResultsTranscriptional levels of particular pss genes differed significantly; the genes encoding glycosyltransferases and enzymes modifying EPS have promoters of weak activities, whereas those encoding proteins involved in EPS polymerization and export possess stronger promoters. Furthermore, RosR affected expression of some pss genes. A mutation in Rt24.2 pssS encoding glucosyltransferase totally abolished EPS synthesis, decreased motility, and increased sensitivity to some stressors. The pssS mutant Rt770 induced formation of nodules on clover roots, which were ineffective in nitrogen fixation.ConclusionEPS secreted by Rt24.2 is required for both adaptation to soil conditions and the establishment of effective symbiosis with clover plants.

Functional analysis of the nifQdctA1y4vGHIJ operon of Sinorhizobium fredii strain NGR234 using a transposon with a NifA-dependent read-out promoter.

Rhizobia are a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes (Fix phenotype). Synthesis of the nitrogenase and its accessory components is under the transcriptional control of the key regulator NifA and is generally restricted to the endosymbiotic forms of rhizobia known as bacteroids. Amongst studied rhizobia, Sinorhizobium fredii strain NGR234 has the remarkable ability to fix nitrogen in association with more than 130 species in 73 legume genera that form either determinate, indeterminate or aeschynomenoid nodules. Hence, NGR234 is a model organism to study nitrogen fixation in association with a variety of legumes. The symbiotic plasmid pSfrNGR234a carries more than 50 genes that are under the transcriptional control of NifA. To facilitate the functional analysis of NifA-regulated genes a new transposable element, TnEKm-PwA, was constructed. This transposon combines the advantages of in vitro mutagenesis of cloned DNA fragments with a conditional read-out promoter from NGR234 (PwA) that reinitiates NifA-dependent transcription downstream of transposition sites. To test the characteristics of the new transposon, the nifQdctA1y4vGHIJ operon was mutated using either the Omega interposon or TnEKm-PwA. The symbiotic phenotypes on various hosts as well as the transcriptional characteristics of these mutants were analysed in detail and compared with the ineffective (Fix(-)) phenotype of strain NGRΔnifA, which lacks a functional copy of nifA. De novo transcription from inserted copies of TnEKm-PwA inside bacteroids was confirmed by qRT-PCR. Unexpectedly, polar mutants in dctA1 and nifQ were Fix(+) on all of the hosts tested, indicating that none of the six genes of the nifQ operon of NGR234 is essential for symbiotic nitrogen fixation on plants that form nodules of either determinate or indeterminate types.

The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with the wild type, the rosR mutant infected host plant roots much less effectively and its nodule occupation was disturbed. At the ultrastructural level, the most striking differences between the mutant and the wild-type nodules concerned the structure of infection threads, release of bacteria, and bacteroid differentiation. This confirms an essential role of RosR in establishment of successful symbiotic interaction of R. leguminosarum bv. trifolii with clover plants.

Differential Expression of Symbiosis-Related Genes in Yellow Lupine

The genus Lupinus comprises approximately 500 plant species widespread in North and South Americas, Africa, and Southern Europe. Reconstruction of lupine phylogeny based on the analysis of symbiosis-related genes, rbcL, and SSU rRNA genes indicates that lupines represent one of the most ancient genera among Papilionideae (Doyle and Doyle, 1997). It is presumed that lupines retained many unique and ancient features. One of such features seems to be a lupinoid type, collar shaped nodule, which shares the characteristics of both determinate and indeterminate nodules (Golinowski et al., 1987). In the course of our studies, we characterised two genes coding for PR10 class proteins. The unique character of these genes can be recognised in distinct expression patterns in different organs as a response to pathogenic or symbiotic bacteria. The linkage between symbiosis and expression of these two genes appears to be especially intriguing. To understand this relationship, the symbiotic region carrying nodulation functions from Bradyrhizobium sp. WM9 (Lupinus) was characterised. Our further objective is to study plant responses using PR10 genes as molecular markers upon inoculation with bacteria carrying mutations in defined nod functions responsible for the host range determination.

Mutation in the pssZ Gene Negatively Impacts Exopolysaccharide Synthesis, Surface Properties, and Symbiosis of Rhizobium leguminosarum bv. trifolii with Clover

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a nitrogen-fixing symbiosis with clover plants (Trifolium spp.). This bacterium secretes large amounts of acidic exopolysaccharide (EPS), which plays an essential role in the symbiotic interaction with the host plant. This polymer is biosynthesized by a multi-enzymatic complex located in the bacterial inner membrane, whose components are encoded by a large chromosomal gene cluster, called Pss-I. In this study, we characterize R. leguminosarum bv. trifolii strain Rt297 that harbors a Tn5 transposon insertion located in the pssZ gene from the Pss-I region. This gene codes for a protein that shares high identity with bacterial serine/threonine protein phosphatases. We demonstrated that the pssZ mutation causes pleiotropic effects in rhizobial cells. Strain Rt297 exhibited several physiological and symbiotic defects, such as lack of EPS production, reduced growth kinetics and motility, altered cell-surface properties, and failure to infect the host plant. These data indicate that the protein encoded by the pssZ gene is indispensable for EPS synthesis, but also required for proper functioning of R. leguminosarum bv. trifolii cells.