Barbara Łotocka

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.

Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia

Pairwise comparisons of Genista tinctoria (dyer’s weed) rhizobium nodA, nodC, and nodZ gene sequences to those available in databanks revealed their highest sequence identities to nodulation loci of Bradyrhizobium sp. (Lupinus) strains and rhizobia from other genistoid legumes. On phylogenetic trees, genistoid microsymbionts were grouped together in monophyletic clusters, which suggested that their nodulation genes evolved from a common ancestor. G. tinctoria nodulators formed symbioses not only with the native host, but also with other plants of Genisteae tribe such as: Lupinus luteus, Sarothamnus scoparius, and Chamaecytisus ratisbonensis, and they were classified as the genistoid cross-inoculation group. The dyer’s weed root nodules were designated as indeterminate with apical meristem consisting of infected and uninfected 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.

Expression Analysis of PIN Genes in Root Tips and Nodules of Medicago truncatula

Polar auxin transport is dependent on the family of PIN-formed proteins (PINs), which are membrane transporters of anionic indole-3-acetic acid (IAA−). It is assumed that polar auxin transport may be essential in the development and meristematic activity maintenance of Medicago truncatula (M. truncatula) root nodules. However, little is known about the involvement of specific PIN proteins in M. truncatula nodulation. Using real-time quantitative PCR, we analyzed the expression patterns of all previously identified MtPIN genes and compared them between root nodules and root tips of M. truncatula. Our results demonstrated significant differences in the expression level of all 11 genes (MtPIN1–MtPIN11) between examined organs. Interestingly, MtPIN9 was the only PIN gene with higher expression level in root nodules compared to root tips. This result is the first indication of PIN9 transporter potential involvement in M. truncatula nodulation. Moreover, relatively high expression level in root nodules was attributed to MtPINs encoding orthologs of Arabidopsis thaliana PIN5 subclade. PIN proteins from this subclade have been found to localize in the endoplasmic reticulum, which may indicate that the development and meristematic activity maintenance of M. truncatula root nodules is associated with intracellular homeostasis of auxins level and their metabolism in the endoplasmic reticulum.

Symbiosis of Astragalus cicer with its microsymbionts: partial nodC gene sequence, host plant specificity, and root nodule structure

Astragalus cicer (cicer milkvetch) nodule bacteria were investigated for host plant specificity and partial nodC gene sequences, whilst their native host was studied for the microscopic structure of root nodules. The strains under investigation formed nodules not only on the original host but also on Astragalus glycyphyllos, Astragalus sinicus, Lotus corniculatus, and Phaseolus vulgaris. The nodules induced on the cicer milkvetch were classified as indeterminate and characterized by apical, persistent meristem, a large bacteroid region with infected and uninfected cells, and elongated bacteroids singly located inside peribacteroid membranes. By comparison of the partial nodC gene sequences of a representative strain of astragali rhizobia to those contained in the GenBank database, a close symbiotic relationship of A. cicer microsymbionts to Rhizobium sp. (Oxytropis) was found.

Phylogeny of Symbiotic Genes and the Symbiotic Properties of Rhizobia Specific to Astragalus glycyphyllos L.

The phylogeny of symbiotic genes of Astragalus glycyphyllos L. (liquorice milkvetch) nodule isolates was studied by comparative sequence analysis of nodA, nodC, nodH and nifH loci. In all these genes phylograms, liquorice milkvetch rhizobia (closely related to bacteria of three species, i.e. Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium ciceri) formed one clearly separate cluster suggesting the horizontal transfer of symbiotic genes from a single ancestor to the bacteria being studied. The high sequence similarity of the symbiotic genes of A. glycyphyllos rhizobia (99–100% in the case of nodAC and nifH genes, and 98–99% in the case of nodH one) points to the relatively recent (in evolutionary scale) lateral transfer of these genes. In the nodACH and nifH phylograms, A. glycyphyllos nodule isolates were grouped together with the genus Mesorhizobium species in one monophyletic clade, close to M. ciceri, Mesorhizobium opportunistum and Mesorhizobium australicum symbiovar biserrulae bacteria, which correlates with the close relationship of these rhizobia host plants. Plant tests revealed the narrow host range of A. glycyphyllos rhizobia. They formed effective symbiotic interactions with their native host (A. glycyphyllos) and Amorpha fruticosa but not with 11 other fabacean species. The nodules induced on A. glycyphyllos roots were indeterminate with apical, persistent meristem, an age gradient of nodule tissues and cortical vascular bundles. To reflect the symbiosis-adaptive phenotype of rhizobia, specific for A. glycyphyllos, we propose for these bacteria the new symbiovar “glycyphyllae”, based on nodA and nodC genes sequences.

Molecular characterization of SCARECROW (CsSCR) gene expressed during somatic embryo development and in root of cucumber (Cucumis sativus L.)

Somatic embryogenesis (SE) in plants can be used as a model for studying genes engaged in the embryogenic transition of somatic cells. The CsSCARECROW (CsSCR) gene was previously identified among a panel of genes upregulated after the induction of SE in cucumber (Cucumis sativus). The putative CsSCR protein contains conserved GRAS family domains and is extremely similar to AtSCR from Arabidopsis thaliana. SCR proteins are transcription factors involved in root radial patterning and are required for maintenance of the quiescent centre and differentiation of the endodermis. In comparison with other GRAS proteins from cucumber, phylogenetic analyses showed that CsSCR belongs to the SCR cluster. Increased CsSCR transcript accumulation was detected in somatic embryos and roots. Southern blot analysis and screening of the draft version of the cucumber genome confirmed the lack of close homologues in this species. CsSCR transcripts were localized by in situ hybridization in undifferentiated cells in the globular and heart stages of somatic embryogenesis, and in the endodermis of torpedo and cotyledonary stage somatic embryos, and developing primary and lateral roots. This localization was supported by the pattern of reporter gene activity driven by the CsSCR promoter in transgenic cucumber organs. These results suggest that CsSCR is likely to act in tissue radial organization during somatic embryogenesis and root development.

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.