Władysław Golinowski

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.

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.

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.

Localization of acid phosphatase activity in the apoplast of pea (Pisum sativum L.) root nodules grown under phosphorus deficiency

ACPase activity was localized in the apoplast of pea root nodules under phosphorus deficiency. Pea plants (Pisum sativum L. cv. Sze ciotygodniowy) where inoculated with Rhizobium leguminosarum bv. viciae 248 and were cultured on nitrogen-free medium with phosphate (−N/+P) or phosphate-deficient (−N/−P) one. In comparison with control nodules, P-deficient nodules showed the increase of ACPase activity in plant cell walls and the infection threads. The increase in bacterial ACPase activity under P-deficiency may reflect higher demand for inorganic phosphorus that is necessary for bacteria multiplication within the infection threads. The increase of ACPase activity in nodule apoplast under P stress may enlarge the availability of phosphate for plant and bacteria.