ABSTRACT
Temporal reverse transcription-polymerase chain reaction (RT-PCR) expression analyses were performed on Trprx2, a new white clover peroxidase, with roots challenged with homologous rhizobia, heterologous rhizobia, and a pathogen, Pseudomonas syringae. Low levels of Trprx2 expression were evident in all rhizobial treatments but in P.syringae-treated clover background expression was dramatically reduced within 1 h and was undetectable in treatments inoculated for more than 3 h. Spraying 4 mM salicylic acid onto seedlings increased Trprx2 expression. These data suggest a defensive role for Trprx2 in white clover and indicate active defense suppression by the pathogen.
Subject(s)
DNA, Complementary/genetics , DNA, Plant/genetics , Fabaceae/enzymology , Fabaceae/genetics , Peroxidase/genetics , Plants, Medicinal , Amino Acid Sequence , Fabaceae/microbiology , Gene Expression , Genes, Plant , Molecular Sequence Data , Plant Roots/enzymology , Plant Roots/microbiology , Pseudomonas/physiology , Rhizobium/physiology , Sequence Homology, Amino Acid , SymbiosisABSTRACT
Traditionally, new loci involved in the Rhizobium-legume symbiosis have been identified by transposon mutagenesis and/or complementation. Wide dispersal of the symbiotic loci in Rhizobium species NGR234, as well as the large number of potential host-plants to be screened, greatly reduces the efficiency of these techniques. As an alternate strategy designed to identify new NGR234 genes involved in the early stages of the symbiosis, we combined data from competitive RNA hybridisation, subtractive DNA hybridisation and shot-gun sequencing. On the assumption that the expression of most nodulation genes is triggered by compounds released by the host-plant, we identified, in the ordered cosmid library of the large symbiotic plasmid pNGR234a, restriction fragments that carry transcripts induced by flavonoids. To target genes not present in the closely related strain R. fredii USDA257, we selected fragments that also carried sequences purified by subtractive DNA hybridisation. Shot-gun sequencing of this subset of fragments lead to the identification of sequences with strong homology to diverse prokaryotic genes/proteins. Amongst these, a symbiotically active ORF from pNGR234a, is highly homologous to the leucine responsive regulatory protein of Escherichia coli (Lrp), is induced by flavonoids, and is not present in USDA257.
Subject(s)
DNA, Bacterial/analysis , Fabaceae/genetics , Nucleic Acid Hybridization , Plants, Medicinal , Rhizobium/genetics , Sequence Analysis, DNA , Symbiosis/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Fabaceae/microbiology , Genetic Complementation Test , Molecular Sequence Data , Mutagenesis, Insertional , Open Reading Frames , Phenotype , Pseudomonas putida/genetics , Restriction Mapping , Rhizobium/physiology , Sequence Analysis, DNA/methods , Sequence Homology, Amino AcidABSTRACT
The diets suitable for the hand rearing of piglets in incubators were examined. Diet A was based on cows' milk heated to 56 degrees C and had bacteriostatic and antiadhesive properties against Escherichia coli. Diet B was based on evaporated cows' milk and did not have these properties. The numbers of coliform bacteria naturally colonising the small intestines of newborn piglets fed entirely on these diets for one week did not differ significantly, however both were significantly higher than in control piglets suckled from birth. Faecal counts of coliforms were similar in all three groups. Examination of the bacteriostatic sensitivity of the isolated strains to sows' milk indicated a predominance of milk-sensitive strains colonising the suckled piglets and a predominance of milk-resistant strains in the piglets fed diet B. This diet-dependent colonisation could not be explained by the in vitro bacteriostatic properties of the diets alone.