Mutational analysis of the Bradyrhizobium japonicum common nod genes and further nod box-linked genomic DNA regions

Summary By insertional and deletional marker replacement mutagenesis the common nod region of Bradyrhizobium japonicum was examined for the presence of additional, essential nodulation genes. An open reading frame located in the 800 bp large intergenic region between nodD1 and nodA did not appear to be essential for nodulation of soybean. Furthermore, a strain with a deletion of the nodI- and nodJ-like genes downstream of nodC had a Nod⁺ phenotype. A mutant with a 1.7 kb deletion immediately downstream of nodD1 considerably delayed the onset of nodulation. This region carried a second copy of nodD (nodD2). A nodD1-nodD2 double mutant had a similar phenotype to the nodD2 mutant. Using a 22-mer oligonucleotide probe partially identical to the nod box sequence, a total of six hybridizing regions were identified in B. japonicum genomic DNA and isolated from a cosmid library. Sequencing of the hybridizing regions revealed that at least three of them represented true nod box sequences whereas the others showed considerable deviations from the consensus sequence. One of the three nod box sequences was the one known to be associated with nodA, whereas the other two were located 60 to 70 kb away from nif cluster I. A deletion of one of these two sequences plus adjacent DNA material mmutant 308) led to a reduced nodulation on Vigna radiata but not on soybean. Thus, this region is probably involved in the determination of host specificity.Dedicated to Prof. Giorgio Semenza on the occasion of his 60th birthday     

Scopolin, a Biochemical Marker for Resistance to Thielaviopsis basicola in Callus and Crown-Gall Tissue Cultures of Tobacco

Scopolin concentration increased in tissue cultures where Thielaviopsis basicola growth ceased (primary and established callus cultures of the resistant tobacco cultivar Ky 170) while it decreased in tissue cultures where fungal growth persisted (primary and established callus cultures of the susceptible tobacco cultivar Ky 151 and crown-gall cultures of Ky 170 and Ky 151). The concentration of chlorogenic acid, scopoletin, and two other unknown soluble phenols varied after inoculation and no, correlation with tissue culture resistance could be established. Incorporation of Agrobacterium tumefaciens T-DNA in the genome of both cultivars induced a drastic reduction of scopolin in inoculated tissue cultures. T-DNA incorporation had less, influence on uninoculated tissues. Scopolin at concentrations found in tissue cultures was not toxic to the fungus.     

Resistance of Tobacco to Thielaviopsis basicola under Tissue Culture Conditions and Increased Susceptibility after Transformation with Agrobacterium tumefaciens T-DNA

Tobacco callus cultures from a Thielaviopsis basicola (Berk. et Br.) Ferraris resistant cultivar were less severely colonized than callus cultures from susceptible cultivars by the pathogen at all concentrations of kinetin and α-indoleacetic acid tested. However, at concentrations where these substances influenced the morphology of the callus cultures they also influenced the degree of colonization by the fungus. Incorporation of T-DNA of Agrobacterium tumefaciens induced susceptibility in tissue culture of the resistant cultivar but did not significantly modify the reaction of the susceptible cultivar tested.