Type three secretion systems (TTSSs) are protein translocation mechanisms associated with bacterial pathogenicity in host plants, and hypersensitive reactions in non-host plants. Distribution and diversity of TTSS-like genes within a collection of saprophytic and phytopathogenic fluorescent pseudomonads were characterized. This collection included 16 strains belonging to 13 pathogenic species, and 87 strains belonging to five saprophytic species isolated from plant rhizosphere and soil. Presence of conserved hypersensitive reaction/pathogenicity (hrp) genes (hrc RST) was assessed both by PCR using primers designed to amplify the corresponding sequence and by dot-blot hybridization using a PCR-amplified hrc RST fragment as a probe. PCR allowed the detection of TTSS-like genes in 75% and 32% of the phytopathogenic and saprophytic strains, respectively, and dot-blot hybridization in 100% and 49% of the phytopathogenic and saprophytic strains, respectively. The restriction fragment length polymorphism (RFLP) of 26 amplified hrc RST fragments revealed a considerable diversity. Twenty-one distinct RFLP types were identified and one hrc RST fragment was sequenced per RFLP type. The obtained hrc RST sequences clustered into three groups. Two of these groups included both phytopathogenic and saprophytic strains. The diversity of 16S rRNA genes, commonly used as an evolution marker, was characterized using PCR-RFLP. Polymorphism of the 16S rRNA genes corresponded to that of hrc RST genes, suggesting that these genes have followed a similar evolution. However, the occurrence of few mismatches suggests that sometimes TTSS-like genes might have undergone horizontal genetic transfer.

The distribution of rhcRST genes encoding the type III secretion system (T3SS) in a collection of Bradyrhizobium strains was characterized by PCR and Southern blot hybridization. The polymorphism of the corresponding sequences amplified by PCR was characterized by RFLP and sequencing together with those available in the databank. Genomic group I is characterized by the presence of Bradyrhizobium elkanii strains and group II by the presence of B. japonicum and B. liaoningense strains. Highly conserved T3SS-like genes were detected by PCR in all Bradyrhizobium strains isolated from soybean belonging to genomic group II, and in none of the strains belonging to genomic group I. These data were confirmed by Southern blot hybridization that further indicated the presence of sequences showing similarity to the rhcRST sequence in B. elkanii strains. The high level of conservation of rhcRST among Bradyrhizobia of genomic group II and sharing the same host-plant suggests that T3SS-like genes might have undergone horizontal genetic transfer within this genomic group. When considering the three Rhizobiaceae genera, a clear congruence was recorded between the rhcRST, rRNA gene and ITS sequences in bacteria harbouring sequences encoding T3SS, suggesting a relatively ancient emergence of the T3SS in these genera.