Early responses of tobacco suspension cells to rhizobacterial elicitors of induced systemic resistance.

Colonization of roots by selected strains of fluorescent Pseudomonas spp. can trigger induced systemic resistance (ISR) against foliar pathogens in a plant species-specific manner. It has been suggested that early responses in cell suspension cultures in response to rhizobacterial elicitors, such as generation of active oxygen species (AOS) and extracellular medium alkalinization (MA), are linked to the development of ISR in whole plants. Perception of flagellin was demonstrated to elicit ISR in Arabidopsis, and bacterial lipopolysaccharides (LPS) have been shown to elicit several defense responses and to act as bacterial determinants of ISR in various plant species. In the present study, the LPS-containing cell walls, the pyoverdine siderophores, and the flagella of Pseudomonas putida WCS358, P. fluorescens WCS374, and P. fluorescens WCS417, which are all known to act as elicitors of ISR in selected plant species, were tested for their effects on the production of AOS, MA, elevation of cytoplasmic Ca(2+) (Ca(2+)), and defense-related gene expression in tobacco suspension cells. The LPS of all three strains, the siderophore of WCS374, and the flagella of WCS358 induced a single, transient, early burst of AOS, whereas the siderophores of WCS358 and WCS417 and the flagella of WCS374 and WCS417 did not. None of the compounds caused cell death. Once stimulated by the active compounds, the cells became refractory to further stimulation by any of the active elicitors, but not to the elicitor cryptogein from the oomycete Phytophthora cryptogea, indicating that signaling upon perception of the different rhizobacterial compounds rapidly converges into a common response pathway. Of all compounds tested, only the siderophores of WCS358 and WCS417 did not induce MA; the flagella of WCS374 and WCS417, although not active as elicitors of AOS, did induce MA. These results were corroborated by using preparations from relevant bacterial mutants. The active rhizobacterial elicitors led to a rapid increase in Ca(2+), peaking at 6 min, whereas the inactive siderophores of WCS358 and WCS417 elicited a single spike at 1 min. Elicitation of the cells by cell-wall LPS of WCS358 or the siderophore of WCS374 induced a weak, transient expression of several defense-related genes, including PAL and GST. The spectrum of early responses of the suspension cells was not matched by the expression of ISR in whole tobacco plants against Erwinia carotovora pv. carotovora. Of the live bacterial strains, only WCS358 elicited significant ISR, but application of the LPS or the siderophore of all three strains also elicited ISR. Notably, the absence of elicitation of AOS and MA in suspension-cultured cells but induction of ISR in whole plants by the siderophore of WCS358, which was lost upon treatment with the siderophore-minus mutant of WCS358, indicates that the early responses in suspension cells are not predictive of the ability to induce ISR in whole plants. Possible explanations for these discrepancies are discussed.