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Plant growth-promoting rhizobacteria (PGPR) are beneficial microorganisms that colonize the rhizosphere of many plant species and confer beneficial effects, such as an increase in plant growth. PGPR are also well known as inducers of systemic resistance to pathogens in plants. However, the molecular mechanisms involved locally after direct perception of these bacteria by plant cells still remain largely unknown. Burkholderia phytofirmans strain PsJN is an endophytic PGPR that colonizes grapevine and protects the plant against the grey mould disease caused by Botrytis cinerea. This report focuses on local defence events induced by B. phytofirmans PsJN after perception by the grapevine cells. It is demonstrated that, after addition to cell suspension cultures, the bacteria were tightly attaching to plant cells in a way similar to the grapevine non-host bacteria Pseudomonas syringae pv. pisi. B. phytofirmans PsJN perception led to a transient and monophasic extracellular alkalinization but no accumulation of reactive oxygen species or cell death were detected. By contrast, challenge with P. syringae pv. pisi induced a sustained and biphasic extracellular alkalinization, a two phases oxidative burst, and a HR-like response. Perception of the PGPR also led to the production of salicylic acid (SA) and the expression of a battery of defence genes that was, however, weaker in intensity compared with defence gene expression triggered by the non-host bacteria. Some defence genes up-regulated after B. phytofirmans PsJN challenge are specifically induced by exogenous treatment with SA or jasmonic acid, suggesting that both signalling pathways are activated by the PGPR in grapevine.

Secondary metabolites (SMs) play key roles in pathogen responses, although knowledge of their precise functions is limited by insufficient characterization of their spatial response. The present study addressed this issue in Arabidopsis leaves by non-targeted and targeted metabolite profiling of Pseudomonas syringae pv. tomato (Pst-AvrRpm1) infected and adjacent uninfected leaf tissues. While overlap was observed between infected and uninfected areas, the non-targeted metabolite profiles of these regions differed quantitatively and clustering analysis underscores a differential distribution of SMs within distinct metabolic pathways. Targeted metabolite profiling revealed that infected tissues accumulate more salicylic acid and the characteristic phytoalexin of Arabidopsis, camalexin, than uninfected adjacent areas. On the contrary, the antioxidant coumarin derivative, scopoletin, was induced in infected tissues while its glucoside scopolin predominated in adjacent tissues. To elucidate the still unclear relationship between the accumulation of SMs and reactive oxygen species (ROS) accumulation and signalling, a catalase-deficient line (cat2) in which ROS signalling is up-regulated, was used. Metabolic analysis of cat2 suggests that some SMs have important interactions with ROS in redox homeostasis during the hypersensitive response to Pst-AvrRpm1. Overall, the study demonstrates that ROS availability influences both the amount and the pattern of infection-induced SM accumulation.

Laminarin, a beta-1,3 glucan with single beta-glucose branches at position 6, was chemically sulfated to produce PS3 with a degree of sulfation of 2.4. PS3 has previously been shown to activate the salicylic acid (SA) signaling pathway in infiltrated tobacco and Arabidopsis thaliana leaf tissues. Here, we investigated whether PS3 induces systemic defense and resistance responses in tobacco. Using a radiolabeled compound, it was first demonstrated that PS3 remains strictly localized to the infiltrated tissues. PS3 is also resistant to beta-glucanase degradation. In transgenic PR1-beta-glucuronidase (GUS) tobacco plants, PS3 causes a strong increase in GUS activity in treated tissues but none in untreated leaves. PS3-infiltrated tissues challenged with tobacco mosaic virus (TMV) 8 d after elicitor application show a decrease in both the lesion number and the lesion size, whereas treatment with laminarin, the unsulfated native glucan, affected only the lesion number. PS3 does not induce systemic acquired resistance to TMV. PS3 and laminarin show synergistic effects in promoting the oxidative burst in tobacco cell suspensions and in increasing the expression of genes encoding O-methyltransferases of the phenylpropanoid pathway in tobacco plants. No synergistic effect was observed on the expression of either the SA-dependent acidic PR1 gene or the ethylene-dependent basic PR5 gene in tobacco plants.

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.

The capacity of H(2)O(2), the most stable of the reactive oxygen species (ROI), to diffuse freely across biological membranes and to signal gene expression suggests that H(2)O(2) could function as a short-lived second messenger diffusing from cell to cell. We tested this hypothesis in tobacco plants treated with a glycoprotein elicitor. Applied at 50 nM, it induces H(2)O(2) accumulation and the hypersensitive response restricted to the infiltrated zone 1 tissue. Stimulation of a set of defense responses also occurs in the surrounding zone 2 tissue without diffusion of the elicitor. ROI levels in zone 1 were modulated using N-acetyl-L-cysteine (NAC) as a ROI scavenger and Rose Bengal (RB) as a ROI generator. We found that ROI appeared to act as signalling intermediates in pathways leading to salicylic acid accumulation, to PR1, PR5 and 3-hydroxy-3-methylglutarylCoA reductase expression in glycoprotein-treated zone 1 tissues. Compared to the treatment with the elicitor alone, co-infiltration of the glycoprotein and NAC increased the surface of zone 2 showing PR1 and O-methyltransferase expression. Application of RB had the opposite effect. The data suggest that, in our system, ROI did not act as a cell-to-cell diffusible signal to activate PR protein and O-methyltransferase expression in zone 2.

In potato ( Solanum tuberosum ) mitochondria, about two-thirds of the tRNAs are encoded by the mitochondrial genome and one-third is imported from the cytosol. In the case of tRNAGly isoacceptors, a mitochondrial-encoded tRNAGly(GCC) was found in potato mitochondria, but this is likely to be insufficient to decode the four GGN glycine codons. In this work, we identified a cytosolic tRNAGly(UCC), which was found to be present in S.tuberosum mitochondria. The cytosolic tRNAGly(CCC) was also present in mitochondria, but to a lesser extent. By contrast, the cytosolic tRNAGly(GCC) could not be detected in mitochondria. This selective import of tRNAGly isoacceptors into S. tuberosum mitochondria raises further questions about the mechanism under-lying the specificity of the import process.

Samsun NN tobacco cells were transformed with chimeric genes for pathogenesis-related (PR) proteins derived from genomic (PR-1a, GRP) or cDNA (PR-S) clones under the transcriptional control of the cauliflower mosaic virus 35S promoter. Regenerated plants were assayed by RNA and protein gel blotting, and plants showing high specific expression of the inserted genes were selected for self-pollination and seed formation. Inspection of second generation transformants showed that constitutive expression of PR-1a, GRP, and PR-S in tobacco in general does not have an effect on the phenotypic appearance of the plants or the expression of other endogenous PR genes. Furthermore, constitutive expression of the above genes does not affect the susceptibility of the plants to infection with tobacco mosaic virus or alfalfa mosaic virus.