Propagation of pathogen mediated signals in the phloem: Analysis of electrical and chemical signals and gene activation in companion cells

Project leader: Rainer Hedrich (PI), Peter Ache (PI), Gerhard Bringmann (PI)

Staff: Natalya Ivashikina (since 01/04), Inga Kajahn (since 01/04)

Objectives: Pathogen infection of plants causes, despite local defense reactions, a systemic response (systemic acquired resistance = SAR). We investigate the interface of the local response and information-long-distance-transport in the bidirectional transporting phloem system, which plays a crucial role in these processes. The phloem is electrically, hydraulically and chemically/metabolically separated from the surrounding tissue. We want to determine the induction, progression and structure of phloem mobile signals in Arabidopsis thaliana during infection with the pathogenPseudomonas syringae.

Approach: We analyze electrical- and Ca²⁺-dependent mechanisms of fast signal transmissions in the phloem, using different methods. Applying the aphid technique (Fig. 2b), we are able to detect electrical signals resulting from ion fluxes in the phloem network. Calcium-signals are monitored by phloem expressed GFP-aequorin or the cameleon system. We analyze changes in the expression patterns of companion cells using molecular methods (Arabidopsis-cDNA-array, companion-cell-macro-Array). Following laser microdissection of phloem areas (Fig. 1) and isolation of GFP-marked companion cell protoplasts (Fig. 2a) we compare expression patterns of infected with non-infected plants.

Phloem-mobile signals based on chemical compounds play an important role in the plant-microbe interaction. We analyse phloem sap obtained by the aphid technique to identify and characterize known and new compounds. Comparing secondary metabolite profiles of plants before and after Pseudomonas-infection we will gain insights into the time course and coordination of the systemic pathogen response. The chemical analyses are carried out by the group of G. Bringmann (for more details see there).

Fig. 1 Laser microdissection of Arabidopsis thaliana phloem. From a inflorescens stalk cross section (left) a phloem region was cut by a laser beam (middle) and catapulted into a vial (right).

Progress: GFP-marked companion cell isolation (Fig. 2a) resulted in a cell specific EST collection. The present patterns of companion cell specific transcripts seem to reflect phloem-mobile signals off different origins. The corresponding profile of characteristic chemical compounds in phloem sap will be analysed by HPLC-MS, HPLC-NMR, and HPLC-CD.

We transformed Arabidopsis plants with a construct, containing the GFP/Aequorin gene, expressed under the control of a phloem specific promoter to determine the long-distance pathogen signal. Aequorin activity was detectable using a luminometer. Moreover, physical stimulation resulted in phloem mobile calcium signals and action potentials (Fig. 2c,d), which encouraged us to use now virulent/avirulent Pseudomonas strains and related elicitors for stimulation. Subsequently we will follow the onset and propagation of ion channel based signals.

Fig. 2 Electrophysiologcal measurements of Arabidopsis phloem. (a) SUC2-promoter-GFP-plant, a tool to access pure companion cell protoplasts (inset). Notice GFP-fluorescence in vascular bundles of the leaf. (b) „Aphid-technique = bio-electrode" - Separated aphid stylet (arrowhead) and attached microelectrode. (c) Cold-induced action potential (d) Phloem-calcium-response to cold shock.

Significance: We will cluster phloem/companioncell specific genes and elaborate a model for the Arabidopsis-Pseudomonas-interaction with respect to long distance signalling. In addition we want to elucidate to what extent the action potential and the transient calcium signal are involved in the pathogen recognition and the SAR.

Future projects: We characterized the extremely sulphur-rich S-cells, which are the major storage site for glucosinolates. The latter might enter the phloem upon infection by a pathogen. Up to now it remained unclear, whether the bulk of glucosinolates are synthesized in the S- or the companion cells. We will test, whether the genes for glucosinolate biosynthesis are active in the phloem and whether they are inducible by Pseudomonas elicitors. Promoter-reportergene-constructs with respect to phloem specific, and/or interaction regulated genes will be constructed. These tools will enable us to monitor the spatial distribution of the induced promoter activity. Finally we want to determine changes in the electrical signalling using pathogen relevant loss of function mutants.

Collaborations: Members of SFB567; J.P. Metraux, Fribourg, CH

Publications:

• Ache P., Becker D., Deeken R., Dreyer I., Weber H., Fromm J., Hedrich R. (2001). VFK1, a Vicia faba K⁺ channel involved in phloem unloading, Plant J. 27, 571-580

• Deeken R., Geiger D., Fromm J., Koroleva O., Ache P., Langenfeld-Heyser R., Sauer N., May S.T., Hedrich R. (2002). Loss of the AKT2/3 potassium channel affects sugar loading into the phloem of Arabidopsis. Planta 216, 334-344.

• Deeken R., Ivashikina N., Czirjak T., Philippar K., Becker D., Ache P., Hedrich R. (2003). Tumour Development in Arabidopsis thaliana involves the Shaker-like K⁺ Channels AKT1 and AKT2/3. Plant J., 34, 778-787

• Koroleva O.A., Davies A., Deeken R., Thorpe M.R., Tomos A.D., Hedrich R. (2000). Identification of a new glucosinolate-rich cell type in Arabidopsis flower stalk. Plant Physiol. 124, 599-608

• Ivashikina N., Deeken R., Ache P., Kranz E., Pommerrenig B., Sauer N, Hedrich R. (2003). Isolation of AtSUC2:GFP-marked companion cells for patch-clamp studies and expression profiling. Plant J., 36, 931-945

Current external funding: SFB 567 (Mechanisms of the interspecific interactions of organisms)