New insights into the immune response of plants

When plants are infected by pathogens, they mount a two-phase immune response, which first develops directly at the infection site and then spreads throughout the entire organism. This prepares the previously unchallenged parts of the plant for a possible attack. Calcium signals play an essential role in this process. When plant tissue is damaged by a pathogen, it triggers calcium signals which are then passed on from cell to cell. In addition, the cells use an NADPH oxidase (an enzyme in the cell membrane) to release reactive oxygen species as further signalling molecules, which then interact with the calcium signals to enable the systemic propagation of the immune response. Until now, researchers did not fully understand this interplay between calcium and reactive oxygen species and the regulation of NADPH oxidase by calcium-dependent phosphorylation.

Jörg Kudla's team showed for the first time that two different kinases, both of which are activated by calcium, have to work together in order to facilitate efficient systemic immune signal proliferation. This "bi-kinase module" sensitises the NADPH oxidase to calcium and enables synergistic activation of this enzyme, which then produces more reactive oxygen species. One of the two calcium-dependent kinases was already known, while the second was identified by the team as part of the recently published study. "Such a calcium-activated bi-kinase module has never been described before," explains Jörg Kudla.

Based on their observations, the biologists proposed a model detailing the mechanisms of systemic immune signalling in plants: Triggered by a pathogen, initially a third kinase inside the infected cell triggers the generation of extracellular reactive oxygen species in the cell, which would then diffuse to the surface of neighbouring cells. Up to this point, the process was understood. The team has now discovered that these reactive oxygen species not only trigger new calcium signals in the neighbouring cells, but also activate the calcium-dependent bi-kinase module, which in turn activates the release of reactive oxygen species.

This causes a renewed influx of calcium into the neighbouring cells. In this way, the signal spreads without the affected cells themselves coming into contact with the pathogen. "Surprisingly, we observed that the intensity of the moving calcium signal is relatively weak and yet sufficient to activate the NADPH oxidase via the bi-kinase module. This is likely caused by sensitisation of this enzyme. We have elucidated the molecular mechanisms of this sensitisation," says Jörg Kudla. "We also suspect that this enables this weak calcium signal to spread from cell to cell without disrupting other signalling calcium-dependent processes that are occurring simultaneously in these cells." How exactly the cells regulate the strength of the calcium signal is not yet known.

For their investigation, the team combined various molecular genetic, cell biological and biochemical methods. The investigation of the propagation of calcium signals in tissues was carried out in transgenic plants of thale cress (Arabidopsis thaliana), in which the researchers analysed biosensor proteins for calcium using high-resolution microscopy. For further investigations, human cell cultures were used in which the plant signalling pathway was reconstituted.

Alongside the Kudla research group, Prof Iris Finkemeier's group from the University of Münster was also involved in the project. The other authors are members of the research group headed by Prof Tina Romeis (formerly at the Free University of Berlin and now at the Leibniz Institute of Plant Biochemistry in Halle).