Damage signalling by endo-parasites: A key role for extracellular ATP and calcium channels

If cells are wounded they need to signal to their neighbours that all of them are potentially under threat and that they must gear up to respond. The signals used are termed "Damage/Danger Associated Molecular Patterns" (DAMPs). One of these is extracellular ATP (eATP). Inside the cell, ATP is used as an energy source to keep cells alive but if the membrane surrounding the cell is breached then it leaks out. Once it's extracellular it can be sensed by neighbouring cells which can then respond by adapting, including launching an immune response. We now know that plants might also use eATP as a DAMP, helping them to respond to wounding and defend themselves against damaging microorganisms and pests. It might also be involved in the regeneration of tissues that can occur after wounding. The problem that we have to solve is how the eATP pathway works. We know that in the model plant Arabidopsis, eATP binds to protein receptors in the cell's outer membrane and that this causes an increase in calcium that acts as the signal to launch a defensive response. We have now found that a protein (CNGC19) that makes up a channel to transport calcium into the cell seems responsible for most of the calcium increase in the root that is caused by eATP. This gives us the opportunity to work out how the eATP receptors make the CNGC channels open to let calcium into the cell. As the receptors can add phosphate to proteins to change their activity, we will start by testing whether purified receptor and CNGC proteins can interact and then test whether the CNGC proteins can be phosphorylated by the receptors. We should then be able to find the individual amino acid building blocks of the CNGC protein that are phosphorylated, enabling us to generate mutants that cannot be phosphorylated. As a good test of whether a protein signalling pathway works, these proteins will be made in another cell type that we can use to measure the calcium that comes into the cell when eATP is added. Receptors and channel should let calcium enter but not receptors and the channel that can't be phosphorylated. To support these studies we will measure the calcium increase in roots of Arabidopsis mutants that lack combinations of receptors and channels and find out which cells this signalling pathway works in. We will add eATP but an additional test is to kill an individual cell to simulate a wounding. This can be done with a laser and this simulates the single cell wounding inflicted by invading cyst nematodes. These cut through outer cell layers of the root to gain access to inner feeding sites. Nematodes inflict significant economic damage worldwide and we shall end this project by testing whether the eATP receptors and CNGCs could be part of the root's resistance responses to their attack.

Extracellular ATP( eATP) is a cross-kingdom "Damage/Danger Associated Molecular Pattern" (DAMP), generated by wounding. DAMPs are now considered to be essential in the co-incident triggering of a full immune response to pathogens. In Arabidopsis, two plasma membrane eATP receptors (P2K1 and P2K2) drive an adaptive transcriptional response, through an increase in cytosolic free calcium as a second messenger. We have discovered that Cyclic Nucleotide-Gated Channel 19 (CNGC19) is a critical calcium channel subunit underpinning the root's eATP-induced calcium increase. We shall use in vitro and in planta tests of receptor-CNGC19 interactions to test the hypothesis that CNGC19 phosphorylation promotes channel opening, augmented by pathway reconstruction in a heterologous expression system. We shall use confocal imaging to determine which root cells use the receptor-CNGC19 module to increase cytosolic free calcium in response to eATP and single cell wounding. This will deliver critical information on the contribution of CNGC19 to longitudinal and radial calcium signalling. The cyst nematode Heterodera schachtii wounds epidermal and cortical root cells as it invades to establish feeding sites. We have found that this wounding phase causes an increase in transcripts for defence and phytohormonal signalling that are also upregulated by eATP, pointing to activation of eATP DAMP signalling. We shall determine whether this transcriptional response requires CNGC19 and also determine whether the receptor-CNGC19 signalling module is required for resistance to H. schachtii infection. Together these studies will deliver fundamental information on the eATP DAMP signalling mechanism, the importance of CNGC19 in eATP and wound signalling and the importance of the eATP receptor-CNGC19 signalling module in protecting plants against cyst nematodes.