P-372 - Involvement of the vacuolar trafficking pathway in CNL-mediated hypersensitive response.

The hypersensitive response (HR) is a type of programmed cell death response mediated by nucleotide-binding leucine-rich repeat (NLR) proteins, which is often required for full resistance against pathogens. It has been previously reported that perturbations in the vacuolar trafficking pathway have an inhibitory effect on HR. We have used a dominant-negative version of the ESCRT-associated protein SKD1 (SKD1^E232Q) to block the late steps of the vacuolar trafficking pathway. Co-expression of SKD1^E232Q with different NLRs in Nicotiana benthamiana revealed that the inhibition of HR is specific to CNL-type NLRs. However, SKD1^E232Q had no effect on the protein levels and subcellular localization of the CNL RPM1. Instead, SKD1^E232Q appeared to partially inhibit Ca²⁺ influx into the cytosol upon RPM1 activation. Interestingly, no effect on ROS production was observed. The Arabidopsis thaliana calcium channel AtCNGC19 is highly up-regulated during both PTI and ETI. N. benthamiana has two homologs, namely NbCNGC22a, and NbCNGC22b. We have found that NbCNGC22a is constitutively degraded but stabilized at the plasma membrane upon expression of the constitutive active RPM1^D505V. Overexpression of NbCNGC22a could also restore the HR response of RPM1 in the presence of SKD1^E232Q. Our results suggest that NbCNGC22a may be required for CNLs to induce a complete HR response. Further studies will focus on assessing the connection between NbCNGC22a and the vacuolar trafficking pathway.