A peroxisomal sub-family of Arabidopsis NDR1 homologs: Molecular characterization of a novel targeting pathway and the proteins' functions in plant immunity
Arabidopsis NDR1 plays an important role in plant innate immunity and represents the best characterized member of the NDR1/HIN1-like (NHL) protein family. Of the 45 NHL proteins identified in Arabidopsis, none had been proven to act in peroxisomes. Three members of one subclade (NHL4, NHL6, and NHL25) are single-pass membrane proteins with the novel feature of carrying a predicted peroxisomal targeting signal type 1 (PTS1), even though this was presumably specific to soluble matrix proteins. Preliminary data of former group members had shown that the three proteins were primarily targeted to small puncta and were possibly further directed to peroxisomes, as observed (occasionally) in Arabidopsis and tobacco mesophyll protoplasts (Kataya, 2011; Crappe, 2016). To verify peroxisome targeting of the three NHL proteins and characterize their postulated vesicular targeting pathway, a new transient expression system of entire Arabidopsis seedlings of very high biogenetic activity was established in the group. Only very specific fluorophore combinations (EYFP or mVenus with mCer) were found to prevent weak fluorophore heterodimerization and unspecific import of cytosolic proteins into peroxisomes by the so-called piggy-back mechanism and were concluded to be suitable for in vivo peroxisome targeting analyses, as published jointly in Falter et al. (2019). Not only NHL4 terminating with the canonical PTS1, AKL>, but also NHL6 (LRL>) and NHL25 (FRL>) with their non-canonical PTS1 tripeptides were shown to carry functional PTS1 domains that were sufficient to direct EYFP to peroxisomes. The PTS1 domain of NHL4 also interacted with the tetratricopeptide repeat (TPR) domain of the receptor of soluble PTS1 proteins, AtPex5, in yeast two-hybrid (Y2H) analyses. Peroxisome targeting of the three full-length NHL proteins remained difficult to reveal and required longer expression times but was reproducibly detected in a considerable number of transformed cells in Arabidopsis seedlings. Representatively for NHL4, ...