A peroxisomal sub-family of Arabidopsis NDR1 homologs: Molecular characterization of a novel targeting pathway and the proteins' functions in plant immunity

Abstract

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, peroxisome targeting was confirmed in stable transgenic Arabidopsis lines upon expression from its endogenous promoter. By a comprehensive set of additional methods, including confocal microscopy of fluorophore-tagged full-length, mutated, and deletion constructs and also transmission electron microscopy, the complex trafficking pathway of the NHL proteins was described in detail. Accordingly, it starts at the ER and continues via vesicle-like cargo structures to so-called “docking structures” between mature peroxisomes and attached vesicles and ends at peroxisomes as the NHL proteins’ final destination and site of physiological activity. The results allowed the deduction of a model for this novel PTS1-dependent, multi-step vesicular trafficking pathway of these signal-anchor proteins to plant peroxisomes. To reveal the proteins’ physiological functions, gene expression analyses using publicly available microarray data were verified by qRT-PCR and demonstrated that NHL4 was strongly induced by abscisic acid (ABA), while NHL6 and NHL25 were highly expressed upon infection by virulent Pseudomonas syringae pv. tomato DC3000 (Pst) and two avirulent Pst strains expressing the effector proteins, AvrRpm1 and AvrRpt2. As revealed by transgenic Arabidopsis GUS reporter lines, the promoters of NHL4, NHL6, and NHL25 were constitutively active specifically in tissue related to defense, including hydathodes and vascular tissue, as well as in stomata and trichomes (for NHL6), which further strengthened the indicated protein functions in pathogen defense. To characterize their functions in innate immunity, single, double, and triple nhl knockout (KO) mutants were infected with the above-mentioned avirulent bacteria. However, bacterial proliferation was not significantly different between the nhl mutants and wild-type plants (WT). In-depth kinetic analyses of electrolyte leakage in KO mutants were carried out to investigate a potential regulatory role of the NHL proteins in the hypersensitive response, as reported for NDR1. Upon infection with Pst expressing avrRpt2, the three single nhl KO mutants showed a higher relative electrolyte leakage compared to WT, and this effect was not additive in the double and triple mutants, indicating that each NHL has an important non-redundant function in restricting the hypersensitive response locally. To study the hypothesized functions of the NHL proteins in ABA-dependent plant responses, the inhibitory effect of ABA on seed germination was investigated. Compared to WT seeds, germination of the single KO mutants, nhl4-1 and nhl6-1, was significantly less inhibited by ABA, indicating that NHL4 and NHL6 might play a role in ABA-mediated inhibition of seed germination and possibly also in ABA-transduced abiotic stress responses. Bacterial growth analyses of E. coli transformants producing soluble NHL4 further confirmed that the protein has typical properties of LEA proteins that are able to confer hypertonic stress resistance in E. coli, most likely by protecting specifically sensitive proteins from osmotic damage. This result implied a similar stress-protective chaperone function of NHL4 in planta. Based on the characterized properties of these three NHLs, their physiological roles in ABA-mediated responses (for NHL4) and plant defense (for NHL6 and NHL25) are proposed.