An Arabidopsis Lipid Flippase Is Required for Timely Recruitment of Defenses to the Host–Pathogen Interface at the Plant Cell Surface

Abstract

Deposition of cell wall-reinforcing papillae is an integral component of the plant immune response. The Arabidopsis PENETRATION 3 (PEN3) ATP binding cassette (ABC) transporter plays a role in defense against numerous pathogens and is recruited to sites of pathogen detection where it accumulates within papillae. However, the trafficking pathways and regulatory mechanisms contributing to recruitment of PEN3 and other defenses to the host-pathogen interface are poorly understood. Here, we report a confocal microscopy-based screen to identify mutants with altered localization of PEN3-GFP after inoculation with powdery mildew fungi. We identified a mutant, aberrant localization of PEN3 3 (alp3), displaying accumulation of the normally plasma membrane (PM)-localized PEN3-GFP in endomembrane compartments. The mutant was found to be disrupted in the P-ATPase AMINOPHOSPHOLIPID ATPASE 3 (ALA3), a lipid flippase that plays a critical role in vesicle formation. We provide evidence that PEN3 undergoes continuous endocytic cycling from the PM to the trans-Golgi network (TGN). In alp3, PEN3 accumulates in the TGN, causing delays in recruitment to the host-pathogen interface. Our results indicate that PEN3 and other defense proteins continuously cycle through the TGN and that timely exit of these proteins from the TGN is critical for effective pre-invasive immune responses against powdery mildews.

Arabidopsis BTB/POZ protein-dependent PENETRATION3 trafficking and disease susceptibility

“…Proteins in the outer domain that function in regulating the transport of inorganic compounds include, for example, the NIP5;1 boric acid uptake channel 7 . Factors required for PEN3 and NIP5;1 trafficking from the transGolgi network to the outer domain have been identified [8][9][10] , and exocyst complex components promote polar tethering of several outer domain proteins 9,11 . However, factors that specifically mediate trafficking of polar outer membrane cargos involved in responses to root-penetrating pathogens remain to be discovered.…”

The mysterious life of the plant trans‐Golgi network: advances and tools to understand it better

“…Some of these components are ABC transporters, such as the Arabidopsis PENETRATION 3 (PEN3) ATP binding cassette, that intervenes in the early stages of infection by fungal and bacterial pathogens (Matern et al ., 2019). In noninfected plants, PEN3 and PENETRATION 1 (PEN1) constantly cycle through the TGN/EE to and from the PM (Underwood et al ., 2017). The trafficking mechanisms for the recruitment and delivery of these transporters to the PM at the site of pathogen attack are largely unknown.…”

“…However, the recently identified mutant of the P4‐ATPase aminophospholipid ATPase 3 (ALA3) has provided some clues in this process. This protein resulted necessary for membrane budding and protein cargo exit from the TGN (Underwood et al ., 2017). ALA3 is a lipid flippase that translocate phospholipids from one face of the membrane bilayer to the other, contributing to the uneven distribution of phospholipids to membrane curvature, which are at the basis of vesicle formation during exocytosis and endocytosis.…”

“…ALA3 localizes at the trans‐Golgi and partially localizes with TGN/EE markers. Upon infection by powdery mildew Almeria grains , in the ala3 mutant PEN3 is retained in the TGN/EE, supporting the possibility that PEN3 is misdistributed due to delay in cargo sorting or vesicle production at the TGN/EE (Underwood et al ., 2017). These results support that endocytic and sorting processes are critical for immune responses and the TGN/EE plays a fundamental role in these processes under biotic stress.…”

“…Whether HLB1 cooperates with MIN7 in pathogen resistance is yet unknown. Interestingly, there is experimental evidence that not all the receptor components involved in the first response to pathogens like PEN1 and PEN3 undergo the same sorting mechanisms and traffic through the same routes to reach their final destination (Underwood et al ., 2017). Although additional work is necessary to understand how cargo is segregated into different exocytic cargo routes for defense responses to biotic stresses, these studies support a critical role of the TGN/EE for their delivery for efficient cell responses to pathogen attack.…”