Homologues of the RPW8 Resistance Protein Are Localized to the Extrahaustorial Membrane that Is Likely Synthesized De Novo

Upon penetration of the host cell wall, the powdery mildew fungus develops a feeding structure named the haustorium in the invaded host cell. Concomitant with haustorial biogenesis, the extrahaustorial membrane (EHM) is formed to separate the haustorium from the host cell cytoplasm. The Arabidopsis resistance protein RPW8.2 is specifically targeted to the EHM where it activates haustorium- targeted resistance against powdery mildew. RPW8.2 belongs to a small family with six members in Arabidopsis (Arabidopsis thaliana). Whether Homologs of RPW8 (HR) 1 to HR4 are also localized to the EHM and contribute to resistance has not been determined. Here, we report that overexpression of HR1, HR2, or HR3 led to enhanced resistance to powdery mildew, while genetic depletion of HR2 or HR3 resulted in enhanced susceptibility, indicating that these RPW8 homologs contribute to basal resistance. Interestingly, we found that N-terminally YFP-tagged HR1 to HR3 are also EHM-localized. This suggests that EHM-targeting is an ancestral feature of the RPW8 family. Indeed, two RPW8 homologs from Brassica oleracea tested also exhibit EHM-localization. Domain swapping analysis between HR3 and RPW8.2 suggests that sequence diversification in the N-terminal 146 amino acids of RPW8.2 probably functionally distinguishes it from other family members. Moreover, we found that N-terminally YFP-tagged HR3 is also localized to the plasma membrane and the fungal penetration site (the papilla) in addition to the EHM. Using this unique feature of YFP-HR3, we obtained preliminary evidence to suggest that the EHM is unlikely derived from invagination of the plasma membrane, rather it may be mainly synthesized de novo.