Host Translocation of Plant Pathogen Effectors

 

• The potato blight pathogen Phytophthora infestans secretes effector proteins that are delivered inside (cytoplasmic) or can act outside (apoplastic) plant cells to neutralize host immunity. Little is known about how and where effectors are secreted during infection, yet such knowledge is essential to understand and combat crop disease.
• We used transient Agrobacterium tumefaciens-mediated in planta expression, transformation of P. infestans with fluorescent protein fusions and confocal microscopy to investigate delivery of effectors to plant cells during infection.
• The cytoplasmic effector Pi04314, expressed as a monomeric red fluorescent protein (mRFP) fusion protein with a signal peptide to secrete it from plant cells, did not passively re-enter the cells upon secretion. However, Pi04314-mRFP expressed in P. infestans was translocated from haustoria, which form intimate interactions with plant cells, to accumulate at its sites of action in the host nucleus. The well-characterized apoplastic effector EPIC1, a cysteine protease inhibitor, was also secreted from haustoria. EPIC1 secretion was inhibited by brefeldin A (BFA), demonstrating that it is delivered by conventional Golgi-mediated secretion. By contrast, Pi04314 secretion was insensitive to BFA treatment, indicating that the cytoplasmic effector follows an alternative route for delivery into plant cells.
• Phytophthora infestans haustoria are thus sites for delivery of both apoplastic and cytoplasmic effectors during infection, following distinct secretion pathways.

PWL2 and BAS1 (for biotrophy-associated secreted protein 1), BIC-localized secreted proteins, were translocated into the rice cytoplasm. By contrast, BAS4, which uniformly outlines the IH, was not translocated into the host cytoplasm. Fluorescent PWL2 and BAS1 proteins that reached the rice cytoplasm moved into uninvaded neighbors, presumably preparing host cells before invasion.

SAP11, contains an N-terminal SP sequence and a eukaryotic bipartite nuclear localization signal (NLS). SAP11 was detected by immunocytology in nuclei of young sink tissues of China aster plants infected with AY-WB.

• Root-knot nematodes (RKNs) are obligate endoparasites that maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors synthesized in the oesophageal glands and injected into the plant tissue through the syringe-like stylet certainly play a central role in these processes.
• In a search for nematode effectors, we used comparative genomics on expressed sequence tag (EST) datasets to identify Meloidogyne incognita genes encoding proteins potentially secreted upon the early steps of infection.
• We identified three genes specifically expressed in the oesophageal glands of parasitic juveniles that encode predicted secreted proteins. One of these genes, Mi-EFF1 is a pioneer gene that has no similarity in databases and a predicted nuclear localization signal. We demonstrate that RKNs secrete Mi-EFF1 within the feeding site and show Mi-EFF1 targeting to the nuclei of the feeding cells.
• RKNs were previously shown to secrete proteins in the apoplasm of infected tissues. Our results show that nematodes sedentarily established at the feeding site also deliver proteins within plant cells through their stylet. The protein Mi-EFF1 injected within the feeding cells is targeted at the nuclei where it may manipulate nuclear functions of the host cell.

We show that Glomus intraradices secretes a protein, SP7, that interacts with the pathogenesis- related transcription factor ERF19 in the plant nucleus. SP7-mRFP is secreted by a Magnaporthe oryzae GFP-expressing strain, translocated into the onion cell, and localized to the nucleus. No red nuclei were detected when the infection assay was performed using the GFP control strain (Guy11-GFP). Images show maximal projections of several confocal planes. Scale bars represent 10 mm.

Rust transferred protein 1 from Uromyces fabae (Uf-RTP1p) was not only detected in the host parasite interface, the extrahaustorial matrix, but also inside infected plant cells by immunofluorescence and electron microscopy. Uf-RTP1p does not exhibit any similarity to sequences currently listed in the public databases. However, we identified a homolog of Uf-RTP1p in the related rust fungus Uromyces striatus (Us-RTP1p). The localization of Uf-RTP1p and Us-RTP1p inside infected plant cells was confirmed, using four independently raised polyclonal antibodies. Depending on the developmental stage of haustoria, Uf-RTP1p was found in increasing amounts in host cells, including the host nucleus.