Figure 1.
PDLP1 is specifically expressed in Hpa infected mesophyll cells.
GUS staining of PDLP1pro:GUS (A), PDLP2pro:GUS (B), PDLP3pro:GUS (C), PDLP5pro:GUS (D) in Arabidopsis leaves 6 days post inoculation with Hpa Waco9 shows that GUS staining correlates with Hpa growth in PDLP1pro:GUS expressing plants. At higher magnification, GUS staining is restricted to cells harbouring haustoria (yellow stars) while no GUS staining was detected in non-infected mesophyll cells. Haustoria are indicated by asterisks. Scale bars are 200 µm unless otherwise indicated.
Figure 2.
PDLPs localise to the extra-haustorial membrane.
PDLP1-GFP is observed at the EHM prior to encasement. (A) In unencased haustoria, or those with a developing encasement at the haustorial neck, PDLP1-GFP is present in the EHM surround the haustorium (n = 50/50). PD-located signal is indicated with solid arrows, while open arrows indicate signal associated with haustoria. (B) As the encasement develops (stained with aniline blue, open arrows) and surrounds the entire haustorium PDLP1-GFP fluorescence remains associated with the haustorium and PDLP1-GFP positive bodies can be seen at the encasement periphery (see E). (C) When encasements are mature PDLP1-GFP is no longer associated with the structure. (D) Scheme of the development of the Hpa haustorial encasement, stages I, II and III are indicated in (A–C) for reference. (E) Enlargement of developing encasements shows PDLP1-GFP positive bodies at the periphery of the encasement (arrows). Scale bars are 20 µm (A–C) and 10 µm (E).
Figure 3.
PDLPs, but not other PD proteins, are located at the EHM.
(A) PDLP1-GFP is located at PD (arrows) in both uninfected and infected tissue. During infection, PDLP1-GFP is also located at haustoria (right). (B) EHM association is specific to PDLPs as the PD markers PCBD1-mCit, TET3-YFP and MP17-GFP do not locate to the EHM. PDCB1-mCit and TET3-YFP locate to the developing encasement (n = 13/13 and n = 12/12 respectively) and MP17-GFP shows no association with haustoria (n = 5/5). Images are fluorescent data (left) and fluorescence/transmitted light overlays (right). (C) PDLP2-GFP and PDLP3-GFP are located at the haustoria periphery (n = 10/10 and n = 5/17 respectively). (D) and (E) Diagrammatic representation of the topology of the PDLP1 and synthetic mCit-TMCT proteins following cleavage of the signal peptide. PDLP1 has 2 extracellular DUF26 domains, the transmembrane (TM) domain and cytoplasmic tail (CT), which projects into the cell cytoplasm (cyt). In the mCit-TMCT variant, the TM and CT is fused to mCitrine. (F) mCit-TMCT localises to the EHM similar to PDLP1-GFP (n = 10/10). Asterisks, haustoria; yellow arrows, developing encasement; white arrows, PD. Scale bars are 10 µm.
Figure 4.
PDLPs are required for plant immunity against Hpa.
Hpa Waco9 sporulation 5 DPI is reduced in PDLP1 OE plants but increased in pdlp1,2,3 mutant plants when compared to wild type Col-0. Error bars represent the standard error of the mean. a and b denote statistical significance (p-value <0.05) when the data is analysed by one-way ANOVA and a Tukey-Kramer test.
Table 1.
Proteins present in PDLP1-GFP containing membranes identified by MS/MS.
Figure 5.
PDLP1 is required for callose deposition in haustorial encasement.
(A) Aniline blue staining of callose in Col-0, PDLP OE, pdlp1,2,3 and pmr4 mutant leaves 5 DPI with Hpa Noco2 identifies that pdlp1,2,3 produces fewer encased haustoria at this stage of infection, similar to the callose synthesis mutant pmr4. Quantification (B) of stained haustoria confirms that pdlp1,2,3 produces significantly fewer aniline blue stained encasements than Col-0. PDLP1 OE plants produce more stained encasements than Col-0 plants per field of view. Error bars represent the standard error of the mean. * p-value <0.05, *** p-value <0.001 by Student's t-test. (C) At higher magnification, aniline blue stained encasements in PDLP1 OE cells appear thicker than Col-0 encasements. The transmitted light image of a pdlp1,2,3 haustorium suggests that there is some encasement (arrow) of the haustorium but this structure does not stain with aniline blue. Asterisks indicate haustoria. Scale bars are 100 µm (A) and 10 µm (C).
Figure 6.
PDLP1 promotes membrane tubule formation at the extra-haustorial interface.
Transmission electron micrographs of Hpa Waco9 haustoria observed in Col-0 (A) and PDLP1 OE (B) plants harvested 6 DPI. Boxes represent regions from which high magnification images (C, D, E, F) were taken. High magnification images of the host-pathogen interface in Col-0 (C–E) and PDLP1 OE (F) show that the EHMx and EHM forms an electron dense structure that has membrane invaginations (arrows) at the host surface. In regions in which the haustorium is encased the EHM is not continuously defined and may comprise the EHM and inner membrane of the encasement, thus this membrane is differentially denoted EHMs to allow for the possibility of multiple membrane layers. (F) Membrane invaginations are longer and more abundant in PDLP1 OE plants. (G) An oblique section of the surface of an haustorium in a PDLP1 OE cell illustrates the density and length of these protrusions. Ha, haustorium; En, encasement; EHMxt, extrahaustorial matrix translucent; EHMxd, extrahaustorial matrix dense. Scale bars are 2 µm (A and B), 100 nm (C–F) and 500 nm (E).