Fig 1.
Penetration peg-derived hyphal neck partitioning the hyphopodium and invasive hypha.
(A, B) Confocal laser scanning microscopy (CLSM) images of the development and penetration of hyphopodium of V. dahliae V592 on cellophane (A) and Arabidopsis thaliana root (B). The fungal cell wall was stained with FITC-WGA. Images were obtained at 7 dpi on cellophane and 1 dpi on roots. (C, D) Transmission electron microscopy analysis of V. dahliae invasion of cellophane (C) and cotton root (D). The dashed lines represent the penetration interface of the penetration peg-derived hyphal neck on cellophane. HP, hyphopodium; HN, hyphal neck; IH, invasive hypha. Bar = 2.5 μm in (C) and Bar = 0.5 μm in (D).
Fig 2.
VdNoxB is required for the formation of a septin ring at the penetration peg and hyphal neck in V. dahliae.
(A, B) Cellular localization of VdSep5-GFP in V592 and VdΔnoxb during development of the penetration peg. Numbers indicate the distance from the center of the hyphopodium where the first column (0 μm) shows the beginning of a continuous z series. The star indicates the septin ring at the hyphopodium pore (2.7 μm). Bar = 2.5μm. (C) Quantitative analysis of the diameter reduction of the VdSep5-GFP ring. In V592, from the widest part at the base of the hyphopodium (the 1.5-μm plane) to the hyphopodium pore (the 2.7-μm plane); in VdΔnoxb, from the 2.1-μm plane to the 3.9-μm plane. The bar chart shows the average diameter of the fluorescence signal ring, and 20 hyphopodia were investigated in each assay with three replicates (*P<0.05; t-test). (D, E) VdSep5-GFP localized at the hyphal neck partitioning the hyphopodium and the invasion hypha on cellophane (D) or A. Thaliana roots (E). Bar = 2.5 μm. (F) The VdSep5-GFP ring localized at two individual penetration sites during root epidermis and cortical cell wall penetration. Bar = 2.5 μm.
Fig 3.
Accumulation of secretory proteins surrounding the hyphal neck on cellophane.
(A) VdSCP8-GFP, VdSCP9-GFP and VdSCP10-GFP but not VdIscI-GFP, showed ring signal accumulation at the hyphal necks. Images were obtained at 8 days after the fungal strains were incubated on M0 medium overlaid with cellophane, in each invasive hyphae were observed. Bar = 2.5 μm. (B) The VdSCP10-GFP ring signal surrounds hyphal neck. The hyphal plasma membrane was stained with FM4-64 (red). VdSCP10-GFP, FM4-64, and fluorescence merge images are z-series stacks. HP, HN and IH are marked in the bright-field picture. Bar = 2.5 μm. (C) VdSCP8-GFP, VdSCP9-GFP and VdSCP10-GFP rings localized outside of the VdSep5-RFP ring. Bar = 2.5 μm. (D) FRAP detection of the dynamic accumulation/delivery of VdSCP10-GFP at the hyphal neck on cellophane. Fluorescence at the penetration interface (Pre-bleach) was photobleached (Bleach) and allowed to recover 100% for 27 min (Recovery). Bar = 2.5 μm. (E) Plot of normalized penetration interface fluorescence intensity recovery over time for VdSCP10-GFP. (F) The average recovery time of VdSCP8-GFP, VdSCP9-GFP and VdSCP10-GFP after bleaching on cellophane. Three FRAP tests were performed for each sample.
Fig 4.
Accumulation of secretory proteins at the hyphal neck during root colonization.
(A) VdSCP8-GFP localized at the hyphal neck (at 7.5 μm, distance from the center of the hyphopodium where the first image was obtained at 0 μm), joining a lightly melanized hyphopodium and an invasive hypha. (B) VdSCP8-GFP ring signal accumulated at the cell junction. (C) VdSCP9-GFP shows a weak ring signal at the first hyphal neck (at 1.8 μm, arrow) and a strong ring signal at the second hyphal neck (at 4.8 μm) in two individual penetrations. (D) Z-series projection showing that VdSCP10-GFP preferentially localized at the hyphal neck on roots. Arrowhead indicates VdSCP10-GFP signal dots inside hyphopodium. Black dashed line outlining the hyphopodium in the first picture and white dashed line marking the beginning of an invasive hypha. Fluorescence micrographs of (A,B,C) were merged from 2–3 continuous z series images. Asterisks indicate the ring signals. Bar = 2.5 μm.
Fig 5.
Deletion of VdSep5 decreased secretory protein delivery to the hyphal neck.
(A, B) VdSCP10-GFP signal ring surrounding the hyphal neck in wild-type V592; mutant strain VdΔsep5 retained most of the VdSCP10-GFP inside the hyphopodium and reduced the VdSCP10-GFP signal at the hyphal neck. The plasma membrane of HP and HN was stained with FM4-64 (red). Bar = 2.5 μm. (C) Quantitative analysis of the effect of VdSep5 on VdSCP10-GFP delivery to the penetration interface. More than 30 hyphal necks with a visible signal were investigated for each strain to determine the intensity of the ring signal at the hyphal neck. Two VdΔsep5 mutant strains obtained from individual VdSCP10-GFP-expressing V592 (S6B Fig) were used for the observation. The mean and SD were calculated from two VdSCP10-GFP-expressing V592 and the corresponding VdΔsep5 strains with two biological repeats (*P<0.05; t-test). (D, E) FRAP assay for the dynamic accumulation/delivery of VdSCP10-GFP at the hyphal neck in VdΔsep5. Three FRAP tests were performed.
Fig 6.
Deletion of VdSec22, VdSyn8 or VdExo70 decreased secretory protein delivery to the hyphal necks.
(A) VdSCP10-GFP signal ring surrounding the hyphal neck in wild-type V592. (B-D) The mutant strains VdΔsec22 (B), VdΔsyn8 (C) and VdΔexo70 (D) retained most of the VdSCP10-GFP inside the hyphopodium and reduced the VdSCP10-GFP signal in the hyphal neck. The plasma membrane of HP and HN was stained with FM4-64 (red). (E) Quantitative analysis of the effect of VdSec22, VdSyn8, and VdExo70 on secretory protein delivery to penetration interfaces. More than 30 hyphal necks with a visible “ring” signal were investigated for each VdSCP10-GFP-expressing mutant strain to determine the intensity of the ring signal at the HN. The mean and SD for (E) were calculated from three independent fungal transformants for each mutant (*P<0.05; t-test). Bar = 2.5 μm. (F) FRAP assay for the dynamic accumulation/delivery of VdSCP10-GFP at the hyphal neck in VdΔsec22, VdΔsyn8 and VdΔexo70 on cellophane. Three FRAP tests were performed for each mutant strain.
Fig 7.
The exocyst subunit VdExo70 and SNAREs VdSec22 and VdSyn8 function in wilt virulence.
(A, B) Disease symptoms (A) and disease grades (B) of cotton plants infected with wild-type V592, and VdΔexo70, VdΔsec22 or VdΔsyn8 mutants and the complementary strains at 21 dpi. The grades were evaluated with three replicates of 36 plants for each inoculum (*P<0.05; t-test).
Fig 8.
Simple schematic of secretory protein preferential delivery to the hyphal neck in V. dahliae.
Simple schematic showing the accumulation/delivery of secretory proteins in the hyphal neck. A hyphopodium-mediated breach through the plant root cell wall and invasive hyphal growth. The penetration peg-derived hyphal neck joining the hyphopodium and the invasive hypha form the fungus-host penetration interface, where small secretory proteins are accumulated and secreted. The septin (VdSep5), F-actin, exocyst (VdExo70 and VdSec8), ER-Golgi traffic (VdSec22) and endosome-mediated traffic (VdSyn8) function in the delivery of secretory proteins to the penetration interface.