Colletotrichum orbiculare Secretes Virulence Effectors to a Biotrophic Interface at the Primary Hyphal Neck via Exocytosis Coupled with SEC22-Mediated Traffic

Abstract

The hemibiotrophic pathogen Colletotrichum orbiculare develops biotrophic hyphae inside cucumber (Cucumis sativus) cells via appressorial penetration; later, the pathogen switches to necrotrophy. C. orbiculare also expresses specific effectors at different stages. Here, we found that virulence-related effectors of C. orbiculare accumulate in a pathogen-host biotrophic interface. Fluorescence-tagged effectors accumulated in a ring-like region around the neck of the biotrophic primary hyphae. Fluorescence imaging of cellular components and transmission electron microscopy showed that the ring-like signals of the effectors localized at the pathogen-plant interface. Effector accumulation at the interface required induction of its expression during the early biotrophic phase, suggesting that transcriptional regulation may link to effector localization. We also investigated the route of effector secretion to the interface. An exocytosis-related component, the Rab GTPase SEC4, localized to the necks of biotrophic primary hyphae adjacent to the interface, thereby suggesting focal effector secretion. Disruption of SEC4 in C. orbiculare reduced virulence and impaired effector delivery to the ring signal interface. Disruption of the v-SNARE SEC22 also reduced effector delivery. These findings suggest that biotrophy-expressed effectors are secreted, via the endoplasmic reticulum-to-Golgi route and subsequent exocytosis, toward the interface generated between C. orbiculare and the host cell.

Figure 1.

Fluorescent Protein–Tagged Effectors Accumulated around the Penetration Site to Form a Ring Signal. (A) Localization pattern of C. orbiculare effectors under the control of the corresponding native promoter. C. orbiculare strains carrying each effector gene tagged with mCherry were incubated on cucumber cotyledons for 4 d. Tagged DN3, NIS1, and MC69 accumulated around the appressorial penetration site to form a ring signal, although DN3 and NIS1 exhibit preferential accumulation compared with MC69. The ring signals are indicated by white arrows. BH, biotrophic hypha; DIC, differential interference contrast; MA, melanized appressorium. Bars = 10 μm. (B) Localization pattern of C. orbiculare effectors under the control of the TEF promoter. C. orbiculare strains carrying each effector gene tagged with mCherry or GFP were incubated on cucumber for 4 d. The ring signals are indicated by white arrows. SP, signal peptide of DN3. Bars = 10 μm. (C) The ring signal ratio of mCherry fluorescence for appressoria that formed biotrophic hyphae. Each reporter line was inoculated on cucumber cotyledons, and the inoculated plants were incubated for 4 d. At least 70 appressoria that formed biotrophic hyphae were investigated for each plant sample. The mean and sd were calculated from three independent plant samples. WT indicates the wild-type strain without mCherry. (D) Quantification of the fluorescence intensity of the detected ring signal in each reporter line. Each reporter line was inoculated on cucumber cotyledons, and the inoculated plants were incubated for 4 d. At least 50 detected ring signals were investigated for each sample. The mean fluorescence intensity value of DN3:mCherry was normalized to 1.0.

Figure 2.

Functionality of the Effector:mCherry Fusion Proteins. (A) Cell death suppression assay using DN3:mCherry and DN3ΔSP:mCherry. Agroinfiltration sites that expressed mCherry (control), DN3:mCherry, or DN3ΔSP:mCherry were challenged with Agrobacterium expressing NIS1 in N. benthamiana. The infiltration sites are represented by dashed circles. Strong cell death induced by NIS1 was observed in infiltration sites that expressed mCherry, whereas it was suppressed in the DN3:mCherry and DN3ΔSP:mCherry sites. The photograph was taken at 5 d after the infiltration challenge. (B) Pathogenicity assay using the mc69Δ mutant that expressed Co-MC69:mCherry under the control of the native promoter. The mc69Δ strain was inoculated onto the upper half of the cucumber cotyledon as a control. The tested strain was inoculated onto the lower half. The inoculated cotyledons were incubated for 7 d. (C) NIS1:mCherry:NLS was secreted by M. oryzae and translocated into rice cells. The M. oryzae strain that carried PWL2pro:NIS1:mCherry:NLS was inoculated onto rice leaf sheath, incubated for 32 h, and observed by confocal laser-scanning microscopy. The NIS1:mCherry:NLS signal localized to the BIC (arrowheads) and nucleus (arrows) of the invaded rice leaf sheath cell. IH, invasive hypha. Bar = 20 μm.

Figure 3.

The Ring Signal of the Effectors Localized at the Biotrophic Hyphal Neck beneath the Appressorium. (A) The effector ring signal was localized at the biotrophic invasive hyphal neck. Cucumber cotyledons were inoculated with a C. orbiculare strain that carried TEFpro:DN3:mCherry and incubated for 4 d. Then, appressoria were removed by brushing the surface of cucumber cotyledons infected with the pathogen. BH, biotrophic hypha; DIC, differential interference contrast. Bar = 10 μm. (B) The signal of DN3:mCherry expressed under the control of the DN3 or TEF promoter was undetectable in C. orbiculare cells during penetration of the cellophane membrane. C. orbiculare strains that carried DN3pro:DN3:mCherry or TEFpro:DN3:mCherry were incubated for 16 h. Co, conidium; MA, melanized appressorium; PH, pseudobiotrophic hypha. Bars = 10 μm. (C) GFP-based promoter assays of the DN3 promoter and the TEF promoter in C. orbiculare, which were incubated on cellophane and cucumber. C. orbiculare strains that carried the GFP gene with the DN3 or TEF promoter were incubated on cellophane for 16 h or on cucumber for 3 d. Bars = 10 μm.

Figure 4.

The Effectors Localized at the Pathogen–Host Interface around the Biotrophic Primary Hyphal Neck. (A) Visualization of fungal cytoplasm by GFP. A C. orbiculare strain that carried both TEFpro:GFP and TEFpro:DN3:mCherry constructs was incubated for 4 d on cucumber. The bottom right panel shows a zoomed image of the dashed box. BH, biotrophic hypha. Bars = 10 μm. (B) Visualization of the fungal cell wall using Calcofluor White M2R. A C. orbiculare strain with TEFpro:DN3:mCherry was incubated for 4 d on cucumber. The infected cucumber sample was treated with Calcofluor White M2R for 5 min before UV excitation. The bottom right panel shows a zoomed image of the dashed box. BF, bright field. Bars = 10 μm. (C) TEM analysis of the biotrophic hypha that developed from the appressorium of C. orbiculare. The C. orbiculare wild-type strain was inoculated on cucumber cotyledons. The arrowheads represent the plant-derived membrane, and the dashed lines represent the surface of the biotrophic hypha around the neck. The asterisks indicate the putative interfacial region with high electron density, related to the effector ring signal, around the biotrophic hyphal neck. MA, melanized appressorium; N, neck of the biotrophic hypha; PP, penetration peg; PW, plant cell wall. Bar = 1 μm.

Figure 5.

The Ring Signal Interface Did Not Include Papillary Callose and Was Associated with Membrane Components. (A) Visualization of callose deposition during the biotrophic invasive stage. C. orbiculare with TEFpro:GFP was incubated for 4 d on cucumber. Staining was performed using aniline blue fluorochrome. The dashed circles represent the position of the appressorium. BH, biotrophic hypha. Bar = 10 μm. (B) Simultaneous observation of the effector ring and callose deposition during the biotrophic invasive stage. C. orbiculare with TEFpro:DN3:mCherry was incubated for 4 d on cucumber. Callose staining was performed with aniline blue fluorochrome. Bars = 5 μm. (C) Staining of the membrane components using FM1-43. C. orbiculare with TEFpro:NIS1:GFP was incubated for 4 d on cucumber. The infected cucumber samples were treated rapidly with FM1-43 before observation in distilled water. The white arrow indicates the GFP-labeled ring signal interface. Bar = 10 μm.

Figure 6.

Accumulation of the Effectors at the Ring Signal Interface Was Dependent on Preferential Gene Expression during Early Biotrophy. (A) GFP-based promoter assay using the LAC2, DN3, and NLP1 promoters in the C. orbiculare infection process. C. orbiculare strains that carried the GFP gene with each promoter were incubated on cucumber for 6 h (preinvasion), 3 d (early biotrophy), 4 d (late biotrophy), and 5 d (necrotrophy). BH, biotrophic hypha; NH, necrotrophic hypha. Bars = 10 μm. (B) Promoter-exchange experiment with DN3:mCherry. Each C. orbiculare strain expressed DN3:mCherry under the control of the LAC2, DN3, or NLP1 promoter. Bars = 10 μm.

Figure 7.

Effectors Are Secreted to the Ring Signal Interface Continuously. (A) FRAP analysis indicated the continuous secretion of DN3:mCherry to the ring signal interface. The fluorescence at the interface at 4 dpi (Pre-bleach) was photobleached (Bleach) and allowed to recover over time. Bar = 10 μm. (B) Plot showing the normalized fluorescence intensity of the effector ring. The arrow indicates the time point when bleaching occurred. The fluorescence intensity of the prebleached effector ring was normalized to 1.0.

Figure 8.

SEC4 and Actin Localized to the Cavity Region of the Effector Ring. (A) Subcellular localization of GFP:SEC4, GFP:ACT1, and GFP:SEC22 in C. orbiculare cells during the biotrophic invasive stage. C. orbiculare strains that carried combinations of the effector:mCherry gene and each GFP-tagged test gene (TEFpro:NIS1:mCherry × SCD1pro:GFP:SEC4, TEFpro:NIS1:mCherry × SCD1pro:GFP:ACT1, and TEFpro:DN3:mCherry × SCD1pro:GFP:SEC22, respectively) were inoculated onto cucumber cotyledons, and the inoculated plants were analyzed at 4 dpi. SEC4 and actin (arrowheads) preferentially localized to the cavity region of the effector ring signal (which is equivalent to the neck region of a biotrophic primary hypha). The arrows indicate the effector ring signal. Bars = 10 μm. (B) Quantification of the GFP signal ratio in the cavity region of the effector ring. At least 50 effector ring cavities were investigated in each plant sample. The mean and sd were calculated from three independent plant samples.

Figure 9.

Effects of SEC4 and SEC22 Disruptions on Pathogenicity and Effector Secretion. (A) Pathogenicity assays of the sec4 and sec22 null mutants (sec4Δ and sec22Δ) on host cucumber. The wild-type strain was inoculated onto the left halves of the cucumber cotyledons as a positive control. The sec4Δ and sec22Δ strains were inoculated onto the right halves. The inoculated plants were incubated for 7 d. (B) Effector localization patterns of the wild-type, sec4Δ, and sec22Δ strains with TEFpro:DN3:mCherry on a glass surface. Each strain was incubated in the presence of the melanin biosynthesis inhibitor carpropamid on glass for 24 h. Bars = 10 μm.

Figure 10.

The sec4Δ and sec22Δ Mutants of C. orbiculare Reduced Effector Delivery to the Ring Signal Interface. (A) Effector localization pattern during the biotrophic stage for wild-type and sec4Δ strains with TEFpro:DN3:mCherry. Each strain was incubated on cucumber for 4 d. The white arrow indicates the mCherry-labeled ring signal interface. Bars = 10 μm. (B) Effector localization pattern during the biotrophic stage for wild-type and sec22Δ strains with TEFpro:DN3:mCherry. The cucumber cotyledons were pretreated by heat shock (H.S.) to enable the host invasion of the sec22Δ mutant. Each strain was incubated on cucumber for 3 d. Bars = 10 μm. (C) Quantitative analysis of the effects of SEC4 and SEC22 disruptions on effector delivery to the ring signal interface. Each genotype with TEFpro:DN3:mCherry was incubated on cucumber for 4 d (no heat shock) or 3 d (heat shock). At least 74 biotrophic hyphae were investigated to determine the presence ratio of the effector ring signal. At least 50 mature biotrophic hyphae were investigated for each plant sample to determine the ratio of effector retention inside biotrophic hyphae. The mean and sd were calculated from three independent plant samples.