Two distinct secretion systems facilitate tissue invasion by the rice blast fungus Magnaporthe oryzae

Abstract

To cause plant diseases, pathogenic micro-organisms secrete effector proteins into host tissue to suppress immunity and support pathogen growth. Bacterial pathogens have evolved several distinct secretion systems to target effector proteins, but whether fungi, which cause the major diseases of most crop species, also require different secretory mechanisms is not known. Here we report that the rice blast fungus Magnaporthe oryzae possesses two distinct secretion systems to target effectors during plant infection. Cytoplasmic effectors, which are delivered into host cells, preferentially accumulate in the biotrophic interfacial complex, a novel plant membrane-rich structure associated with invasive hyphae. We show that the biotrophic interfacial complex is associated with a novel form of secretion involving exocyst components and the Sso1 t-SNARE. By contrast, effectors that are secreted from invasive hyphae into the extracellular compartment follow the conventional secretory pathway. We conclude that the blast fungus has evolved distinct secretion systems to facilitate tissue invasion.

Figure 1. The biotrophic interfacial complex is a plant-derived membrane-rich structure.

(a) Schematic representation of the differentiation of a filamentous primary invasive hypha (left, ~22–25 h post inoculation, h.p.i.) into a pseudohyphal-like bulbous invasive hypha (middle, ~26–30 h.p.i.) in a first-invaded rice cell. This differentiation occurs for each new hypha invading a living neighbour cell (right, ~36–40 h.p.i.). Cytoplasmic effectors show preferential accumulation in the BIC (black arrows), which is first located in front of the growing primary hyphal tips, and then remains behind beside the first-differentiated bulbous IH cell. Typical accumulation patterns for cytoplasmic (magenta) and apoplastic (blue) effectors are shown within the EIHM (tan) compartment enclosing the IH. The EIHM appears to lose integrity when the fungus has moved into neighbour cells (dotted line). (b–d) Live cell imaging of M. oryzae infection of rice sheath epidermal cells with the BIC (red) visualized by accumulation of IH-secreted Pwl2:mRFP. Clockwise, images are DIC; mRFP (white arrow indicates BIC); merged GFP (green) and mRFP (red); and GFP alone. Below, white arrow in the inset shows the path for fluorescence intensity distribution linescans on the left. (b) The BIC (red) is located outside the fungal plasma membrane (green), which was visualized by expression of M. oryzae ATPase Pma1:GFP, here imaged at 23 h.p.i. Lack of co-localization between Pwl2:mRFP and Pma1:GFP, indicated by separate subcellular distribution maxima, confirms that the BIC does not contain fungal plasma membrane. (c) The BIC (red) co-localized with intense fluorescence from a rice plasma membrane marker LTi6B:GFP (green), visualized after infection of transgenic rice (at 24 h.p.i.). Note that the plant plasma membrane marker also outlined the entire IH, consistent with invagination around the fungus during cell invasion. Co-localization of fluorescence intensity maxima indicates that the BIC contains material derived from plant plasma membrane. (d) The BIC (red) co-localized with fluorescence from rice ER marker GFP:HDEL (green) expressed in transgenic plants and imaged at 24 h.p.i. Fluorescence intensity maxima demonstrate plant ER localized with and closely surrounding the BIC. White asterisks indicate appressoria. Scale bars, 10 μm.

Figure 2. Secretion components localize similarly in vegetative and primary hyphae.

Fluorescent secretory components () were imaged in M. oryzae filamentous vegetative hyphae in vitro (a,b,d,f) and in tubular primary hyphae growing in rice cells (c,e,g). BICs (white arrows) were observed adjacent to the primary hyphal tips even without fluorescent labelling. (a) Spitzenkörper marker Mlc1:GFP (green) co-localizes (yellow) with the endocytic tracker dye FM4-64 (red) at tips of vegetative hyphae. This confocal image was acquired 10 min after FM4-64 addition. Images clockwise from upper left are bright-field; GFP; merged GFP and FM4-64; and FM4-64 alone. Similarly, Mlc1:GFP identifies Spitzenkörper in primary hyphae inside rice cells (Fig. 3a). (b–g) Images from left to right are bright-field; and GFP alone. (b) Polarisome component Spa2:GFP localizes to the tip of a vegetative hypha. (c) Polarisome component Spa2:GFP concentrates at the primary hyphal tip behind the BIC at 22 h.p.i. (d) v-SNARE GFP:Snc1 localizes at the vegetative hyphal tip. (e) v-SNARE GFP:Snc1 localizes in a bright fluorescent punctum near the primary hyphal tip at 24 h.p.i. (f) Exocyst component Exo70:GFP localizes at the vegetative hyphal tip. (g) Exocyst component Exo70:GFP localizes at the primary hyphal tip at 24 h.p.i. Scale bars, 5 μm.

Figure 3. BIC-associated IH cells contain components of the secretory machinery.

All images are representative of at least five biological replicates with >50 images each. BICs are labelled by arrows in all panels and by red fluorescence from Pwl2:mRFP in all panels except d and g. Unless mentioned otherwise, images left to right are: merged bright-field and mRFP (red); and GFP alone (green). (a) The myosin regulatory light chain Mlc1:GFP () labels the Spitzenkörper at the primary hyphal tip before hyphal differentiation at 24 h.p.i. Images left to right: merged bright-field, GFP and mRFP; merged GFP and mRFP; merged bright-field and GFP; and GFP alone. Scale bar, 5 μm. (b) After differentiation of bulbous IH at 27 h.p.i., Mlc1:GFP accumulates in a large fluorescent punctum in the BIC-associated bulbous IH cell () and near septa (*). Spitzenkörper-like fluorescence is not observed in growing bulbous IH tips. (c) Polarisome component Spa2:GFP localizes as a distinct punctum at the tip of growing IH () at 27 h.p.i. Spa2 fluorescence is not observed in the subapical BIC-associated IH cell. In this image, saturated fluorescence from Pwl2:mRFP labels the rice cytoplasm and nucleus (*). This saturated Pwl2:mRFP fluorescence is seen in the EIHM compartment surrounding the BIC-associated cells, as previously reported. (d) v-SNARE GFP:Snc1 localizes to a large fluorescent punctum () in the BIC-associated IH cell and to smaller vesicles in growing IH at 27 h.p.i. Images left to right: bright-field; and GFP alone. (e) Faint fluorescence from exocyst component Exo70:GFP can be observed in the subapical BIC-associated cell at 28 h.p.i. (f) t-SNARE Sso1:GFP () localizes in the BIC-associated IH cell near the BIC in a first-invaded rice cell at 27 h.p.i. (g) t-SNARE Sso1:GFP () in a second-invaded cell accumulated near BICs, as crescents at the tips of five primary IH (white arrows), and as puncta in two BIC-associated IH cells after differentiation (outline arrows). Shown at 40 h.p.i. (as described in Fig. 1a, right panel). Images left to right: bright-field; and GFP alone. Scale bars, 10 μm unless stated otherwise.

Figure 4. Brefeldin A blocks secretion of apoplastic but not of cytoplasmic effectors.

(a–c) Images left to right: either merged bright-field, mCherry and GFP or merged mCherry and GFP; mCherry alone; and GFP alone. (a) After secretion, cytoplasmic effector Pwl2:mCherry:NLS (red) shows preferential BIC localization (arrow) and translocation into the rice cell, where it accumulates in the rice nucleus (*). Bas4:GFP shows apoplastic localization outlining the IH. (b) In the presence of BFA, Pwl2:mCherry:NLS remains BIC-localized (arrow), but Bas4:GFP (green) is retained in the fungal ER, imaged with the same transformant in (a) 10 h after exposure to BFA. (c) Cytoplasmic effectors Bas1:mRFP (red, middle) and AVR-Pita:GFP (green, right) still co-localized in the BIC (arrow) after 5 h exposure to BFA. (d) Fluorescence recovery after photobleaching (FRAP) demonstrates continuous secretion of Pwl2:GFP into the BIC in the presence of BFA. Rice tissue infected by a fungal strain expressing Pwl2:GFP and Bas4:mRFP was incubated in BFA for 3 h before photobleaching of Pwl2:GFP in the BIC. Secretion of Bas4:mRFP had been blocked at this point. FRAP results were identical in the presence or absence of BFA (P=0.019). Bars show mean fluorescence intensity recovery after bleaching (mean±s.d., four FRAP experiments). Images left to right before photobleaching: merged bright-field and GFP; GFP alone; and mRFP alone. Images left to right after photobleaching and recovery: merged bright-field and GFP; and GFP alone. Scale bars, 10 μm.

Figure 5. Secretion of cytoplasmic effectors involves exocyst components Exo70 and Sec5.

(a–c) Wild-type strain Guy11 and corresponding mutants expressed Pwl2:mCherry:NLS and Bas4:GFP. Images clockwise from the upper left: bright-field; merged mCherry (red) and GFP (green); fluorescence intensity linescans for mCherry (red) and GFP (green) along the path of the white arrow; single channel GFP or mCherry fluorescence shown as black and white inverse images, respectively. (a) Pwl2:mCherry:NLS in wild type shows preferential BIC localization, translocation and accumulation in the rice nucleus (white asterisk) with no fluorescence observed in the BIC-associated IH cell. Bas4:GFP localizes to the EIHM compartment. (b) Δexo70 mutant shows partial retention of Pwl2:mCherry:NLS, predominantly in the BIC-associated IH cell. Bas4:GFP secretion appears normal. Internal red fluorescence in the BIC-associated IH cell is further visualized by a black and white inverse image (lower left) and by fluorescence intensity scans, which show red fluorescence between the peaks of green fluorescence marking the boundary of the EIHM. Faint red fluorescence in the rice nucleus (white asterisk) is consistent with effector secretion being only partially blocked in the mutants. (c) Δsec5 mutant showing partial retention of Pwl2:mCherry:NLS inside the BIC-associated IH cell, but no retention of Bas4:GFP, as further visualized by black and white inverse images and fluorescence intensity scans. (d–f) Images left to right: merged bright-field and mRFP; single channel mRFP fluorescence as a black and white inverse image; and corresponding fluorescence intensity linescan. (d) Wild type Guy11 expressing cytoplasmic effector Bas1:mRFP. Bas1:mRFP fluorescence is not observed inside wild type IH cells. (e) Bas1:mRFP expressed by an Δexo70 mutant shows significant retention inside the IH, predominantly in the BIC-associated cells. (f) Bas1:mRFP expressed by an Δsec5 mutant strain shows significant retention of Bas1:mRFP inside the IH, predominantly in the BIC-associated cells. Linescans shown are representative of wild type (n=20) and knockout mutants (n=20 for each). Scale bars, 10 μm.

Figure 6. Exocyst component and t-SNARE mutants suggest roles in pathogenicity.

(a) Targeted deletions of the SEC5, EXO70 and SSO1 genes in the aggressive Chinese field isolate O-137 (WT) resulted in a significant reduction in pathogenicity on a fully susceptible rice cultivar YT-16 in whole plant spray inoculation assays at Kansas State University. Numbers of lesions formed and lesion sizes were both reduced. (b) Similar pathogenicity defects were observed after inoculation of mutants in the Guy11 (WT) background on rice cultivar CO-39 at the University of Exeter. These data are presented as a bar chart showing the frequency of lesions formed per 5 cm of CO-39 leaf surface (P<0.05 for all mutants; n=30 for each mutant; mean±s.d., three experiments).

Figure 7. t-SNARE mutants suggest a role in BIC development.

(a) Δsso1 mutant showing inappropriate secretion of Pwl2:mCherry:NLS during plant infection. In addition to BICs in the expected location, second fluorescent foci occurred midway along the primary hyphae. Images left to right: bright-field; GFP (green); and mCherry (red). (b) Another infection site in which Pwl2:mCherry:NLS expressed by an Δsso1 mutant strain shows inappropriate secretion. Images clockwise from the upper left: bright-field; merged mCherry (red) and GFP (green); fluorescence intensity linescans for mCherry (red) and GFP (green) along the white arrow; single channel GFP or mCherry fluorescence shown as black and white inverse images. (c) Bas1:mRFP expressed by an Δsso1 mutant shows inappropriate secretion. Images left to right: merged bright-field and mRFP; mRFP fluorescence shown as black and white inverse image; and fluorescence intensity linescan along the white arrow. Rice nuclei are indicated by a white asterisk. Linescans shown are representative of wild type (n=20) and knockout mutants (n=20 for each). Scale bars, 10 μm.

Figure 8. Model for effector secretion by M. oryzae.

This cartoon shows a bulbous IH at 26–30 h.p.i. inside a rice cell. The EIHM (tan) continues around the BIC. Apoplastic effectors (blue), including Bas4, Bas113 and Slp1, accumulate in the EIHM compartment surrounding the IH, resulting in uniform outlining of the IH. These apoplastic effectors follow the conventional, BFA-sensitive, Golgi-dependent secretion pathway. In contrast, cytoplasmic effectors (magenta), including Pwl2, AVR-Pita, Bas1 and Bas107, accumulate in the BIC beside the first-differentiated bulbous IH cell. To a lesser extent, as observed at saturated fluorescence exposure levels (Fig. 3c), cytoplasmic effectors (magenta dotted line) also accumulate inside the EIHM surrounding the BIC-associated cells (the primary hypha and first-differentiated bulbous IH cell), but they do not outline the subsequently formed bulbous IH cells. These cytoplasmic effectors follow a nonconventional, BFA-insensitive secretion pathway involving exocyst and SNARE proteins.