Nuclear and structural dynamics during the establishment of a specialized effector-secreting cell by Magnaporthe oryzae in living rice cells
- Emma N. Shipman†1,
- Kiersun Jones†1,
- Cory B. Jenkinson1,
- Dong Won Kim1,
- Jie Zhu1 and
- Chang Hyun Khang1Email author
© The Author(s). 2017
Received: 27 September 2016
Accepted: 18 January 2017
Published: 26 January 2017
To cause an economically important blast disease on rice, the filamentous fungus Magnaporthe oryzae forms a specialized infection structure, called an appressorium, to penetrate host cells. Once inside host cells, the fungus produces a filamentous primary hypha that differentiates into multicellular bulbous invasive hyphae (IH), which are surrounded by a host-derived membrane. These hyphae secrete cytoplasmic effectors that enter host cells presumably via the biotrophic interfacial complex (BIC). The first IH cell, also known as the side BIC-associated cell, is a specialized effector-secreting cell essential for a successful infection. This study aims to determine cellular processes that lead to the development of this effector-secreting first IH cell inside susceptible rice cells.
Using live-cell confocal imaging, we determined a series of cellular events by which the appressorium gives rise to the first IH cell in live rice cells. The filamentous primary hypha extended from the appressorium and underwent asymmetric swelling at its apex. The single nucleus in the appressorium divided, and then one nucleus migrated into the swollen apex. Septation occurred in the filamentous region of the primary hypha, establishing the first IH cell. The tip BIC that was initially associated with the primary hypha became the side BIC on the swollen apex prior to nuclear division in the appressorium. The average distance between the early side BIC and the nearest nucleus in the appressorium was estimated to be more than 32 μm. These results suggest an unknown mechanism by which effectors that are expressed in the appressorium are transported through the primary hypha for their secretion into the distantly located BIC. When M. oryzae was inoculated on heat-killed rice cells, penetration proceeded as normal, but there was no differentiation of a bulbous IH cell, suggesting its specialization for establishment of biotrophic infection.
Our studies reveal cellular dynamics associated with the development of the effector-secreting first IH cell. Our data raise new mechanistic questions concerning hyphal differentiation in response to host environmental cues and effector trafficking from the appressorium to the BIC.
KeywordsAppressorium Biotrophic interfacial complex Biotrophy Effector Live-cell imaging Mitosis Rice blast fungus Hyphal differentiation
During the biotrophic invasion, the primary hypha and IH secrete both cytoplasmic effector proteins (effectors; i.e., Pwl2, Bas1, Bas107, and AvrPiz-t) that enter the host cytoplasm and apoplastic effectors (i.e., Bas4, Bas113, and Slp1) that are retained in the EIHM compartment [10, 12–17]. These effectors are known or presumed to suppress host defense and facilitate infection [10, 14, 16]. Cytoplasmic effectors are preferentially localized in the biotrophic interfacial complex (BIC), which has been hypothesized as the site of effector translocation into the host cytoplasm [12, 18]. Two stages of BICs have been previously described  (Fig. 1a). The tip BIC is associated with the apex of the primary hypha. As the hypha develops, the BIC is positioned on the side of the first IH cell, and thus it is called the side BIC. The first IH cell, also known as the side BIC-associated cell, is a specialized effector-secreting cell essential for a successful infection. Giraldo et al. (2013) have shown that this IH cell uses two distinct pathways for effector secretion. One is the conventional secretory pathway for secretion of apoplastic effectors into the EIHM compartment; the other is the exocyst components Exo70 and Sec5-involved pathway for secretion of cytoplasmic effectors into the BIC. In addition, this first IH cell is critical in its role as the mother cell of the subsequent biotrophic IH cells.
Although our knowledge of early biotrophic invasion has increased, questions remain to be answered, such as how the first IH cell develops inside live host cells, what mechanisms control hyphal differentiation, and what cellular processes are involved in BIC development and effector trafficking. In this study, we provide insights into some of these questions by using live-cell imaging of M. oryzae invasion of susceptible rice cells. Our studies reveal a series of cellular events associated with the first IH cell development. These studies lead to new mechanistic questions concerning in planta hyphal differentiation and effector trafficking.
Results and discussion
Apical swelling of the primary hypha precedes mitosis
We generated M. oryzae transformants constitutively expressing EYFP (labeling the cytoplasm with green fluorescence) and histone H1-tdTomato fusion protein (H1-tdTomato; labeling nuclei with red fluorescence). These transformants exhibited wild-type morphology and pathogenicity (Additional file 1). To characterize the development of the first IH cell upon host penetration (transition between the first two stages in Fig. 1a), we used one of these transformants, CKF2138, and imaged 124 random infection sites from 20 separate inoculations and microscopy sessions at 22–28 h post inoculation (hpi). Quantitative analysis of these images revealed three sequential growth stages (Fig. 1b): (1) the filamentous primary hypha with one nucleus in the appressorium, (2) the apically bulbous primary hypha with one nucleus in the appressorium, and (3) the apically bulbous primary hypha that contains a nucleus with another nucleus in the appressorium. We quantified these observations by measuring the diameter of the primary hypha close to the appressorium and the diameter at the apical region of the primary hypha before or after swelling (Fig. 1c). At all growth stages, the diameter of the primary hypha close to the appressorium was conserved with a mean of 2.3 μm (standard deviation = 0.4 μm, n = 124). In infection sites with the anucleate primary hypha and a single nucleus present in the appressorium, the diameter of the primary hypha tip had a mean of 3.1 μm (standard deviation = 1.0 μm, n = 55). In infection sites displaying one nucleus in the appressorium and another one in the bulbous portion of the primary hypha, the diameter of the bulbous portion had a mean of 5.6 μm (standard deviation = 0.9 μm, n = 69), frequently ranging from 4.0 to 6.9 μm. Taken together, these data indicate that nuclear division and migration typically occur after the apical tip of the primary hypha has swollen to over 4 μm in diameter.
Mitosis in the appressorium and subsequent migration of a single nucleus into the first bulbous IH cell
Formation of the first septum after nuclear division and migration
Development of BICs during hyphal differentiation
Regular growth from the appressorium penetration to the side BIC
The filamentous primary hypha appeared to extend from the appressorium to a consistent distance before widening at its apical region. We reasoned that the side BIC would be positioned at a defined distance from the appressorium. To test this, we measured the distance from the appressorium penetration point to the side BIC using M. oryzae transformant CKF1616, expressing the BIC-localized cytoplasmic effector Pwl2 fused to mCherry:NLS and also IH-outlining apoplastic effector Bas4 fused to EGFP . The penetration point was identified by examining the EGFP fluorescence outlining the primary hypha in conjunction with the bright-field, and the BIC was identified based on the strongly localized mCherry fluorescence. We conducted two-dimensional measurements of these images using the Zen Black open Bezier tool (Version 8.1, Zeiss). The distance along the primary hypha from the penetration point to the side BIC has an average of 36 μm, ranging from 30 to 45 μm (n = 22) (Fig. 4b top; Fig. 4c). To obtain more accurate measurements of the same infection sites, we also used the three-dimensional dendrite measurement algorithm in Imaris (Version 7.6, Bitplane). An additional movie file shows the three-dimensional measurement in more detail (see Additional file 2: Movie S1). The average distance was 43 μm, ranging from 32 to 54 μm (n = 22) (Fig. 4b bottom; Fig. 4c; Additional file 2: Movie S1). Our finding of this considerable distance raises an intriguing question concerning effector secretion. Cytoplasmic effectors such as Pwl2 are secreted into the tip BIC and the side BIC, which are located more than 32 μm from the appressorial nucleus (Fig. 4a left and middle; Fig. 4b-c) in which the PWL2 gene is expressed (Zhu and Khang, unpublished). There must be a mechanism to transport effectors from the appressorium through the primary hypha for their secretion to the distantly located BIC, but the mechanism remains to be determined.
Additional file 2: Movie S1. Three-dimensional measurement from the appressorium penetration point to the BIC of M. oryzae strain CKF1616 growing in a rice cell at 29 hpi. Apoplastic effector Bas4 with a translational fusion to EGFP (green) outlined the invasive hypha. The BIC and the rice nucleus were visualized by BIC-accumulating cytoplasmic effector Pwl2 with a translational fusion to mCherry:NLS (red). Overlap of green and red signals is shown in yellow. Source image is a series of 1 μm confocal optical sections taken over a depth of 23 μm. Movie was created using the key frame animation feature in Imaris software (Version 7.6, Bitplane). At the start of the animation, the penetration point is on the left, and the BIC is on the right. As the animation rotates, a penetration point-to-BIC measurement (gray line) is overlaid on the image. The measurement was performed with the dendrite creation algorithm in the Imaris software (described in the Methods section) and has a length of 33.2 μm. A maximum intensity projection of the same infection site in this movie is shown in Fig. 4b (top), and a mirrored still of the last frame in this movie is shown in Fig. 4b (bottom). Bar = 5 μm. (MOV 4288 kb)
Hyphal growth inside heat-killed rice cells
Plasmids, strains, and fungal transformation
For nuclear-localized tandem-dimer Tomato (tdTomato), the binary plasmid pCK1287 was constructed by cloning the histone H1 gene from N. crassa (H1) at the 5′ end of the tdTomato gene under control of the M. oryzae ribosomal protein 27 (P27) promoter as follows: the 2.2 kb EcoRI-BamHI fragment (P27:H1) isolated from pBV229 and the 1.7 kb BamHI-HindIII fragment (tdTomato:Nos terminator) isolated from pBV359 were ligated between EcoRI and HindIII sites of the binary vector pBV1 (pBHt2) . For the membrane-localized GFP, the binary plasmid pCK1417 was constructed by cloning the following three fragments between EcoRI- HindIII sites of pBV141 (pBGt) : (a) the 0.5 kb EcoRI-NheI fragment (P27) isolated from pCK1374, (b) the 1.4 kb NheI-HpaI fragment (GFP-PLCdelta) isolated from pGFP-C1-PLCdelta:PH  (Addgene plasmid # 21179), and (c) the 0.5 kb PvuII-HindIII fragment of pBV210 (N. crassa β-tubulin terminator). To construct pCK1374, the 0.5 kb PCR product containing the P27 promoter was amplified from pBV167 using the primers CKP23 (5′-GAATTCGAATTGGGTACTCAAATTGG-3′) and CKP355 (5′- GCTAGCTTTGAAGATTGGGTTCCTAC-3′) and subsequently cloned in pJET1.2 (Thermo Scientific). The underlined sequences in CKP23 and CKP355 correspond to EcoRI and NheI sites, respectively. pBV plasmids (pBV1, pBV141, pBV167, pBV210, pBV229, and pBV359 as well as pBV377 described below) were obtained from Dr. Barbara Valent (Kansas State University).
Plasmids were transformed into M. oryzae O-137 or O-137-derived transformants using Agrobacterium tumefaciens-mediated transformation . O-137 is a highly aggressive isolate collected from rice (Oryza sativa) in Hangzhou, Zhejiang, China . CKF2138 (used in Figs. 1b, c and 5) was made by transforming KV1 with pCK1287 (P27:H1-tdTomato). KV1 is an O-137-derived strain that constitutively expresses cytoplasmic EYFP (P27:EYFP) . CKF1962 (used in Fig. 2a) was made by sequentially transforming O-137 with pCK1288 (P27:3xGFP:nuclear localization signal or NLS) and pCK1287 . M. oryzae Guy11 (H1-RFP; used in Fig. 2b and c) has been described in Fernandez et al. (2014). CKF2686 (used in Fig. 3) was made by sequentially transforming O-137 with pCK1287 and pCK1417 (P27:GFP-PLCdelta:PH). CKF1651 (used in Fig. 4a) was made by transforming the O-137-derived CKF110 (P27:EYFP and P27:H1-mRFP) with pBV377 (expressing cytoplasmic effector protein Pwl2 fused to mRFP with the native PWL2 promoter) . CKF1616 (alias KV121; used in Fig. 4b) is an O-137-derived strain that expresses cytoplasmic effector Pwl2 fused to mCherry:NLS with the native PWL2 promoter and apoplastic effector Bas4 fused to EGFP with the native BAS4 promoter . Fungal transformants were purified by the isolation of single germinating spores. Wild-type strain O-137 and transformants were stored dehydrated and frozen at −20 °C to maintain full pathogenicity and cultured on oatmeal agar plates at 24 °C under continuous light .
Rice sheath inoculations were performed as previously described [11, 31]. Briefly, excised leaf sheaths (5–9 cm long) from 17- to 21-day old plants were inoculated with a spore suspension (5 × 104 spores/mL in sterile water). The inoculated sheaths were hand-trimmed at 22–28 hpi and immediately used for confocal microscopy. For heat-killed sheath inoculation assays, we first optimized heat-treatment conditions as follows: Trimmed sheaths were incubated in 70 °C water for 10 or 25 min, immersed in 0.75 M sucrose, and then visually examined under a 20× objective lens for indications of plasmolysis. Sheath cells treated for 10 min underwent some plasmolysis, but those treated for 25 min did not, indicating the 25 min incubation was sufficient to kill cells. In subsequent heat-killed inoculations, pre-trimmed sheaths were incubated in 70 °C water for 25 min, allowed to cool to room temperature, and then inoculated with a spore suspension.
Microscopy and image analysis
Confocal microscopy was performed on a Zeiss Axio Imager M1 microscope equipped with a Zeiss LSM 510 META system using Plan-Apochromat 20×/0.8 NA and Plan-Neofluor 40×/1.3 NA (oil) objectives. Excitation/emission wavelengths were 488 nm/505 to 530 nm (EGFP and YFP), and 543 nm/560 to 615 nm (mRFP, mCherry, and tdTomato). Images were acquired and processed using LSM 510 software (Version 3.2). Confocal images of CKF1616 used for measuring the distance from the appressorial penetration point to the side BIC were obtained from Dr. Barbara Valent. For 2-dimensional measurements, confocal z-stack images were processed using the Zen Black software (Version 8.1, Zeiss). Each image was converted to a maximum intensity projection. The appressorial penetration point was identified based on brightfield visualization of the appressorium and on where the green fluorescence (Bas4-EGFP) outline of the primary hypha stopped. The BIC was identified from the overlap of red (Pwl2-mCherry:NLS) and green fluorescence. The distance between these two points was measured along the center of the primary hypha using the open Bezier tool. For 3-dimensional measurements, confocal z-stack images were imported into Imaris software (Version 7.6, Bitplane). The penetration point and BIC were identified as described for 2D measurements. The dendrite creation algorithm was used to produce a filament between the two points based on green fluorescence outline of the primary hypha for determining the path, and the length of the created dendrite was measured.
Biotrophic interfacial complex
Extra-invasive hyphal membrane
Hours post inoculation
We thank Drs. Seogchan Kang, Barbara Valent, and Richard Wilson for sharing materials. We thank current and former members of the Khang Lab (http://www.khanglab.org/) for support and discussions. We acknowledge the assistance of the Biomedical Microscopy Core at the University of Georgia with imaging using a Zeiss LSM 510 confocal microscope.
This work was supported by the Agriculture and Food Research Initiative competitive grants program, Award number 2014-67013-21717 from the USDA National Institute of Food and Agriculture.
Availability of data and materials
The data sets supporting the results of this article are included within the article and two additional files.
CHK conceived and designed the experiments. ENS, KJ, CBJ, DWK, JZ, and CHK performed the experiments. ENS, KJ, CBJ, CHK analyzed the data and wrote the paper. All authors have read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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