The penetration ring is a novel infection structure formed by the penetration peg for invading plant cell membrane in rice blast fungus

Joint Public Review:

This study presents novel insights into the formation and characterization of a penetration ring during host infection by Magnaporthe oryzae. Based on the solid genetic evidence and localization data, the authors demonstrate the structural presence of the penetration ring and the contribution of Ppe1 to fungal virulence. Nevertheless, the mechanisms through which the penetration ring influences host-pathogen interaction, including its potential function in effector translocation, remain only partially resolved. Further work using higher-resolution imaging and functional assays will help address this knowledge gap. Overall, the findings are valuable for advancing our understanding of plant-pathogen interactions, though important mechanistic questions remain open.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study focuses on characterizing a previously identified gene, encoding the secreted protein Ppe1, that may play a role in rice infection by the blast fungus Magnaporthe oryzae. Magnaporthe oryzae is a hemibiotrophic fungus that infects living host cells before causing disease. Infection begins with the development of a specialized infection cell, the appressorium, on the host leaf surface. The appressorium generates enormous internal turgor that acts on a thin penetration peg at the appressorial base, forcing it through the leaf cuticle. Once through this barrier, the peg elaborates into bulbous invasive hyphae that colonizes the first infected cell before moving to neighboring cells via plasmodesmata. During this initial biotrophic growth stage, invasive hyphae invaginate the host plasma membrane, which surrounds growing hyphae as the extra-invasive hyphae membrane (EIHM). To avoid detection, the fungus secretes apoplastic effectors into the EIHM matrix via the conventional ER-Golgi secretion pathway. The fungus also forms a plant-derived structure called the biotrophic interfacial complex (BIC) that receives cytoplasmic effectors through an unconventional secretion route before they are delivered into the host cell. Together, these secreted effector proteins act to evade or suppress host innate immune responses. Here the authors contribute to our understanding of M. oryzae infection biology by showing how Ppe1, which localizes to both the appressorial penetration peg and to the appressorial-like transpressoria associated with invasive hyphal movements into adjacent cells, maximizes host cell penetration and disease development and is thus a novel contributor to rice blast disease.

We sincerely appreciate the reviewer’s thoughtful evaluation of our work. We are grateful for your recognition of Ppe1 as a novel contributor to M. oryzae infection biology and your insightful summary of its spatio-temporal localization and functional importance in host penetration. We also appreciate devoting your time to provide us with constructive feedback, which greatly strengthens our manuscript.

Strengths:

A major goal of M. oryzae research is to understand how the fungus causes disease, either by determining the physiological underpinnings of the fungal infection cycle or by identifying effectors and their host targets. Such new knowledge may point the way to novel mitigation strategies. Here, the authors make an interesting discovery that bridges both fungal physiology and effector biology research by showing how a secreted protein Ppe1, initially considered an effector with potential host targets, associates with its own penetration peg (and transpressoria) to facilitate host invasion. In a previous study, the authors had identified a small family of small secreted proteins that may function as effectors. Here they suggest Ppe1 (and, later in the manuscript, Ppe2/3/5) localizes outside the penetration peg when appressoria develops on surfaces that permit penetration, but not on artificial hard surfaces that prevent peg penetration. Deleting the PPE1 gene reduced (although did not abolish) penetration, and a fraction of those that penetrated developed invasive hyphae that were reduced in growth compared to WT. Using fluorescent markers, the authors show that Ppe1 forms a ring underneath appressoria, likely where the peg emerges, which remained after invasive hyphae had developed. The ring structure is smaller than the width of the appressorium and also lies within the septin ring known to form during peg development. This so-called penetration ring also formed at the transpressorial penetration point as invasive hyphae moved to adjacent cells. This structure is novel, and required for optimum penetration during infection. Furthermore, Ppe1, which carries a functional signal peptide, may form on the periphery of the peg, together suggesting it is secreted and associated with the peg to facilitate penetration. Staining with aniline blue also suggests Ppe1 is outside the peg. Together, the strength of the work lies in identifying a novel appressorial penetration ring structure required for full virulence.

We are deeply grateful to the reviewer for the clear understanding and insightful evaluation of our work. Your recognition of the novel contribution and scientific merit of our study is both encouraging and motivating. We sincerely appreciate the time, expertise and constructive feedback dedicated to reviewing our manuscript, as the comments have been instrumental in enhancing the quality of this work.

Weaknesses:

The main weakness of the paper is that, although Ppe1 is associated with the peg and optimizes penetration, the function of Ppe1 is not known. The work starts off considering Ppe1 a secreted effector, then a facilitator of penetration by associating with the peg, but what role it plays here is only often speculated about. For example, the authors consider at various times that it may have a structural role, a signaling role orchestrating invasive hyphae development, or a tethering role between the peg and the invaginated host plasma membrane (called throughout the host cytoplasmic membrane, a novel term that is not explained). However, more effort should be expended to determine which of these alternative roles is the most likely. Otherwise, as it stands, the paper describes an interesting phenomenon (the appressorial ring) but provides no understanding of its function.

We sincerely appreciate the reviewer’s comments. We have revised "host cytoplasmic membrane" to "host plasma membrane" throughout the manuscript for consistency. To further investigate the role of the Ppe1 in the interaction between M. oryzae and rice, we overexpressed PPE1 in rice ZH11. A pCXUN-SP-GFP-Ppe1 vector containing a signal peptide and an N-terminal GFP tag was constructed (pCXUN-SP-GFP-Ppe1), and 35 GFP-PPE1-OX plants (T0) were subsequently obtained through Agrobacterium-mediated rice transformation. Subsequently, PCR and qRT-PCR validation were performed on the T0 transgenic plants. The PCR results showed that the inserted plasmid could be amplified from the genomic DNA extracted from the leaves of all the resulting T0 plants (Author response image 1A). qRT-PCR results indicated that most T0 transgenic plants could transcriptionally express PPE1 (Author response image 1B). T0 plants with higher expression levels were selected for western blot analysis, which confirmed the presence of GFP-Ppe1 bands of the expected size (Author response image 1C). To further explore the targets of Ppe1 in rice, the leaf sheaths of T0 plants were inoculated with M. oryzae strain Guy11. Total proteins were extracted at 24 hours post-inoculation (hpi) and subjected to immunoprecipitation using GFP magnetic beads. Silver staining revealed more interacting protein bands in T0 plants compared to ZH11 and GFP-OX controls (Author response image 1D). These samples were then analyzed by mass spectrometry in which 331 rice proteins that potentially interact with Ppe1 were identified (Author response image 1E). Subsequently, yeast two-hybrid assays were performed on 13 putative interacting proteins with higher coverage, but no interaction was detected between Ppe1 and these proteins (Author response image 1F-G). Considering that the identification and functional validation of interacting proteins is a labor-intensive and time-consuming endeavor, we will focus our future efforts on in-depth studies of Ppe1's function in rice.

Author response image 1.

Screening of Ppe1 candidate targets in rice. (A) The determination of GFP-PPE1 construct in transgenic rice. (B) The expression of PPE1 transgenic rice (T0) was verified by qRT-PCR. (C) Western blot analysis of Ppe1 expression in transgenic rice. (D) Rapid silver staining for detection of the purified proteins captured by the GFP-beads. (E) Venn diagram comparing the number of proteins captured in the different samples. (F) Identity of the potential targets of Ppe1 in rice. (G) Yeast two-hybrid assay showing negative interaction of Ppe1 with rice candidate proteins.

The inability to nail down the function of Ppe1 likely stems from two underlying assumptions with weak support. Firstly, the authors assume that Ppe1 is secreted and associated with the peg to form a penetration ring between the plant cell wall and cytoplasm membrane. However, the authors do not demonstrate it is secreted (for instance by blocking Ppe1 secretion and its association with the peg using brefeldin A).

To investigate the secretion pathway of Ppe1 in M. oryzae, we determined the inhibitory effects of Brefeldin A (BFA) on conventional ER-to-Golgi secretion in fungi as suggested by the reviewer. We inoculated rice leaf sheaths with conidia suspensions from the Ppe1-mCherry and PBV591 strains (containing a Pwl2-mCherry-NLS and Bas4-GFP co-expressing constructs) and treated them with BFA. We found that, even after exposure to BFA for 5 to 11 hours, the Ppe1-mCherry still formed its characteristic ring conformation (Author response image 2). Similarly, in the BFA-treated samples, the cytoplasmic effector Pwl2-mCherry accumulated at the BIC, while the apoplastic effector Bas4-GFP was retained in the invasive hyphae (Author response image 2). These results indicate that Ppe1 is not secreted through the conventional ER-Golgi secretion pathway.

Author response image 2.

The secretion of Ppe1 is not affected by BFA treatment. (A) and (B) The Ppe1-mCherry fluorescent signal was still observed both in the presence and absence of BFA. (C) Following BFA treatment, the secretion of the apoplastic effector Bas4-GFP was blocked while that of the cytoplasmic effector Pwl2-mCherry was not affected. The rice leaf sheath tissue was inoculated with 50 μg/mL BFA (0.1% DMSO) at 17 hpi. Images were captured at 22 hpi for A and 28 hpi for B and C. Scale bars = 10 µm.

Also, they do not sufficiently show that Ppe1 localizes on the periphery of the peg. This is because confocal microscopy is not powerful enough to see the peg. The association they are seeing (for example in Figure 4) shows localization to the bottom of the appressorium and around the primary hyphae, but the peg cannot be seen. Here, the authors will need to use SEM, perhaps in conjunction with gold labeling of Ppe1, to show it is associating with the peg and, indeed, is external to the peg (rather than internal, as a structural role in peg rigidity might predict). It would also be interesting to repeat the microscopy in Figure 4C but at much earlier time points, just as the peg is penetrating but before invasive hyphae have developed - Where is Ppe1 then? Finally, the authors speculate, but do not show, that Ppe1 anchors penetration pegs on the plant cytoplasm membrane. Doing so may require FM4-64 staining, as used in Figure 2 of Kankanala et al, 2007 (DOI: 10.1105/tpc.106.046300), to show connections between Ppe1 and host membranes. Note that the authors also do not show that the penetration ring is a platform for effector delivery, as speculated in the Discussion.

We sincerely appreciate the reviewer's valuable suggestion regarding SEM with immunogold labeling to precisely visualize Ppe1's association with penetration peg. While we fully acknowledge this would be an excellent approach, after consulting several experts in the field, we realized that the specialized equipment and technical expertise required for fungal immunogold-SEM are currently unavailable to us. We sincerely hope that the reviewer will understand this technical limitation.

To further strengthen our evidence for the role of Ppe1's in anchoring penetration peg to the plant plasma membrane, we provided new co-localization images of Ppe1 and penetration peg (Fig. S7). At 16 hours post-inoculation (hpi), when the penetration peg was just forming and prior to the development of invasive hyphae, the Ppe1-mCherry fluorescence forms a tight ring-like structure closely associated with the base of the appressorium. As at 23 hpi, the circular Ppe1-mCherry signal was still detectable beneath the appressorium, and around the penetration peg which differentiated into the primary invasive hyphae. Furthermore, we obtained 3D images of the strain expressing both Ppe1-mCherry and Lifeact-GFP during primary invasive hyphal development. The results revealed that Ppe1 forms a ring-like structure that remains anchored to the penetration peg during fungal invasion (Fig. S6).

We also conducted FM4-64 staining experiment as recommended by the reviewer. Although the experiment provided valuable insights, we found that the resolution was insufficient to precisely delineate the spatial relationship between Ppe1 and host membranes at the penetration peg (Author response image 3). To optimize this colocalization, we tested the localization between Ppe1-mCherry ring and rice plasma membrane marker GFP-OsPIP2 (Fig. S8). These new results provide compelling complementary evidence supporting our conclusion that Ppe1 functions extracellularly at the host-pathogen interface. We hope these additional data will help address the reviewer's concerns regarding Ppe1's localization.

Author response image 3.

FM4-64-stained rice leaf sheath inoculated with M. oryzae strain expressing Ppe1-GFP. Ppe1-GFP ring was positioned above the primary invasive hyphae. Scale bar = 5 µm.

Secondly, the authors assume Ppe1 is required for host infection due to its association with the peg. However, its role in infection is minor. The majority of appressoria produced by the mutant strain penetrate host cells and elaborate invasive hyphae, and lesion sizes are only marginally reduced compared to WT (in fact, the lesion density of the 70-15 WT strain itself seems reduced compared to what would be expected from this strain). The authors did not analyze the lesions for spores to confirm that the mutant strains were non-pathogenic (non-pathogenic mutants sometimes form small pinprick-like lesions that do not sporulate). Thus, the pathogenicity phenotype of the knockout mutant is weak, which could contribute to the inability to accurately define the molecular and cellular function of Ppe1.

We appreciate the reviewer’s comments. To ensure the reliability of our findings, we conducted spray inoculation experiments with multiple independent repeats. Our results consistently demonstrated that deletion of the PPE1 gene significantly attenuates the virulence of M. oryzae. Further analysis of lesion development and sporulation in the Δppe1 mutant revealed that it retains the ability to produce conidia. To validate these observations, we generated a PPE1 knockout in the wild-type reference strain Guy11. Similarly, we observed a significant decrease in the pathogenicity of the Δppe1 mutants generated from the wild-type Guy11 strain compared to Guy11 in the spray assay (Fig S2). These results collectively indicate the importance of Ppe1 in the pathogenicity of M. oryzae to rice.

In summary, it is important that the role of Ppe1 in infection be determined.

Reviewer #2 (Public review):

The article focuses on the study of Magnaporthe oryzae, the fungal pathogen responsible for rice blast disease, which poses a significant threat to global food security. The research delves into the infection mechanisms of the pathogen, particularly the role of penetration pegs and the formation of a penetration ring in the invasion process. The study highlights the persistent localization of Ppe1 and its homologs to the penetration ring, suggesting its function as a structural feature that facilitates the transition of penetration pegs into invasive hyphae. The article provides a thorough examination of the infection process of M. oryzae, from the attachment of conidia to the development of appressoria and the formation of invasive hyphae. The discovery of the penetration ring as a structural element that aids in the invasion process is a significant contribution to the understanding of plant-pathogen interactions. The experimental methods are well-documented, allowing for reproducibility and validation of the results.

We sincerely appreciate the thoughtful and insightful evaluation of our work. Thank you for recognizing the significance of our findings regarding the penetration ring and the functional role of Ppe1 during host invasion.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Line 48: "after appressorium- or transpressorium-mediated penetration of plant cell wall" - transpressoria do not penetrate the plant cell wall.

Thank you for your valuable suggestion. For improved clarity, we have rephrased the sentence as follows: In this study, we showed that a penetration ring is formed by penetration pegs after appressorium-mediated penetration of plant cell wall.

Line 143: "approximately 25% of the 143 appressoria formed by the Δppe1 mutant had no penetration peg" - It is not possible to see the penetration peg by confocal microscopy.

Thank you for your valuable suggestion. We have revised the sentence as follows: In contrast, approximately 25% of the appressoria formed by the Δppe1 mutant had no penetration.

Line 159: "inner cycle" -should be inner circle?

We gratefully acknowledge the reviewer's careful reading. The typographical error has been corrected throughout the revised manuscript.

Line 255: "These results indicate that initiation of penetration peg formation is necessary for the formation of the penetration ring." Actually, more precisely, they indicate that penetration is necessary.

We appreciate this suggestion and have revised the text to be more concise: These results indicate that penetration is necessary for the formation of the penetration ring.

Line 282: "unlike subcellular localizations of other effectors"- is this an effector if no plant targets are known?

We appreciate this suggestion and have revised the text as follows: unlike subcellular localizations of Bas4, Slp1, Pwl2, and AvrPiz-t.

Line 299: "it may function as a novel physical structure for anchoring penetration pegs on the surface of plant cytoplasm membrane after cell wall penetration" - an interaction with the plant plasma membrane was not shown and this is speculative.

We have provided new evidence to show the spatial positioning of Ppe1-mCherry ring with the rice plasma membrane (see figure S8)

Line 301: "It is also possible that this penetration ring functions as a collar or landmark that is associated with the differentiation of penetration pegs (on the surface of cytoplasm membrane) into primary invasive hyphae enveloped in the EIHM cytoplasm membrane (Figure 7)." The alternative conclusions for Ppe1 function, either interacting with host membranes or acting as a developmental landmark, need to be resolved here.

We appreciate this suggestion and have revised the text as follows: It is also possible that this penetration ring functions as a collar that is associated with the differentiation of penetration pegs into primary invasive hyphae enveloped in the EIHM (Figure 7).

Line 317: "is likely a structural feature or component for signaling the transition of penetration pegs to invasive hyphae",- if the authors think Ppe1 has these roles, why do they refer to Ppe1 as an effector?

Many thanks for these comments. We have revised this and refer to Ppe1 as a secreted protein throughout the revised manuscript.

Line 337: "After the penetration of plant cell wall, the penetration ring may not only function as a physical structure but also serve as an initial effector secretion site for the release of specific effectors to overcome plant immunity in early infection stages"- which is it? Also, no evidence is provided to suggest it is a platform for effector secretion.

We sincerely appreciate your valuable suggestion. We have revised this sentence as follows: After the penetration of plant cell wall, the penetration ring may not only function as a physical structure but also serve as a secretion site for the release of specific proteins to overcome plant immunity during the early infection stages.

Reviewer #2 (Recommendations for the authors):

(1) While the study suggests the penetration ring as a structural feature, it remains unclear whether it also serves as a secretion site for effectors. Further exploration of this aspect would strengthen the conclusions.

We thank the reviewer for this useful suggestion. In this study, we demonstrated that Ppe1 proteins form a distinct penetration ring structure at the site where the penetration peg contacts the plant plasma membrane prior to differentiation into primary invasive hyphae (Figs. 2 and 7). Thus, we reasoned that penetration ring may function as a novel physical structure. Notably, additional Ppe family members (Ppe2, Ppe3, and Ppe5) were also found to localize to this penetration ring (Fig. 6B), suggesting that it also serves as a secretion site for releasing proteins. To test whether Ppe1 and Ppe2 label to the same site, we analyzed the colocalization between Ppe1-GFP and Ppe2-mCherry. The results showed that Ppe1-GFP and Ppe2-mCherry are well colocalized (Author response image 4). This study primarily focuses on the discovery and characterization of the penetration ring. The potential role of this structure in effector translocation will be investigated in future studies.

Author response image 4.

Ppe1 co-localizes with Ppe2 at the penetration ring in M. oryzae. Line graphs were generated at the directions pointed by the white arrows. Scale bar = 2μm.

(2) The article could benefit from a discussion on the broader implications of these findings for developing resistant crop varieties or new fungicidal strategies.

We have incorporated this discussion as suggested (lines 358-360).

(3) What is the significance of the formation of the penetration ring in the pathogenicity of the rice blast fungus? Or, how does it assist the fungus in its infection process?

Our findings have several significant implications. First, we believe that the discovery of the penetration ring as a novel physical structure associated with the differentiation of invasive hyphae represents a breakthrough in plant-pathogen interactions that will be of interest to fungal biologists, pathologists and plant biologists. Secondly, our study presents new role of the peg as a specialized platform for secretory protein deployment, in addition to its commonly known role as a physical penetration tool for the pathogen. Thirdly, we identify Ppe1 as a potential molecular target for controlling the devastating rice blast disease, as Ppe homologs are absent in plants and mammals. We have incorporated this discussion in the revised manuscript (lines 354-362).