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

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.

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.

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.

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). 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.

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.

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.