Rgs1 is a regulator of effector gene expression during plant infection by the rice blast fungus Magnaporthe oryzae.

To cause rice blast disease, the filamentous fungus Magnaporthe oryzae secretes a battery of effector proteins into host plant tissue to facilitate infection. Effector-encoding genes are expressed only during plant infection and show very low expression during other developmental stages. How effector gene expression is regulated in such a precise manner during invasive growth by M. oryzae is not known. Here, we report a forward-genetic screen to identify regulators of effector gene expression, based on the selection of mutants that show constitutive effector gene expression. Using this simple screen, we identify Rgs1, a regulator of G-protein signaling (RGS) protein that is necessary for appressorium development, as a novel transcriptional regulator of effector gene expression, which acts prior to plant infection. We show that an N-terminal domain of Rgs1, possessing transactivation activity, is required for effector gene regulation and acts in an RGS-independent manner. Rgs1 controls the expression of at least 60 temporally coregulated effector genes, preventing their transcription during the prepenetration stage of development prior to plant infection. A regulator of appressorium morphogenesis is therefore also required for the orchestration of pathogen gene expression required for invasive growth by M. oryzae during plant infection.

A Basic Guide to the Growth and Manipulation of the Blast Fungus, Magnaporthe oryzae.

The blast fungus, Magnaporthe oryzae, is a devastating plant pathogen that threatens global food security. The social and economic importance of blast disease has contributed to this filamentous fungus becoming a model organism for the study of host-pathogen interactions. Availability of the complete genome sequences of many strains of the pathogen, as well as rice and wheat cultivars, coupled with the tractability of M. oryzae to classical and molecular genetic manipulation have contributed to its widespread study. Although M. oryzae has been extensively investigated for the past two decades, procedures for storing, maintaining, and manipulating the blast fungus in the laboratory had not been compiled and updated. As a consequence, there is considerable disparity in how the fungus is stored and manipulated between studies. In this article, we present a collection of protocols providing clear explanations of how to preserve filter stocks of M. oryzae; how to grow the fungus in both liquid and solid media; how to extract genomic DNA from fungal mycelium; how to induce appressorium formation on coverslips for visualization and tissue collection; and how to perform two distinct types of plant infection assay for virulence assessment. By sharing our most used laboratory procedures, we aim to address some of the knowledge gaps in current M. oryzae protocols and contribute to uniformity and robustness in studies by the Magnaporthe research community. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Storage of M. oryzae strains Basic Protocol 2: Revival and regular maintenance of M. oryzae cultures in solid medium Alternate Protocol 1: Regular maintenance of M. oryzae cultures in liquid medium Basic Protocol 3: Genomic DNA extraction from M. oryzae mycelium Alternate Protocol 2: Quick DNA extraction from M. oryzae mycelium Basic Protocol 4: M. oryzae induction of appressorium development on glass coverslips for microscopy Alternate Protocol 3: M. oryzae induction of appressorium development on glass coverslips for tissue collection Basic Protocol 5: M. oryzae rice infection assay through spray inoculation Alternate Protocol 4: M. oryzae leaf-drop plant infection assay.

The appressorium at a glance.

Many plant pathogenic fungi have the capacity to infect their plant hosts using specialised cells called appressoria. These structures act as a gateway between the fungus and host, allowing entry to internal tissues. Appressoria apply enormous physical force to rupture the plant surface, or use a battery of enzymes to digest the cuticle and plant cell wall. Appressoria also facilitate focal secretion of effectors at the point of plant infection to suppress plant immunity. These infection cells develop in response to the physical characteristics of the leaf surface, starvation stress and signals from the plant. Appressorium morphogenesis has been linked to septin-mediated reorganisation of F-actin and microtubule networks of the cytoskeleton, and remodelling of the fungal cell wall. In this Cell Science at a Glance and accompanying poster, we highlight recent advances in our understanding of the mechanisms of appressorium-mediated infection, and compare development on the leaf surface to the biology of invasive growth by pathogenic fungi. Finally, we outline key gaps in our current knowledge of appressorium cell biology.

Appressorium-mediated plant infection by Magnaporthe oryzae is regulated by a Pmk1-dependent hierarchical transcriptional network.

Rice blast is a devastating disease caused by the fungal pathogen Magnaporthe oryzae that threatens rice production around the world. The fungus produces a specialized infection cell, called the appressorium, that enables penetration through the plant cell wall in response to surface signals from the rice leaf. The underlying biology of plant infection, including the regulation of appressorium formation, is not completely understood. Here we report the identification of a network of temporally coregulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which regulates genes associated with induction of major physiological changes required for appressorium development-including cell-cycle control, autophagic cell death, turgor generation and melanin biosynthesis-as well as controlling a additional set of virulence-associated transcription factor-encoding genes. Pmk1-dependent phosphorylation of Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organization, polarized exocytosis and effector gene expression, which are necessary for plant tissue invasion. Identification of this regulatory cascade provides new potential targets for disease intervention.

The Biology of Invasive Growth by the Rice Blast Fungus Magnaporthe oryzae.

This introductory chapter describes the life cycle of Magnaporthe oryzae, the causal agent of rice blast disease. During plant infection, M. oryzae forms a specialized infection structure called an appressorium, which generates enormous turgor, applied as a mechanical force to breach the rice cuticle. Appressoria form in response to physical cues from the hydrophobic rice leaf cuticle and nutrient availability. The signaling pathways involved in perception of surface signals are described and the mechanism by which appressoria function is also introduced. Re-polarization of the appressorium requires a septin complex to organize a toroidal F-actin network at the base of the cell. Septin aggregation requires a turgor-dependent sensor kinase, Sln1, necessary for re-polarization of the appressorium and development of a rigid penetration hypha to rupture the leaf cuticle. Once inside the plant, the fungus undergoes secretion of a large set of effector proteins, many of which are directed into plant cells using a specific secretory pathway. Here they suppress plant immunity, but can also be perceived by rice immune receptors, triggering resistances. M. oryzae then manipulates pit field sites, containing plasmodesmata, to facilitate rapid spread from cell to cell in plant tissue, leading to disease symptom development.

Investigating the cell and developmental biology of plant infection by the rice blast fungus Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent of rice blast disease, the most widespread and serious disease of cultivated rice. Live cell imaging and quantitative 4D image analysis have provided new insight into the mechanisms by which the fungus infects host cells and spreads rapidly in plant tissue. In this video review article, we apply live cell imaging approaches to understanding the cell and developmental biology of rice blast disease. To gain entry to host plants, M. oryzae develops a specialised infection structure called an appressorium, a unicellular dome-shaped cell which generates enormous turgor, translated into mechanical force to rupture the leaf cuticle. Appressorium development is induced by perception of the hydrophobic leaf surface and nutrient deprivation. Cargo-independent autophagy in the three-celled conidium, controlled by cell cycle regulation, is essential for appressorium morphogenesis. Appressorium maturation involves turgor generation and melanin pigment deposition in the appressorial cell wall. Once a threshold of turgor has been reached, this triggers re-polarisation which requires regulated generation of reactive oxygen species, to facilitate septin GTPase-dependent cytoskeletal re-organisation and re-polarisation of the appressorium to form a narrow, rigid penetration peg. Infection of host tissue requires a further morphogenetic transition to a pseudohyphal-type of growth within colonised rice cells. At the same time the fungus secretes an arsenal of effector proteins to suppress plant immunity. Many effectors are secreted into host cells directly, which involves a specific secretory pathway and a specialised structure called the biotrophic interfacial complex. Cell-to-cell spread of the fungus then requires development of a specialised structure, the transpressorium, that is used to traverse pit field sites, allowing the fungus to maintain host cell membrane integrity as new living plant cells are invaded. Thereafter, the fungus rapidly moves through plant tissue and host cells begin to die, as the fungus switches to necrotrophic growth and disease symptoms develop. These morphogenetic transitions are reviewed in the context of live cell imaging studies.

Periodontal Side Effect During Orthopedic Face Mask Therapy.

A 7-year-old patient exhibited gingival recession of tooth #41 and severe plaque accumulation after 3 months of face mask therapy. The recession improved only slightly after appropriate oral hygiene instructions and motivation. Decisive improvement began when the vertical chin pad extension was reduced to avoid pressure on the affected area.

Dental and alveolar arch forms in a Caucasian population compared with commercially available archwires.

INTRODUCTION: The aim of this study was to identify an arch form, comprising dentition and alveolus, representative of the Caucasian population, and to compare it with the shape of the main archwires on the market. MATERIALS AND METHODS: The study sample comprised 35 pairs of dental casts, taken from the arches of 35 Southern Europeans with ideal natural occlusion. After the dental casts were scanned, the reference points of the dental arches (FA points) and alveolar bone (WALA ridges) were identified with 3D software. Cartesian coordinates and intercanine and intermolar diameters were calculated for each patient, and curves representing the upper and lower dentition, as well as those showing the shape of the lower supporting bone, were traced and compared with those pertaining to different types of commonly used orthodontic archwires. RESULTS: Ideal shapes of the mandibular alveolus and of the upper and lower archwires were calculated and compared with those actually on the market. Statistical analysis showed significant differences between the two, particularly at the upper and lower molars and canines. Likewise, there were significant differences between the shapes of the available archwires and the WALA ridge, except for the intercanine width. CONCLUSIONS: None of the commercial archwires examined faithfully represented the shape of the 'ideal' dentition we calculated, particularly at the molars and canines. The bone structure of the mandibular support cannot be used as a guide to the shape of the arch form during orthodontic treatment.

Intra-arch widths: a meta-analysis.

OBJECTIVE: To determine and compare the mean values of various intra-arch distances in Class I, Class II division 1 and Class II division 2 patients. MATERIALS AND METHODS: The search engines PubMed, Embrace and Cochrane were employed to select articles for further study, and the sample thereby selected comprised adult subjects with full permanent dentition, without missing teeth, malformations or previous orthodontic treatment. Only subjects from articles published during the last 10 years were included in the study. RESULTS: The search located a total of 628 articles regarding arch form. However, only eight articles were chosen for the study. We observed that: the distance between mandibular canines is smaller in Class I than in Class II division 1; mandibular inter-molar distance is similar in Class I and Class II division 1; maxillary inter-canine distance is similar in Class I, Class II division 2 and Class II division 1; maxillary inter-premolar width is greater in Class I than in Class II division 1; and that maxillary inter-molar width is similar in Class I and Class II division 2. CONCLUSIONS: No statistically significant differences in arch width were found between the different classes analysed (there are only slightly differences between classes), except in the case of mandibular inter-canine width (L3), which was smaller in Class I than in Class II,1, and maxillary inter-premolar width (U4), which was smaller in Class II-I than in Class I.