The phosphorylation landscape of infection-related development by the rice blast fungus.

Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.

A molecular mechanosensor for real-time visualization of appressorium membrane tension in Magnaporthe oryzae.

The rice blast fungus Magnaporthe oryzae uses a pressurized infection cell called an appressorium to drive a rigid penetration peg through the leaf cuticle. The vast internal pressure of an appressorium is very challenging to investigate, leaving our understanding of the cellular mechanics of plant infection incomplete. Here, using fluorescence lifetime imaging of a membrane-targeting molecular mechanoprobe, we quantify changes in membrane tension in M. oryzae. We show that extreme pressure in the appressorium leads to large-scale spatial heterogeneities in membrane mechanics, much greater than those observed in any cell type previously. By contrast, non-pathogenic melanin-deficient mutants, exhibit low spatially homogeneous membrane tension. The sensor kinase ∆sln1 mutant displays significantly higher membrane tension during inflation of the appressorium, providing evidence that Sln1 controls turgor throughout plant infection. This non-invasive, live cell imaging technique therefore provides new insight into the enormous invasive forces deployed by pathogenic fungi to invade their hosts, offering the potential for new disease intervention strategies.

The transcriptional landscape of plant infection by the rice blast fungus Magnaporthe oryzae reveals distinct families of temporally co-regulated and structurally conserved effectors.

The rice blast fungus Magnaporthe oryzae causes a devastating disease that threatens global rice (Oryza sativa) production. Despite intense study, the biology of plant tissue invasion during blast disease remains poorly understood. Here we report a high-resolution transcriptional profiling study of the entire plant-associated development of the blast fungus. Our analysis revealed major temporal changes in fungal gene expression during plant infection. Pathogen gene expression could be classified into 10 modules of temporally co-expressed genes, providing evidence for the induction of pronounced shifts in primary and secondary metabolism, cell signaling, and transcriptional regulation. A set of 863 genes encoding secreted proteins are differentially expressed at specific stages of infection, and 546 genes named MEP (Magnaportheeffector protein) genes were predicted to encode effectors. Computational prediction of structurally related MEPs, including the MAX effector family, revealed their temporal co-regulation in the same co-expression modules. We characterized 32 MEP genes and demonstrate that Mep effectors are predominantly targeted to the cytoplasm of rice cells via the biotrophic interfacial complex and use a common unconventional secretory pathway. Taken together, our study reveals major changes in gene expression associated with blast disease and identifies a diverse repertoire of effectors critical for successful infection.

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.

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.

Contrasting Patterns of Colonization with Verticillium longisporum in Winter- and Spring-Type Oilseed Rape (Brassica napus) in the Field and Greenhouse and the Role of Soil Temperature.

Oilseed rape, an important source of vegetable plant oil, is threatened by Verticillium longisporum, a soil-borne vascular fungal pathogen so far occurring in oilseed rape growing regions in Europe and Canada. Despite intensive research into V. longisporum in the last decades in controlled conditions, basic knowledge is still lacking about the time course of infection, temporal pattern of colonization, and disease development on field-grown plants. In this study, colonization of roots, stem bases, and stems with V. longisporum was followed by real-time PCR from the seedling until mature plant stages in 2-year field experiments with microsclerotia-infested plots and either spring-type or autumn-sown (winter-type) oilseed rape cultivars. The temporal pattern of plant colonization differed between greenhouse and field-grown oilseed rape and between spring- and winter-type plants in the field. Within 28 to 35 days, a continuous systemic colonization with V. longisporum was detected in roots and shoots of young plants in the greenhouse associated with significant stunting. In contrast, real-time PCR analysis of V. longisporum in field-grown winter oilseed rape plants displayed a strongly discontinuous colonization pattern with low fungal growth in roots during juvenile growth stages until flowering, whereas in spring oilseed rape, no root colonization was observed until early flowering stages. Hence, stem colonization with the pathogen required 6 months in winter oilseed rape and 1 month in spring oilseed rape from the time of initial root infection. The different patterns of stem colonization were related to soil temperature. Average soil temperatures in 5-cm depth during 7 days before sampling time points from 2 years of field experiments displayed a significant relationship with fungal colonization in the root. A climate chamber inoculation trial with soil temperature levels that varied from 6 to 18°C revealed a threshold temperature of >12°C in the soil to enable root invasion. This soil condition is reached in winter-type oilseed rape in the field in Germany either until the eight-leaf stage in early autumn or after pod stage in spring, whereas in spring-sown oilseed rape early root infection is delayed owing to the cool conditions during juvenile growth stages. The delay of stem colonization in field-grown oilseed rape may explain the lack of stunting as observed in the greenhouse and the previously reported inconsistent effects of V. longisporum on yield levels and seed quality, which were confirmed in this study.

Potential for Seed Transmission of Verticillium longisporum in Oilseed Rape (Brassica napus).

Verticillium longisporum is a soilborne vascular fungal pathogen that has spread throughout the European oilseed rape cultivation area since the 1980s and was detected in canola fields in Canada in 2014. In a series of greenhouse and field inoculation experiments using V. longisporum-resistant and susceptible cultivars of winter and spring types of oilseed rape, the present study investigated the potential of V. longisporum dissemination by seeds of Brassica napus. Greenhouse inoculation studies with a DsRed-labeled isolate of V. longisporum confirmed the systemic growth of the pathogen from roots to seeds. Further monitoring of plant colonization in the greenhouse with a species-specific real-time polymerase chain reaction assay verified the pathogen growth from roots to stem bases, pods, and seeds in root-inoculated plants. The frequency of recovery of viable colonies of V. longisporum from seeds harvested from greenhouse-grown inoculated plants ranged from 0.08 to 13.3%. The frequency of seed transmission in the greenhouse differed in oilseed rape cultivars varying in susceptibility to V. longisporum. Subsequent studies on transmission of the disease into the offspring revealed that only 1.7 to 2.3% of plants showed disease symptoms as confirmed by the formation of microsclerotia in the stems. Results from field-grown plants differed from the greenhouse studies. The degree of seed transmission in the field was dependent on the crop type. Although only low concentrations of DNA of V. longisporum were detectable in seeds harvested from severely infected winter oilseed rape, significantly greater concentrations of fungal DNA were found in seeds of spring-type oilseed rape, at similar soil conditions and inoculum densities. Correspondingly, plating seeds that were harvested from infected plants on agar yielded viable V. longisporum colonies only from seeds of the spring-type but not of the winter-type plants. Lack of seed infection in the winter-type crop was confirmed in two seasons. Equally, none of the offspring grown from seeds from severely diseased winter oilseed rape plants developed symptoms of Verticillium stem striping. The results suggest that the rate of seed transmission of V. longisporum depends on the degree of plant colonization, which is significantly faster under greenhouse than field conditions and in a spring-sown crop compared with an autumn-sown oilseed rape crop. According to our studies, disease transmission by seeds from European winter oilseed rape production cannot be confirmed.