Two independent S-phase checkpoints regulate appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae

To cause rice blast disease, the fungal pathogen Magnaporthe oryzae develops a specialized infection structure called an appressorium. This dome-shaped, melanin-pigmented cell generates enormous turgor and applies physical force to rupture the rice leaf cuticle using a rigid penetration peg. Appressorium-mediated infection requires septin-dependent reorientation of the F-actin cytoskeleton at the base of the infection cell, which organizes polarity determinants necessary for plant cell invasion. Here, we show that plant infection by M. oryzae requires two independent S-phase cell-cycle checkpoints. Initial formation of appressoria on the rice leaf surface requires an S-phase checkpoint that acts through the DNA damage response (DDR) pathway, involving the Cds1 kinase. By contrast, appressorium repolarization involves a novel, DDR-independent S-phase checkpoint, triggered by appressorium turgor generation and melanization. This second checkpoint specifically regulates septin- dependent, NADPH oxidase-regulated F-actin dynamics to organize the appressorium pore and facilitate entry of the fungus into host tissue

The Exocyst Complex in Health and Disease

Exocytosis involves the fusion of intracellular secretory vesicles with the plasma membrane, thereby delivering integral membrane proteins to the cell surface and releasing material into the extracellular space. Importantly, exocytosis also provides a source of lipid moieties for membrane extension. The tethering of the secretory vesicle before docking and fusion with the plasma membrane is mediated by the exocyst complex, an evolutionary conserved octameric complex of proteins. Recent findings indicate that the exocyst complex also takes part in other intra-cellular processes besides secretion. These various functions seem to converge toward defining a direction of membrane growth in a range of systems from fungi to plants and from neurons to cilia. In this review we summarize the current knowledge of exocyst function in cell polarity, signaling and cell-cell communication and discuss implications for plant and animal health and disease

CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus

The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species

Chitosan inhibits septin-mediated plant infection by the rice blast fungus Magnaporthe oryzae in a protein kinase C and Nox1 NADPH oxidase-dependent manner

Chitosan is a partially deacetylated linear polysaccharide composed of β-1,4-linked units of d-glucosamine and N-acetyl glucosamine. As well as a structural component of fungal cell walls, chitosan is a potent antifungal agent. However, the mode of action of chitosan is poorly understood. Here, we report that chitosan is effective for control of rice blast disease. Chitosan application impairs growth of the blast fungus Magnaporthe oryzae and has a pronounced effect on appressorium-mediated plant infection. Chitosan inhibits septin-mediated F-actin remodelling at the appressorium pore, thereby preventing repolarization of the infection cell. Chitosan causes plasma membrane permeabilization of M. oryzae and affects NADPH oxidase-dependent synthesis of reactive oxygen species, essential for septin ring formation and fungal pathogenicity. We further show that toxicity of chitosan to M. oryzae requires the protein kinase C-dependent cell wall integrity pathway, the Mps1 mitogen-activated protein kinase and the Nox1 NADPH oxidase. A conditionally lethal, analogue (PP1)-sensitive mutant of Pkc1 is partially remediated for growth in the presence of chitosan, while ∆nox1 mutants increase their glucan : chitin cell wall ratio, rendering them resistant to chitosan. Taken together, our data show that chitosan is a potent fungicide which requires the cell integrity pathway, disrupts plasma membrane function and inhibits septin-mediated plant infection

Chitosan inhibits septin-mediated plant infection by the rice blast fungus Magnaporthe oryzae in a protein kinase C and Nox1 NADPH oxidase-dependent manner

Chitosan is a partially deacetylated linear polysaccharide composed of β-1,4-linked units of d-glucosamine and N-acetyl glucosamine. As well as a structural component of fungal cell walls, chitosan is a potent antifungal agent. However, the mode of action of chitosan is poorly understood. Here, we report that chitosan is effective for control of rice blast disease. Chitosan application impairs growth of the blast fungus Magnaporthe oryzae and has a pronounced effect on appressorium-mediated plant infection. Chitosan inhibits septin-mediated F-actin remodelling at the appressorium pore, thereby preventing repolarization of the infection cell. Chitosan causes plasma membrane permeabilization of M. oryzae and affects NADPH oxidase-dependent synthesis of reactive oxygen species, essential for septin ring formation and fungal pathogenicity. We further show that toxicity of chitosan to M. oryzae requires the protein kinase C-dependent cell wall integrity pathway, the Mps1 mitogen-activated protein kinase and the Nox1 NADPH oxidase. A conditionally lethal, analogue (PP1)-sensitive mutant of Pkc1 is partially remediated for growth in the presence of chitosan, while ∆nox1 mutants increase their glucan : chitin cell wall ratio, rendering them resistant to chitosan. Taken together, our data show that chitosan is a potent fungicide which requires the cell integrity pathway, disrupts plasma membrane function and inhibits septin-mediated plant infection

Clathrin-mediated endocytosis facilitates the internalization of Magnaporthe oryzae effectors into rice cells

Fungi and oomycetes deliver effectors into living plant cells to suppress defenses and control plant processes needed for infection. Little is known about the mechanism by which these pathogens translocate effector proteins across the plasma membrane into the plant cytoplasm. The blast fungus Magnaporthe oryzae secretes cytoplasmic effectors into a specialized biotrophic interfacial complex (BIC) before translocation. Here, we show that cytoplasmic effectors within BICs are packaged into punctate membranous effector compartments that are occasionally observed in the host cytoplasm. Live cell imaging with fluorescently labeled proteins in rice (Oryza sativa) showed that these effector puncta colocalize with the plant plasma membrane and with CLATHRIN LIGHT CHAIN 1, a component of clathrin-mediated endocytosis (CME). Inhibiting CME using virus-induced gene silencing and chemical treatments resulted in cytoplasmic effectors in swollen BICs lacking effector puncta. By contrast, fluorescent marker colocalization, gene silencing, and chemical inhibitor studies failed to support a major role for clathrin-independent endocytosis in effector translocation. Effector localization patterns indicated that cytoplasmic effector translocation occurs underneath appressoria before invasive hyphal growth. Taken together, this study provides evidence that cytoplasmic effector translocation is mediated by CME in BICs and suggests a role for M. oryzae effectors in coopting plant endocytosis

An HLA-E-targeted TCR bispecific molecule redirects T cell immunity against Mycobacterium tuberculosis

Peptides presented by HLA - E, a molecule with very limited polymorphism, represent attractive targets for T cell receptor (TCR) - based immunotherapies to circumvent the limitations imposed by the high polymorphism of classical HLA genes in the human population. Here, we describe a TCR - based bispecific molecule that potently and selectively binds HLA - E in complex with a peptide encoded by the inhA gene of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans. We reveal the biophysical and structural bases underpinning the potency and specificity of this molecule and demonstrate its ability to redirect polyclonal T cells to target HLA - E - expressing cells transduced with mycobacterial inhA as well as primary cells infected with virulent Mtb. Additionally, we demonstrate elimination of Mtb - infected cells and reduction of intracellular Mtb growth. Our study suggests an approach to enhance host T cell immunity against Mtb and provides proof of principle for an innovative TCR - based therapeutic strategy overcoming HLA polymorphism and therefore applicable to a broader patient population

Metabolic rewiring induced by ranolazine improves melanoma responses to targeted therapy and immunotherapy

Resistance of melanoma to targeted therapy and immunotherapy is linked to metabolic rewiring. Here, we show that increased fatty acid oxidation (FAO) during prolonged BRAF inhibitor (BRAFi) treatment contributes to acquired therapy resistance in mice. Targeting FAO using the US Food and Drug Administration-approved and European Medicines Agency-approved anti-anginal drug ranolazine (RANO) delays tumour recurrence with acquired BRAFi resistance. Single-cell RNA-sequencing analysis reveals that RANO diminishes the abundance of the therapy-resistant NGFRhi neural crest stem cell subpopulation. Moreover, by rewiring the methionine salvage pathway, RANO enhances melanoma immunogenicity through increased antigen presentation and interferon signalling. Combination of RANO with anti-PD-L1 antibodies strongly improves survival by increasing antitumour immune responses. Altogether, we show that RANO increases the efficacy of targeted melanoma therapy through its effects on FAO and the methionine salvage pathway. Importantly, our study suggests that RANO could sensitize BRAFi-resistant tumours to immunotherapy. Since RANO has very mild side-effects, it might constitute a therapeutic option to improve the two main strategies currently used to treat metastatic melanoma

Combining ChIP-chip and Expression Profiling to Model the MoCRZ1 Mediated Circuit for Ca2+/Calcineurin Signaling in the Rice Blast Fungus

Significant progress has been made in defining the central signaling networks in many organisms, but collectively we know little about the downstream targets of these networks and the genes they regulate. To reconstruct the regulatory circuit of calcineurin signal transduction via MoCRZ1, a Magnaporthe oryzae C2H2 transcription factor activated by calcineurin dephosphorylation, we used a combined approach of chromatin immunoprecipitation - chip (ChIP-chip), coupled with microarray expression studies. One hundred forty genes were identified as being both a direct target of MoCRZ1 and having expression concurrently differentially regulated in a calcium/calcineurin/MoCRZ1 dependent manner. Highly represented were genes involved in calcium signaling, small molecule transport, ion homeostasis, cell wall synthesis/maintenance, and fungal virulence. Of particular note, genes involved in vesicle mediated secretion necessary for establishing host associations, were also found. MoCRZ1 itself was a target, suggesting a previously unreported autoregulation control point. The data also implicated a previously unreported feedback regulation mechanism of calcineurin activity. We propose that calcium/calcineurin regulated signal transduction circuits controlling development and pathogenicity manifest through multiple layers of regulation. We present results from the ChIP-chip and expression analysis along with a refined model of calcium/calcineurin signaling in this important plant pathogen