Biology subjects

Martin-Urdiroz, M.

Publications and source records attributed to Martin-Urdiroz, M..

4 recordsLinked to original sources

bioRxiv2021

Clathrin-mediated Endocytosis Facilitates 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 vesicles that are occasionally observed in the host cytoplasm. Live cell imaging with fluorescently-labeled rice showed that effector vesicles colocalize with plant plasma membrane and with clathrin light chain-1, a marker for clathrin-mediated endocytosis (CME). Inhibition of CME using Virus-Induced Gene Silencing (VIGS) and chemical treatments results in cytoplasmic effectors in swollen BICs lacking vesicles. In contrast, fluorescent marker co-localization, VIGS and chemical inhibitor studies failed to support a major role for clathrin-independent endocytosis in effector vesicle formation. Localization studies of two novel effectors, and of known effectors after CME inhibition, indicate that cytoplasmic effector translocation occurs underneath appressoria before invasive hyphal growth. Taken together, this study provides evidence that cytoplasmic effector translocation is mediated by clathrin-mediated endocytosis in BICs and suggests a role for M. oryzae effectors in co-opting plant endocytosis.

plant biology↗

bioRxiv2020

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

O_LIChitosan is a partially deacetylated linear polysaccharide composed of {beta}-1,4-linked units of D-glucosamine and N-acetyl glucosamine. As well as acting as a structural component of fungal cell walls, chitosan can be applied as a potent antifungal agent. However, the mode-of-action of chitosan in fungal pathogens is poorly understood. C_LIO_LIHere, 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 re-modelling at the appressorium pore, thereby preventing re-polarisation of the infection cell and rice leaf cuticle penetration. C_LIO_LIWe found that 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. Our data further show that the toxicity of chitosan to M. oryzae requires the protein kinase C-dependent cell wall integrity pathway and the Nox1 NADPH oxidase. A conditionally lethal, analogue (PP1)-sensitive mutant of Pkc1 is partially remediated for growth in the presence of chitosan and PP1, while {Delta}nox1 mutants increase their glucan/chitin cell wall ratio, rendering them resistant to chitosan. C_LIO_LITaken together, our data show that chitosan is a potent fungicide for control of the rice blast fungus which involves the cell wall integrity pathway, disrupts plasma membrane and inhibits septin-mediated plant infection. C_LI

plant biology↗

bioRxiv2020

A hierarchical transcriptional network controls appressorium-mediated plant infection by the rice blast fungus Magnaporthe oryzae

Rice blast is a pervasive and devastating disease that threatens rice production across the world. In spite of its importance to global food security, however, the underlying biology of plant infection by the blast fungus Magnaporthe oryzae remains poorly understood. In particular, it is unclear how the fungus elaborates a specialised infection cell, the appressorium, in response to surface signals from the rice leaf. Here, we report the identification of a network of temporally co-regulated transcription factors that act downstream of the Pmk1 mitogen-activated protein kinase pathway to regulate gene expression during appressorium-mediated plant infection. We have functionally characterised this network of transcription factors and demonstrated the operation of a hierarchical transcriptional control system. We show that this tiered regulatory mechanism involves Pmk1-dependent phosphorylation of the Hox7 homeobox transcription factor, which represses hyphal-associated gene expression and simultaneously induces major physiological changes required for appressorium development, including cell cycle arrest, autophagic cell death, turgor generation and melanin biosynthesis. Mst12 then regulates gene functions involved in septin-dependent cytoskeletal re-organisation, polarised exocytosis and effector gene expression necessary for plant tissue invasion.

microbiology↗

bioRxiv2018

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 gene editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based gene editing method. It has previously been reported in M. oryzae that transformation with plasmids expressing Cas9 can generate specific mutations using sgRNAs, directing the endonuclease to specific genes. We show, however, that expression of Cas9 is highly toxic to M. oryzae, rendering this approach impractical. We demonstrate that using purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs), provides an alternative and very effective gene editing procedure. 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. 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.

microbiology↗