Autophagy-dependent TOR reactivation drives fungal growth in living host rice cells

Eukaryotic filamentous plant pathogens with biotrophic growth stages like the devastating hemibiotrophic rice blast fungus Magnaporthe oryzae grow for extended periods in living host plant cells without eliciting defense responses. M. oryzae elaborates invasive hyphae (IH) that grow in and between living rice cells while separated from host cytoplasm by plant-derived membrane interfaces. However, although critical to the plant infection process, the molecular mechanisms and metabolic strategies underpinning this intracellular growth phase are poorly understood. Eukaryotic cell growth depends on activated target-of-rapamycin (TOR) kinase signaling, which inhibits autophagy. Here, using live-cell imaging coupled with plate growth tests and RNAseq, proteomic, quantitative phosphoproteomics and metabolic approaches, we show how cycles of autophagy in IH modulate TOR reactivation via α-ketoglutarate to sustain biotrophic growth and maintain biotrophic interfacial membrane integrity in host rice cells. Deleting the M. oryzae serine-threonine protein kinase Rim15-encoding gene attenuated biotrophic growth, disrupted interfacial membrane integrity and abolished the in planta autophagic cycling we observe here for the first time in wild type. δrim15 was also impaired for glutaminolysis and depleted for α-ketoglutarate. α-ketoglutarate treatment of Δrim15- infected leaf sheaths remediated Δrim15 biotrophic growth. In WT, α-ketoglutarate treatment suppressed autophagy. α-ketoglutarate signaling is amino acid prototrophy- and GS-GOGAT cycle-dependent. We conclude that, following initial IH elaboration, cycles of Rim15-dependent autophagic flux liberate α-ketoglutarate - via the GS-GOGAT cycle - as an amino acid-sufficiency signal to trigger TOR reactivation and promote fungal biotrophic growth in nutrient-restricted host rice cells.

Disruption of the Interfacial Membrane Leads to Magnaporthe oryzae Effector Re-location and Lifestyle Switch During Rice Blast Disease

To cause the devastating rice blast disease, the hemibiotrophic fungus Magnaporthe oryzae produces invasive hyphae (IH) that are enclosed in a plant-derived interfacial membrane, known as the extra-invasive hyphal membrane (EIHM), in living rice cells. Little is known about when the EIHM is disrupted and how the disruption contributes to blast disease. Here we show that the disruption of the EIHM correlates with the hyphal growth stage in first-invaded susceptible rice cells. Our approach utilized GFP that was secreted from IH as an EIHM integrity reporter. Secreted GFP (sec-GFP) accumulated in the EIHM compartment but appeared in the host cytoplasm when the integrity of the EIHM was compromised. Live-cell imaging coupled with sec-GFP and various fluorescent reporters revealed that the loss of EIHM integrity preceded shrinkage and eventual rupture of the rice vacuole. The vacuole rupture coincided with host cell death, which was limited to the invaded cell with presumed closure of plasmodesmata. We report that EIHM disruption and host cell death are landmarks that delineate three distinct infection phases (early biotrophic, late biotrophic, and transient necrotrophic phases) within the first-invaded cell before reestablishment of biotrophy in second-invaded cells. M. oryzae effectors exhibited infection phase-specific localizations, including entry of the apoplastic effector Bas4 into the host cytoplasm through the disrupted EIHM during the late biotrophic phase. Understanding how infection phase-specific cellular dynamics are regulated and linked to host susceptibility will offer potential targets that can be exploited to control blast disease.

Exploring the Biophysical Interaction of 3-Pentadecylphenol with Head Group Region of Lipid Membrane using Fisetin as an Interfacial Membrane Probe

3-Pentadecylphenol (PDP) is a phenolic lipid easily available from natural sources. This compound has different pharmacological, biological and industrial applications. A molecular level understanding on the membrane modification properties of...

Effect of Pea Protein Isolate, Carboxymethyl Cellulose, Pectin and Their Interaction on Physicochemical and Oxidative Stability of Oil-in-Water Emulsions

Biopolymer interaction in oil-in-water (o/w) emulsions has been demonstrated to positively modify the emulsion physicochemical properties which lead to desirable stability. The present work focused on the effect of pea protein isolate (PPI), pectin, carboxymethyl cellulose (CMC) and their interaction on physicochemical properties and oxidative stability of o/w emulsions using a mixture design approach. The emulsions were prepared with 40 % sunflower oil stabilized with 1 % of PPI, pectin and CMC, respectively, as well as their mixtures according to a simplex-centroid design (10 points). The pH values for all emulsions were within acidic condition (3.22 to 4.66) and increased significantly (p<0.05) as the PPI-CMC level increased. Regression modelling revealed that ternary mixture of PPI-pectin-CMC had the strongest significant (p<0.05) synergism on 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity (85.06 to 91.17 %). Besides, interaction between PPI and CMC significantly (p<0.05) reduced the interfacial tension and at the same time thickened the interfacial membrane to provide the emulsion with desirable small droplet size (10.56 μm). This synergistic interaction effect also significantly (p<0.05) improved oxidative stability of the emulsion resulting in low total oxidation value (<7) due to decreased oxygen transportation rate across the thick interfacial membrane surrounding the emulsion droplets. Moreover, with high coefficients of determination (R2 > 96%) and insignificant lack of fit (p>0.05) of the fitted models, this study also proved that the mixture design with regression modelling was useful in elucidating PPI, CMC and pectin interactions and also able to empirically predict the responses to any blend of combination of the components.

Systematic Characterization of DMPC/DHPC Self-Assemblies and Their Phase Behaviors in Aqueous Solution

Self-assemblies composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) form several kinds of structures, such as vesicle, micelle, and bicelle. Their morphological properties have been studied widely, but their interfacial membrane properties have not been adequately investigated. Herein, we report a systematic characterization of DMPC/DHPC assemblies at 20 °C. To investigate the phase behavior, optical density OD500, size (by dynamic light scattering), membrane fluidity 1/PDPH (using 1,6-diphenyl-1,3,5-hexatriene), and membrane polarity GP340 (using 6-dodecanoyl-N,N-dimethyl-2-naphthylamine) were measured as a function of molar ratio of DHPC (XDHPC). Based on structural properties (OD500 and size), large and small assemblies were categorized into Region (i) (XDHPC < 0.4) and Region (ii) (XDHPC ≥ 0.4), respectively. The DMPC/DHPC assemblies with 0.33 ≤ XDHPC ≤ 0.67 (Region (ii-1)) showed gel-phase-like interfacial membrane properties, whereas DHPC-rich assemblies (XDHPC ≥ 0.77) showed disordered membrane properties (Region (ii-2)). Considering the structural and interfacial membrane properties, the DMPC/DHPC assemblies in Regions (i), (ii-1), and (ii-2) can be determined to be vesicle, bicelle, and micelle, respectively.

Influence of Oxidants on the Stability of Tocopherol in Model Nanoemulsions: Role of Interfacial Membrane Organized by Nonionic Emulsifiers

Nanoemulsions were prepared by using emulsifiers with various sizes of hydrophilic and hydrophobic groups to determine the impact of interfacial characteristics on the stability of α-tocopherol incorporated into the nanoemulsions. The α-tocopherol concentration remaining after 3 weeks of storage at 25°C depended greatly on the type of oxidative stress, which indicated that the environment surrounding the oil droplets could determine the stability of α-tocopherol in nanoemulsions. α-Tocopherol was gradually degraded by radical-mediated oxidation over storage, and approximately 60% of its initial concentration remained after 3 weeks of storage. However, under acid- and iron-mediated oxidation, α-tocopherol concentration steeply decreases for the initial 3-day storage, but the degradation rate of α-tocopherol decreased after 3 days of storage and over 90% of the initial α-tocopherol remained after 3 weeks of storage. Interestingly, and contrary to our expectations, the thickness and/or density of the droplet interfacial membrane rarely affected the stability of α-tocopherol incorporated into nanoemulsions. Although it is difficult to generalize beyond α-tocopherol, we conclude that the properties of oil droplet surfaces had no influence on the storage stability of α-tocopherol encapsulated in the droplets.