Characterizing Roles for the Glutathione Reductase,Thioredoxin Reductase and Thioredoxin Peroxidase-Encoding Genes of Magnaporthe oryzae during Rice Blast Disease

Understanding how pathogenic fungi adapt to host plant cells is of major concern to securing global food production. The hemibiotrophic rice blast fungus Magnaporthe oryzae, cause of the most serious disease of cultivated rice, colonizes leaf cells asymptomatically as a biotroph for 4–5 days in susceptible rice cultivars before entering its destructive necrotrophic phase. During the biotrophic growth stage, M. oryzae remains undetected in the plant while acquiring nutrients and growing cell-to-cell. Which fungal processes facilitate in planta growth and development are still being elucidated. Here, we used gene functional analysis to show how components of the NADPH-requiring glutathione and thioredoxin antioxidation systems of M. oryzae contribute to disease. Loss of glutathione reductase, thioredoxin reductase and thioredoxin peroxidase-encoding genes resulted in strains severely attenuated in their ability to grow in rice cells and that failed to produce spreading necrotic lesions on the leaf surface. Glutathione reductase, but not thioredoxin reductase or thioredoxin peroxidase, was shown to be required for neutralizing plant generated reactive oxygen species (ROS). The thioredoxin proteins, but not glutathione reductase, were shown to contribute to cell-wall integrity. Furthermore, glutathione and thioredoxin gene expression, under axenic growth conditions, was dependent on both the presence of glucose and the M. oryzae sugar/ NADPH sensor Tps1, thereby suggesting how glucose availability, NADPH production and antioxidation might be connected. Taken together, this work identifies components of the fungal glutathione and thioredoxin antioxidation systems as determinants of rice blast disease that act to facilitate biotrophic colonization of host cells by M. oryzae.     

The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the β-TrCP homologue slimb

The ubiquitin–proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase—an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of β-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family.     

A New Paradigm for Enzymatic Control of α-Cleavage and β-Cleavage of the Prion Protein

The cellular form of the prion protein (PrP^C) is found in both full-length and several different cleaved forms in vivo. Although the precise functions of the PrP^C proteolytic products are not known, cleavage between the unstructured N-terminal domain and the structured C-terminal domain at Lys-109↓His-110 (mouse sequence), termed α-cleavage, has been shown to produce the anti-apoptotic N1 and the scrapie-resistant C1 peptide fragments. β-Cleavage, residing adjacent to the octarepeat domain and N-terminal to the α-cleavage site, is thought to arise from the action of reactive oxygen species produced from redox cycling of coordinated copper. We sought to elucidate the role of key members of the ADAM (a disintegrin and metalloproteinase) enzyme family, as well as Cu²⁺ redox cycling, in recombinant mouse PrP (MoPrP) cleavage through LC/MS analysis. Our findings show that although Cu²⁺ redox-generated reactive oxygen species do produce fragmentation corresponding to β-cleavage, ADAM8 also cleaves MoPrP in the octarepeat domain in a Cu²⁺- and Zn²⁺-dependent manner. Additional cleavage by ADAM8 was observed at the previously proposed location of α-cleavage, Lys-109↓His-110 (MoPrP sequencing); however, upon addition of Cu²⁺, the location of α-cleavage shifted by several amino acids toward the C terminus. ADAM10 and ADAM17 have also been implicated in α-cleavage at Lys-109↓His-110; however, we observed that they instead cleaved MoPrP at a novel location, Ala-119↓Val-120, with additional cleavage by ADAM10 at Gly-227↓Arg-228 near the C terminus. Together, our results show that MoPrP cleavage is far more complex than previously thought and suggest a mechanism by which PrP^C fragmentation responds to Cu²⁺ and Zn²⁺.     

Cells in cells: morphogenetic and metabolic strategies conditioning rice infection by the blast fungus Magnaporthe oryzae

The rice blast fungus Magnaporthe oryzae is a global food security threat due to its destruction of cultivated rice. Of the world's rice harvest, 10–30 % is lost each year to this pathogen, and changing climates are likely to favor its spread into new areas. Insights into how the fungus might be contained could come from the wealth of molecular and cellular studies that have been undertaken in order to shed light on the biological underpinnings of blast disease, aspects of which we review herein. Infection begins when a three-celled spore lands on the surface of a leaf, germinates, and develops the specialized infection structure called the appressorium. The mature appressorium develops a high internal turgor that acts on a thin penetration peg, forcing it through the rice cuticle and into the underlying epidermal cells. Primary then invasive hyphae (IH) elaborate from the peg and grow asymptomatically from one living rice cell to another for the first few days of infection before host cells begin to die and characteristic necrotic lesions form on the surface of the leaf, from which spores are produced to continue the life cycle. To gain new insights into the biology of rice blast disease, we argue that, conceptually, the infection process can be viewed as two discrete phases occurring in markedly different environments and requiring distinct biochemical pathways and morphogenetic regulation: outside the host cell, where the appressorium develops in a nutrient-free environment, and inside the host cell, where filamentous growth occurs in a glucose-rich, nitrogen-poor environment, at least from the perspective of the fungus. Here, we review the physiological and metabolic changes that occur in M. oryzae as it transitions from the surface to the interior of the host, thus enabling us to draw lessons about the strategies that allow M. oryzae cells to thrive in rice cells.     

Mechanism of proteasome gate modulation by assembly chaperones Pba1 and Pba2

The active sites of the proteasome are housed within its central core particle (CP), a barrel-shaped chamber of four stacked heptameric rings, and access of substrates to the CP interior is mediated by gates at either axial end. These gates are constitutively closed and may be opened by the regulatory particle (RP), which binds the CP and facilitates substrate degradation. We recently showed that the heterodimeric CP assembly chaperones Pba1/2 also mediate gate opening through an unexpected structural arrangement that facilitates the insertion of the N terminus of Pba1 into the CP interior; however, the full mechanism of Pba1/2-mediated gate opening is unclear. Here, we report a detailed analysis of CP gate modulation by Pba1/2. The clustering of key residues at the interface between neighboring α-subunits is a critical feature of RP-mediated gate opening, and we find that Pba1/2 recapitulate this strategy. Unlike RP, which inserts at six α-subunit interfaces, Pba1/2 insert at only two α-subunit interfaces. Nevertheless, Pba1/2 are able to regulate six of the seven interfacial clusters, largely through direct interactions. The N terminus of Pba1 also physically interacts with the center of the gate, disrupting the intersubunit contacts that maintain the closed state. This novel mechanism of gate modulation appears to be unique to Pba1/2 and therefore likely occurs only during proteasome assembly. Our data suggest that release of Pba1/2 at the conclusion of assembly is what allows the nascent CP to assume its mature gate conformation, which is primarily closed, until activated by RP.     

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Mitotic checkpoint gene expression is tuned by codon usage bias

The mitotic checkpoint (also called spindle assembly checkpoint, SAC) is a signaling pathway that safeguards proper chromosome segregation. Correct functioning of the SAC depends on adequate protein concentrations and appropriate stoichiometries between SAC proteins. Yet very little is known about the regulation of SAC gene expression. Here, we show in the fission yeast Schizosaccharomyces pombe that a combination of short mRNA half‐lives and long protein half‐lives supports stable SAC protein levels. For the SAC genes mad2 ⁺ and mad3 ⁺, their short mRNA half‐lives are caused, in part, by a high frequency of nonoptimal codons. In contrast, mad1 ⁺ mRNA has a short half‐life despite a higher frequency of optimal codons, and despite the lack of known RNA‐destabilizing motifs. Hence, different SAC genes employ different strategies of expression. We further show that Mad1 homodimers form co‐translationally, which may necessitate a certain codon usage pattern. Taken together, we propose that the codon usage of SAC genes is fine‐tuned to ensure proper SAC function. Our work shines light on gene expression features that promote spindle assembly checkpoint function and suggests that synonymous mutations may weaken the checkpoint.