Pepsin IHC staining of the laryngeal mucosa appears to be a sensitive and specific test for diagnosing LPR in a clinical application.

Magnaporthe oryzae, the ascomycete fungus that causes rice blast disease, initiates conidiation in response to light when grown on Prune-Agar medium containing both carbon and nitrogen sources. Macroautophagy/autophagy was shown to be essential for M. oryzae conidiation and induced specifically upon exposure to light but is undetectable in the dark. Therefore, it is inferred that autophagy is naturally induced by light, rather than by starvation during M. oryzae conidiation. However, the signaling pathway(s) involved in such phototropic induction of autophagy remains unknown. We identified an M. oryzae ortholog of GCN5 (MGG_03677), encoding a histone acetyltransferase (HAT) that negatively regulates light- and nitrogen-starvation-induced autophagy, by acetylating the autophagy protein Atg7. Furthermore, we unveiled novel regulatory mechanisms on Gcn5 at both transcriptional and post-translational levels, governing its function associated with the unique phototropic response of autophagy in this pathogenic fungus. Thus, our study depicts a signaling network and regulatory mechanism underlying the autophagy induction by important environmental clues such as light and nutrients.

Summary Ferroptosis, an iron‐dependent cell death process, was found to occur in Magnaporthe oryzae, and plays a key role in infection‐related development therein. Ferroptosis in the rice‐blast fungus was confirmed based on five basic criteria. We confirmed the dependence of ferroptosis on ferric ions, and optimized ratio‐fluorescence imaging of C11‐BODIPY581/591 as a precise sensor for lipid peroxides that mediate ferroptosis in M. oryzae. We uncovered an important regulatory function for reduced glutathione and NADPH oxidases in modulating the superoxide moieties required for ferroptotic cell death. We found ferroptosis to be necessary for the developmental cell death of conidia during appressorium maturation in rice blast. Such ferroptotic cell death initiated first in the terminal cell and progressed sequentially to the entire conidium. Iron chelation or chemical inhibition of ferroptosis caused conidial cells to remain viable, and led to strong defects in host invasion by M. oryzae. Ferroptosis induction exclusively in the host severely constrained the invasive growth of M. oryzae. We found inter‐reliant and independent roles for ferroptosis and autophagy in controlling such precise cell death in M. oryzae during pathogenic differentiation. Our study provides significant molecular insights into the role of developmental cell death and iron homeostasis in fungal pathogenesis.

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AbstractWe identified that ferroptosis, an iron-dependent non-apoptotic cell death process, occurs in the rice blast fungus Magnaporthe oryzae, and plays a key role in infection-related development therein. Ferroptosis in the blast fungus was confirmed based on the four basic criteria. We confirmed the dependence of ferroptosis on ferric ions, and optimized C11-BODIPY581/591 as a key sensor for subcellular detection and quantification of lipid peroxides that mediate ferroptotic cell death during the pathogenic growth phase of M. oryzae. In addition, we uncovered an important regulatory function for reduced glutathione and the NADPH oxidases in generating/modulating the superoxide moieties for ferroptotic cell death in Magnaporthe. Ferroptosis was found to be necessary for the specific developmental cell death in conidia during appressorium maturation in rice blast. Such ferroptotic cell death initiated first in the terminal cell and progressed sequentially to the entire conidium. Chelation of iron or chemical inhibition of ferroptosis caused conidial cells to remain viable and led to strong defects in host invasion by M. oryzae. Precocious induction of ferroptosis in a blast-susceptible rice cultivar led to resistance against M. oryzae invasion. Interestingly, ferroptosis and autophagy were found to play inter-reliant or codependent roles in contributing to such precise cell death in M. oryzae conidia during pathogenic differentiation. Our study provides significant molecular insights into understanding the role of developmental cell death and iron homeostasis in infection-associated morphogenesis and in fungus-plant interaction in the blast pathosystem.

The secretory cavity is a typical structure in Citrus fruit and is formed by schizolysigeny. Previous reports have indicated that programmed cell death (PCD) is involved in the degradation of secretory cavity cells in the fruit, and that the spatio-temporal location of calcium is closely related to nuclear DNA degradation in this process; however, the molecular mechanisms underlying this Ca2+ regulation remain largely unknown. Here, we identified CgCaN that encodes a Ca2+-dependent DNase in the fruit of Citrus grandis ‘Tomentosa’, the function of which was studied using calcium ion localization, DNase activity assays, in situ hybridization, and protein immunolocalization. The results suggested that the full-length cDNA of CgCaN contains an ORF of 1011 bp that encodes a protein 336 amino acids in length with a SNase-like functional domain. CgCaN digests dsDNA at neutral pH in a Ca2+-dependent manner. In situ hybridization signals of CgCaN were particularly distributed in the secretory cavity cells. Ca2+ and Ca2+-dependent DNases were mainly observed in the condensed chromatin and in the nucleolus. In addition, spatio-temporal expression patterns of CgCaN and its protein coincided with the time-points that corresponded to chromatin degradation and nuclear rupture during the PCD in the development of the fruit secretory cavity. Taken together, our results suggest that Ca2+-dependent DNases play direct roles in nuclear DNA degradation during the PCD of secretory cavity cells during Citrus fruit development. Given the consistency of the expression patterns of genes regulated by calmodulin (CaM) and calcium-dependent protein kinases (CDPK) and the dynamics of calcium accumulation, we speculate that CaM and CDPK proteins might be involved in Ca2+ transport from the extracellular walls through the cytoplasm and into the nucleus to activate CgCaN for DNA degradation.

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