Rapid translation of genome sequences into meaningful biological information hinges on the integration of multiple experimental and informatics methods into a cohesive platform. Despite the explosion in the number of genome sequences available, such a platform does not exist for filamentous fungi. Here we present the development and application of a functional genomics and informatics platform for a model plant pathogenic fungus, Magnaporthe oryzae. In total, we produced 21,070 mutants through large-scale insertional mutagenesis using Agrobacterium tumefaciens-mediated transformation. We used a high-throughput phenotype screening pipeline to detect disruption of seven phenotypes encompassing the fungal life cycle and identified the mutated gene and the nature of mutation for each mutant. Comparative analysis of phenotypes and genotypes of the mutants uncovered 202 new pathogenicity loci. Our findings demonstrate the effectiveness of our platform and provide new insights on the molecular basis of fungal pathogenesis. Our approach promises comprehensive functional genomics in filamentous fungi and beyond.

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Background: Sanguinarine is a benzophenanthridine alkaloid that is known to have antimicrobial, anti-inflammatory, antioxidant and anticancer properties. In this study, we examined the effects of this compound on reactive oxygen species (ROS) production and the association of these effects with apoptotic tumor cell death using a human breast carcinoma MDA-MB-231 cell line. Methods: Cytotoxicity was evaluated by trypan blue exclusion methods. Apoptosis was detected using DAPI staining, agarose gel electrophoresis and flow cytometer. The expression levels of proteins were determined by Western blot analyses and caspase activities were measured using colorimetric assays. The ROS production and mitochondrial membrane potential (MMP, ΔΨ_m) changes were measured fluorimetrically. Results: The results of this study demonstrate that sanguinarine mediates ROS production, and that this mediation is followed by a decrease in MMP, the release of cytochrome c, activation of caspase-9 and caspase-3, and downregulation of antiapoptosis factor XIAP and cIAP-1. Sanguinarine also promoted the activation of caspase-8 and truncation of Bid (tBid). Moreover, the quenching of ROS generation by N-acetyl-L-cysteine administration, a scavenger of ROS, reversed the sanguinarine-induced apoptosis effects via inhibition of ROS production, MMP collapse, tBid expression and the subsequent activation of caspases. These observations clearly indicate that ROS are involved in the early molecular events in the sanguinarine-induced apoptotic pathway. Conclusion: Our data imply that sanguinarine-induced ROS are key mediators of MMP collapse, which leads to the release of cytochrome c followed by caspase activation, culminating in apoptosis.

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