Transcripts and tumors: regulatory and metabolic programming during biotrophic phytopathogenesis [version 1; referees: 3 approved]

Biotrophic fungal pathogens of plants must sense and adapt to the host environment to complete their life cycles. Recent transcriptome studies of the infection of maize by the biotrophic pathogen Ustilago maydis are providing molecular insights into an ordered program of changes in gene expression and the deployment of effectors as well as key features of nutrient acquisition. In particular, the transcriptome data provide a deeper appreciation of the complexity of the transcription factor network that controls the biotrophic program of invasion, proliferation, and sporulation. Additionally, transcriptome analysis during tumor formation, a key late stage in the life cycle, revealed features of the remodeling of host and pathogen metabolism that may support the formation of tremendous numbers of spores. Transcriptome studies are also appearing for other smut species during interactions with their hosts, thereby providing opportunities for comparative approaches to understand biotrophic adaptation

Grx4 : a key regulator of secondary metabolism, nitrogen uptake, and iron homeostasis in the corn smut fungus, Ustilago maydis

The corn smut fungus, Ustilago maydis, is the premier basidiomycete model for the study of biotrophic plant-pathogen interactions. In fungi, monothiol glutaredoxins are central regulators of key cellular functions such as iron homeostasis, cell wall integrity and redox status via their interactions with other proteins as well as iron-sulfur clusters and glutathione. In this study I characterized the novel roles of the monothiol glutaredoxin Grx4 in the biology of U. maydis. In addition to its roles identified in other fungi, Grx4 is necessary for normal pathogenesis by U. maydis on its plant host, Zea mays. Mutants expressing a conditional allele of grx4 under the control of the arabinose-induced/glucose-repressed promoter Pcrg, exhibited decreased virulence on maize which correlated with grx4 transcript levels at the time of infection. Additionally, perturbations were detected in homeostasis and perception of the essential nutrients iron and nitrogen following grx4 repression in glucose-containing media. Furthermore, grx4 repression strongly altered secondary metabolism, leading to induction of melanin and itaconic acid biosynthetic genes and accumulation of these metabolites in vitro. A subset of Grx4-regulated multicopper oxidase genes with possible roles in secondary metabolism, (particularly genes potentially involved in melanin biosynthesis) were targeted for further reverse genetic study. This aspect of the work identified the key laccase enzyme required for melanization and laccase activity by U. maydis in a variety of conditions, Lac2. It also identified hitherto unknown roles in virulence for this gene family. Together, these data suggest that glutaredoxins could play important roles in the virulence of plant pathogenic fungi in addition to their established roles as key regulators of fundamental cellular processes. The involvement of Grx4 in the production by U. maydis of industrially relevant secondary metabolites (e.g., itaconic acid) also renders these findings of biotechnological interest.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

The Monothiol Glutaredoxin Grx4 Influences Iron Homeostasis and Virulence in Ustilago maydis

The corn smut fungus, Ustilago maydis, is an excellent model for studying biotrophic plant-pathogen interactions, including nutritional adaptation to the host environment. Iron acquisition during host colonization is a key aspect of microbial pathogenesis yet less is known about this process for fungal pathogens of plants. Monothiol glutaredoxins are central regulators of key cellular functions in fungi, including iron homeostasis, cell wall integrity, and redox status via interactions with transcription factors, iron-sulfur clusters, and glutathione. In this study, the roles of the monothiol glutaredoxin Grx4 in the biology of U. maydis were investigated by constructing strains expressing a conditional allele of grx4 under the control of the arabinose-inducible, glucose-repressible promoter Pcrg1. The use of conditional expression was necessary because Grx4 appeared to be essential for U. maydis. Transcriptome and genetic analyses with strains depleted in Grx4 revealed that the protein participates in the regulation of iron acquisition functions and is necessary for the ability of U. maydis to cause disease on maize seedlings. Taken together, this study supports the growing appreciation of monothiol glutaredoxins as key regulators of virulence-related phenotypes in pathogenic fungi.Science, Faculty ofNon UBCMicrobiology and Immunology, Department ofReviewedFacultyResearche

An Autonomous Molecular Bioluminescent Reporter (AMBER) for Voltage Imaging in Freely Moving Animals.

Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When co-expressed with the luciferin-producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several-fold increase in the bioluminescence output (ΔL/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons of Caenorhabditis elegans is expressed. Using the compressed sensing approach, the electropharingeogram of the C. elegans pharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition