These authors contributed equally to this work. SummaryTo facilitate glucocorticoid-inducible transgene expression from the pOp promoter in Arabidopsis the ligandbinding domain of a rat glucocorticoid receptor (GR LBD) was fused to the amino terminus of the synthetic transcription factor LhG4 to generate LhGR-N. Fusions bearing the GR LBD at other positions in LhG4 exhibited incomplete repression or inefficient induction. LhGR-N was stringently repressed in the absence of exogenous glucocorticoid but was fully activated by addition of 2 lM dexamethasone which resulted in 1000-fold increase in GUS reporter activity. Half maximal induction was achieved with 0.2 lM dexamethasone. Reporter transcripts were detectable within 2 h of dexamethasone application and peaked 4-10 h later. Neither LhGR-N nor dexamethasone affected seedling development although ethanol retarded development when used as a solvent for dexamethasone. The efficiency of the pOp target promoter was improved 10-to 20-fold by incorporating six copies of the ideal lac operator with sufficient inter-operator spacing to allow simultaneous occupancy. Introduction of the TMV X sequence into the 5¢UTR resulted in a further 10-fold increase in dexamethasone-inducible reporter activity and an increase in the induction factor to 10 4 . Although promoters containing the TMV X sequence exhibited slightly increased basal expression levels in the absence of dexamethasone, stringent regulation of the cytokinin biosynthetic gene ipt was achieved with all promoters. Despite the severity of the induced ipt phenotypes, transcripts for the KNOX homoeodomain transcription factors BREVIPEDICELLUS and SHOOTMERISTEMLESS were not significantly increased within 48 h of dexamethasone application to seedlings.

The major cause of athlete’s foot is Trichophyton rubrum, a dermatophyte or fungal pathogen of human skin. To facilitate molecular analyses of the dermatophytes, we sequenced T. rubrum and four related species, Trichophyton tonsurans, Trichophyton equinum, Microsporum canis, and Microsporum gypseum. These species differ in host range, mating, and disease progression. The dermatophyte genomes are highly colinear yet contain gene family expansions not found in other human-associated fungi. Dermatophyte genomes are enriched for gene families containing the LysM domain, which binds chitin and potentially related carbohydrates. These LysM domains differ in sequence from those in other species in regions of the peptide that could affect substrate binding. The dermatophytes also encode novel sets of fungus-specific kinases with unknown specificity, including nonfunctional pseudokinases, which may inhibit phosphorylation by competing for kinase sites within substrates, acting as allosteric effectors, or acting as scaffolds for signaling. The dermatophytes are also enriched for a large number of enzymes that synthesize secondary metabolites, including dermatophyte-specific genes that could synthesize novel compounds. Finally, dermatophytes are enriched in several classes of proteases that are necessary for fungal growth and nutrient acquisition on keratinized tissues. Despite differences in mating ability, genes involved in mating and meiosis are conserved across species, suggesting the possibility of cryptic mating in species where it has not been previously detected. These genome analyses identify gene families that are important to our understanding of how dermatophytes cause chronic infections, how they interact with epithelial cells, and how they respond to the host immune response.

SummaryWe describe pOp/LhGR, a dexamethasone-inducible derivative of the pOp/LhG4 transcription activation system, and its use in tobacco to regulate expression of uidA (encoding b-glucuronidase; GUS) and the cytokinin-biosnythetic gene ipt. The pOp/LhGR system exhibited stringent regulation and strong induced phenotypes in soil and tissue culture. In conjunction with an improved target promoter, pOp6, that carries six copies of an optimized lac operator sequence the pOp6/LhGR system directed induced GUS activities that exceeded those obtained with pOp/LhG4 or the CaMV 35S promoter but without increased uninduced activity. A single dose of dexamethasone was sufficient to direct cytotoxic levels of ipt expression in soil-grown plants although uninduced plants grew normally throughout a complete life cycle. In vitro, induced transcripts were detectable within an hour of dexamethasone application and 1 nM dexamethasone was sufficient for half maximal induction of GUS activity. Various methods of dexamethasone application were successfully applied under tissue culture and greenhouse conditions. We observed no inhibitory effects of dexamethasone or LhGR on plant development even with the highest concentrations of inducer, although tobacco seedlings were adversely affected by ethanol used as a solvent for dexamethasone stock solutions. The pOp/LhGR system provides a highly sensitive, efficient, and tightly regulated chemically inducible transgene expression system for tobacco plants.

SummarySeveral vector systems are available for tissue-specific transactivation or chemical induction of transgene expression in plants. The choice facing researchers is which promoter system to commit to as this determines the range and characteristics of the expression resources available. The decision will not be the same for all species or applications. We present some general discussion on the use of these technologies and review in detail the properties in various (mainly angiosperm) species of the most promising: mGal4:VP16/UAS and pOp/LhG4 for transactivation, and the alc-switch, GVE/VGE, GVG, pOp6/LhGR, and XVE systems for chemical induction.

SummaryPlant-derived nitric oxide (NO) triggers defence, priming the onset of the hypersensitive response and restricting pathogen ingress during incompatibility. However, little is known about the role of pathogen-produced NO during pre-infection development and infection. We sought evidence for NO production by the rice blast fungus during early infection.NO production was measured using fluorescence of DAR-4M and the role of NO assessed using NO scavengers. The synthesis of NO was investigated by targeted knockout of genes potentially involved in NO synthesis, including nitric oxide synthase-like genes (NOL2 and NOL3) and nitrate (NIA1) and nitrite reductase (NII1), generating single and double Dnia1Dnii1, Dnia1Dnol3, and Dnol2Dnol3 mutants.We demonstrate that Magnaporthe oryzae generates NO during germination and in early development. Removal of NO delays germling development and reduces disease lesion numbers. NO is not generated by the candidate proteins tested, nor by other argininedependent NO systems, by polyamine oxidase activity or non-enzymatically by low pH. Furthermore, we show that, while NIA1 and NII1 are essential for nitrate assimilation, NIA1, NII1, NOL2 and NOL3 are all dispensable for pathogenicity.Development of M. oryzae and initiation of infection are critically dependent on fungal NO synthesis, but its mode of generation remains obscure.

Fluorescent protein markers are widely used to report plant membrane traffic; however, effective protocols to quantify fluorescence or marker expression are lacking. Here the 20 residue self-cleaving 2A peptide from Foot and Mouth Disease Virus was used to construct polyproteins that expressed a trafficked marker in fixed stoichiometry with a reference protein in a different cellular compartment. Various pairs of compartments were simultaneously targeted. Together with a bespoke image analysis tool, these constructs allowed biosynthetic membrane traffic to be assayed with markedly improved sensitivity, dynamic range and statistical significance using protocols compatible with the common plant transfection and transgenic systems. As marker and effector expression could be monitored in populations or individual cells, saturation phenomena could be avoided and stochastic or epigenetic influences could be controlled. Surprisingly, mutational analysis of the ratiometric assay constructs revealed that the 2A peptide was dispensable for efficient cleavage of polyproteins carrying a single internal signal peptide, whereas the signal peptide was essential. In contrast, a construct bearing two signal peptide/anchors required 2A for efficient separation and stability, but 2A caused the aminoterminal moiety of such fusions to be mis-sorted to the vacuole. A model to account for the behaviour of 2A in these and other studies in plants is proposed.

SummaryPlants respond to pathogen attack via a rapid burst of reactive oxygen species (ROS). However, ROS are also produced by fungal metabolism and are required for the development of infection structures in Magnaporthe oryzae.To obtain a better understanding of redox regulation in M. oryzae, we measured the amount and redox potential of glutathione (E GSH ), as the major cytoplasmic anti-oxidant, the rates of ROS production, and mitochondrial activity using multi-channel four-dimensional (x,y,z,t) confocal imaging of Grx1-roGFP2 and fluorescent reporters during spore germination, appressorium formation and infection.High levels of mitochondrial activity and ROS were localized to the growing germ tube and appressorium, but E GSH was highly reduced and tightly regulated during development. Furthermore, germlings were extremely resistant to external H 2 O 2 exposure ex planta. E GSH remained highly reduced during successful infection of the susceptible rice cultivar CO39. By contrast, there was a dramatic reduction in the infection of resistant (IR68) rice, but the sparse hyphae that did form also maintained a similar reduced E GSH .We conclude that M. oryzae has a robust anti-oxidant defence system and maintains tight control of E GSH despite substantial oxidative challenge. Furthermore, the magnitude of the host oxidative burst alone does not stress the pathogen sufficiently to prevent infection in this pathosystem.

GTPases of the Rab1 subclass are essential for membrane traffic between the endoplasmic reticulum (ER) and Golgi complex in animals, fungi and plants. Rab1-related proteins in higher plants are unusual because sequence comparisons divide them into two putative subclasses, Rab-D1 and Rab-D2, that are conserved in monocots and dicots. We tested the hypothesis that the Rab-D1 and Rab-D2 proteins of Arabidopsis represent functionally distinct groups. RAB-D1 and RAB-D2a each targeted fluorescent proteins to the same punctate structures associated with the Golgi stacks and trans-Golgi-network. Dominant-inhibitory N121I mutants of each protein inhibited traffic of diverse cargo proteins at the ER but they appeared to act via distinct biochemical pathways as biosynthetic traffic in cells expressing either of the N121I mutants could be restored by coexpressing the wild-type form of the same subclass but not the other subclass. The same interaction was observed in transgenic seedlings expressing RAB-D1 [N121I]. Insertional mutants confirmed that the three Arabidopsis Rab-D2 genes were extensively redundant and collectively performed an essential function that could not be provided by RAB-D1, which was non-essential. However, plants lacking RAB-D1, RAB-D2b and RAB-D2c were short and bushy with low fertility, indicating that the Rab-D1 and Rab-D2 subclasses have overlapping functions.