SUMMARY Arabidopsis seedlings display rhythmic growth when grown under diurnal conditions, with maximal elongation rates occurring at the end of the night under short-day photoperiods. Current evidence indicates that this behavior involves the action of the growth-promoting bHLH factors PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PHYTOCHROME-INTERACTING FACTOR 5 (PIF5) at the end of the night, through a coincidence mechanism that combines their transcriptional regulation by the circadian clock with control of protein accumulation by light. To assess the possible role of PIF3 in this process, we have analyzed hypocotyl responses and marker gene expression in pif single- and higher-order mutants. The data show that PIF3 plays a prominent role as a promoter of seedling growth under diurnal light/dark conditions, in conjunction with PIF4 and PIF5. In addition, we provide evidence that PIF3 functions in this process through its intrinsic transcriptional regulatory activity, at least in part by directly targeting growth-related genes, and independently of its ability to regulate phytochrome B (phyB) levels. Furthermore, in sharp contrast to PIF4 and PIF5, our data show that the PIF3 gene is not subject to transcriptional regulation by the clock, but that PIF3 protein abundance oscillates under diurnal conditions as a result of a progressive decline in PIF3 protein degradation mediated by photoactivated phyB, and consequent accumulation of the bHLH factor during the dark period. Collectively, the data suggest that phyB-mediated, post-translational regulation allows PIF3 accumulation to peak just before dawn, at which time it accelerates hypocotyl growth, together with PIF4 and PIF5, by directly regulating the induction of growth-related genes.

A mechanism for integrating light perception and the endogenous circadian clock is central to a plant's capacity to coordinate its growth and development with the prevailing daily light/dark cycles. Under short-day (SD) photocycles, hypocotyl elongation is maximal at dawn, being promoted by the collective activity of a quartet of transcription factors, called PIF1, PIF3, PIF4, and PIF5 (phytochromeinteracting factors). PIF protein abundance in SDs oscillates as a balance between synthesis and photoactivated-phytochrome-imposed degradation, with maximum levels accumulating at the end of the long night. Previous evidence shows that elongation under diurnal conditions (as well as in shade) is also subjected to circadian gating. However, the mechanism underlying these phenomena is incompletely understood. Here we show that the PIFs and the core clock component Timing of CAB expression 1 (TOC1) display coincident cobinding to the promoters of predawn-phased, growthrelated genes under SD conditions. TOC1 interacts with the PIFs and represses their transcriptional activation activity, antagonizing PIF-induced growth. Given the dynamics of TOC1 abundance (displaying high postdusk levels that progressively decline during the long night), our data suggest that TOC1 functions to provide a direct output from the core clock that transiently constrains the growth-promoting activity of the accumulating PIFs early postdusk, thereby gating growth to predawn, when conditions for cell elongation are optimal. These findings unveil a previously unrecognized mechanism whereby a core circadian clock output signal converges immediately with the phytochrome photosensory pathway to coregulate directly the activity of the PIF transcription factors positioned at the apex of a transcriptional network that regulates a diversity of downstream morphogenic responses.PIFs | photoperiod | TOC1 | circadian clock | gating of growth G iven the importance of solar energy to plants, they have evolved sophisticated photosensory-response systems to monitor and adapt to the diurnal photoperiod (1). This environmental parameter provides a precise index of the progression of the earth's seasons and the time of the day and thereby a signal that regulates a spectrum of growth and developmental responses (such as elongation growth, flowering, and dormancy) appropriate to the prevailing conditions.The photoreceptors in the phytochrome family (phyA-E in Arabidopsis) are the primary sensors of this signal (2, 3). These chromoproteins regulate two pathways in parallel that converge to control the morphogenic response: (i) the phytochromeinteracting factor (PIF) pathway, whereby the photoactivatedphytochrome molecules bind to and induce the degradation of the PIF proteins (notably the PIF1, PIF3, PIF4, and PIF5 quartet, a subfamily of basic helix-loop-helix transcription factors), thereby altering the expression of the PIF direct-target genes and the cognate downstream transcriptional network (4, 5), and (ii) the circadian clock, whereby the phytochromes entrain t...

In the course of an analysis of the functions and assembly of the cell wall of Candida albicans, we have cloned and characterized a gene, which we designated CSP37 (cell surface protein), encoding a 37-kDa polypeptide which is a membrane-associated protein. Candida albicans is an imperfect fungus capable of causing life-threatening infections in immunocompromised patients as well as a variety of mucosal infections in generally healthy individuals (50). Due to its importance as a human pathogen and the limited number of safe drugs to control deep-rooted infections, many laboratories have undertaken cellular, molecular, and genetic studies to understand the mechanisms governing C. albicans biology. However, the diploid nature of this fungus and the failure to identify a sexual cycle have hampered many classical genetic approaches (59).A number of factors are thought to contribute to the virulence of C. albicans, but their relative importance during pathogenesis remains unclear (14). Potential virulence determinants include the ability to switch from a yeast to a mycelial form (31,62) and also between different colonial morphologies (64), the production of extracellular hydrolytic enzymes (28, 36), synthesis of receptor-ligand molecules required for recognition, and adhesion to the host tissues (7, 8), etc. Since the cell wall is involved in these processes, some groups have attempted to elucidate the mechanism of synthesis of its different constituents, their assembly, and the modifications that occur during the morphological transition. Thus, a number of cell surface antigens have been reported to be preferentially expressed in hyphal or blastoconidial cell morphologies (10,52,66,67), and several genes related to cell wall architecture (assembly and functions) have been cloned. These genes have been identified by various methods, including differential hybridization screening (27), complementation in Saccharomyces cerevisiae (3), cross-hybridization with genes cloned from other organisms (heterologous genes) (20, 65), hybridization with sequence-specific oligonucleotides (9), and PCR amplification (11,12). We have used another approach to clone morphologyspecific C. albicans genes related to the cell wall. We have isolated cDNA clones by screening with polyclonal antibodies raised against isolated cell walls of blastoconidia and mycelial cells (17,61).In this paper, a cDNA clone that reacted with polyclonal antibodies specific for mycelial cell walls was studied. It encodes a novel protein with no significant homology to known sequences and absent from the S. cerevisiae genome. ⌬csp37 null mutants were constructed and subsequently phenotypic analysis and virulence testing were conducted. Location of the protein codified and potential functions are discussed. MATERIALS AND METHODSMicroorganisms and growth conditions. The C. albicans strains used in this study are listed in Table 1. Cells were routinely grown in YPD (2% glucose, 1% yeast extract, 2% Bacto Peptone [Difco, Detroit, Mich.]) with shaking at the selecte...

The phytochrome (phy)-interacting basic helix-loop-helix transcription factors (PIFs) constitutively sustain the etiolated state of dark-germinated seedlings by actively repressing deetiolation in darkness. This action is rapidly reversed upon light exposure by phy-induced proteolytic degradation of the PIFs. Here, we combined a microarray-based approach with a functional profiling strategy and identified four PIF3-regulated genes misexpressed in the dark (MIDAs) that are novel regulators of seedling deetiolation. We provide evidence that each one of these four MIDA genes regulates a specific facet of etiolation (hook maintenance, cotyledon appression, or hypocotyl elongation), indicating that there is branching in the signaling that PIF3 relays. Furthermore, combining inferred MIDA gene function from mutant analyses with their expression profiles in response to light-induced degradation of PIF3 provides evidence consistent with a model where the action of the PIF3/MIDA regulatory network enables an initial fast response to the light and subsequently prevents an overresponse to the initial light trigger, thus optimizing the seedling deetiolation process. Collectively, the data suggest that at least part of the phy/ PIF system acts through these four MIDAs to initiate and optimize seedling deetiolation, and that this mechanism might allow the implementation of spatial (i.e., organ-specific) and temporal responses during the photomorphogenic program.

Screening cDNA libraries with polyclonal antibody preparations against Candida albicans yeast or mycelial cell walls resulted in isolation of several positive clones. Some of them encoded enolase; others encoded a protein of the 70 kDa heat-shock protein family (Hsp70p), etc. The presence of these cytosolic proteins in the cell wall of actively growing C. albicans was discovered by analytical (SDS-PAGE and Western blot) and cytological (indirect immunofluorescence) experiments. Supplementation of cell cultures with papulacandin B, an antibiotic that inhibits formation of the -glucan skeleton, resulted in the release of enolase to the supernatant fluids; this release was prevented when 0.6 M KCI was present as an osmotic stabilizer. The cell wall of C. albicans incorporated exogenously added proteins (enolase and Escherichia coli and C albicans cytosolic proteins). The presence in the C. albicans cell wall of enolase, Hsp70p, and probably other intracellular proteins that are highly immunogenic might help the fungal cells to evade the host defences, and consequently could represent a survival mechanism for C. albicans in vivo.

Candida albicans is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of C. albicans was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designatedPRA1, for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The PRA1 protein was homologous to surface antigens of Aspergillus spp. which react with serum from aspergillosis patients, suggesting that thePRA1 protein may have a role in the host-parasite interaction during candidal infection.

Borna disease virus (BDV) is a noncytolytic

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