Elsevier

Fungal Genetics and Biology

Volume 121, December 2018, Pages 56-64
Fungal Genetics and Biology

The contribution of the White Collar complex to Cryptococcus neoformans virulence is independent of its light-sensing capabilities

https://doi.org/10.1016/j.fgb.2018.09.008Get rights and content

Highlights

  • The blue light sensing complex is required for virulence of Cryptococcus neoformans.

  • Mutation of a specific cysteine residue for transmission of the light signal blocks light responses.

  • Strains with the cysteine substituted with other amino acid residues have wild type levels of virulence.

  • The contribution of the White Collar 1 homolog to virulence is independent of light signaling.

Abstract

The White Collar complex is responsible for sensing light and transmitting that signal in many fungal species. In Cryptococcus neoformans and C. deneoformans the complex is involved in protection against damage from ultraviolet (UV) light, repression of mating in response to light, and is also required for virulence. The mechanism by which the Bwc1 photoreceptor contributes to virulence is unknown. In this study, a bwc1 deletion mutant of C. neoformans was transformed with three versions of the BWC1 gene, the wild type, BWC1C605A or BWC1C605S, in which the latter two have the conserved cysteine residue replaced with either alanine or serine within the light-oxygen-voltage (LOV) domain that interacts with the flavin chromophore. The bwc1+ BWC1 strain complemented the UV sensitivity and the repression of mating in the light. The bwc1+ BWC1C605A and bwc1+ BWC1C605S strains were not fully complemented for either of the phenotypes, indicating that these BWC1 alleles impair the light responses for strains with them. Transcript analysis showed that neither of the mutated strains (bwc1+ BWC1C605A and bwc1+ BWC1C605S) showed the light-inducible expression pattern of the HEM15 and UVE1 genes as occurs in the wild type strain. These results indicate that the conserved flavin-binding site in the LOV domain of Bwc1 is required for sensing and responding to light in C. neoformans. In contrast to defects in light responses, the wild type, bwc1+ BWC1, bwc1+ BWC1C605A and bwc1+ BWC1C605S strains were equally virulent, whereas the bwc1 knock out mutant was less virulent. Furthermore, pre-exposure of the strains to light prior to inoculation had no influence on the outcome of infection. These findings define a division in function of the White Collar complex in fungi, in that in C. neoformans the role of Bwc1 in virulence is independent of light sensing.

Introduction

The effects of light on fungi impact aspects of their biology as diverse as sporulation, circadian clock function, primary metabolism, and the biosynthesis of secondary metabolites such as toxins or pigments (Fuller et al., 2016, Tisch and Schmoll, 2010). Those responses are controlled by a small number of photosensory proteins (Fischer et al., 2016, Idnurm et al., 2010, Rodriguez-Romero et al., 2010). Among them the blue light receptor White Collar-1 (WC-1), first characterized in the model ascomycete Neurospora crassa, appears to have originated early and then been well-conserved during the evolution of the fungi.

In N. crassa the light-regulated processes are controlled by WC-1 and WC-2, which form a GATA-type zinc finger transcription factor complex. WC-1 contains a number of domains: for DNA binding, two PAS domains (named after the Per, Arnt and Sim proteins, in which they were first observed) involved in protein-protein interactions, two putative transcriptional activation domains, a nuclear localization signal, and a LOV domain which is a specialized type of PAS domain involved in environmental sensing of light, oxygen and voltage (Froehlich et al., 2002, He et al., 2002, Wang et al., 2015). The chromophore flavin adenine dinucleotide (FAD), accommodated in the LOV domain, is essential for the light sensing activity of WC-1. A motif of NCRFLQ amino acids that contributed to the α’A helix is a highly conserved region among LOV domains, as the cysteine (C) residue is essential for the covalent attachment of the flavin chromophore to the sensor protein in the presence of light (Cheng et al., 2003). WC-1 interacts with a partner protein WC-2, another GATA-type transcription factor, to form the White Collar complex (WCC) that activates the expression of light-responsive genes upon light stimulation (Collett et al., 2002, Froehlich et al., 2002, Liu et al., 2003).

The availability of fungal genome sequences has allowed the identification of photoreceptors, especially wc-1 and wc-2 orthologs, in varied fungal species. Mutation of these white collar genes in most cases abolishes the effects of blue light on the species. However, analysis of such mutants has also revealed phenotypes in the absence of light, such as changes in sporulation or secondary metabolite production (Fuller et al., 2016, Fuller et al., 2015), leading to questions about how this complex functions above and beyond its perception of light.

One property that may be unrelated to light status is virulence in pathogenic species. In the human pathogenic fungi Cryptococcus neoformans and C. deneoformans [their previous names are C. neoformans var. grubii/serotype A and C. neoformans var. neoformans/serotype D, respectively (Hagen et al., 2015)], light inhibits mating and induces a protective response against ultraviolet light. Mutation of the orthologs of N. crassa wc-1 and wc-2 impairs these effects (Idnurm and Heitman, 2005, Lu et al., 2005, Verma and Idnurm, 2013). An additional phenotype associated with mutating the BWC1 or BWC2 genes in C. neoformans and the related species C. deuterogattii is a reduction in virulence in animal models of disease (Idnurm and Heitman, 2005, Zhu et al., 2013). Cryptococcosis refers to a set of diseases caused by the C. neoformans species complex, currently made up of seven species that are most problematic when growing in the lungs and central nervous systems of people (Heitman et al., 2011). While a number of factors and genes have been identified that control virulence, mutation of the WCC does not impact on any of these that are known to date (Alspaugh, 2015, Brown et al., 2014).

The role of light or the WCC in fungal virulence is more widespread than the Cryptococcus species. For instance, mutation of the WC homologs also alters the pathogenicity in plant pathogens. In the maize leaf pathogen Cercospora zeae-maydis, the WC-1 ortholog is required for tropism toward the host stomata and lesion formation (Kim et al., 2011a). In the rice blast pathogen Pyricularia (Magnaporthe) oryzae, light represses asexual spore releases and a dark-phase immediately after pathogen–host contact plays a critical role in successful disease development, with spore release and the light-dependent repression accomplished by the photoreceptor MGWC-1 (Kim et al., 2011b, Lee et al., 2006). A T-DNA insertion within the WC-2 ortholog also causes a decrease in pathogenicity of P. oryzae (Jeon et al., 2007). Mutation of wc1 in Fusarium oxysporum, which is normally a plant pathogen, causes a decrease in virulence in a mouse model of disease (Ruiz-Roldán et al., 2008). In the gray mold pathogen Botrytis cinerea, loss of BcWCL1 results in attenuated virulence in the host plant when light is present, and this is due to BcWCL1 being required for coping with excessive reactive oxygen species generated by the host’s oxidative burst and photooxidative stress (Canessa et al., 2013). A light-responsive transcription factor, BcLTF1, downstream of BcWCL1 is responsible for anti-oxidative stress activities and is thus required for advanced host infection by B. cinerea (Schumacher et al., 2014). In addition, the influence of the circadian clock, which is controlled by light, on the disease outcome in the Botrytis cinerea-Arabidopsis thaliana interaction is dependent on the effects of the clock and light from the fungal pathogen, rather than changes in plant defense systems (Hevia et al., 2015). In addition to the genetic contributions of the WCC to virulence, pre-exposing fungi to light can impact subsequent virulence outcomes (Campbell and Berliner, 1973, Oliveira et al., 2018).

It is not yet clear in the pathogenic fungi how light or the light-sensing proteins contribute to virulence. This study used C. neoformans as the model organism to test if light sensing is responsible for the function of the conserved White Collar complex in virulence.

Section snippets

Generation of plasmids

The LITMUS 28i vector (New England Biolabs, Ispwich, MA) was double digested with the restriction enzymes KpnI and BamHI. The BWC1 open reading frame and adjacent 5′ and 3′ regulatory regions were amplified from the genomic DNA of strain KN99α with the primer pair PK003/PK004, and then cloned into linearized LITMUS 28i using T4 DNA ligase, yielding the LITMUS 28i-BWC1 vector. Primer sequences are given in Table 1. A plasmid without errors in BWC1 was identified by sequencing the inserts. The

The flavin-binding site in the LOV domain of Bwc1 is required for full light responses in C. neoformans

Plasmid vectors were created with three versions of the BWC1 gene: a wild type copy, and those that cause a cysteine to alanine or serine substitution in the flavin-binding part of the encoded protein. These substitutes were based on previous work on the Neurospora crassa WC-1 and oat phototropin proteins (Cheng et al., 2003, Salomon et al., 2000), with the rationale that the new isoforms would retain their other functions apart from a loss in the ability to transmit the light signal. These

Discussion

The LOV domains are essential for the light sensing capabilities of photosensory proteins in plants, fungi and bacteria (Briggs and Spudich, 2005), and LOV-domain containing proteins are implicated as virulence factors in pathogenic fungi and bacteria. In fungi, the most common and best-characterized LOV domain protein is White Collar 1 (Fischer et al., 2016). This gene, and a second gene wc-2, were first identified and cloned based on mutant phenotypes in the ascomycete N. crassa (Ballario et

Acknowledgments

We thank Barbara Howlett for suggestions and edits on the manuscript. We are grateful for support from the Chinese Scholarship Council to P.Z. and National Institutes of Health NIAID (grant AI094364) and Australian Research Council (grant FT130100146) to A.I.

References (65)

  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method

    Methods.

    (2001)
  • A.S. Oliveira et al.

    Metarhizium robertsii illuminated during mycelial growth produces conidia with increased germination speed and virulence

    Fungal Biol.

    (2018)
  • J. Purschwitz et al.

    Functional and physical interaction of blue- and red-light sensors in Aspergillus nidulans

    Curr. Biol.

    (2008)
  • C.L. Baker et al.

    The circadian clock of Neurospora crassa

    FEMS Microbiol. Rev.

    (2012)
  • P. Ballario et al.

    White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein

    EMBO J.

    (1996)
  • W.R. Briggs et al.

    Handbook of Photosensory Receptors

    (2005)
  • C.C. Campbell et al.

    Virulence differences in mice of type A and B Histoplasma capsulatum yeasts grown in continuous light and total darkness

    Infect. Immun.

    (1973)
  • P. Canessa et al.

    Assessing the effects of light on differentiation and virulence of the plant pathogen Botrytis cinerea: characterization of the White Collar Complex

    PLoS One

    (2013)
  • M. Castrillo et al.

    The flavoproteins CryD and VvdA cooperate with the white collar protein WcoA in the control of photocarotenogenesis in Fusarium fujikuroi

    PLoS One

    (2015)
  • P. Cheng et al.

    Functional conservation of light, oxygen, or voltage domains in light sensing

    Proc. Natl. Acad. Sci. USA

    (2003)
  • J.M. Christie et al.

    Phototropin LOV domains exhibit distinct roles in regulating photoreceptor function

    Plant J.

    (2002)
  • M.A. Collett et al.

    Light and clock expression of the Neurospora clock gene frequency is differentially driven by but dependent on WHITE COLLAR-2

    Genetics

    (2002)
  • S. Crosson et al.

    Structure of a flavin-binding plant photoreceptor domain: insights into light-mediated signal transduction

    Proc. Natl. Acad. Sci. USA

    (2001)
  • S. Crosson et al.

    Photoexcited structure of a plant photoreceptor domain reveals a light-driven molecular switch

    Plant Cell.

    (2002)
  • R. Fischer et al.

    The complexity of fungal vision

    Microbiol. Spectrum.

    (2016)
  • M.A. Friedl et al.

    Photostimulation of Hypocrea atroviridis growth occurs due to a cross-talk of carbon metabolism, blue light receptors and response to oxidative stress

    Microbiology

    (2008)
  • A.C. Froehlich et al.

    White Collar-1, a circadian blue light photoreceptor, binding to the frequency promoter

    Science

    (2002)
  • K.K. Fuller et al.

    Fungal photobiology: visible light as a signal for stress, space and time

    Curr. Genet.

    (2015)
  • M. Gyalai-Korpos et al.

    Relevance of the light signaling machinery for cellulase expression in trichoderma reesei (hypocrea jecorina)

    BMC Res. Notes

    (2010)
  • Q. He et al.

    White collar-1, a DNA binding transcription factor and a light sensor

    Science

    (2002)
  • J. Herrou et al.

    Function, structure and mechanism of bacterial photosensory LOV proteins

    Nat. Rev. Microbiol.

    (2011)
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