Abstract
Many organisms harbor circadian clocks that promote their adaptation to the rhythmic environment. While a broad knowledge of the molecular mechanism of circadian clocks has been gained through the fungal model Neurospora crassa, little is known about circadian clocks in other fungi. N. crassa belongs to the same class as many important plant pathogens including the vascular wilt fungus Verticillium dahliae. We identified homologs of N. crassa clock proteins in V. dahliae, which showed high conservation in key protein domains. However, no evidence for an endogenous, free-running and entrainable rhythm was observed in the daily formation of conidia and microsclerotia. In N. crassa the frequency (frq) gene encodes a central clock protein expressed rhythmically and in response to light. In contrast, expression of Vdfrq is not light-regulated. Temporal gene expression profiling over 48 h in constant darkness and temperature revealed no circadian expression of key clock genes. Furthermore, RNA-seq over a 24 h time-course revealed no robust oscillations of clock-associated transcripts in constant darkness. Comparison of gene expression between wild-type V. dahliae and a ΔVdfrq mutant showed that genes involved in metabolism, transport and redox processes are mis-regulated in the absence of Vdfrq. In addition, VdΔfrq mutants display growth defects and reduced pathogenicity in a strain dependent manner. Our data indicate that if a circadian clock exists in Verticillium, it is based on alternative mechanisms such as post-transcriptional interactions of VdFRQ and the WC proteins or the components of a FRQ-less oscillator. Alternatively, it could be that whilst the original functions of the clock proteins have been maintained, in this species the interactions that generate robust rhythmicity have been lost or are only triggered when specific environmental conditions are met. The presence of conserved clock genes in genomes should not be taken as definitive evidence of circadian function.
Highlights
Circadian clocks are endogenous timekeepers that enable organisms to anticipate cyclic changes in the environment and confer adaptive advantage (Dunlap and Loros, 2006; Baker et al, 2012)
Homologs of clock genes are present in all Verticillium species tested: V. albo-atrum, V. alfalfae, V. nonalfalfae, V. dahliae, V. nubilum, V, tricorpus, V. isaacii, V. klebahnii, and V. zaregamsianum
With regard to the existence of a circadian clock in V. dahliae our results suggest three possibilities; (i) the clock is absent, (ii) the clock is post-transcriptional and constitutive gene expression leads to oscillation at the protein level, (iii) the clock is only active during specific developmental stages and/or specific conditions, e.g., the clock is activated when a host is detected and is only functional in planta
Summary
Circadian clocks are endogenous timekeepers that enable organisms to anticipate cyclic changes in the environment and confer adaptive advantage (Dunlap and Loros, 2006; Baker et al, 2012). The defining characteristics of circadian clocks are: rhythmicity that persists in constant conditions (absent cyclic conditions) with a period of approximately 24 h; the ability to entrain to external signals such as light and temperature; and temperature and nutritional compensation of period (Pittendrigh, 1960) Such oscillations have been widely observed in most branches of life, and are well characterized in Neurospora crassa. FRQ is progressively phosphorylated by the kinase CK1, and degraded by the FWD-1 protein (He et al, 2003) This leads to a reduced FFC-mediated inhibition of the WCC which results in initiation of a new cycle (Dunlap and Loros, 2006). The activity of nitrate reductase oscillates in the absence of FRQ (Christensen et al, 2004), and a cryptochrome-dependent oscillator (CDO) (Nsa et al, 2015) has been described
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