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Homologues of Key Circadian Clock Genes Present in Verticillium Dahliae Do bioRxiv preprint doi: https://doi.org/10.1101/2019.12.20.883116; this version posted December 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Homologues of key circadian clock genes present in Verticillium dahliae do 2 not direct circadian programs of development or mRNA abundance. 3 4 Emma Cascant-Lopez1, Susan K. Crosthwaite1, Louise J. Johnson2 and Richard J. 5 Harrison1,3* 6 7 1 Genetics, Genomics and Breeding, NIAB EMR, East Malling, ME19 6BJ, UK. 8 2 School of Biological Sciences, University of Reading, Reading, RG6 6AH, UK. 9 3NIAB, Huntingdon Road, Cambridge, CB3 0LE, UK. 10 11 *Correspondence: 12 [email protected] 13 14 Abstract 15 Many organisms harbour circadian clocks that promote their adaptation to the rhythmic 16 environment. While a broad knowledge of the molecular mechanism of circadian clocks 17 has been gained through the fungal model Neurospora crassa, little is known about circadian 18 clocks in other fungi. N. crassa belongs to the same class as many important plant pathogens 19 including the vascular wilt fungus Verticillium dahliae. We identified homologues of N. crassa 20 clock proteins in V. dahliae, which showed high conservation in key protein domains. 21 However, no evidence for an endogenous, free-running and entrainable rhythm was observed 22 in the daily formation of conidia and microsclerotia. In N. crassa the frequency (frq) gene 1 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.20.883116; this version posted December 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 23 encodes a central clock protein expressed rhythmically and in response to light. In contrast, 24 expression of Vdfrq is not light-regulated. Temporal gene expression profiling over 48 hours 25 in constant darkness and temperature revealed no circadian expression of key clock genes. 26 Furthermore, RNA-seq over a 24 h time-course revealed no robust oscillations of RNA in 27 constant darkness. Comparison of gene expression between wild-type V. dahliae and a ΔVdfrq 28 mutant showed that genes involved in metabolism, transport and redox processes are mis- 29 regulated in the absence of Vdfrq. In addition, VdΔfrq mutants display growth defects and 30 reduced pathogenicity in a strain dependent manner. Our data indicate that if a circadian clock 31 exists in Verticillium, it is based on alternative mechanisms such as post-transcriptional 32 interactions of VdFRQ and the WC proteins or the components of a FRQ-less oscillator. 33 Alternatively, it could be that whilst the original functions of the clock proteins have been 34 maintained, in this species the interactions that generate rhythmicity have been lost or are only 35 triggered when specific environmental conditions are met. The presence of conserved clock 36 genes in genomes should not be taken as definitive evidence of circadian function. 37 38 Author summary 39 Circadian clocks are used by organisms to orchestrate the activity of cellular processes such 40 that they occur at an optimal time of day. Research carried out in the filamentous fungus 41 Neurospora crassa has revealed a huge amount of information about the components its 42 circadian clock, its interactions with the environment and how it drives cellular biochemistry 43 and physiology. Although homologues of the Neurospora clock genes are present in a number 44 of fungi, functional clocks have been demonstrated in a just a handful. Importantly, a link 45 between the circadian clock of the plant pathogen Botrytis cinerea and virulence has recently 46 been reported. We report that another significant plant pathogen, Verticillium dahliae, contains 2 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.20.883116; this version posted December 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 47 well-conserved homologues of all key clock genes. We find that diurnal development of 48 conidia and microsclerotia is not influenced by a circadian clock. Furthermore, in a constant 49 environment we find no evidence of rhythmic transcript accumulation. However, deletion of 50 the central clock component results in altered growth and reduced virulence. This led us to 51 question the role of clock genes in Verticillium. We are forced to consider that in this species 52 the interactions that generate rhythmicity have been lost, are generated purely via post- 53 transcriptional modification of clock proteins, are only triggered when specific environmental 54 conditions are met or never evolved. 55 56 Introduction 57 Circadian clocks are endogenous timekeepers that enable organisms to anticipate cyclic 58 changes in the environment and thus confer adaptive advantage [1-2]. The defining 59 characteristics of circadian clocks are: rhythmicity that persists in constant conditions (absent 60 cyclic conditions) with a period of approximately 24 hours; the ability to entrain to external 61 signals such as light and temperature; and temperature and nutritional compensation of period 62 [3]. Such oscillations have been widely observed in most branches of life, and are particularly 63 well characterized in Neurospora crassa. 64 In N. crassa, the circadian clock is based on a transcription-translation negative feedback loop 65 [4], initiated by the photoreceptor and transcription factor White Collar-1 (WC-1) [5]. In the 66 dark, WC-1 and White Collar-2 (WC-2) dimerize through their PAS domains, forming the 67 White-Collar Complex (WCC) [6]. The WCC binds to the clock-box motif in the promoter of 68 the frequency (frq) gene, activating its transcription [7]. When FRQ is synthesized, it associates 69 with FRQ-interacting RNA helicase (FRH) forming the FCC complex. FCC inhibits the 70 activity of the WCC, and consequently transcription of frq is reduced. FRQ is progressively 3 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.20.883116; this version posted December 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 71 phosphorylated by the kinase CK1, and degraded by the FWD-1 protein [8]. This leads to a 72 reduced FCC-mediated inhibition of the WCC, which results in initiation of a new cycle [1]. 73 In addition to the FRQ/WC-based oscillator (FWO) described above, N. crassa contains other 74 FRQ-independent oscillators known as FLOs [9-11]. For example, the activity of nitrate 75 reductase oscillates in the absence of FRQ [12], and a cryptochrome-dependent oscillator 76 (CDO) [13] has also been described. 77 Both light and temperature can entrain the clock. Light rapidly induces transcription of frq, 78 which leads to clock resetting and entrainment to the external stimulus [14]. The WCC complex 79 activates frq transcription by binding to light responsive elements (LREs) present in the frq 80 promoter [7]. Two LREs have been identified in the frq promoter; the proximal LRE is 81 necessary for light induction of frq, whereas the distal LRE (known as the Clock-Box) is 82 required for both light and circadian clock regulation [15-16]. Additionally, the WCC is 83 essential for the transcriptional activation of numerous light-regulated genes, including other 84 transcription factors, leading to a genetic regulatory cascade [17]. A subset of the downstream 85 genes are known to be regulated by both the circadian clock and light. Included in this subset 86 is the photoreceptor VVD, which is involved in photo-adaptation [18]. Despite not being 87 essential for circadian function, VVD enhances the robustness of the clock by preventing clock 88 resetting at dawn and promoting clock resetting at dusk [11,17-20]. Other targets include genes 89 that function in carotenoid synthesis and spore development [15]. Importantly, rhythmic 90 mRNA levels in N. crassa are not only controlled by light and the circadian clock at the 91 promoter level, but through post-transcriptional regulation [21]. In N. crassa the circadian 92 clock regulates approximately 40 % of the genome and a quarter of expressed proteins [21-22]. 93 Temperature compensation and temperature resetting of the clock is mediated post- 94 transcriptionally [23]. frq transcript levels remain the same at different temperatures but FRQ 95 protein cycles remain at a higher mean level at high temperatures [24]. VVD is also temperature 4 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.20.883116; this version posted December 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 96 regulated, and plays a role in temperature compensation of the clock [25]. In addition, the 97 existence of a temperature-compensated FLO that requires the components of the WCC has 98 been described [10,25]. An example of a temperature-compensated FLO-output is the clock- 99 controlled gene ccg-16, which has been demonstrated to oscillate rhythmically in the absence 100 of FRQ [10]. 101 Despite detailed knowledge of the N.
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