Homologues of key circadian clock genes present inVerticillium dahliaedo not direct circadian programs of development or mRNA abundance
Authors: Cascant-Lopez Emma , Crosthwaite Susan K. , Johnson Louise J. , Harrison Richard J. 
Published: December 2019

Many organisms harbour 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 modelNeurospora crassa, little is known about circadian clocks in other fungi.N. crassabelongs to the same class as many important plant pathogens including the vascular wilt fungusVerticillium dahliae.We identified homologues ofN. crassaclock proteins inV. 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. InN. crassathefrequency(frq) gene encodes a central clock protein expressed rhythmically and in response to light. In contrast, expression ofVdfrqis not light-regulated. Temporal gene expression profiling over 48 hours 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 RNA in constant darkness. Comparison of gene expression between wild-typeV. dahliaeand aΔVdfrqmutant showed that genes involved in metabolism, transport and redox processes are mis-regulated in the absence ofVdfrq. In addition,VdΔfrqmutants 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 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.

Author summary

Circadian clocks are used by organisms to orchestrate the activity of cellular processes such that they occur at an optimal time of day. Research carried out in the filamentous fungusNeurospora crassahas revealed a huge amount of information about the components its circadian clock, its interactions with the environment and how it drives cellular biochemistry and physiology. Although homologues of the Neurospora clock genes are present in a number of fungi, functional clocks have been demonstrated in a just a handful. Importantly, a link between the circadian clock of the plant pathogenBotrytis cinereaand virulence has recently been reported. We report that another significant plant pathogen,Verticillium dahliae,contains well-conserved homologues of all key clock genes. We find that diurnal development of conidia and microsclerotia is not influenced by a circadian clock. Furthermore, in a constant environment we find no evidence of rhythmic transcript accumulation. However, deletion of the central clock component results in altered growth and reduced virulence. This led us to question the role of clock genes in Verticillium. We are forced to consider that in this species the interactions that generate rhythmicity have been lost, are generated purely via post-transcriptional modification of clock proteins, are only triggered when specific environmental conditions are met or never evolved.