Abstract/Summary

The circadian clock is an internal timekeeper mechanism that allows organisms to anticipate and synchronize to daily environmental changes. The circadian clock is present in most branches of life; in plants, it modulates defence mechanisms impacting plant-pathogen interactions. This suggests that pathogens may also time their biological events in order to optimize infection. A broad knowledge of the molecular mechanism of circadian clocks has been gained through the fungal model Neurospora crassa. Nevertheless, little is known about circadian clocks in other fungi. We sought to characterize, therefore, the presence of a circadian clock in the fungal plant-pathogen Verticillium dahliae, and assess its influence on pathogenicity. To characterize the presence of a circadian system in V. dahliae, we first performed comparative genetics studies, and identified homologs of all the N. crassa circadian clock genes in V. dahliae. High conservation of clock protein domains between N. crassa and V. dahliae was observed. We studied whether the daily formation of conidia and miscrosclerotia (infective propagules) were regulated in a circadian manner in the plant pathogen. However, no evidence for an entrainable, free-running rhythm was observed. Moreover, temporal gene expression profiling with qRT-PCR in constant darkness showed a lack of rhythmic frq expression. Thus, in order to assess the role of the putative clock genes in V. dahliae, gene deletion mutants were produced. Deleting the core clock gene frq did not have an obvious effect on fungal morphology. However, the deletion of the transcription factor and photoreceptor wc-1 resulted in the abolishment of microsclerotia, and stopped the light-regulated spore production, confirming that wc-1 is involved in the production of the daily developmental rhythm by mediating transcriptional responses to white light. Finally, through in vitro pathogenicity tests we observed that Arabidopsis plants inoculated with the wild-type fungus showed more severe disease symptoms than the plants inoculated with the clock mutant strains. In conclusion, V. dahliae presents all the necessary genetic loci for a functional clock, but there is no evidence of rhythmicity in either morphological traits or in gene expression. However, putative clock genes play an important role in the overall fitness of the fungus, suggesting that they have additional non circadian roles. Therefore, their study can provide a better understanding of the hostpathogen interaction.