No Evidence That Homologs of Key Circadian Clock Genes Direct Circadian Programs of Development or mRNA Abundance in Verticillium dahliae

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, freerunning 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 Vdfrq mutant showed that genes involved in metabolism, transport and redox processes are mis-regulated in the absence of Vdfrq. In addition, Vd frq 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.

Circadian oscillations in Trichoderma atroviride and the role of core clock components in secondary metabolism, development, and mycoparasitism against the phytopathogen Botrytis cinerea

“…In fungi, little is known about FRQ function besides N. crassa and B. cinerea . A recent study of frq in the phytopathogen V. dahliae found no significant phenotypic differences in the deletion strain regarding sporulation and microsclerotia formation in DD or LD ( Cascant-Lopez et al, 2020 ). Similarly, in the entomopathogenic fungi Metarhizium robertsii , FRQ is necessary for ring formation under LD 12:12, although neither the WT nor the mutant strain displayed such rings under DD conditions.…”

“…These findings reinforce the importance of defined nutrient/temperature conditions to monitor the clockworks and help future fungal circadian studies visualize otherwise weak oscillations, like those observed in Pyronema confluens and Ophiocordyceps kimflemingiae ( de Bekker et al, 2017 ; Traeger and Nowrousian, 2015 ). This could also help monitor frq molecular oscillations in fungi where previous attempts have failed, like in Verticillium dahliae, Aureobasidium pullulans, and Magnaporthe oryzae ( Cascant-Lopez et al, 2020 ; Deng et al, 2015 ; Franco et al, 2017 ).…”

“…Studies on BLR1 and BLR2 and the presence of a frq homolog that is induced by light, and whose expression depends on BLR1 ( Garcia-Esquivel et al, 2016 ; Steyaert et al, 2010a ), suggest the presence of a functional clock and underlying circadian regulation in this fungus. However, to date, neither circadian oscillations of tafrq /TaFRQ levels nor circadian phenotypes have been reported in T. atroviride; meanwhile, various studies have failed at detecting overt or molecular circadian rhythms in several fungi across fungal clades ( Cascant-Lopez et al, 2020 ; Hevia et al, 2016 ; Larrondo and Canessa, 2019 ).…”

Identification and expression of functionally conserved circadian clock genes in lichen-forming fungi

“…This phenotype of light-induced frq expression has been observed in other Ascomycota such as the soil-living saprophyte Pyronema confluens (Pezizomycetes) 59 , as well as in the Mucoromycota, such as the arbuscular mycorrhizal fungus R. irregularis 26 . The presence of frq, wc-1 , and wc-2 alone, however, is not enough to infer a functional FWO oscillator: in the plant pathogen Verticillium dahliae, the frq homolog is present, but is largely unresponsive to light 60 . It is thus noteworthy that, in the present study, we observe that lichen-forming fungi from two highly diverged lineages, the Lecanoromycetes ( U. pustulata ) and the Eurotiomycetes ( D. miniatum ) both display frq light - dependent responses similar to the canonical responses of N. crassa , pointing to a functionally-conserved core circadian clock mechanism in at least two highly-diverged groups of lichen-forming fungi.…”

Effect of Different Light Wavelengths on Zymoseptoria tritici Development and Leaf Colonization in Bread Wheat

“…Similar to other Ascomycetes, Z. tritici can sense and respond to light by inducing morphological changes during its mycelial development [ 24 , 25 , 26 ]. Its light-sensing capacity could be ascribable to the multiple photoreceptor homolog genes coded in the Z. tritici genome such as the blue-light receptor white collar-1 ( Ztwco-1 ), VIVID, cryptochromes, photolyases, and phytochromes [ 27 , 28 ]. In particular, Ztwco-1 regulates light detection and controls the hyphal morphology, production of pycnidia, and micropycnidiospores [ 25 , 29 ].…”