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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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:
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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. crassa circadian clock, little is known about the existence
102 of functional circadian clocks and their molecular basis in other fungi, although homologues
103 of the N. crassa core circadian clock proteins, especially WC-1 and WC-2, have been found in
104 other fungal species [26-27]. It has been hypothesized that FRH and FWD-1 were present in
105 the common ancestor of all fungi [28]. WC-1 and WC-2 were probably gained in the common
106 ancestor of Zygomycetes, Basidiomycetes and Ascomycetes and subsequently lost in the
107 Saccharomycetes. FRQ was likely gained in the ancestor of Ascomycetes and lost in
108 Eurotiomycetes, remaining in Sordariomycetes, Leotiomycetes and Dothideomycetes [28-29].
109 Interestingly, FRQ is the least conserved of the clock proteins. While examples of functional
110 frq-dependent circadian oscillators are present in the Leotiomycete Botrytis cinerea [30] and
111 in the Pezizomycete Pyronema confluens [29], other fungi, such as the Dothideomycete
112 Aureobasidium pullulans, show no rhythmic frq expression, although they do display a
113 circadian developmental rhythm [31]. Additional circadian rhythms have been demonstrated
114 in species lacking a frq homologue, such as in Aspergillus flavus and Aspergillus nidulans [32].
115 Furthermore, the yeast Saccharomyces cerevisiae was shown to exhibit circadian entrainment
116 of metabolism despite the absence of a frq homologue [33], indicating a widespread presence
117 of circadian oscillatory molecular processes across fungal species.
118 N. crassa is phylogenetically related to many important plant pathogens including Verticillium
119 dahliae. V. dahliae is an asexual soil-borne Sordariomycete that causes wilt disease on more
120 than 200 plant species worldwide, including high-value agricultural crops [34]. Verticillium
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121 sp. can persist in soil for long periods in the form of melanized resting bodies. In V. dahliae,
122 these are clusters of thick-walled, septate and dark pigmented hyphal cells called microsclerotia
123 [35]. Microsclerotia germinate upon the detection of plant root exudates, and colonize the
124 vascular system. Once inside the vessels, conidia are produced and transported upwards
125 reaching extensive portions of the plant. Blockage of the transport system results in wilting of
126 the plant and the subsequent formation of microsclerotia in the dead tissue, concluding the
127 disease cycle.
128 The aim of this work was to determine whether a circadian clock is present in V. dahliae. As
129 previously described [36], under 24 hour light/dark cycles V. dahliae displays concentric rings
130 of conidia and microsclerotia. We report that this phenotype is directly driven by external cues,
131 rather than entrained. Comparative genetic studies between N. crassa and V. dahliae reveal
132 that key clock components are conserved not only at the domain level, but down to individual
133 phosphorylation sites. Similar sequences to the proximal and distal LRE motifs are present
134 upstream of the VdFRQ ORF, although they are very widely spaced. These binding motifs may
135 be important for a WC-mediated light-response. However, qRT-PCR studies over a 48-hour
136 time-course revealed no rhythmic Vdfrq expression under constant conditions or in cycles of
137 light/dark and high/low temperature. RNA-sequencing gene expression studies in the WT
138 revealed large-scale changes in gene expression driven by changes from light to dark, but time-
139 course RNA-sequencing revealed no strong signature of gene expression indicative of
140 circadian rhythmicity. Our results show that while there is high conservation of clock
141 components between V. dahliae and N. crassa, there is no strong evidence of a functional
142 circadian clock in V. dahliae, at either the physiological or the molecular level.
143
144 Methods
6 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.
145 Identification of V. dahliae putative clock-genes
146 N. crassa OR74A and V. dahliae JR2 genomes were downloaded from Ensembl Fungi [37].
147 Homologues were identified using BLASTp against the V. dahliae JR2 genome. Whole gene
148 alignments between the query N. crassa gene and the V. dahliae JR2 hit were carried out in
149 Geneious R10 using the MUSCLE algorithm. Next, V. dahliae JR2 hits were aligned to the
150 genomes of five V. dahliae strains isolated and sequenced at NIAB EMR (12253, 12158,
151 12251, 12161 and 12008), available at DDBJ/EMBL/GenBank database using the following
152 numbers: PRJNA344737 and PRJNA352681. Domains were identified within predicted
153 proteins using InterProScan. Nuclear localization signals (NLS) were identified using cNLS
154 Mapper. Identification of clock gene homologues in V. albo-atrum (PD747), V. alfalfae
155 (PD683), V. nonalfalfae (TAB2), V. longisporum subgenome A (VLB2), V. longisporum
156 subgenome D, V. nubilum (PD621), V. tricorpus (PD593), V. isaacii (PD660), V. klebahnii
157 (PD401) and V. zaregamsianum (PD739) was performed using BLASTp in collaboration with
158 the Thomma Lab (Wageningen University).
159
160 Orthology analysis
161 The study involved 25 species, mostly plant pathogens, sampled from across the phylum Fungi.
162 The respective genome (repeat-masked), CDS, and protein sequences were downloaded from
163 Ensembl Fungi [37]. The orthology relationships between the genes in each genome were first
164 established using the OrthoFinder ver. 1.0.7 [38] and OrthoMCL ver. 2.0.9 [39] pipelines. The
165 genomes of 25 fungal species were searched for homologues of frq (NCBI ID: 3876095), wc-
166 1 (NCBI ID: 3875924), wc-2 (NCBI ID: 3879968), frh (NCBI ID: 3872445), fwd-1 (NCBI ID:
167 3872130) and vvd (NCBI ID: 3873728). The phylogenetic tree was built using a concatenated
168 alignment of DI/D2 regions of large subunit (LSU) rDNA and ITS regions that were identified
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169 through BLAST and extracted from the genomes. The Tamura-Nei method [40] was used to
170 build trees, as implemented within Geneious R10.
171
172 Promoter Motif Identification
173 2000 bp upstream and downstream of each gene ORF were extracted. MEME suite ver. 4.11
174 was subsequently used to analyse the motif content of promoter regions [41]. For motif
175 scanning purposes, the program FIMO [42] was used to deal with ungapped motifs (ACE motif
176 in ccg2 promoter), while gapped motif search for sequences showing similarity to the Clock-
177 box element in frequency promoters was carried out with GLAM2SCAN [43]. For de novo
178 motif discovery, DREME [44] and GLAM2 were used for ungapped and gapped motifs,
179 respectively. Motif enrichment analysis was carried out with the AME program. A manual
180 screen of consensus LRE motifs described in [15, 45] on the 2000 bp upstream and 2000 bp
181 downstream regions of the frq orthologues across 13-species was performed using Geneious
182 R10.
183
184 V. dahliae isolates and growth conditions
185 Strains of V. dahliae (S1 Table) were stored at -80 °C as conidial suspensions preserved in 50
186 % glycerol. Isolates were cultivated on petri dishes containing Prune Lactose Yeast Agar
187 (PLYA) [46] at 24 °C in constant darkness. After a week, plates were flooded with 2 ml sterile
188 water and gently rubbed using a plastic spreader to create a spore suspension.
189
190 Plate assays
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191 Conidial suspensions were filtered using filter paper with 3 to 5μm pore size and 1 µL of a 3 x
192 105 spores/mL suspension was point-inoculated in the centre of a PLYA plate and incubated
193 for 14 days under the appropriate light and/or temperature conditions. For light entrainment
194 experiments, after 14 days at 24 ℃ in 12:12 LD, plates were marked at the end of the dark
195 cycle before transfer to constant darkness (DD) for 7 days. For temperature entrainment assays,
196 the cultures were incubated under 12 h at 20 ℃ followed by 12 h at 28 ℃ in the dark during
197 14 days prior to transfer to constant temperature. To assess the effect of entrainment by both
198 light and temperature, plates were incubated in 12 h light at 28 ℃/12 h dark at 20 ℃, before
199 transfer to constant darkness and temperature conditions. To examine nutritional compensation
200 cultures were inoculated onto a high-nitrogen medium (PDA), low-nitrogen medium (Czapek
201 DOX Agar), minimal medium (MM) [47] and basal minimal medium (BMM) [48] and
202 incubated in 12:12 LD cycles for 14 days. Strains were incubated in Panasonic MLR-352 or
203 MIR-154 incubators equipped with broad spectrum fluorescent lamps FL40SSENW37. The
204 light intensity was 75 µmol s-1 m-2 (approximately 5600 lux). Colony size was measured using
205 an electronic calliper. Each experiment was repeated at least three times and contained three
206 replicas of each strain and treatment.
207
208 Time-course and light pulse experiments
209 Rhythmic expression analysis by qRT-PCR in free-running conditions (constant darkness and
210 constant temperature) was similar to the protocol described in [49]. A mycelial mat was formed
211 by inoculating 1 x 108 spores of V. dahliae into Petri dishes containing 20 ml of half strength
212 PDB medium and incubated at 25 °C under constant light for 96 hours. Individual mycelial
213 disks (1 cm diameter) of mycelium were cut and inoculated in 100 ml Erlenmeyer flasks
214 containing 25 ml of half strength PDB medium. A total of 36 flasks were used to cover 12
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215 sequential time-points throughout two circadian cycles (4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h,
216 32 h, 36 h, 40 h, 44 h, 48 h), each replicated three times. The flasks were incubated at 25 °C in
217 constant light for at least 24 hours under agitation at 120 rpm. Cultures were staggered into
218 constant darkness at different intervals, so that when harvested cultures were of similar ages.
219 Cultures were harvested under red light. Disks were dried with filter paper, placed into 2 ml
220 Eppendorf tubes, frozen in liquid nitrogen and stored at -80 °C.
221 A similar methodology to that described in [50] was performed for light and temperature
222 entrainment analysis. A total of 36 flasks were used to cover 12 sequential time-points
223 throughout one LD cycle (L2, L4, L6, L8, L10, L11.5, D2, D4, D6, D8, D10,
224 D11.5). Similarly, a total of 36 flasks were used to cover 12 sequential time-points throughout
225 one cycle of 12 h at 20 ℃ (low temperature, LT) /12 h at 28 ℃ (high temperature, HT) (L2,
226 L4, L6, L8, L10, L11.5, H2, H4, H6, H8, H10, H11.5).
227 For light pulse experiments, flasks containing half strength PDB medium were inoculated with
228 V. dahliae or N. crassa mycelial discs and subsequently grown in constant light for 24 hours
229 at 25 ℃ (120 rpm), after which they were transferred to constant darkness for 30 hours prior
230 to a 15 minutes exposure to white light (75 µmol s-1 m-2). After the light pulse, cultures were
231 harvested under red light. Control flasks were not exposed to light pulses and were harvested
232 under red safe-light conditions.
233
234 RNA extraction
235 Frozen mycelium of N. crassa and V. dahliae were ground with a mortar and pestle and total
236 RNA extracted using the RNeasy Plant Mini Kit (Qiagen GmbH, Germany) according to the
237 manufacturer’s instructions. The integrity of RNA samples was assessed using the Agilent
238 4200 TapeStation system (Agilent, Santa Clara, USA).
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239
240 Gene expression analysis by Quantitative Real Time PCR (qRT-PCR)
241 cDNA from 1 µg of RNA was synthesized using the QuantiTect Reverse Transcription Kit
242 (Qiagen, Germany) following the manufacturer’s instructions. qRT-PCR was performed with
243 SYBR green (qPCRBIO SyGreen Mix Lo-Rox, PCR Biosystems, UK) and amplification
244 followed using the CFX96™ Real-Time PCR detection system (Bio-rad). Normalisation was
245 carried out against V. dahliae β-tubulin and Elongation factor 1-⍺ transcripts. For N. crassa, β-
246 tubulin and TATA binding box-encoding gene served as housekeeping genes. Primers are
247 listed in S2 Table. PCR reactions were carried out in 10 µl containing 400 nM of each primer,
248 5 µl of 2x qPCRBIO SyGreen Mix and 2 µL of 1:4 diluted cDNA sample. The PCR conditions
249 were 95 °C 2 min; 39 cycles of 95 °C 10 sec, 60 °C 10 sec, 72 °C 30 sec and 1 cycle of 95 °C
250 10 sec, 65 °C 5 sec 95 °C 5 sec. Relative gene expression was calculated using the comparative
-(∆∆Ct) 251 cycle threshold (CT) method (2 ) method [51]. Expression values for free running time-
252 course experiments were normalized to the cultures grown 4 h in the dark (D4). Expression
253 values for time-course experiments under LD cycles were normalized to the cultures grown 2
254 h in the dark (D2), and cultures grown 2 h at 20 °C (2L) were the reference samples for the
255 temperature cycle/entrainment time-courses. The expression values for light pulsed cultures
256 were normalized to cultures maintained in control conditions (DD).
257 Expression values derived from three biological replicates (unless otherwise stated) each
258 containing three technical repeats, were analysed using one-way Analysis of Variance
259 (ANOVA). The residuals were tested for normality and if required, data were log-transformed.
260 Statistical analyses were carried out using R Studio software. Statistical analysis of rhythmicity
261 of time-course experiments was achieved with JTK-CYCLE [52] in the R software (version
262 3.3.0) using the 2-(∆∆Ct) normalized data of all the replicates. The analysis was performed as
263 described in the JTK-CYCLE manual, looking for period lengths of 24 h.
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264
265 RNA-Seq
266 For the time-course RNA-seq experiment in free-running conditions, samples of V. dahliae
267 12253, 12008 and ΔVdfrq_12253 were harvested under red light after 6 h, 12 h, 18 h and 24 h
268 in the dark. Concurrently, for light versus dark analysis, cultures were incubated in constant
269 darkness and then were transferred to white-light and harvested after 6 hours. The experiment
270 contained three biological replicates of each strain and condition.
271 Total RNA was extracted as described above. For transcriptome sequencing, samples with a
272 minimum of 1 µg of RNA (100 ng/µl), ≥ 6.8 RIN and 260/280 nm values > 1.8 were sent to
273 Novogene Technology Co. Ltd. (Wan Chai, Hong Kong). Sequencing was performed on
274 Illumina HiSeq P150.
275 Quality control of the RNA-seq reads was carried out by fastQC and adapters were trimmed
276 using fastq-MCF (available from https://expressionanalysis.github.io/ea-utils/). STAR
277 software [53] was used to align RNA-seq reads to the reference Verticillium dahliae JR2
278 genome (Ensembl Fungi). Mapped read counts were calculated using the program
279 featureCounts [54]. The analysis of expression of all predicted genes was performed with the
280 DESeq2 package in R. A grouping command was used to assess differentially expressed genes
281 (DEG) between conditions (L or D) and strains (Vd12253, Vd12008 and ΔVdfrq_12253). The
282 false discovery rate (FDR) cut-off was set to 0.05. Genes were considered to be significantly
283 differentially expressed when p-value < 0.05 and presented more than 1-log2 fold change
284 (LFC) in transcript level.
285 For rhythmic expression analysis, the raw counts of the time-course samples were normalized
286 to fragments per kilobase of transcript per million mapped fragments (FPKM). The time-course
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287 of each individual strain was analyzed using JTK-CYCLE, looking for rhythms of 24 h [52].
288 Genes with a p-value < 0.05 and a false discovery rate of q < 0.1 were considered rhythmic.
289 Principal component analysis (PCA) plot and samples distances plot used rlog-transformed
290 read counts, and were carried out using R. Gene ontology (GO) terms were retrieved from
291 GO.db in R. Gene enrichment analysis for GO terms was performed using topGO in R with
292 Fisher’s exact test to retrieve significantly enriched processes of the DEG. The analysis of
293 secondary metabolite biosynthetic gene clusters in V. dahliae was performed using
294 antiSMASH [55].
295
296 Construction of V. dahliae frq replacement cassette
297 The 5’ and 3’ flanking regions of the Vdfrq locus were amplified from genomic DNA using
298 primer pairs HRFrq1-F/R and HRFrq2-F/R respectively (S2 Table). Core USER Bricks and
299 the hygromycin resistance cassette were amplified from pRF-HU2 plasmid and the vector
300 bricks were assembled through USER reaction and transformed into E. coli DH5α competent
301 cells [56]. The resulting vector junctions were amplified using B1.B2-F/R, B1.B2.F1.H-F/R
302 and H.F2.B1-R to confirm the correct assembly.
303
304 Agrobacterium tumefaciens transformation
305 Agrobacterium tumefaciens EHA105 was transformed with the Vdfrq replacement vector as
306 described in [57]. Putative transformants were checked by PCR using primers B1.B2-F/R,
307 B1.B2.F1.H-F/R and H.F2.B1-R (S2 Table).
308
309 Agrobacterium-mediated transformation of V. dahliae
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310 A. tumefaciens EHA105 containing the pEcFrq-D1 binary vector was grown at 28 oC for 48 h
311 in MM containing 50 μg ml-1 of rifampicin and 50 μg ml-1 of kanamycin on a rotary shaker
312 (200 rpm). At an optical density of OD600 = 0.5, bacterial cells were diluted to OD600 = 0.15
313 with IM [58] containing 200 μM acetosyringone (AS). Cells were grown for an additional
314 period of 12-15 h before being mixed with an equal volume of a spore suspension (1x106 spores
315 ml-1) of the required strain of V. dahliae, previously grown for three days in flasks containing
316 PDB liquid media in a rotary shaker 180 rpm in the dark. From this mixture aliquots of 200 μl
317 were plated on a cellophane membrane placed on an IM agar plate. After incubation at 25 oC
318 for 48 h the membrane was transferred onto a PDA plate containing 200 μg/ml tricarcillin and
319 50 μg/ml hygromicin B and incubated at 24 oC for a further 5 to 7 days. Discrete colonies were
320 selected and grown for 24 h in PDB liquid cultures containing 50 μg/ml hygromycin B. Single-
321 spore colonies were obtained by plating 100 μL of the culture on PDA with 50 μg/ml
322 hygromycin B. PCR was carried out on selected single-spore mutants to confirm insertion of
323 hygromicin using the primers TestHygr-F/R and FrqUS_Hygr-F/R, and lack of product from
324 deleted gene using the primers TestFrq-F/R (S1 Fig.). The PCR conditions using Taq 5x Master
325 Mix (New England Biolab) were 95 oC 30 sec; 35 cycles of 95 oC 20 sec, 60 oC 20 sec, 68 oC
326 2 min and 68 oC for 5 min.
327
328 Arabidopsis thaliana and strawberry in vitro propagation
329 For pathogenicity tests, seeds of Arabidopsis thaliana ecotype Columbia (Col), were surface-
330 sterilized by sequential immersion in 70% ethanol for 1 min and 10% (v/v) bleach containing
331 0.1% (v/v) Tween-20 for 5 minutes with gentle shaking. Seeds were then washed with
332 autoclaved distilled water for 5 minutes five times, re-suspended in sterile 0.1% agarose, and
333 subsequently stratified at 4 oC over a period of 2-3 days in darkness [59]. 5 to 6 seeds were
334 pipetted into 120 x 120 mm square petri dishes (Thermo Fisher Scientific) containing half-
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335 strength Murashige and Skoog salts (MS), pH 5.7; 0.8% (wt/vol) Phytagel (Sigma-Aldrich);
336 1% sucrose; and grown at 22oC under 16:8 LD cycles in a Panasonic MLR-352 incubator for
337 3-4 weeks.
338 Strawberry (Fragaria x ananassa) cultivar Hapil plantlets were preserved in sterile jars
339 containing with SMT proliferation medium (MS salts 4.41 g, 177.5 μM Benzylamino purine
340 (BAP), 254 μM Gibberellic acid (GA), 255 μM Indole butyric acid (IBA), sucrose 30 g, agar
341 (Oxoid 3) 7.5 g, adjust pH = 5.6). The optimal growth conditions were 24 oC in the light and
342 19 oC in the dark, in a photoperiod of 16 h light and 8 h dark. For long-term in vitro material,
343 plantlets were transferred to fresh SMT medium in sterile conditions every 6 weeks. For root
344 induction, SMR rooting medium (MS salts 4.41 g, 254 μM GA, 255 μM IBA, sucrose 30 g,
345 agar (Oxoid 3) 7.5 g, adjust pH = 5.6) was used. Four weeks prior to a pathogenicity test, roots
346 were carefully removed from the media just below the crown and the tips were moved to square
347 petri dishes containing ATS medium as specified in [60], where subsequent roots were
348 produced.
349
350 Arabidopsis thaliana and strawberry root inoculation assay
351 V. dahliae conidial suspensions were harvested from 7 day old liquid cultures and diluted to 1
352 x 106 spores/ml with PDB. Three to four-week-old Arabidopsis seedlings were moved under
353 aseptic conditions to new plates containing half-strength MS salts pH 5.7; 0.8% (wt/vol)
354 Phytagel (Sigma-Aldrich) without sucrose, and root tips were trimmed using sterilized scissors.
355 Each plate contained five Arabidopsis seedlings that were inoculated with 200 μL of the spore
356 suspension spread onto the roots. In mock plates seedlings were inoculated with 200 μL of
357 autoclaved distilled water. Plates were subjected to a light-dark cycle of 12:12 h and constant
358 temperature of 24 oC for the duration of the experiment.
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359 For the strawberry inoculation, plants were removed from the plates and the roots were
360 carefully cleaned free of agar with a sterile blade. Before the inoculation, 1 cm of roots was
361 trimmed from the bottom with sterile scissors. Plants were inoculated by dipping the root
362 systems into a flask containing 1 x 106 spores/ml suspension (6 plants/100 ml inoculum) for 5
363 minutes. A fresh spore suspension was used for each batch of 6 plants. Plants were then placed
364 to new ATS medium plates sealed with tape. Uninfected plants were inoculated with sterile
365 distilled water following the same procedure as stated before. Plates were subjected to a light-
366 dark cycle of 12:12 h and temperature of 24 oC in a Panasonic MLR-352 incubator.
367
368 Disease assessment
369 Arabidopsis and strawberry cv. Hapil seedlings were photographed and scored for disease
370 symptoms using a scale with nine categories modified from [61]; Score 1: Healthy plant, 2:
371 Slight symptoms on older leaves (yellowing), 3: 1-2 outer leaves affected, 4: >2 leaves affected,
372 5: 50% leaves affected, 6:> 50% leaves affected, 7: 75% leaves affected, 8: >75% leaves
373 affected, 9: Plant dead.
374
375 Results
376 Distribution of core clock gene homologues in Verticillium species and other
377 Sordariomycete, Dothideomycetes, and Leotiomycetes of economic importance.
378 The genomes of 25 Sordariomycete, Dothideomycete and Leotiomycete species were searched
379 for homologues of frq, wc-1, wc-2, frh, fwd-1 and vvd. The predicted proteins from these
380 genomes were clustered and proteins in the same orthogroup cluster as the N. crassa genes
381 were considered orthologous genes. Orthologues of all six clock oscillator genes are present in
382 most species of Sordariomycetes tested in this study, including important plant-pathogenic
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383 fungi such as Glomerella graminicola and Neonectria ditissima (Fig 1A). Homologues of
384 clock genes are present in all Verticillium species tested: V. albo-atrum, V. alfalfae, V.
385 nonalfalfae, V. dahliae, V. nubilum, V, tricorpus, V, isaacii, V. klebahnii and V.
386 zaregamsianum (pers. comm. B. Thomma). Fusarium oxysporum has three copies of frq and
387 vvd. Conversely, members of the chaetomium family appear to have lost frq, wc-1, and vvd
388 homologues. In contrast to Sclerotinia sclerotiorum and Botrytis cinerea, the Leotiomycete
389 Blumeria graminis lacks a vvd homologue, but contains copies of the other clock components.
390 vvd is absent in the Dothideomycetes Cercospora zeae-maydis, Alternaria alternata and
391 Venturia inaequalis, but present in the wheat pathogen Zymoseptoria tritici that contains
392 homologues of all the clock genes (Fig 1A). The genome of the Ustilaginomycetes Ustilago
393 maydis, on the other hand, does not harbour an orthologue of frq, in agreement with the findings
394 in [28].
395 Alignments of core clock proteins between N. crassa and V. dahliae revealed sequence
396 identities greater than 43% and query coverages greater than 39%. Consistent with previous
397 reports [29], V. dahliae FRQ is the least conserved (43.82% identity), and FRH is the best
398 conserved (68.69% identity) of the core clock proteins. Clock proteins are strongly conserved
399 at the domain level (Fig 1B). Although the FRQ protein alignment created between N. crassa
400 and V. dahliae sequences shows moderate conservation overall, a number of important regions
401 are highly conserved (S2 Fig). In N. crassa PEST1 and PEST2 are involved in determination
402 of period length and cytoplasmic accumulation of the WCC [62-63]. Whilst a high degree of
403 variation within VPEST2 exists, VPEST1 is better conserved. The formation of FRQ
404 homodimers, essential for clock function, is mediated through its coiled-coil domain [64].
405 Between N. crassa and V. dahliae this domain shares 69.69% identity. FRQ-CK1a interaction
406 domains, FCD1 and FCD2 [65-66] share 87.5% identity between N. crassa and V. dahliae. In
407 contrast, low sequence conservation of the FRQ-FRH interaction domain (FFD) [67] is
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408 observed between N. crassa and V. dahliae, with 4 of 10 sites conserved. V. dahliae FRQ
409 protein contains two predicted nuclear localization signal (NLS) sequences, one almost
410 identical to the N. crassa NLS (DLLKRDKLFEIKVHGLPKPKKRELE). The NLS present in
411 N. crassa FRQ is required for its entrance into the nucleus and down-regulation of frq
412 transcription [68]. Furthermore, of the 73 in vivo and in vitro identified phosphorylation sites
413 in N. crassa FRQ [69], 40 sites are conserved in V. dahliae FRQ. Of note Ser 513, a determinant
414 of period length [70] and FRQ degradation, is present in VdFRQ.
415 Alignment of frq homologues from six V. dahliae strains (JR2, 12253, 12251, 12008, 12161
416 and 12158) revealed several single nucleotide polymorphisms (SNPs) between isolates that are
417 associated with their vegetative compatibility (VC) background (data not shown). For instance,
418 associated with VC subclade II-2 (12161 and 12158) or VC subclade II-1 (JR2, 12253, 12251
419 and 12008). Consequently, VdFRQ protein alignment indicates four amino acid substitutions.
420 However, none of these substitutions are in the known functional domains.
421 In N. crassa, WC-1 and WC-2 interact through their PAS domains to form a heterodimeric
422 complex (WCC complex). The WCC is essential for light-induced gene expression of frq and
423 other light-regulated genes, and is required for the generation of circadian rhythms [64,71]. V.
424 dahliae WC-1 and WC-2 contain all the domains required for their interaction, light perception
425 and transcription factor (TF) activity (Fig 1B). VdWC-1 and VdWC-2 share 45.22% and
426 51.12% sequence identity with the N. crassa homologues, respectively. Nevertheless, VdWC-
427 1 is highly conserved (79.0% to 94.34% identity) in the known protein domains. Vd WC-1
428 contains a conserved Light-oxygen-voltage (LOV) domain. This domain is related to the
429 Period-ARNT-Single-minded (PAS) domain family, and is distinguished by binding a flavin
430 cofactor and the presence of a GXNCRFLQ motif [72]. Furthermore, VdWC-1 contains two
431 highly conserved PAS domains required for protein interaction, and a GATA-type zinc finger
432 DNA binding domain commonly present in GATA-type transcription factors (S3 Fig). A
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433 region of basic amino acids (LLSNKKKRKRRKGVG) required for rhythmicity and circadian
434 expression of the Neurospora circadian clock gene frequency is also present [73]. Despite the
435 high level of conservation of most WC-1 domains between N. crassa and V. dahliae, the
436 conservation is poor in the first 480 aa. In N. crassa, this region contains a poly-glutamine
437 (Poly-Q) region expansion aa 16-61 that has previously been observed in transcription factors
438 and is reported to play a role in transcription activation and to affect period length [74-75].
439 Other reports suggest that neither the N nor C-terminal polyQ stretches are required for
440 activation of frq transcription, and it is an adjacent region spanning aa 100-200 in WC-1 that
441 is necessary for clock control of frq expression in the dark but not for light responsiveness of
442 frg [76]. In V. dahliae, however, the polyQ stretch is not continuous and conservation is
443 minimal in the aa 100-200 region. Furthermore, V. dahliae WC-1 lacks the C-terminal (Poly-
444 Q) region present at the aa 1091-1133 in N. crassa.
445 VdWC-2 contains a single PAS domain, a GATA-type zinc finger transcription factor domain
446 with 65.71 % and 79.24 % sites conserved. A putative NLS (RGRKRKRQW) sequence
447 identified in V. dahliae, lacks a homologous sequence in N. crassa (S4 Fig).
448
449 V. dahliae conidiation and microsclerotia production are light-regulated but not under
450 control of a circadian clock
451 V. dahliae produces visible concentric rings of conidia and microsclerotia when cultured in
452 cyclic environments, such as light/dark (LD) and high/low temperature cycles [36]. Under 24
453 hour LD cycles at constant temperature, hyphal growth occurs under both light and darkness,
454 however conidiation from hyphae is induced after a period of light (Fig 2A and B). After a
455 minimum of 48 hours of growth microsclerotia form but in LD cycles they are mainly produced
456 in the dark [36]. Nevertheless, in either constant darkness (DD) or constant light (LL)
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457 conditions V. dahliae cultures contain conidia and microsclerotia but they are not produced in
458 a ring fashion (Fig 2B). To test if development is controlled by a circadian clock we
459 investigated whether the morphological rhythms found in this pathogen are endogenous and
460 can be entrained. We found that V. dahliae cultures transferred to DD after an entrainment
461 period of 12:12 LD for 14 days at 22 °C cease to produce rings of conidia and microsclerotia.
462 In 12:12 temperature cycles of 20 °C and 28 °C in DD or LL the banding pattern is synchronized
463 to the temperature oscillations, and ceases when cultures are transferred to constant
464 temperature (Fig 2C). Thus, the developmental rhythms are induced by light and temperature
465 cycles, rather than being self-sustainable.
466 Circadian clocks are able to entrain to cyclical cues in the environment and in all organisms
467 studied to-date, light experienced late in the subjective day causes phase delays of clock time
468 whilst light experienced late in the subjective night causes phase advances. This affects
469 rhythms that are clock-controlled such that in changing day-lengths clock-controlled outputs
470 occur at the correct time of day [3]. This ability to reset also allows circadian clocks to entrain
471 to environmental cycles shorter and longer than 24 hours (T cycles). A phenomenon known as
472 frequency demultiplication emerges when organisms are exposed to very short or long periods
473 (e.g. 6:6 or 24:24 LD or temperature cycles). That is, they display a 24-hour rhythm as if the
474 oscillation is entrained to a 12:12 cycle [3]. In contrast, if a rhythm is simply a direct response
475 to external cues, it assumes the periodicity of the driving light or temperature cycle [3]. In V.
476 dahliae, cultures grown under 6:6 LD and 6:6 temperature cycles show narrow conidial and
477 microsclerotia bands, whereas cultures under 28:28 LD and 28:28 temperature cycles form
478 widely spaced bands (Fig 2D). Thus, the developmental rhythm shows no evidence of
479 frequency demultiplication. Instead, conidial and microsclerotia production follows each
480 light/dark and temperature transition.
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481 To establish whether the lack of free-running morphological rhythms was representative of a
482 wider set of V. dahliae isolates, 12 different isolates were tested under light/dark and
483 temperature 20 °C /28 °C cycles (S5 Fig). Although all the isolates respond to both light and
484 temperature cycles, in the absence of cyclic light or temperature there is no evidence of free-
485 running rhythms. Data collected from one isolate of a species is not necessarily representative
486 of a species [77], yet alone different species. For this reason, we characterized the photobiology
487 and tested for the presence of circadian rhythms in three additional species in the Verticillium
488 genus: Verticillium albo-atrum, Verticillium nubilum and Verticillium tricorpus. When cultures
489 were treated in an alternating 12 h light/12 h dark photocycle, or 20 °C /28 °C cycles in constant
490 darkness, concentric rings of conidia and resting structures were formed. However, as in V.
491 dahliae, there is no apparent circadian rhythm of development on transfer to constant
492 conditions in any of the Verticillium species tested in this study (S6 Fig).
493
494 V. dahliae lacks rhythmic Vdfrq gene expression in LD and DD
495 A characteristic property of circadian rhythms is their persistence in constant conditions, and
496 rhythmic expression of frq in continuous darkness has been widely used as a marker of
497 circadian time [78]. In V. dahliae isolates 12253, 12008 and 12158, Vdfrq mRNA levels under
498 free-running conditions are arrhythmic (p-value=0.94, p-value=1, p-value=0.41, respectively).
499 In contrast, exposed to the same conditions, robust oscillation of N. crassa frq is seen (Fig 3A)
500 and, in agreement with previous studies [79], frq expression peaked at 12 h and 36 h after
501 transfer to DD (subjective morning). Another feature of circadian rhythms is that they
502 anticipate cyclic changes in the environment. Therefore, we assessed the expression of several
503 clock genes (frq, wc-1, wc-2 and vvd) in a 12:12 LD cycle, with high time-point resolution
504 before and after “lights on” (Fig 3B). Vdfrq transcript levels under DD seemed to anticipate
505 the transition to light as the expression slightly increased from D10 to D11.5, albeit with less
21 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.
506 than a 1 log2 fold change. However, Vdfrq transcript levels do not exhibit significant rhythmic
507 oscillation (p-value=1) and drop after the first time-point in the light. In contrast, the expression
508 of Vdwc-1, Vdwc-2 and Vdvvd do not anticipate the dark/light transition. There is a slight
509 increase in the expression of Vdwc-1 and Vdwc-2 after 2 hours in the light, whereas Vdvvd is
510 highly expressed after the dark to light transition, and drops to basal levels at a later time-points
511 possibly due to photoadaptation [80] (Fig 3B). In conclusion, there is no strong evidence for
512 anticipatory behaviour that could indicate light entrainment of a V. dahliae circadian clock, nor
513 is there significant rhythmic gene expression in LD. Similarly, there is no significant difference
514 in the expression of Vdfrq, Vdwc-1, Vdwc-2 or Vdccg-16 before or after the transition between
515 a high temperature (HT) period of 28 °C or a low temperature (LT) period of 20 °C (Fig 3C).
516
517 Promoter Clock box and LRE motif identification
518 In N. crassa the expression of frq is rapidly induced by pulses of light, mediated by the WCC,
519 enabling light entrainment of the clock and synchronization to external conditions [5,14]. The
520 presence of key clock genes in Verticillium but absence of observable clock-driven
521 developmental rhythms prompted us to look at the promoter regions of Vdfrq and homologues
522 of several other genes regulated by the WCC in Neurospora. For comparison we included in
523 our search the frq promoters of a subset of fungal species known to have a functional circadian
524 clock, such as Botrytis [30] and Magnaporthe [81]. Two experimentally verified WCC binding
525 motifs in the promoters of Neurospora clock-related genes containing two imperfect GATN
526 repeats [7,16], were identified in our in silico analysis: the proximal and distal light regulatory
527 element (LRE) motifs containing the sequence 5’GATNC--CGATN3’, where N is the same in
528 both repeats [45], and 5’GATCGA3’ [15].
22 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.
529 Putative proximal or distal motifs containing the sequence 5’GATNC--CGATN3’ are found in
530 the frq promoter region (2000 bp upstream the ORF) of all fungal species analysed in this study
531 (S7 Fig) . However, in most species, including V. dahliae, the distance between the two LRE
532 motifs is more than 70 bp, very far from the 3 and 11 nucleotide gap found in the functionally
533 verified proximal and distal N. crassa frq promoter motifs [45]. The clock-containing organism
534 B. cinerea presents a putative proximal motif with a 5 nucleotide gap and N. ditissima shows
535 two 4 nucleotide-gapped putative motifs within the first 400 nucleotides upstream the
536 ORF. Interestingly, V. tricorpus, V. zaregamsianum and V. albo-atrum also present putative
537 motifs with a short gap of maximum 14 nucleotides but they strongly differ in the position
538 within the promoter. On the other hand, Magnaporthe poae presents a putative motif with a
539 gap length of 55 nucleotides.
540 A perfect match to the proposed WCC binding motif, 5’GATCGA3’, described by Smith et
541 al., [15] was found several times in the majority of the promoters. In N. crassa, the motif is
542 found in the antisense sequence and close to the gene start site, which is also observed in M.
543 poae, V. nonalfalfae, V. alfalfae, V. dahliae and V. albo-atrum. At the terminator level, N.
544 crassa presents a WCC binding motif that induces the transcription of an antisense frq sequence
545 known as qrf [82]. In our study, only N. ditissima and V. isaaci show a gapped motif in the
546 antisense sequence, although they are considerably distant from the stop codon. Overall, our
547 analysis shows that the conservation in number, position and sequence of promoter motifs does
548 not necessarily correlate with the presence of an active circadian clock.
549
550 Light does not activate the transcription of Vdfrq but regulates expression of
551 photoreceptors Vdcry and Vdvvd
23 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.
552 We then tested the ability of light to induce expression of frq in V. dahliae. The mycelium of
553 V. dahliae 12253 strain was grown on shaking liquid media in constant darkness (DD). Then,
554 the cultures were either kept in the dark (dark control) or transferred to white light for 15
555 minutes after which RNA levels were assessed by qRT-PCR (see methods). Our results reveal
556 that V. dahliae frq expression is not induced by pulses of light (Fig 3D), whereas, as expected,
557 N. crassa frq transcript increases 10-fold. Furthermore, in contrast to N. crassa [74], the
558 expression of Vdwc-1 also lacks induction by light (Fig 3D). In contrast, orthologues of the
559 light-inducible N. crassa genes vvd and cry are both up-regulated. Vdcry mRNA increases 14-
560 fold and Vdvvd mRNA 5-fold in response to light (Fig 3D).
561
562 Time-course RNA-Seq reveals lack of transcriptional rhythmicity in constant conditions
563 Because V. dahliae clock gene homologues show no evidence of circadian regulation we
564 undertook an unbiased approach to identify rhythmically expressed genes. Following light
565 entrainment and subsequent transfer to dark, RNA-Seq analysis was carried out in three
566 biological replicates harvested at DD6, DD12, DD18 and DD24 in wild-type (12008 and
567 12253) strains. The normalized data (FPKM) of each strain was analysed using JTK-Cycle
568 [52]. The parameters were set to look for rhythms with a period of 24 hours. An important
569 point to bear in mind is that JTK-cycle duplicates the 24-hour time series and then analyses the
570 data for rhythmicity. Thus, as well as transcripts that reach their peak or nadir at DD12 and
571 DD18, transcripts that decrease or increase with time after the light to dark transfer will be
572 identified as rhythmic.
573 In V. dahliae 12253, significant oscillation in expression is observed for 568 genes
574 (approximately 4.9 % of the genome; p < 0.05) although the q value (the minimum false
575 discovery rate at which the test is considered significant) ranges from 0.2 to 0.8. The amplitude
24 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.
576 of the cycle is lower than 10 in 432 genes, which suggests a very weak oscillation. In V. dahliae
577 12008, 884 genes are identified with rhythmic expression, with a p-value < 0.05 and a q value
578 ranging from 0.1 to 0.5. In this isolate vvd, cry-dash and putative clock-controlled genes ccg-
579 9 and ccg-16 are significantly rhythmic, with amplitudes ranging from 1.4 to 21.5. However,
580 most oscillations are weak with amplitudes lower than 10 in 83% of the rhythmically expressed
581 genes. Of the 568 and 884 rhythmic genes identified from V. dahliae 12253 and 12008, only
582 34 genes are rhythmic in both strains (S8 Fig). For those genes, the cycle amplitude is low with
583 q values higher than 0.1 (S3 Table) and several (Chr1g02750, Chr1g18780, Chr2g02830,
584 Chrg300650, Chr3g11370, Chr4g07950, Chr5g05660, Chr7g00930) display different
585 expression patterns in each strain (S8 Fig). The expression of most genes encoding
586 orthologues of the central clock-oscillator (frq, wc-1, wc-2, frh, fwd-1), clock-controlled genes
587 (ccg-1, ccg-6, ccg-7, ccg-8, ccg-9, ccg-14) and photoreceptors (phr, phy, rgs-lov, nop-1, lov-u)
588 are not significantly rhythmic according to the JTK Cycle analysis. Exceptions are cry-dash
589 (Chr1g17210) and vvd (Chr3g10380) genes (Fig 4). In order to validate the RNA-seq results,
590 V. dahliae 12253 and 12008 samples harvested every 4 hours over a 48 hour time-course were
591 analysed by qRT-PCR (Fig 3A). In both strains, cry-dash and vvd were arrhythmic. This result
592 highlights the possible misinterpretation of transcripts as rhythmic by JTK that are simply
593 falling or rising after the light to dark transfer.
594
595 V. dahliae frq affects fungal growth
596 Having found no strong evidence of circadian rhythmicity we wondered what function V.
597 dahliae FRQ might have. To assess whether Vdfrq plays a role in development, we assessed
598 whether the banding pattern of conidia and microsclerotia is generated by the Vdfrq knockout
599 mutants Δfrq_12253 and Δfrq_12008. The Δfrq mutants grown in either 12:12 h LD cycles or
600 temperature cycles (20 oC - 28 oC) present the same characteristic banding pattern of
25 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.
601 microsclerotia and conidia as the wild type (WT) strains (Fig 5A). Despite the variability
602 among replicates we observed a reduction in total colony growth for both Δfrq mutants
603 (Δfrq_12253 and Δfrq_12008) with respect to the WT (12253 and 12008) strains (p-value <
604 0.01, p-value < 0.01, respectively). The reduction in daily growth was independent of lighting
605 conditions (Fig 5A) but dependent on the nutritional composition of the culture medium (S9
606 Fig). Δfrq_12008 showed reduced growth compared to WT_12008 on PLYA media, but not
607 on a minimal medium (MM) (p-value=0.99), Czapek Dox agar (DOX) (p-value=1) or basal
608 modified medium (BMM) (p-value=1). Colonies of Δfrq_12253 were significantly larger than
609 WT_12253 when incubated on BMM and MM (p-value < 0.01, p-value < 0.01, respectively).
610 Bands of microsclerotia were observed on all media types, but conidial rings were masked by
611 masses of mycelium when grown on DOX and BMM media (S9 Fig). Although a large
612 heterogeneity exists between Vdfrq mutants of different V. dahliae isolates, the results suggest
613 that Vdfrq plays a role in normal fungal growth.
614
615 V. dahliae Δfrq leads to widespread transcriptional differences.
616 Because LREs are present in the Vdfrq promoter and to uncover the possible function of Vdfrq
617 we compared gene expression in dark-grown WT and Δfrq cultures after exposure to 6 hours
618 of white light. Of 11388 genes with nonzero total counts, 235 genes are up-regulated and 162
619 genes are down-regulated in the WT strain (L vs D in V.d 12253). In the Δfrq mutant, 65 genes
620 are up-regulated and 30 genes are down-regulated (L vs D in Δfrq_12253). It is interesting that
621 in the Δfrq strain, light has a reduced effect on induction/repression of gene expression
622 suggesting that VdFRQ may indirectly affect light signaling.
623 The deletion of Vdfrq has a large effect on gene expression. Interestingly, the difference in
624 gene expression between the Δfrq and the WT is greater in the light than in the dark: 278 genes
26 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.
625 are up-regulated and 195 genes are down-regulated in the Δfrq_12253 mutant grown under
626 dark conditions (Δfrq vs WT in D), whereas 435 genes are up-regulated and 233 genes are
627 down-regulated in the Δfrq_12253 mutants harvested after 6 h in the light (Δfrq vs WT in L).
628 Functional enrichment analysis was carried out to investigate the function of genes
629 differentially expressed between the WT and the Δfrq_12253 mutant. Regardless of light
630 conditions Δfrq_12253 up-regulated genes are involved in metabolic processes, translation,
631 protein secretion and nucleotide metabolic processes. The down-regulated genes in
632 Δfrq_12253 grown in either light or dark were involved in redox processes, heme oxidation,
633 circadian rhythms and glutamate biosynthetic processes. Additionally, genes that are down-
634 regulated only in the light are functionally enriched for phosphate ion transport, nitrate
635 assimilation transport, superoxide anion generation, one-carbon metabolic processes as well as
636 pathogenesis processes (S4 Table).
637
638 V. dahliae frq regulates the expression of photoreceptor, TF- and SM- encoding genes.
639 In Neurospora, the transcription factor complex of the clock oscillator machinery activates the
640 transcription of many other TFs which results in a gene regulatory cascade [84]. As a result, the
641 circadian clock modulates the expression of genes involved in many processes, especially those
642 involved in physiology and metabolism [21]. Therefore, we hypothesized that disruption of
643 Vdfrq would affect the expression of photoreceptors, transcription factor-encoding genes and
644 genes involved in secondary metabolism.
645 We looked at how the absence of VdFRQ affects expression of clock, photoreceptor,
646 transcription factor and secondary metabolism-encoding genes in V. dahliae under different
647 light conditions (6 h dark and 6 h light). The expression of fwd-1, wc-1 and wc-2 are
648 significantly down-regulated in Δfrq_12253 in both light and dark conditions, although the
27 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.
649 LFC in expression does not reach the threshold of 1 (S5 Table). Interestingly, when exposed
650 to light vvd is significantly up-regulated in the Δfrq_12253 mutant, indicating that VdFRQ has
651 a negative effect on vvd transcription. In the case of cry-dash gene, its expression is light-
652 regulated in both the WT and Δfrq_12253, although the absolute expression level in the Vdfrq
653 mutant is significantly lower than in the WT. The expression profile of the rgs-lov, cry-1, phy,
654 phr and nop-1 genes does not change in the absence of Vdfrq.
655 We found that the absence of Vdfrq also affects the expression of several TF-encoding genes.
656 A total of 50 genes containing functional annotations associated with TF exhibited differences
657 in expression due to the lighting conditions or strain background. Most of the differences in
658 expression were due to the Vdfrq mutation. 26 TF-encoding genes were up-regulated, and 8
659 TF-encoding genes were down-regulated in Δfrq_12253 regardless of the light conditions.
660 However, there were 6 TF-encoding genes that were no longer light-induced or light-repressed
661 in Δfrq_12253 mutant strain (S5 Table).
662 Deletion of Vdfrq also affected core secondary metabolite-biosynthetic gene expression. The
663 lovastatine nonaketide synthase encoding gene (VDAG_JR2_Chr1g23880) [84] alongside 7
664 other members of the PKS gene cluster (cluster 14) are strongly affected in the absence of
665 Vdfrq. Interestingly, the light-induced expression of these genes was compromised in
666 Δfrq_12253 (S6 Table). Additionally, 6 genes of cluster 14 are the top most down-regulated
667 genes in Δfrq_12253 (LFC between -1.5 to -3.4), including the lovastatine nonaketide synthase
668 encoding gene, TOXD protein-encoding gene and a hydrolase encoding gene. An additional
669 cluster of PKS encoding genes, the putative aflatoxin biosynthetic cluster (cluster 17), exhibit
670 10 up-regulated genes in the absence of Vdfrq. Several members of cluster 24 (PKS), where
671 the core biosynthetic gene encodes a fatty acid synthase, also exhibit overexpression in
672 Δfrq_12253. Furthermore, 4 genes of the 5 non-ribosomal peptide synthases (NRPS, cluster
673 78) are highly up-regulated (LFC > 2) in the absence of Vdfrq (S6 Table). Therefore, Vdfrq is
28 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.
674 crucial for the regulation of expression of secondary-metabolism-encoding genes in V.
675 dahliae.
676
677 V. dahliae Δfrq mutants display reduced pathogenicity in a strain-dependent manner
678 In Botrytis cinerea the circadian clock regulates virulence [85] and consistent with this finding,
679 in a small number of fungi, circadian clock mutants show altered pathogenicity. To assess
680 whether the loss of Vdfrq would influence the process of infection of V. dahliae we evaluated
681 pathogenicity of two wild-type isolates (V. dahliae 12253 and 12008) as well as Δfrq_12253
682 and Δfrq_12008 on A. thaliana and Fragaria x ananassa in vitro-grown plants. Both wild type
683 isolates were isolated from UK strawberries and fall within the VC group subclade II-2, 12008
684 being a highly virulent isolate and 12253 being a moderately virulent isolate. The infected
685 seedlings were incubated in a 12:12 LD cycle for 28 days. Symptoms were visually rated at 0,
686 7, 14, 21 and 28 dpi on a scale of 1 to 9, in which 1 was equal to no symptoms and 9 equalled
687 a dead plant. In A. thaliana seedlings infected with the wild-type strain WT_12253 present
688 symptoms in up to 75% of the leaves after 21 days of inoculation (Fig 5C). At the same time
689 post inoculation plants infected with Δfrq_12253 show symptoms of wilt on 20% of leaves.
690 The difference is more obvious at 28 dpi, when most plants infected with WT_12253 are dead
691 whilst Δfrq_12253 infected plants display slight chlorotic symptoms in several outer leaves.
692 The AUDPC confirmed a significant difference in pathogenicity between Δfrq_12253 and
693 WT_12253 strains (p-value < 0.01). Contrary to this observation, WT_12008 and Δfrq_12008
694 do not present differences in virulence (Fig 5D). Similar results are obtained from
695 pathogenicity tests on a susceptible strawberry cultivar (Hapil). After 6 weeks of inoculation,
696 plantlets infected with the Δfrq_12253 strain show fewer disease symptoms than the
697 WT_12253 strain (Fig 5E), whereas the WT_12008 and Δfrq_12008 strains do not show
698 differences in the ability to cause disease (Fig 5F). These results indicate that pathogenicity of
29 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.
699 the Vdfrq mutant is impaired in an isolate-dependent manner, with isolates with greater degrees
700 of pathogenicity showing non-significant differences.
701
702 Discussion
703 Evidence that the outcome of a plant-pathogen interaction can depend on the time of day at
704 which the interaction occurs [86-88], has recently put the spotlight on the study of the circadian
705 clock in plant pathogenic fungi. There are multiple examples of important fungal species
706 harbouring the genetic components of the circadian clock, but the role of the clock on
707 pathogenicity is not well understood. Optimising the processes of infection to be in synchrony
708 with a plant’s most susceptible time could be advantageous to some fungal species for more
709 efficient infection. Similarly, understanding the daily changes in the developmental stages of
710 pathogenic organisms could help design more precise disease control strategies in agriculture.
711 In agreement with the findings of Salichos and Rokas [28], core clock orthologues were found
712 in most of the tested Sordariomycetes species, including important plant-pathogenic fungi.
713 Homologues of clock genes were identified in all species of the Verticillium genus: V. albo-
714 atrum, V. alfalfae, V. nonalfalfae, V. dahliae, V. longisporum subgenome D, V. nubilum, V.
715 tricorpus, V. isaacii, V. klebahnii and V. zaregamsianum. This result contrasts with previous
716 analysis in which loss of the wc-2 homologue in V. albo-atrum was reported [28], probably
717 due to the poor quality at this time of the publicly available V. albo-atrum genome. Several
718 species of the Dothideomycetes and Leotiomycetes, such as Blumeria graminis, Cercospora
719 zeae-maydis, Alternaria alternata and Venturia ineaqualis lack a homologue of the blue-light
720 receptor vvd, but do contain homologues of the other clock components.
721 The V. dahliae core clock homologues display strong conservation at the domain level.
722 Remarkably, VdFRQ contains all the domains identified in N. crassa FRQ and shares similar
30 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.
723 NLS sequences required for import of the protein to the nucleus. In addition, VdFRQ exhibits
724 conservation of phosphorylation sites, crucial for regulated activity and degradation and that
725 determine periodicity in N. crassa [70]. However, small changes in domains may be crucial for
726 function. For instance, although a coil-coil domain is present in VdFRQ the probability that it
727 can form dimers is much lower than that of the N. crassa FRQ coil-coil domain. Without
728 dimerisation FRQ does not interact with the WHITE COLLAR proteins and overt rhythmicity
729 is lost [64]. Proteins forming the WCC are also conserved in V. dahliae, but VdWC-1 lacks the
730 C-terminal polyQ region and has lost conservation at the N-terminal. The N-terminal of WC-
731 1 contains important domains required for protein-protein interaction and subsequent
732 transcriptional activation in N. crassa [76]. Nevertheless, zinc fingers and basic regions of both
733 WC-1 and WC-2 required for binding DNA are present and WCC phosphosites that govern
734 circadian repression in Neurospora [70] are conserved. Thus, V. dahliae contains all the
735 components required for a TTFL but changes in some important domains may compromise
736 their ability to generate an oscillator.
737 In Neurospora the WCCs that activate transcription in response to light and rhythmically in
738 the dark differ in composition, in the DNA motifs they bind, and in the regions of the WCC
739 proteins required for DNA binding. The WCC that responds to light is composed of two WC-
740 1 proteins and one WC-2 protein. The complex binds close to the transcriptional start site of
741 frq and binding requires only the zinc finger and proximal basic region of WC-2. In contrast
742 the heterodimer of WC-1 and WC-2, responsible for transcriptional activation of frq in the
743 dark, binds to the clock-box over 1 kb upstream of the transcriptional start site. Binding to the
744 DNA requires zinc fingers and basic regions of both proteins and recruitment of chromatin
745 modifiers SWI/SNF to initiate transcription [73]. Although several putative LREs were
746 identified in the Vdfrq promoter, core sequences of the clock box that have previously been
747 found within 1-3 bp of each other, in V. dahliae are separated by 80 bp. The lack of a classic
31 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.
748 clock box may underlie the lack of circadian rhythmicity of Vdfrq expression. However, ChIP-
749 Seq studies have revealed diverse motifs bound by light-activated WCC [15,83] and report that
750 the human GATA3 protein can bind palindromic GATA sites and GATA sites located on
751 different molecules of DNA, indicating that perhaps proximity of binding motifs is not
752 necessarily limiting.
753 In order to determine whether the V. dahliae morphological rhythm was under the control of a
754 circadian clock, a variety of tests were performed. Although rhythms of conidiation and
755 microsclerotia development are observed under light-dark and temperature cycles, they do not
756 persist in the absence of external stimuli and thus lack a key characteristic of circadian rhythms
757 [3]. This result, repeated on different media, supports the tentative conclusion that the lack of
758 sustainable developmental rhythms in constant conditions is unlikely to be due to media
759 composition. Possible explanations for the absence of observable clock-controlled free-running
760 developmental rhythms in V. dahliae include dampening of an existing rhythm in the absence
761 of external signals or the lack of a functional clock.
762 If the former is true we theorized that the existence of a circadian clock could be revealed
763 through analysis of development under different entrainment regimens. Frequency
764 demultiplication effects whereby clock-controlled outputs occur once every 24 h when external
765 periods are close to half of the endogenous period (T=12) are observed in circadian rhythms
766 [3,32]. Exposure to long periods (T=48) have the opposite effect, resulting in a reduction in the
767 frequency of the output to once every 24 hours [3,89]. V. dahliae grown under under 6:6 LD
768 or 28:28 LD cycles did not exhibit frequency demultiplication and produced rings of
769 development every 12 or 56 hours, respectively.
770 A defining characteristic of circadian clocks is their ability to entrain to external stimuli such
771 as light and temperature [3]. In N. crassa, short pulses of light trigger a rapid induction of frq
772 transcription that result in the resetting of the clock [14]. WC-1 is required for photoinduction
32 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.
773 of frq in response to light not only in N. crassa [7] but in other fungal species [29-31]. However,
774 our results show that whereas V. dahliae photoreceptor-encoding genes Vdvvd and Vdcry-dash
775 rapidly respond to light, Vdfrq expression is not light-induced. Furthermore, Vdfrq, Vdwc-1,
776 Vdwc-2, Vdvvd and Vdccg-16 transcript levels do not show robust anticipatory behaviour nor
777 the significant rhythmicity in light or temperature cycles seen in other fungi with circadian
778 clocks [29-30,90]. Constitutive expression of Vdfrq under cyclic environmental conditions
779 could be a symptom of a dysfunctional FRQ-WC clock. Alternatively, if a post-transcriptional
780 FRQ-WC clock runs in V. dahliae it may represent an ancestral oscillator that in some fungi
781 has subsequently been reinforced through additional feedback regulation acting on
782 transcription and mRNA abundance.
783 RT-PCR analysis of V. dahliae gene expression in constant darkness after light entrainment
784 revealed no circadian oscillation of Vdfrq mRNA in either of the isolates tested. However,
785 RNA-seq gene expression studies over a 24-hour period revealed putative rhythmic expression
786 in approximately 7% of genes, yet none of these differed more than the accepted cut-off of 1.5-
787 fold change in expression for oscillating genes [91]. A 1.5 fold change is the threshold above
788 which JTK_cycle reliably distinguishes between rhythmic and non-rhythmic samples. The
789 putative rhythmic genes were also not well-correlated in the two wild-type isolates and those
790 that were significantly rhythmic in both strains mainly showed an up or down regulation trend
791 after the light to dark transition. It is likely that higher resolution sampling over a 48 hour time-
792 course would greatly improve the accuracy of rhythmic transcript identification [92]. In
793 conclusion, no strong signature of rhythmic gene expression that would indicate possible
794 regulation of mRNA levels by a circadian clock was observed in V. dahliae.
795 In order to determine the impact of Vdfrq on the morphology of V. dahliae it was deleted in
796 two different isolates. The absence of Vdfrq does not lead to the abolishment of developmental
797 rhythms but results in reduced colony growth on most media. Moreover, pathogenicity tests
33 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.
798 reveal reduced infectivity of Vdfrq mutants of a weakly-pathogenic isolate but normal disease
799 progression of a highly virulent isolate. Interestingly, this observation is repeatable across plant
800 species (A. thaliana and Fragaria x ananassa). These data suggest that the growth penalty
801 and/or specific changes in the expression of genes unrelated to growth in the Vdfrq deletion
802 mutants influence infection and disease symptoms in a strain-dependent manner. Further study
803 of a wide range of isolates will be needed to determine if the influence Vdfrq has on
804 pathogenicity is correlated with virulence of the wildtype parent. Transcriptional profiling of a
805 Vdfrq knockout mutant revealed possible roles for VdFRQ in metabolic and signaling
806 processes and in pathogenicity. This result is in agreement with the observation that circadian
807 control has a major impact on metabolism in N. crassa [21]. Interestingly, the absence of Vdfrq
808 has an effect on the light response. One reason for this could be that in ΔVdfrq expression of
809 Vdwc-1 is down-regulated. This prompts speculation that a Vdwc-1 deletion would also affect
810 pathogenicity. To summarise, our data reveal large changes in gene expression, altered growth
811 and pathogenicity in the Vdfrq deletion mutant. Whether or not these phenotypes result from
812 VdFRQ functioning outwith a circadian clock cannot at present be ascertained.
813 With regard to the existence of a circadian clock in V. dahliae our results suggest three
814 possibilities; i) the clock is absent, ii) the clock is post-transcriptional and constitutive gene
815 expression leads to oscillation at the protein level, iii) the clock is only active during specific
816 developmental stages and /or specific conditions e.g. the clock is activated when a host is
817 detected and is only functional in planta. As V. dahliae infects and moves through host tissue
818 it is likely that an ability to anticipate time-of-day changes in host immunity would be
819 beneficial.
820 At least three circumstances can be envisaged where circadian rhythmicity might be absent.
821 The first is when an organism is always ready to respond to the rhythmic environment. It has
822 been reported that despite the presence of homologues of most clock genes in Picea abies
34 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.
823 (Norway Spruce) they found no evidence of circadian gene expression in constant conditions
824 [93]. The authors note that because gymnosperms make chlorophyll in the dark the strong
825 adaptive pressure to anticipate dawn is lacking. Indeed, there is little evidence to support
826 circadian gene transcription/expression in gymnosperms [94-95]. Nevertheless, night break
827 experiments indicate that a circadian rather than an hour-glass clock is used in photoperiodism
828 [96-98]. Secondly, in an environment where the absence of a circadian clock reduces the
829 organism’s ability to anticipate and respond to a changing environment but this has no adverse
830 effect. In a mutant form of einkorn wheat, rhythmicity of known clock and clock-regulated
831 genes is lost. Counter-intuitively, rather than having a detrimental effect, in certain
832 environments this mutant is more productive and less variable than the wild-type [99].
833 Importantly, the presence of an alternative circadian clock running under these conditions
834 cannot be ruled out. Thirdly, in a predominantly arrhythmic environment, for example in
835 underground caves and burrows where changes in temperature and humidity are minimal, or
836 during the long winter night and perpetual daylight during mid-summer at high latitudes. Some
837 organisms living under such conditions on initial inspection have indeed shown little or no
838 evidence of circadian rhythmicity however, when studied in more detail these early conclusions
839 have been overturned [100].
840 While definitive proof of the absence of a circadian clock is difficult to obtain, the evidence
841 for post-transcriptional clocks and the importance of post-transcriptional modification of clock
842 proteins is abundant and strong. A classic demonstration that post-transcriptional processes can
843 generate a circadian clock was provided by [101] who showed that cyanobacterial clock
844 proteins KaiA, KaiB and KiaC in vitro in the presence of ATP exhibit cycles of
845 phosphorylation and dephosphorylation that have a period of approximately 24 hours, are self-
846 sustainable and temperature compensated [101-102]. Many post-transcriptional processes act
847 on clock gene transcripts and proteins and are key to the generation of circadian rhythmicity
35 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.
848 [103]. Indeed, it is long known that rhythms persist in enucleated Acetabularia crenulata [104]
849 but there are also numerous examples of rhythmically expressed animal and plant clock genes
850 that when constitutively expressed do not ablate rhythmicity. Rather posttranscriptional
851 mechanisms maintain rhythmic expression and activity of the clock proteins [105-106] i.e.
852 rhythmic transcription enhances the amplitude of rhythmic post-transcriptional processing.
853 Indeed, even some rhythms generated post-transcriptionally are not necessarily essential parts
854 of circadian clocks. For example, in Neurospora circadian rhythms of FRQ abundance can be
855 decoupled from its activity [107].
856 The results in this study demonstrate conservation of the core clock proteins between V. dahliae
857 and N. crassa. However, rhythmic gene expression in V. dahliae was not detected in either LD
858 or free-running conditions. Thus, if a circadian clock is absent in V. dahliae then, at least in
859 this fungus, the other function(s) of VdFRQ must require a very similar domain structure. On
860 the other hand, if constitutive levels of mRNA give rise to a solely protein-based circadian
861 clock in V. dahliae our data also indicate that rhythmic outputs are not regulated at the level of
862 mRNA abundance. An alternative possibility is that generation of circadian rhythmicity in V.
863 dahliae is conditional on specific environmental conditions. The recent characterization of a
864 frq-dependent circadian oscillator in the Leotiomycetes Botrytis cinerea suggests that frq is a
865 component of circadian oscillators in fungal groups that evolved concurrently with N. crassa
866 [29]. By extrapolation, when frq and other key clock genes are represented in a genome the
867 expectation is that a FRQ-WC clock is present. This is true even when no overt rhythms in
868 behaviour or development can be detected because clock-regulated timing of cellular
869 biochemistry can confer a competitive advantage [108-109]. In the wild V. dahliae
870 microsclerotia germinate in the presence of root exudates [36] and it is possible that this signal
871 initiates oscillations of a circadian clock. Future studies will determine whether or not a
36 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.
872 circadian clockwork emerges in planta and if so what advantages this confers on the V. dahliae
873 infection cycle.
874
875 Acknowledgements
876 The authors acknowledge funding from BBSRC BB/R00935X/1 and BB/RR008191/1
877
878 Author Contributions statement
879 EC and RH designed the experiments. EC performed the experiments. EC, SC, LJ and RH
880 analysed the data. EC, SC, LJ and RH wrote and edited the manuscript.
881
882 Conflict of interest statement
883 The authors declare no competing interests.
884
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1198 Figure captions
1199 Figure 1. The circadian clock components are conserved across fungal species. A. N.
1200 crassa clock gene homologues across 25 fungal genomes. The presence, absence and copy
1201 number is shown for core clock genes (frq, wc-1, wc-2, frh, fwd-1, vvd) on the heatmap on the
1202 right. The phylogenetic tree was constructed based on the concatenated D1/D2 and ITS
1203 sequence alignment using the Neighbour-Joining method based on the Tamura-Nei model with
1204 Geneious R10 software. Bootstrap support values from 1000 replicates are shown at the nodes.
1205 The fungal classes are shown in color boxes on the tree. The Basidiomycetes Ustilago maydis
1206 was used as outgroup species. B. Protein domain structure of core clock proteins in N. crassa
1207 and V. dahliae based on InterProScan database. FRQ, WC-1, WC-2, FRH, FWD-1 and VVD
1208 are highly conserved at the domain level. Protein length is displayed at the end of each protein
50 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.
1209 sequence. CC (coiled-coil), FCD and FRQ-CK1a (interaction domains), PEST (protein
1210 association), polyQ (poly-glutamine stretch domain), LOV (Light-oxygen-voltage sensing),
1211 PAS (Per-Arnt-Sim, protein binding domain), GATA (Zinc finger, GATA-type, DNA binding
1212 domain), Helicase (ATPase activity), F-box (protein-protein interaction motif), WD40 repeat
1213 (interacting domain).
1214 Figure 2. Verticillium dahliae morphological rhythms do not free-run. A. Development of
1215 V. dahliae in 12 h light: 12 h dark cycles (LD 12:12). After a light period, conidiophores (white
1216 bands) are developed above the hyphae, but never on the leading edge. Microsclerotia start to
1217 develop 48 h after inoculation, and are produced during the dark phase of the cycle. This leads
1218 to concentric zones of conidia and microsclerotia. B. V. dahliae morphological rhythm under
1219 different light regimes. V. dahliae hyaline isolate 12158 and the microsclerotia producer
1220 isolates 12253 and 12008 were point-inoculated on PLYA plates and grown in constant
1221 darkness (DD); constant white light (LL); cycles of 12 h white-light/12 h dark (LD 12:12). All
1222 plates were incubated at 24 °C. Experiments were performed five times. C. Strains 12158,
1223 12253, and 12008 point-inoculated on PLYA plates and grown under light cycles of 12 h white-
1224 light:12 h dark at 24 °C or under temperature cycles of 12 h 20 °C: 12 h 28 °C (T20/28 12:12)
1225 for 14 days, were transferred to constant darkness (LD12:12-DD) or constant temperature at
1226 24 °C (T20/28 12:12 - Ct24) for 7 days. Red horizontal lines indicate the period of growth
1227 under constant conditions. D. V. dahliae entrainment to short and long T cycles. V. dahliae
1228 isolates 12158, 12253 and 12008 were point inoculated onto PYLA plates and incubated for
1229 14 days at 24 °C under T cycles of LD 6:6 and LD 28: 28.
1230 Figure 3. Clock-gene expression profile in Verticillium dahliae. A. Time-course expression
1231 of N. crassa frq and V. dahliae frq, vvd and cry under free-running conditions (DD, 24 °C) in
1232 different isolates (12253, 12008 and 12158). VdFrq, Vdvvd and Vdcry transcript levels were
1233 assessed by qRT-PCR every four hours over a period of 48 hours in constant darkness (DD).
51 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.
1234 N. crassa 30-7 bd (first graph) was utilised as an experimental control. The β-tubulin and
1235 Elongation factor 1-⍺ genes were the V. dahliae housekeeping genes. The N. crassa
1236 housekeeping genes were the β-tubulin and TATA binding box-encoding genes. Transcript
1237 levels are normalized to ddCt of DD4 samples in each strain (control = 1). Data is presented as
1238 mean (∓SEM) from three independent biological replicates. B. Gene expression analysis of
1239 four V. dahliae putative clock genes (Vdfrq, Vdwc-1, Vdwc-2 and Vdvvd). Cultures were
1240 entrained to 12 h:12 h LD cycles and V. dahliae 12253 tissue was harvested at different time-
1241 points in the dark (D) or light (L) over 24-hours. The dashed vertical lines represent the
1242 transition from dark to light conditions. Black-white bars indicate the dark-light conditions
1243 respectively. β-tubulin and Elongation factor 1-⍺ genes were used as housekeeping genes
1244 against which clock gene signals were normalised. Transcript levels are normalized to ddCt of
1245 D2 conditions for each gene (control = 1). Data is presented as mean (∓SEM) from three
1246 independent biological replicates. C. Gene expression analysis of putative clock and clock-
1247 controlled genes under (Vdfrq, Vdwc-1, Vdwc-2 and Vdccg-16) cyclic temperature conditions.
1248 Cultures were entrained to 12 h/12 h 20 ℃/28 ℃ temperature cycles, and V. dahliae 12253
1249 mycelial tissues were harvested at different time-points at low (20 ℃, LT) or high (28 ℃, HT)
1250 temperatures over a 24-hour period. The dashed vertical lines represent the transition from L
1251 to H conditions. Blue-red bars indicate the low-high temperature conditions respectively. β-
1252 tubulin and Elongation factor 1-⍺ genes were used as housekeeping genes. Transcript levels
1253 are normalized to ddCt of L2 conditions for each gene (control = 1). Data is presented as mean
1254 (∓SEM) from three independent biological replicates. D. qRTPCR expression analysis of
1255 Vdfrq, Vdwc-1, Vdcry and Vdvvd in the dark and in response to a 2 minute light pulse. After 36
1256 hours in constant dark at 25 °C, half of the cultures were given a 15 minute light pulse after
1257 which they were cultures were harvested. Statistical analysis of circadian expression was
1258 assessed by JTK-Cycle software.
52 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.
1259 Figure 4. Expression pattern of N. crassa homologues of clock oscillator and clock-
1260 controlled genes (ccg) in both V. dahliae 12253 and 12008 over 24-hour in the dark. The
1261 scales are different for every gene. Statistical significant rhythmic genes assessed by JTK-
1262 Cycle are marked with an asterisk (*).
1263 Figure 5. The effect of Vdfrq deletion mutants on fungal growth and pathogenicity. A.
1264 Phenotype of V. dahliae frq deletion in 12253 and (B) 12008 background. PYLA plates point
1265 inoculated with WT 12253, Δfrq_12253, WT 12008 and Δfrq_12008 and incubated in LD
1266 12:12 and temperature (20/28) 12:12 cycles for 14 days. Boxplot graphs depict WT 12253,
1267 Δfrq_12253, WT 12008 and Δfrq_12008 colony diameters in DD and LD, n=6. Letters
1268 indicate statistical differences (p-value < 0.05), Tukey’s HSD test. C. Disease score of A.
1269 thaliana in vitro seedlings inoculated with V. dahliae isolates WT_12253 or Δfrq_12253 at 0,
1270 7, 14, 21 and 28 days post-inoculation under 12:12 LD cycle. The area under the disease
1271 progression curve (AUDPC) was calculated. Different letters indicate statistical differences
1272 (p-value < 0.05), Tukey’s HSD test. Disease symptoms 28 days after inoculation of A.
1273 thaliana seedlings with V. dahliae isolates and a mock (control) inoculation are shown on the
1274 right. D. Disease score of A. thaliana in vitro seedlings inoculated with V. dahliae isolates
1275 WT_12008 or Δfrq_12008. E. Disease score of the susceptible strawberry cultivar Hapil
1276 inoculated with WT_12253 and Δfrq_12253, 6 weeks after inoculation. A one-way ANOVA
1277 was performed. Data are presented as mean (∓SEM) with different letters indicating statistical
1278 differences (p-value < 0.05). Disease symptoms 6 weeks after inoculation of strawberry
1279 plantlets with V. dahliae isolates and a mock (control) inoculation are shown on the right. F.
1280 Disease score of the susceptible strawberry cultivar Hapil inoculated with WT 12008 and
1281 Δfrq_12008.
1282
53 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.
1283 Supporting information
1284 S1 Fig. Generation of V. dahliae Δfrq mutants. A. Strategy scheme of the replacement of
1285 frq gene in V. dahliae. Genomic regions utilized for homologous recombination (black
1286 fragments) are shown. Black arrows symbolise primer pairs used for the validation PCR. B.
1287 Gel of the validation PCRs for the correct V. dahliae knockout transformants. C. Primer pairs
1288 utilized in PCR validation.
1289 S2 Fig. Alignment of FRQ homologous sequences from N. crassa and V. dahliae JR2.
1290 Protein domains characterized in N. crassa FRQ are annotated in red: Coiled-coil domain,
1291 FCD, FFD and PEST domains. Green bar, nuclear localization signal (NLS). Blue circle,
1292 conserved serine residue at position 513 (Ser-513). Green circles, additional phosphorylation
1293 sites previously identified in N. crassa.
1294 S3 Fig. Alignment of N. crassa and V. dahliae WC-1. Protein domains: Dark blue poly-
1295 glutamine stretch sequences. Blue, LOV domain with black box indicating the GXNCRFLQ
1296 motif. Red, PAS domains. Yellow, predicted nuclear localization signal (NLS). Green, GATA-
1297 type zinc-finger.
1298 S4 Fig. Alignment of N. crassa and V. dahliae WC-2. Protein domains: Red, PAS
1299 domain. Yellow, NLS (V.dahliae only). Green GATA-type zinc-finger.
1300 S5 Fig. The morphological rhythms of 12 V. dahliae isolates do not free- run. V. dahliae
1301 strains isolated from multiple hosts were point- inoculated on PLYA plates and incubated for
1302 14 days in an alternating 12 h dark/ 12 h white-light cycle (12:12 LD) (row 1) or under 12 h at
1303 20 °C/ 12 h at 28°C (12:12 20/28) (row 3). Plates were transferred to constant darkness (12:12
1304 LD - DD) (row 2) or constant temperature (12:12 20/28 -Ct 24) (row 4) for 7 days following
1305 the initial 14 days incubation in cyclic environments.
54 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.
1306 S6 Fig. The morphological rhythm of Verticillium albo-atrum isolate 11001 and 11006,
1307 Verticillium nubilum isolate 15001 and Verticillium tricorpus isolate 20001 do not free run.
1308 The isolates were incubated on PYLA plates for 14 days under alternating 12 h white-light/12
1309 h dark cycles (12:12 LD) (row 1) or under 12 h at 20 °C/ 12 h at 28°C (12:12 20/28) (row 3).
1310 Plates were transferred to constant darkness (12:12 LD - DD) (row 2) or constant temperature
1311 (12:12 20/28 -Ct 24) (row 4) for 7 days following the initial 14 days incubation in cyclic
1312 environments. Red lines indicate the period of growth under constant conditions.
1313 S7 Fig. Schematic representation of frq promoter and LRE motifs in N. crassa, M. poae,
1314 Verticillium spp. and B. cinerea. The confirmed distal LRE (dLRE) and proximal LRE
1315 (pLRE) motifs in the promoter and the qrf LRE (qLRE) motif in the terminator of N. crassa
1316 frq gene are marked with arrows. The putative promoter motifs containing the sequence
1317 5’GATNC--CGATN3’ He and Liu, (2005) in the promoters (2000 bp upstream the 5’ UTR)
1318 and terminator (2000 bp downstream the 3’ UTR) are shown in blue. The 5’GATCGA3’ (Smith
1319 et al., 2010) sequences are displayed in purple
1320 S8 Fig. Expression profile of cycling genes detected by JTK Cycle from a 24-hour time-
1321 course RNA dataset under constant dark. A. Venn diagram of putative rhythmic genes in V.
1322 dahliae 12253 and 12008 detected by JTK_Cycle. Samples were collected every 6 hours over
1323 a 24 h time-course in constant darkness at 24 °C. B. Expression pattern of the 34 genes
1324 identified as rhythmic by JTK-Cycle in both V. dahliae 12253 and 12008.
1325 S9 Fig. Verticillium dahliae growth and developmental phenotypes differ under different
1326 culture medias. A. Growth media composition affects growth rates of WT and clock mutant
1327 strains. Quantification of colony size of the strains presented in B. Boxplots represent
1328 distribution of colony diameters after 14 days of incubation in 12:12 LD cycles. Two
1329 independent experiments, each containing three replicates. Analysis of variances (ANOVA)
1330 was performed. Letters indicate significant differences (p-value<0.05) for each media type,
55 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.
1331 Tukey’s HSD test. B. Morphological phenotype of WT_12253, WT_12008, Δfrq_12008,
1332 Δfrq_12253 strains were incubated on PLYA, Czapek DOX, MM and BMM plates under 12:12
1333 LD conditions for 14 days.
1334 S1 Table. List of fungal strains.
1335 S2 Table. List of primers.
1336 S3 Table. List of rhythmically expressed genes in both Verticillium dahliae WT 12253,
1337 WT 12008. Data was assessed utilising JTK-Cycle and genes with p-value < 0.05 are shown.
1338 S4 Table. List of significantly enriched GO terms related to biological processes in
1339 Δfrq_12253 versus the WT_12253 strain in light and dark.
1340 S5 Table. Expression of putative core clock genes, photoreceptor- and TF-encoding genes
1341 in WT 12253 and Δfrq_12253 in both light and dark conditions. Transcripts displaying a
1342 Log fold change (LFC) >1 were classified as pink, dark red if the LFC > 2, light green if the
1343 LFC < -1 and dark green if the LFC < -2. Yellow boxes indicate p- values < 0.05.
1344 S6 Table. Most differentially expressed genes in Δfrq_12253. Transcripts displaying a Log
1345 fold change (LFC) >1 were classified as pink, dark red if the LFC > 2, light green if the LFC
1346 < -1 and dark green if the LFC < -2. Yellow boxes indicate p- values < 0.05.
56 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.
Fig 1 A B
1 frq wc-1wc-2frh fwd-1vvd CC FCD2 FFD N.c 78.7 Verticillium klebahnii 1 FCD1 PEST1 PEST2 82.3 Verticillium isaacii CC FCD2 FFD 989 FRQ V.d 66 Verticillium tricorpus FCD1 PEST1 PEST2 983 Verticillium zaregamisianum 1 polyQ LOV PAS PAS GATA polyQ 50.2 N.c 91.7 Verticillium nonalfalfae NLS 99.6 Verticillium alfalfae 1 1167 polyQ LOV PAS PAS GATA WC-1 100 Verticillium dahliae V.d 99.1 Verticillium nubilum NLS 1112 99.8 1 PAS GATA Verticillium albo-atrum N.c
Glomerella graminicola 1 Gene copies PAS GATA 530 WC-2 99.9 81.9 Thialevia terrestris Sordariomycetes V.d 0 100 Chaetomium globosum 463 1 1 ATP-binding Helicase Ski2 100 Sordaria macrospora N.c 2 54.8 Neurospora crassa rRNA-processing arch 1 1106 ATP-binding Helicase Ski2 3 FRH 59.7 Neonectria ditissima 100 V.d Fusarium oxysporum rRNA-processing arch 95.9 1106 1 Trichoderma reesei F-Box WD40 repeat WD40 repeat N.c 100 Sclerotinia sclerotiorum F-Box WD40 repeat 1 997 Botrytis cinerea F-Box WD40 repeat 99.2 FWD-1 V.d Blumeria graminis Leotiomycetes F-Box WD40 repeat WD40 repeat 1079 Zymoseptoria tritici 1 100 LOV 32.4 Cercospora zeae-maydis N.c
70.9 1 186 Alternaria alternata LOV VVD Venturia ineaqualis V.d
Ustilago maydis Dothideomycetes 183 0.03 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.
Figure 2 A B Vd 12158 Vd 12253 Vd 12008
DD
LL
C Vd 12158 Vd 12253 Vd 12008 LD 12:12
LD 12:12 to DD D Vd 12158 Vd 12253 Vd 12008
T20/28 12:12 LD 6:6
T20/28 12:12 LD 28:28 to Ct T24 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.
Figure 3 V. dahliae frq mRNA V. dahliae ccg-16 mRNA A N. crassa frq mRNA C 14 p-value=0.08 p-value=0.13 NcFrq 3 3 12 p-value=0.00 CT) 10
∆∆ 2 2 8
6 1 1 4 Normalized Normalized relative expression ( Normalized relative Normalized relative expression (∆∆CT) 2 0 0 0 DD 4 8 12 16 20 24 28 32 36 40 44 48 LT2 LT6 LT10LT11HT2 HT6HT10HT11 LT2 LT6 LT10LT11HT2 HT6HT10HT11 Hours Hours V. dahliae frq mRNA V. dahliae wc-1 mRNA V. dahliae wc-2 mRNA 14 VdFrq 12253 VdFrq 12008 VdFrq 12158 12 p-value=0.94 p-value=1 p-value=0.41 3 p-value=0.31 3 p-value=1 10 CT)
8 ∆∆ 2 2 6 4 1 1 Normalized relative Normalized relative expression (∆∆CT)
2 Normalized relative expression ( 0 0 0 DD 4 8 12 16 20 24 28 32 36 40 44 48 LT2 LT6 LT10LT11HT2 HT6HT10HT11 LT2 LT6 LT10LT11HT2 HT6HT10HT11 V. dahliae vvd mRNA Hours Hours 5 VdVvd 12253 VdVvd 12008 D N. crassa frq V. dahliae frq p-value=1 p-value=0.12 4 mRNA mRNA 25 25 3 b 20 20 2 15 15 1 10 10 expression(∆∆CT) Normalized relative 0 5 5 a a DD 4 8 12 16 20 24 28 32 36 40 44 48 Normalized relative expression (∆∆CT) a 0 0 V. dahliae cry mRNA DD LP DD LP 5 VdCry 12253 VdCry 12008 gghdhdhdhdhdhdhdhdV. dahliae wc-1 V. dahliae cry V. dahliae vvd p-value=1 25 hmRNA 25 mRNA 25 mRNA 4 p-value=1 b 20 20 20 3 15 15 15 2 10 10 10 b 1 5 5 5 Normalized relative Normalized relative expression (∆∆CT) a a a a Normalized relative Normalized relative expression (∆∆CT) 0 0 0 0 DD 4 8 12 16 20 24 28 32 36 40 44 48 DD LP DD LP DD LP
B V. dahliae frq mRNA V. dahliae wc-1 mRNA V. dahliae wc-2 mRNA V. dahliae cry mRNA p-value=1 p-value=0.7 p-value=0.48 p-value=0.39 7 7 7 7 6 6 6 6 CT) 5 5 5 5 ∆∆ (∆∆CT) 4 4 4 4 3 3 3 3 2 2 2 2 xpresson Normalized Normalized relative e
Normalized Normalized relative expression ( 1 1 1 1 0 0 0 0
D2 D6 D10D11 L2 L6 L10L11 D2 D6 D10D11 L2 L6 L10L11 D2 D6 D10D11 L2 L6 L10L11 D2 D6 D10D11 L2 L6 L10L11 Hours Hours Hours Hours 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.
Figure 4
frq wc-1 wc-2 frh fwd-1 4.5 4.8 6.6 4.5 4.2 4.4 6.5 4.4 4.1 4.6 6.4 4.3 4.3 4.4 6.3 4.2 4.2 4.0 6.2 4.1 4.1 4.2
D6 D6 D6 D6 D6 D12D18D24 D12D18D24 D12D18D24 D12D18D24 D12D18D24 vvd * cry-dash * phr phy rgs-lov 3.6 6.6 3.00 4.85 5.0 6.4 2.75 4.80 3.4 4.9 6.2 2.50 4.8 6.0 4.75 3.2 2.25 4.7 5.8 2.00 4.70 3.0 5.6 4.65 4.6 Strain D6 D6 D6 D6 D6 D12D18D24 D12D18D24 D12D18D24 D12D18D24 D12D18D24 V.dahliae_12008 nop-1 lov-u ccg-16 ccg-1 ccg-6 V.dahliae_12253 5.00 16.2 4.75 3.2 5.0 11.00 4.5 16.0 4.50 3.0 10.75 4.0 4.25 10.50 15.8 2.8 3.5 10.25 15.6
Normalized expressionNormalized FPKM) (Log2 D6 D6 D6 D6 D6 D12D18D24 D12D18D24 D12D18D24 D12D18D24 D12D18D24
ccg-7 ccg-8 ccg-9 ccg-14 10.8 7.00 13.0 6.0 10.6 6.75 12.5 10.4 5.8 6.50 12.0 10.2 6.25 10.0 5.6 11.5 6.00
D6 D6 D6 D6 D12D18D24 D12D18D24 D12D18D24 D12D18D24 Hours in DD E A Disease score Disease score C Figure 5 Figure 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 T20/28 12:12 LD 12:12 Strawberry (cv. (cv. Strawberry‘ WT Mock d 0 a bioRxiv preprint 12253 ∆frq 12253 WT 12253 Mock preprint (whichwasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplayin asatr inoculationDays after d 7 12253 WT b Hapil 4 d 14 Δfrq_12253 ’) 12253 doi: ∆frq ab 1 d 21 https://doi.org/10.1101/2019.12.20.883116 perpetuity. Itismadeavailableundera 8 d 28 Colony diameter (mm) 20 30 40 50 60 6 weeks piweeks6 a DD 8dy pi 28days b a
LD
WT 12253 WT Mock WT 12253 WT b Mock 12253 frq ∆ 12253 frq ∆ B D F Disease score T20/28 12:12 LD 12:12 2 3 4 5 6 7 8 9 0 1 Disease score ; this versionpostedDecember21,2019. 0 1 2 3 4 5 6 7 8 9 CC-BY-NC-ND 4.0Internationallicense WT Strawberry d 0 Mock 12008 a ∆frq 12008 WT 12008 Mock asatr inoculationDays after d 7 (cv. (cv. 12008 WT b ‘ 4 d 14 Hapil Δ frq_12008 ’) 12008 ∆frq b 1 d 21 8 d 28 Colony diameter (mm) 20 30 40 50 60 The copyrightholderforthis . 6 weeks piweeks6 a DD b 8dy pi 28days a LD
b
WT 12008 WT Mock 12008 frq ∆ WT 12008 WT 12008 frq ∆ Mock