Effect of light and darkness on the growth and development of downy mildew pathogen Hyaloperonospora arabidopsidis

Disease development in plants requires a susceptible host, a virulent pathogen, and a favourable environment. Oomycete pathogens cause many important diseases and have evolved sophisticated molecular mechanisms to manipulate their hosts. Day length has been shown to impact plant-oomycete interactions but a need exists for a tractable reference system to understand the mechanistic interplay between light regulation, oomycete pathogen virulence, and plant host immunity. Here we present data demonstrating that light is a critical factor in the interaction between Arabidopsis thaliana and its naturally occurring downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We investigated the role of light on spore germination, mycelium development, sporulation and oospore formation of Hpa, along with defence responses in the host. We observed abundant Hpa sporulation on compatible Arabidopsis under day lengths ranging from 10 to 14 hours. Contrastingly, exposure to constant light or constant dark suppressed sporulation. Exposure to constant dark suppressed spore germination, mycelial development and oospore formation. Interestingly, exposure to constant light stimulated spore germination, mycelial development and oospore formation. A biomarker of plant immune system activation was induced under both constant light and constant dark. Altogether, these findings demonstrate that Hpa has the molecular mechanisms to perceive and respond to light and that both the host and pathogen responses are influenced by the light regime. Therefore, this pathosystem can be used for investigations to understand the molecular mechanisms through which oomycete pathogens like Hpa perceive and integrate light signals, and how light influences pathogen virulence and host immunity during their interactions.

pathogen of grapevine, continuous light did not have any effect on the growth of the 23 mycelium and formation of sporangiophores, but the shape of sporangia was observed 24 to be immature (Rumbolz et al., 2002). In the lettuce downy mildew pathogen Bremia 25 recorded ( Figure 2B). When seedlings were exposed to constant light or constant dark 1 beginning immediately after inoculation for 3 days, then shifted to a normal light regime, 2 light sporulation was recovered 7dpi in samples exposed to constant dark. Abundant 3 sporulation was observed 7dpi in samples exposed to constant light, similar to plants 4 grown under a normal light regime ( Figure 2D). These experiments demonstrated that 5 the suppression of sporulation by constant light treatment of varying durations was not 6 a permanent effect and that sporulation could be recovered by returning the plants to 7 a normal regime. 8 9

Different light conditions affect mycelial growth of Hpa in leaves 10
Considering that plants grown under constant light for 7d supported abundant Hpa 11 sporulation after they were returned to a normal 12h L / 12 D regime (Figure 2), it 12 seemed likely that mycelium may have grown inside the leaf during exposure to 13 constant light but did not produce sporangia until a normal light regime was restored. 14 To check this possibility, infected At seedlings were stained with trypan blue 3dpi. 15 Trypan blue staining highlights mycelial growth along with sexual spore (oospores) 16 that are produced in the interior of the leaf and asexual fruiting bodies (sporangia) that 17 form on the exterior of the leaf. 18

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In plants grown under the normal light cycle, mycelia had grown throughout 20 cotyledons, sporangia had formed, and sporulation was observed over the whole 21 surface of the cotyledon (Figure 3a). In contrast to the normal light cycle, in cotyledons 22 exposed to constant light, there were extensive mycelia 3dpi and abundant oospores 23 but no conidiophores (Figure 3b). These results indicate that vegetative growth and 24 sexual sporulation can proceed under constant light, but asexual sporulation is 25 suppressed. 26 8 1 In cotyledons exposed to constant dark, less mycelial development was observed in 2 those that were exposed to either a normal light cycle or constant light (Figure 3c). A 3 small number of oospores were observed, similar to that observed under the constant 4 light experiment. 5

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To precisely assess Hpa growth in planta, we used a quantitative PCR assay in which 7 Hpa DNA is quantified as a proxy for pathogen biomass. During evaluation over three 8 days, mycelium biomass showed an increase in all groups ( Figure 4). However, the 9 lowest biomass was observed with constant dark exposure, whilst the constant light 10 gave the highest biomass production in every day. Constant light conditions produced 11 a significant increase in biomass compared to that observed with normal light The germination assay was first carried out with the reference light regime (12h L / 12h 1 D). Under this regime, spores germinate after six to eight hours and a germ tube 2 emerges ( Figure 5a). After 12 and 24h, germ tubes have extended on the surface of 3 the cellophane (Figure 5b and c). After 48 hours, formation of mycelial branches was 4 obvious and most branches were laterally oriented as they covered the surface ( Figure  5 5d). reference regime was 33% after 24 hours. The spores which were exposed to 24 10 hours constant dark showed a 22% germination rate, which was the lowest 11 percentage observed within this time period. Under constant light after 24 hours, 37% 12 of Hpa spores were germinated on cellophane ( Figure 6). After 48h, the germination 13 percentage increased for all treatments. The germination rate under constant dark 14 was the lowest with 31%, the reference regime was 57% and constant light was 49%. 15 After 72h, interestingly, the percentage of germination under constant dark and 16 constant light was the same. However, in the reference light regime, germination 17 increased and reached the highest percentage. At the end of 3d, germination seemed 18 to be completed and spores appeared to have lost their viability. These results 19 indicate that light is an important factor for spore germination independently of the 20 host, and that optimal germination of spores occurs under a normal light/dark regime. 21 22

Hpa mycelial biomass growth is affected by inoculation time 23
If there is a synchronized circadian regulation of Hpa development and host defence, 24 the inoculation time should be important for optimal colonization. Accordingly, previous 25 reports have demonstrated that the time of day for inoculation can impact the degree 26 to which Hpa can successfully colonize Arabidopsis, due at least in part to circadian 1 upregulation of host immune responses during a time period that encompasses 2 subjective dawn. Due to these observations, the optimal infection time for Hpa 3 development was not obvious. Therefore, biomass productions between two 4 inoculation times was compared using qPCR. Two zeitgeber time points were 5 determined to observe the effect of day and night (or light and dark) on the 6 development of pathogenicity. ZT0 refers to the beginning of daylight in an entrained 7 cycle and ZT12 is the beginning of night, under experimental conditions of 12h L/ 12h 8 D). One sample was inoculated at dawn (ZT0); beginning of the light period then

Sporulation assay 22
Inoculated Col-rpp4 seedlings were exposed to 3 different light (L) /dark (D) periods; 23 12h L/12h D, 14h L/10h D and 10h L/14h D for 7 d at 16˚C and the amount of 24 sporulation was assessed. 25 26 Another experiment was designed to understand the effect of extreme light regimes on 1 Hpa sporulation. The inoculated samples were exposed to 4 different light regimes; 7 d 2 constant light, 7 d constant dark, constant light after 3dpi and constant dark after 3dpi, 3 and control light/dark regime (12h L/ 12h D) was also included. As a light source, white 4 fluorescent bulbs (300 mmol m±2 s±1, 10 Osram HQIL 400 W-lamps plus four Osram 5 L40/ W60 fluorescent bulbs; Osram, Berlin, Germany) were used. To quantify 6 sporulation, 10 infected seedlings from each replicate were taken and placed into an 7 Eppendorf tube containing 250µl H20. Samples were vortexed and conidiospores were 8 counted using a haemocytometer. All experiments had minimum three replicas and 9 were repeated 3 times. All results were evaluated and compared statistically. 10 11 Trypan blue staining 12 Cotyledons of 7 d old Col-rpp4 were spray inoculated with Hpa-Emoy2 and were 13 exposed to a normal 12h L / 12h D cycle, constant light or constant dark and examined 14 at 3 dpi after staining with Trypan Blue as described at below; 15 Seedlings were taken from infected samples at the 0 hrs, 12 hrs, 1d, 2d, 3d, 4d, 5d, 16 6d, 7d post inoculation (dpi). Infected leaf segments were placed in an Eppendorf tube, 17 covered with 1 ml or enough amount trypan blue solution (10 g phenol, 10 ml glycerol, 18 10 ml lactic acid, 10ml water and 0.02 g of trypan blue (Merck) in ethanol (96%; 1:2 19 v/v) and boiled at 100 o C for 1 min. The leaf segments were then de-stained for an 20 hour in chloral hydrate (2mg/ml) (Sigma). All steps were carried out in a fume hood. 21 Pathogen structures were viewed under a CARL Zeiss Axioskop 4 plus microscope. 22

GUS assay 24
Transgenic PR1-GUS lines were used. Three-week-old seedlings were exposed to 1 constant light or dark for 1 to 3 days. Then, seedlings were transferred to 24 well replica 2 plates that contained 1 ml X-Gluc histochemical staining solution (50 mM X-Gluc in 50 3 mM NaPO4 pH 7.0) and incubated overnight at 37 0 C. After staining, leaves were treated 4 with 70% methanol up to 4 h. The samples were washed with ethanol, immersed in 5 glycerol and tissues were examined for GUS staining under dissecting microscope. The number of germinated Hpa spores was counted using a haemocytometer. 16 17 Determining biomass growth using qPCR. 18 The biomass of mycelium produced by Hpa-Emoy2 up to 3dpi was measured from 19 samples exposed to three different light regimes by Real-Time Quantitative PCR (RT-20 qPCR). The Hpa-Actin gene and At-Actin gene were used for quantification and its 21 relative protocol was followed as described (Anderson and McDowell, 2015). After 22 Col-rpp4 seedlings were inoculated with Hpa-Emoy2, samples were separated and Using Hpa-Arabidopsis reference system, we showed that light regimes significantly 10 affect several stages of the Hpa disease cycle, including spore germination, mycelial 11 development, oospore formation and sporulation. We also obtained preliminary result 12 suggesting that light regimes can also influence the immune status of the host. These observations have been reported for other downy mildew pathogens, for which a 20 "recovery" period of four hours in the dark was sufficient to enable sporulation 21 (reviewed in Rotem et al., 1978). The mechanism behind this recovery is unknown We also tested whether the timing of inoculation affected Hpa's capacity to colonize 18 the plant. A previous report demonstrated that effector-triggered immunity and basal 19 immunity against Hpa is more efficient early in the day (Wang et al., 2011), and we 20 confirmed this observation by using a different virulent isolate of Hpa. Our results 21 demonstrate that plants inoculated at dusk supported significantly more mycelial 22 growth than plants inoculated at dawn, even at three dpi. Our experiments do not 23 point directly to an underlying mechanism, but we hypothesize that this might reflect 24 a difference in timing of basal defense mechanisms that limit growth of virulent Hpa. 25 Wang et al (2011) noted that SA-dependent gene expression was stronger in the day 26 than at night; accordingly, it was reported that morning and midday inoculations lead 1 to higher salicylic acid accumulation, quicker and more intense PR (pathogen-related) 2 gene activation and expression, and HR responses than inoculations in the dusk or 3 at night (Griebel and Zeier, 2008). These previous reports on different systems 4 support our data and help to explain why night time inoculation is more efficient than 5 day time inoculation (Figure 8). In conclusion, we have reported several lines of evidence that light is a critical factor 19 during development of downy mildew disease on Arabidopsis and can influence 20 responses in the pathogen and the host. We can now exploit this system to understand 21 the mechanistic basis of these effects, using the well-developed tools for Arabidopsis 22 in combination with a new protocol for reverse genetics in Hpa. Our future studies will 23 focus on circadian regulation on both the host and pathogen side. While it is well-24 established that circadian regulation of host immunity is an important factor in immunity 25 against Hpa and other pathogens in Arabidopsis, the role of circadian regulation in 26 oomycete virulence is unexplored and therefore could be an enlightening area for 1 future inquiries.  Jimenez-Quiros from the University of Worcester are gratefully acknowledged. 14 15

Data availability statement 16
The data that support the findings of this study are available from the corresponding 17 author on reasonable request.  Samples were exposed to the reference light regime during the first 3 days (D/L black 20 column), then were exposed to constant light (light grey column) or constant dark (dark 21 grey column) over the subsequent 4 days (4dpi-7dpi). After end of the 7dpi, the 22 samples were transferred to the reference light regime again and sporulation was 23 recorded until 11dpi. c) Samples were exposed to constant light or constant dark for 24 7days immediately after inoculation. After 7dpi samples were transferred to the normal 25 light regime again and sporulation was recorded until 11 dpi. d) Samples were 26 exposed to constant light or constant dark beginning immediately after inoculation for 3 1 days, then shifted to a normal light regime, with sporulation recorded at 4 and 7 dpi. All 2 experiments were repeated 3 times. All results were evaluated and compared 3 statistically. *P < 0.05, **P < 0.01, ***P < 0.001, paired Student's t-test Spores were placed on cellophane strips and examined at regular intervals. a) after 19 6h, spore was germinated and germ tube was produced, b and c) after 12 and 24h, 20 respectively, germ tube became longer, d) after 48h, lateral mycelial branches were 21 obvious and hyphae began to cover the surface of the cellophane. cellophane. The spore germination on cellophane, which exposed to constant light 25 (blank column), constant dark (black column) and 12h L/ 12h D (lined column) regime was determined after 24h, 48h and 72h. Values represent means of three 1 experiments, and error bars correspond to the standard error of the means. Asterisks 2 indicate statistically significant differences to the reference regime in two-tailed 3 Student's t-test (p <0,05). Seedlings grown under normal 12h L / 12h D regime. b) Seedlings exposed to 13 constant light. c) Seedlings exposed to constant dark. After 48 hours of exposure to 14 these regimes, histochemical GUS assays were carried out. These experiments were 15 repeated 3 times with similar results. 16