A simplified model for crosstalk between the circadian clock and plant innate immunity.

<p>(<b>A</b>) Timing of stomata-dependent and -independent defense in a day. At night, plants might rely more on closed stomata to provide physical constrains to limit pathogen invasion but have relatively lower levels of stomata-independent defense. Once pathogens bypass such constrains (i.e. via infiltration infection in the laboratory), they encounter a plant host that is more susceptible than during the day. During the day, most stomata are wide open. In the presence of pathogens, plants can only transiently reduce stomatal aperture for a few hours (this study and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Melotto1" target="_blank">[1]</a>). Thus, during the day plants might depend more on stomata-independent defense to restrict pathogen invasion. Stomata-dependent defense could be stronger at night while stomata-independent defense could be stronger during the day. (<b>B</b>) The circadian clock regulates both stomata-dependent and -independent defense pathways to restrict pathogen growth in Arabidopsis. In a stomata-dependent pathway, CCA1 and LHY act, at least in part, through GRP7 as a direct downstream target to regulate stomatal aperture and thereby defense. Other downstream targets of CCA1 and LHY and other components of the central oscillator of the circadian clock might also be involved in regulating stomata-dependent and –independent defense. On the other hand, pathogen infection can activate PTI, ETI and other defense signaling in the host. PTI induced by flg22 feeds back to regulate clock activity. In addition, flg22-triggered signaling is under circadian clock control <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Bhardwaj1" target="_blank">[27]</a>. Thus, we conclude that the clock-defense crosstalk involves flg22-mediated signaling. Flg22 can affect stomatal aperture <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Zhang1" target="_blank">[91]</a>. However, whether this function of flg22 is through its regulation of the circadian clock or through a direct regulation of stomata is unclear. Other questions, such as whether additional PAMPs, effectors, and other defense signaling molecules are involved in clock-defense crosstalk, remain to be answered.</p

Clock activity of plants misexpressing <i>CCA1</i> and/or <i>LHY</i> is disrupted in LD.

<p>Eight-day-old seedlings of Col-0, <i>cca1-1</i>, <i>lhy-20</i>, <i>cca1-1lhy-20</i>, and <i>CCA1ox</i> expressing <i>ProCCA1:LUC</i> reporter were grown from germination in 12 hr light/12 hr dark cycles at 22°C. Luciferase activity was recorded with a Packard TopCount luminometer in LD at 22°C. (<b>A</b>) Mean circadian traces for <i>ProCCA1:LUC</i> activity. (<b>B</b>) Summary of phase value for <i>ProCCA1:LUC</i> in each genotype. Standard error of the mean (SEM) (n = 12–24) was used for (<b>A</b>) and (<b>B</b>). Letters indicate significant difference among the samples (P<0.05; Student's t-test).</p

CCA1 and LHY conferred disease resistance is SA-independent.

<p>(<b>A</b>) Picture of 25-day-old plants. (<b>B</b>) SA quantitation. Twenty-five-day old plants grown in 12 hr light/12 hr dark cycle (LD) at 22°C were harvested at ZT1, 7, 13, 19 and 25. Total SA were extracted and measured as described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Wang6" target="_blank">[90]</a>. (<b>C</b>) Infection with <i>PmaDG3</i>. Twenty five-day-old plants were infected by spraying with the virulent strain <i>PmaDG3</i> (1×10⁸ CFU/ml) at ZT13. Bacterial growth was assessed at 3 dpi. Data represent the mean bacterial numbers ± SEM (n = 6). Letters indicate significant difference among the samples (P<0.05; Student's t-test). These experiments were repeated three times with similar results.</p

The clock period is shortened by treatment with flg22 but not with BTH.

<p>(<b>A</b>) Mean circadian period of the <i>ProCCA1:LUC</i> reporter. Eight-day-old Col-0 seedlings expressing the <i>ProCCA1:LUC</i> reporter were grown from germination in 12 hr light/12 hr dark cycles at 22°C. At ZT7, eight-day-old seedlings were treated with flg22 (1 µM or 10 µM) or BTH (10 µM or 300 µM) and transferred to 96-well plates containing 200 µl of MS media and 30 µl of a 2.5 mM D-luciferin solution. Luciferase activity was recorded with a Packard TopCount luminometer in LL at 22°C. (<b>B</b>) Cotyledon movement assay with <i>acd6-1</i>. Eight-day-old <i>acd6-1</i> seedlings grown in a 12 hr light/121 hr dark cycle at 22°C were transferred to 24-well cloning plates and recorded in LL at 22°C for cotyledon movement. SEM (n = 12–24) was used for (<b>A</b>) and (<b>B</b>). These experiments were repeated twice with similar results.</p

Bacterial growth in plants infiltrated with <i>Pseudomonas syringae pv.</i><i>maculicola</i> strain DG3 (<i>PmaDG3</i>).

<p>(<b>A</b>) Time scheme used in this report. The white box indicates the light period and black boxes indicate dark periods. (<b>B</b>) ZT1 infection. (<b>C</b>) ZT13 infection. In 12 hr L/12 hr D (LD), 25-day-old plants were grown and infected by infiltration with <i>PmaDG3</i> at 1×10⁵ colony forming unit (CFU)/ml. Bacterial growth was assessed at 3 dpi. Data represent the average of bacterial numbers in six samples ± standard error. Log transformed bacterial growth was used in statistical analysis (Student's t-test). Letters indicate significant difference among the samples (P<0.05). These experiments were repeated three times with similar results.</p

CCA1 and LHY contribute synergistically to resistance to <i>Hyaloperonospora arabidopsidis</i> (<i>Hpa</i>).

<p>(<b>A</b>) Infection with <i>Hpa</i> Emco5. (<b>B</b>) Infection with <i>Hpa</i> Emoy2. Seven-day-old seedlings were spray-infected at ZT7 in LD with the virulent strain <i>Hpa</i> Emco5 or the avirulent strain <i>Hpa Emoy2</i> (5×10⁴ spores/ml in water). Sporangiophore production in cotyledons of each genotype was counted at 7 dpi. Data represent the average number of sporangiophores from 20 seedlings for <i>CCA1ox</i> and 50 seedlings for other genotypes ± SEM. Letters indicate significant difference among the samples (P<0.01; Mann-Whitney test). These experiments were repeated three times with similar results.</p

Overexpression of the <i>LHY</i> gene confers enhanced disease susceptibility to <i>P.</i><i>syringae</i>.

<p>(<b>A</b>) Infiltration with DC3000. (<b>B</b>) Spray with DC3000. 30-day-old plants were infected with <i>P. syringae pv. tomato</i> strain DC3000 (DC3000) by infiltration (1×10⁵ CFU/ml) or spray (1×10⁸ CFU/ml) at ZT1 or ZT13 in LD. Bacterial growth was assessed at 3 dpi. (<b>C</b>) Cell death staining. The fourth to fifth leaves of Ler and <i>LHYox</i> were stained with trypan blue to visualize cell death <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003370#ppat.1003370-Ng1" target="_blank">[54]</a>. (<b>D</b>) SA quantification. Total SA was extracted from 20- and 30-day old plants. Data represent the average of SA levels (n = 3) ± standard deviation. Statistical analysis was performed with Student's t-test (StatView 5.0.1). Asterisks indicate significant difference between L<i>er</i> and <i>LHYox</i> at the same time point (P<0.05). These experiments were repeated three times with similar results.</p

Defense activation by <i>P.</i><i>syringae</i> infection shortens the period of the <i>ProCCA1:LUC</i> reporter activity.

<p>(<b>A</b>) Mean circadian traces for <i>ProCCA1:LUC</i> activity. (<b>B</b>) Mean circadian period of the <i>ProCCA1:LUC</i> reporter. Col-0 seedlings expressing the <i>ProCCA1:LUC</i> reporter were grown from germination in 12 hr light/12 hr dark cycles at 22°C. At ZT7, eight-day-old seedlings were incubated with <i>PmaDG3</i> or <i>PmaDG6</i> (1×10⁸ or 1×10⁷ CFU/ml, labeled as 0.1 or 0.01, respectively) for 3 mins, blot dried, and transferred to 96-well plates containing 200 µl of MS media and 30 µl of a 2.5 mM D-luciferin solution. Luciferase activity was recorded with a Packard TopCount luminometer in LL at 22°C. RAE: relative amplitude error. RAE values close to zero indicate strong rhythms while those close to 1 indicate the limit of statistically significant rhythmicity. SEM (n = 12–24) was used for (<b>A</b>) and (<b>B</b>). These experiments were repeated twice with similar results.</p

Disruption of <i>CCA1</i> and <i>LHY</i> leads to altered stomatal activity.

<p>(<b>A</b>) Stomatal aperture at ZT1 (left) or ZT13 (right) for Col-0, <i>cca1-1</i>, <i>lhy-20</i>, <i>cca1-1lhy-20</i>, and <i>CCA1ox</i>. (<b>B</b>) Stomatal aperture at ZT1 or ZT13 for L<i>er</i> and <i>LHYox</i>. (<b>C</b>) Stomatal aperture at 1 hr (top) or 3 hr (bottom) after exposure to <i>PmaDG3</i> or mock for Col-0, <i>cca1-1</i>, <i>lhy-20</i>, <i>cca1-1lhy-20</i>, and <i>CCA1ox</i>. (<b>D</b>) Stomatal aperture at 1 hr or 3 hr after exposure to <i>DC3000</i> or mock for L<i>er</i> and <i>LHYox</i>. For (<b>A</b>) and (<b>B</b>), three leaves from uninfected 25-day-old plants grown in 12 hr light/12 hr dark at 22°C were taken at the indicated times for the measurement of stomatal aperture. For (<b>C</b>) and (<b>D</b>), <i>P. syringae</i> treatment was conducted at ZT4 to ensure that most stomata were open upon treatment. Leaves were immersed in bacterial suspension (10⁸ cfu/ml) or water as mock treatment. At least three leaves of a genotype were collected at the indicated times for stomatal aperture measurement. Data represents the average of three experiments ± SEM. Each of these experiments contains at least 70 randomly chosen stomata. Different letters in (<b>A</b>) indicate significant difference among the samples. Asterisks in (<b>C</b>) and (<b>D</b>) indicate significant difference between mock-treated and infected plants of the same genotype (P<0.001; Student's t-test). These experiments were repeated three times with similar results.</p

CCA1-regulated <i>GRP7</i> affects disease resistance to <i>P.</i><i>syringae</i> and stomatal activity.

<p>(<b>A</b>) Expression of <i>GRP7</i> is disrupted in <i>CCA1ox</i> in LD. Twenty five-day-old Col-0 and <i>CCA1ox</i> plants grown in a chamber with a 12 hr light/12 hr dark cycle and 22°C were harvested at ZT1 at 6 hr interval for 24 hrs followed by RNA extraction and northern blotting. 18S rRNA was used as a loading control. These experiments were repeated twice with similar results. (<b>B</b>) Stomatal aperture at ZT1 or ZT13. (<b>C</b>) Stomatal aperture at 1 hr (left) or 3 hr (right) after exposure to <i>PmaDG3</i> or mock solution. (<b>D</b>) Bacterial growth assay with ZT1 or ZT13 infection in LD. Asterisks indicate significant difference among the samples at the indicated times in panels (<b>B</b>) and (<b>D</b>) or within the same genotypes in panel (<b>C</b>) (P<0.05; Student's t-test). These experiments were repeated twice with similar results.</p