The impaired virulence of <i>cks1</i> correlates with enhanced induction of defense genes and can be partially restored by exogenous cytokinin.

<p>The transcriptional regulation of defense-marker genes was evaluated upon inoculation with <i>cks1</i> mutant and complemented control strain. Nipponbare plants were inoculated with spore suspension (in gelatin 0.5%) of either the <i>cks1</i> mutant or <i>cks1</i>^<i>CKS1</i> control strain. Gene expression (normalized by plant Actin gene) was measured at different times after inoculation. <i>PBZ1</i> is a classical disease-related marker coding for a PR10 protein [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#ppat.1005457.ref056" target="_blank">56</a>], <i>PR5</i> and <i>PR10</i> are classical disease-related markers [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#ppat.1005457.ref057" target="_blank">57</a>], <i>CHI</i> and <i>CHI7</i> are chitinases [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#ppat.1005457.ref058" target="_blank">58</a>]. (A) Gene expression was measured before the first indications of fungal penetration (< 6hpi). (B) Kinetin (50 μM) was applied (KIN) or not (mock) at 24 hpi and gene expression was also measured at 48 hpi. A t-test was used to compare the means between <i>cks1</i> and <i>cks1</i>^<i>CKS1</i>; for one given gene * indicate significant differences between <i>cks1</i> and complemented strain; for (A) *, p-value < 0.04 and **, p-value<0.002 and for (B) *, pvalue<0.03; **, p-value<0.008. The values presented are the means calculated from four independent replicates. The experiments were repeated twice with similar results.</p

The virulence of the <i>cks1</i> strain is fully restored by an exogenous application of cytokinin.

<p>One day after inoculation with the <i>cks1</i> mutant or the <i>cks1</i>^<i>CKS1</i> control strain, plants were treated with 50 μM of the CK compound, kinetin, or buffer alone. Kinetin alone without infection had no visible effect on leaf aspect. (A) The symptoms were observed 6 dpi. The number of lesion per leaf (B) and the size of lesions (C) were measured. The values represent the mean and SD of three biological replicates of 10 individuals. The entire experiment was repeated 3 times with similar results. The different letters indicate significant differences between values (p-value < 0.01) as estimated by a generalized linear model (B) or a mixed model (C) using ANOVA analysis (See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#sec019" target="_blank">Materials and Methods</a>).</p

The impaired virulence of <i>cks1</i> correlates with an enhanced induction of the oxidative burst and can be partially restored by inhibiting NAD(P)H oxidase activity.

<p>The relationship between virulence and reactive oxygen species accumulation was evaluated in the <i>cks1-</i>infected leaves. (A) The oxidative burst was detected 48 h after inoculation using DAB stain that turns brown upon reaction with H₂O₂. Brown spots correspond to sites where the wild-type blast fungus penetrated (see inlet), whereas a global browning was visible with infection with <i>cks1</i> mutant. This experiment was repeated two times and gave similar results. (B, C) DPI, an NAD(P)H oxidase inhibitor partially restores the virulence of the <i>cks1</i> mutant. One day after inoculation (once appressorium formation was initiated), plants were treated with DPI (0.5μM diluted in DMSO as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#ppat.1005457.ref054" target="_blank">54</a>]. The symptoms were observed 6 dpi (B) and the number of lesions per leaf was measured (C). The letters indicate significantly different values according to a generalized linear model and ANOVA analysis (p-value < 0.04), see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#sec019" target="_blank">Materials and Methods</a>.</p

The <i>cks1</i> mutant strain is less virulent than wild-type and complemented control strains.

<p>Nipponbare plants were inoculated with <i>Magnaporthe cks1</i> mutants and control strains to evaluate virulence. The results for control GY11 and the complemented strain were similar and are only shown for symptoms. (A) Disease symptoms were observed 6 days after inoculation. Grey spots represent susceptible lesions whereas brown spots represent failed penetration events. The number of susceptible lesions per leaf (generalized linear model, p-value = 0.02) and size of lesions (mixt model, p-value = 0.003) were measured as shown in (B) and (C) respectively (for more details see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#sec019" target="_blank">materials & methods</a>). The values represent the mean and SD from four biological replicates each composed of 6 plants. The percentage of spores from the <i>cks1</i> and <i>cks1</i>^<i>CKS1</i> that penetrated the leaf was measured (D) under the microscope at different time points (hpi: hour post-inoculation). The data presented is the mean and SD of three biological replicates (>100 infection sites/replicate). A t-test was used to compare the penetration of the mutant and control strains, *, p-value < 0.01; **, p-value < 0.002; ***, p-value < 3.10⁻⁵. All experiments were repeated three times with similar results and one representative experiment is shown here.</p

Plant cytokinin signaling is differently affected during <i>cks1</i> infection.

<p>The transcriptional regulation of CK marker genes (<i>OsRR6</i> (Os04g57720) and <i>OsRR1</i> (Os11g04720) as named by Pareek et al., (2006)[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#ppat.1005457.ref053" target="_blank">53</a>] was evaluated by quantitative RT-PCR using the Actin gene for normalization. Nipponbare plants were inoculated with spore suspension (in gelatin 0.5%) of either the <i>cks1</i> mutant (black bars) or <i>cks1</i>^<i>CKS1</i> control strain (white bars) and gene expression was measured at 2, 4 and 6 hours post inoculation (hpi), before penetration of the leaf tissues but at stages where there was no significant difference in growth of the <i>cks1</i> mutant compared to the complemented strain (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#ppat.1005457.s005" target="_blank">S3C Fig</a>). The values presented are the Log2 ratios (infected/not infected) of the means calculated from four independent replicates. Uninfected plants were sprayed with gelatin 0.5% but without spore suspension. This experiment was repeated three times and showed similar results. A t-test was used to compare the means of expression quantified in <i>cks1</i> (black bars) and <i>cks1</i>^<i>CKS1</i> (white bars) inoculated plants. *: values significantly different at p-value < 0.05.</p

High fertilization levels restored <i>cks1</i> virulence without inhibiting defense induction.

<p>Plants were fertilized (high fertilization) or not (low fertilization) 24h before inoculation. Fertilization was done as in Ballini et <i>al</i>, 2013 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#ppat.1005457.ref061" target="_blank">61</a>] to test the effect of plant nutritional status on <i>cks1</i> virulence. (A) Symptoms 6 days after inoculation and the number of lesion per leaf in plants inoculated with <i>cks1</i> mutant or <i>cks1</i>^<i>CKS1</i> under low or high nitrogen fertilization. Three biological replicates composed of 10 plants were analyzed per strain/condition. The different letters indicate significant differences between values (p-value < 0.03) as estimated by a t-test. (B) The expression of defense-marker genes was measured 48hpi and first normalized with <i>Actin</i> in Nipponbare plants inoculated with <i>cks1</i>^<i>CKS1</i> (white bars) or with <i>cks1</i> (black bars), fertilized (High Nitrogen, HN) or not (Low Nitrogen, LN) 24h before inoculation. For each gene, the mean and the SD of relative expression obtained from 4 biological replicates (each of 3 plants) are presented. A t-test was done on raw data to compare relative expression in <i>cks1</i> and <i>cks1</i>^<i>CKS1</i> inoculated plants. *, p-value<0.04; **, p-value<0.003; ***, p-value<0.0005.</p

The virulence of <i>cks1</i> can be restored in immuno-deficient rice mutants.

<p>The <i>cks1</i> strain is more virulent on rice mutants deficient for basal defenses. KO-<i>cebip</i> (A) and <i>KO-nh1</i> (B) rice mutants and control plants (WT) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005457#ppat.1005457.ref057" target="_blank">57</a>], all in Nipponbare genetic background, were spray-inoculated with <i>cks1</i>. Symptoms were measured 6 days after inoculation on three replicates containing 6 plants. The values are the mean and SD from three biological replicates. A t-test was used to compare the percentage of susceptible lesions of <i>cks1</i> on immune-deficient mutant and respective control plants, p-value < 0.02.</p