Cytokinin Production by the Rice Blast Fungus Is a Pivotal Requirement for Full Virulence

Abstract

Plants produce cytokinin (CK) hormones for controlling key developmental processes like source/sink distribution, cell division or programmed cell-death. Some plant pathogens have been shown to produce CKs but the function of this mimicry production by non-tumor inducing pathogens, has yet to be established. Here we identify a gene required for CK biosynthesis, CKS1, in the rice blast fungus Magnaporthe oryzae. The fungal-secreted CKs are likely perceived by the plant during infection since the transcriptional regulation of rice CK-responsive genes is altered in plants infected by the mutants in which CKS1 gene was deleted. Although cks1 mutants showed normal in vitro growth and development, they were severely affected for in planta growth and virulence. Moreover, we showed that the cks1 mutant triggered enhanced induction of plant defenses as manifested by an elevated oxidative burst and expression of defense-related markers. In addition, the contents of sugars and key amino acids for fungal growth were altered in and around the infection site by the cks1 mutant in a different manner than by the control strain. These results suggest that fungal-derived CKs are key effectors required for dampening host defenses and affecting sugar and amino acid distribution in and around the infection site.

Fig 1. The cks1 mutant strain is less virulent than wild-type and complemented control strains.

Nipponbare plants were inoculated with Magnaporthe cks1 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 materials & methods). The values represent the mean and SD from four biological replicates each composed of 6 plants. The percentage of spores from the cks1 and cks1 ^CKS1 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.

Fig 2. Plant cytokinin signaling is differently affected during cks1 infection.

The transcriptional regulation of CK marker genes (OsRR6 (Os04g57720) and OsRR1 (Os11g04720) as named by Pareek et al., (2006)[53] 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 cks1 mutant (black bars) or cks1 ^CKS1 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 cks1 mutant compared to the complemented strain (S3C Fig). 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 cks1 (black bars) and cks1 ^CKS1 (white bars) inoculated plants. *: values significantly different at p-value < 0.05.

Fig 3. The virulence of the cks1 strain is fully restored by an exogenous application of cytokinin.

One day after inoculation with the cks1 mutant or the cks1 ^CKS1 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 Materials and Methods).

Fig 4. The impaired virulence of cks1 correlates with an enhanced induction of the oxidative burst and can be partially restored by inhibiting NAD(P)H oxidase activity.

The relationship between virulence and reactive oxygen species accumulation was evaluated in the cks1-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 cks1 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 cks1 mutant. One day after inoculation (once appressorium formation was initiated), plants were treated with DPI (0.5μM diluted in DMSO as previously described [54]. 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 Materials and Methods.

Fig 5. The impaired virulence of cks1 correlates with enhanced induction of defense genes and can be partially restored by exogenous cytokinin.

The transcriptional regulation of defense-marker genes was evaluated upon inoculation with cks1 mutant and complemented control strain. Nipponbare plants were inoculated with spore suspension (in gelatin 0.5%) of either the cks1 mutant or cks1 ^CKS1 control strain. Gene expression (normalized by plant Actin gene) was measured at different times after inoculation. PBZ1 is a classical disease-related marker coding for a PR10 protein [56], PR5 and PR10 are classical disease-related markers [57], CHI and CHI7 are chitinases [58]. (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 cks1 and cks1 ^CKS1; for one given gene * indicate significant differences between cks1 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.

Fig 6. The virulence of cks1 can be restored in immuno-deficient rice mutants.

The cks1 strain is more virulent on rice mutants deficient for basal defenses. KO-cebip (A) and KO-nh1 (B) rice mutants and control plants (WT) [57], all in Nipponbare genetic background, were spray-inoculated with cks1. 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 cks1 on immune-deficient mutant and respective control plants, p-value < 0.02.

Fig 7. High fertilization levels restored cks1 virulence without inhibiting defense induction.

Plants were fertilized (high fertilization) or not (low fertilization) 24h before inoculation. Fertilization was done as in Ballini et al, 2013 [61] to test the effect of plant nutritional status on cks1 virulence. (A) Symptoms 6 days after inoculation and the number of lesion per leaf in plants inoculated with cks1 mutant or cks1 ^CKS1 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 Actin in Nipponbare plants inoculated with cks1 ^CKS1 (white bars) or with cks1 (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 cks1 and cks1 ^CKS1 inoculated plants. *, p-value<0.04; **, p-value<0.003; ***, p-value<0.0005.

Fig 8. The impaired virulence of cks1 is associated with altered contents of nutritional elements essential for the fungus.

Metabolomic analysis of plants infected with cks1 and complemented mutant strains. Glucose, aspartate and glutamate contents were quantified (nmol/mg of fresh weight) as well as other sugars and amino acids, during infection (times are indicated), at the site of inoculation corresponding to the “infected zone” and one centimeter apart on both sides (upper and lower) with respect to the inoculated zone (S7 Fig). Only the lower “non-inoculated, neighboring part of the leaf” is shown but all data relative to the “upper non-infected zone” can be found in S7 Fig. For more details see also Materials and Methods. A t-test (*, p-value < 0.05) was used to compare amino acid contents in leaf fragments from plants inoculated with the cks1 (black bars) and cks1 ^cks1 control complemented strain (white bars). The dashed arrows point to the significant changes (*t-test, p-value < 0.05) of amino-acid contents in cks1-infected plants between 24 and 48 hpi. For each time point four replicates composed of three leaf fragments were analyzed, mean and SD are indicated.