Overexpression of a Cytochrome P450 Monooxygenase Involved in Orobanchol Biosynthesis Increases Susceptibility to Fusarium Head Blight

Abstract

Fusarium Head Blight (FHB) is a cereal disease caused primarily by the ascomycete fungus Fusarium graminearum with public health issues due to the production of mycotoxins including deoxynivalenol (DON). Genetic resistance is an efficient protection means and numerous quantitative trait loci have been identified, some of them related to the production of resistance metabolites. In this study, we have functionally characterized the Brachypodium distachyon BdCYP711A29 gene encoding a cytochrome P450 monooxygenase (CYP). We showed that BdCYP711A29 belongs to an oligogenic family of five members. However, following infection by F. graminearum, BdCYP711A29 is the only copy strongly transcriptionally induced in a DON-dependent manner. The BdCYP711A29 protein is homologous to the Arabidopsis thaliana MAX1 and Oryza sativa MAX1-like CYPs representing key components of the strigolactone biosynthesis. We show that BdCYP711A29 is likely involved in orobanchol biosynthesis. Alteration of the BdCYP711A29 sequence or expression alone does not modify plant architecture, most likely because of functional redundancy with the other copies. B. distachyon lines overexpressing BdCYP711A29 exhibit an increased susceptibility to F. graminearum, although no significant changes in defense gene expression were detected. We demonstrate that both orobanchol and exudates of Bd711A29 overexpressing lines stimulate the germination of F. graminearum macroconidia. We therefore hypothesize that orobanchol is a susceptibility factor to FHB.

Keywords: Brachypodium distachyon; Fusarium Head Blight; cytochrome P450; orobanchol; susceptibility.

FIGURE 1

The Bradi1g75310 (BdCYP711A29) gene is transcriptionally induced during FHB and following DON treatment. Relative quantification of Bradi1g75310 transcripts in the Bd21-3 (WT) ecotype of B. distachyon following F. graminearum infections or following DON treatment. (A) Bradi1g75310 expression level (fold-change. log₂) following point infection with the FgDON⁺ (solid line) or with the FgDON^– (dashed line) strain of F. graminearum compared to mock treatment. (B) Bradi1g75310 expression level (fold-change. log₂) following DON treatment compared to mock treatment. The relative quantity of the Bradi1g75310 transcripts compared to mock condition was calculated using the comparative cycle threshold (Ct) method (2^–ΔΔCt). The B. distachyon UBC18 and ACT7 genes (Bradi4g00660 and Bradi4g41850) were used as endogenous controls to normalize the data for differences in input RNA between the different samples. Mean of three independent biological replicates ± standard deviation. Asterisks indicate significant differences between conditions (Student’s t-test p value < 0.05).

FIGURE 2

Molecular phylogenetic analysis of A. thaliana, B. distachyon, H. vulgare, O. sativa, and S. moellendorffii CYP711As. The protein evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model (Jones et al., 1992). The bootstrap consensus tree inferred from 500 replicates (Felsenstein, 1985) is taken to represent the evolutionary history of the taxa analyzed (Felsenstein, 1985). Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches (Felsenstein, 1985). Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. This analysis involved 16 amino acid sequences. There were a total of 567 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). The tree has been rooted with Selaginella moellendorffii CYP711A1 (SmCYP711A1). At: A. thaliana; Bd: B. distachyon; Hv: Hordeum vulgare; Os: O. sativa; Sm: S. moellendorffii. Protein sequences used in this analysis are available under the following accession numbers: AtCYP711A1, OAP07831.1; BdCYP711A5, XP_003571126.1; BdCYP711A6: XP_003560652.1; BdCYP711A29, XP_003562092.2; BdCYP711A30: XP_003575594.2; BdCYP711A31, XP_010237353.2; HvCYP711A5, BAJ97619.1; HvCYP711A6, KAE87888859.1; HVCYP711A29, BAJ98237.1; HvCYP711A30, KAE8810993.1; OsCYP711A2, XP_015633367.1; OsCYP711A3, XP_015644699.2; OsCYP711A4, XP_015642272.1; OsCYP711A5, XP_015626073.1; OsCYP711A6, XP_015644019.1; SmCYP711A1, XP_002972055.1.

FIGURE 3

Expression pattern of the 5 BdCYP711A genes in roots, leaves, and spikes. (A), (B), and (C) correspond to different expression patterns, mentioned as categories in the main text. Relative quantification of transcripts of the BdCYP711A5 (Bradi3g08360), BdCYP711A6 (Bradi1g37730), BdCYP711A29 (Bradi1g75310), BdCYP711A30 (Bradi4g08970), and BdCYP711A31 (Bradi4g09040) genes in different organs of the Bd21-3 (WT) ecotype of B. distachyon: roots and leaves were collected from 3 week-old plants under hydroponic conditions in ½ MS liquid medium and spikes were collected at mid-anthesis on plants grown in pots under standard conditions. The relative quantity of transcripts was calculated using the comparative cycle threshold (Ct) method (2^{– ΔCt}) using the B. distachyon UBC18 and ACT7 genes (Bradi4g00660 and Bradi4g41850) as endogenous controls to normalize the data for differences in input RNA between the different samples. Mean of three independent biological replicates ± standard deviation. For each gene, asterisks indicate significant differences between organs (*p value < 0.05, Student’s t-test).

FIGURE 4

Molecular characterization of B. distachyon lines overexpressing the BdCYP711A29 gene. Relative expression of the BdCYP711A29 gene in spikes. leaves and roots of overexpressing lines OE-CYP11.29 and OE-CYP12.20. The relative quantity of BdCYP711A29 transcripts (fold-change. log₂) of OE lines as compared with the Bd21-3 WT line was calculated using the comparative cycle threshold method (2^{– ΔΔCt}). The B. distachyon UBC18 and ACT7 genes (Bradi4g00660 and Bradi4g41850) were used as endogenous controls to normalize the data for differences in input RNA between the different samples. Data represent mean values of three independent biological experiments (n = 3) and two technical repetitions ± standard deviation. Different letters indicate significant differences between conditions (Student’s t-test p value < 0.01).

FIGURE 5

Quantification of orobanchol in B. distachyon exudates. Relative quantification of orobanchol in root exudates of WT (Bd21-3), overexpressing (OE-CYP12.20), and mutant (M5374#135) lines after 7 days of phosphorus starvation. AUC, Area under the curve. MRM transitions 321 > 224 and 347 > 97 were used to quantify GR24 and orobanchol signals, respectively. Mean of three biological replicates ± standard deviation. The asterisk indicates significant differences between conditions (p-value < 0.05, Tukey’s test).

FIGURE 6

Overexpression of the BdCYP711A29 gene increases FHB susceptibility following spray inoculation of F. graminearum. (A) Typical FHB symptoms at 7 and 14 days following spray inoculation of whole spikes of the different lines by the F. graminearum FgDON⁺ strain. Bars equal 1 cm. (B) Percentage of spikelets exhibiting FHB symptoms on at least 50% of the florets of the inoculated spikes at 7 and 14 dpi by the PH-1 strain. Mean of four independent biological replicates (n = 52). Different letters indicate significant differences between conditions; upper case and lower case letters indicate the statistical comparison at 7 and 14 dpi, respectively (p-value < 0.05, Tukey’s test).

FIGURE 7

Overexpression of the BdCYP711A29 gene promotes F. graminearum development on B. distachyon spikes following spray inoculation. Relative quantification of fungal DNA by qPCR compared to total DNA 7 and 14 days after spray inoculation of different B. distachyon lines with the FgDON⁺ strain of F. graminearum. Mean of three independent biological replicates ± standard error. Different letters indicate significant differences between conditions (p-value < 0.05, one way ANOVA and pairwise t-tests with Bonferroni correction).

FIGURE 8

Relative defense gene expression levels in B. distachyon lines altered in BdCYP711A29 following F. graminearum infection compared to mock condition. Relative quantification (fold-change, log₂) of the PR9 (Bradi1g39190), (A), BdPAL6 (Bradi3g47110), (B), PR1-5 (Bradi1g57590), (C), and Bradi5gUGT3300 (Bradi5g03300), (D) expression levels in the B. distachyon lines altered in the BdCYP711A29 locus or gene expression 96 hours after F. graminearum infection (FgDON⁺ strain) compared to mock treatment. The relative quantity of gene transcripts compared to mock condition was calculated using the comparative cycle threshold (Ct) method (2^–ΔΔCt). The B. distachyon UBC18 and ACT7 genes (Bradi4g00660 and Bradi4g41850) were used as endogenous controls to normalize the data for differences in input RNA between the different samples. Mean of three independent biological replicates ± standard deviation. The asterisk (A) indicates significant differences (Student’s t-test, p value < 0.05). No difference was shown to be statistically significant under more stringent conditions (Student’s t-test, p value < 0.01).

FIGURE 9

Orobanchol promotes spore germination of F. graminearum. (A) Counting of germinating tubes developed by macroconidia of the F. graminearum FgDON⁺ strain 12h after incubation on agar medium containing different concentrations of orobanchol. 0.01% DMSO was used as a negative control as it corresponds to the dilution solution of orobanchol. Counts were performed on at least 500 macroconidia per condition and repeated in three biological replicates. Different letters indicate statistically significant differences (p-value < 0.01, Tukey’s test). (B) Percentage of macroconidial germination of the F. graminearum FgDON⁺ strain 12 h after incubation in exudates of the different B. distachyon lines. Asterisks indicate significant differences compared to the wild-type line Bd21-3 (*p-value < 0.05, **p-value < 0.001, Student’s t-test).