Ngly, Psta-induced WRKY33 did not bind for the W5 area upstream of CYP82C2 (Fig. 3b, c), a W-box region that does not include any WRKY33-specific motifs and is just upstream of neighboring gene of unknown function At4g31960. WRKY33 reportedly binds to W5 in response to flg2249 and Botrytis cinerea35. By contrast, Psta-induced WRKY33 bound strongly towards the W1 region upstream of CYP71BNATURE COMMUNICATIONS | (2019)ten:3444 | 41467-019-11406-3 | www.nature.comnaturecommunicationswrkCy3 3 Le r-0 Le r-1 D i-Gol-D i-GCamalexinICN + ICA-ME4OH-ICA + 4OH-ICA-MED EXb:WR KYwrky33 DEX:WRKY33-mycD i-G 33 -m yc #2Le r-f lsNATURE COMMUNICATIONS | 41467-019-11406-ARTICLEa70 60 Relative fold expression 50 40 30 B 20 1 10 c Cw rk y3 3 42 4 31 three T WWRKY33 aFOX1 a2.CYP82C2 9h 12 h B a BA2.ab3 A two c B d Cb1.5 a1.0 B 0.five b A 0.42 four 31 3 w rk y3 three w rk y3 3 31 three W WAc CT Twrky33 DEX:WRKY33-flagwrky33 DEX:WRKY33-flagwrky33 DEX:WRKY33-flagbTAG 000 ATG FOX1 TAAcFOX1 promoterCYP82C2 promoter100 W2 W1 ATG 000 000 ATG CYP82C2 TGAFold enrichmentW5 WWW1 0.1 W2 W1 0.1 W5 W4 W3 W2 WWFig. three WRKY33 directly activates 4OH-ICN biosynthetic genes. a qPCR analysis of 4OH-ICN regulatory and biosynthetic genes in seedlings co-treated with 20 M dex and Psta for 9 and 12 h. Different letters denote statistically important differences (P 0.05, one-factor ANOVA coupled to Tukey’s test). Lowercase and uppercase letters denote comparisons across 9 and 12 h time points, respectively. Data represent imply SE of 4, five, four, five (9 h) and six, six, 6, five (12 h) replicates of 15 two seedlings every single. b Schematic of FOX1 and CYP82C2 loci, indicating nt positions of W-box-containing regions (W). c ChIP-PCR analysis of W-box-containing regions upstream of FOX1 and CYP82C2 in wrky33DEX:WRKY33-flag plants co-treated with 20 M dex (D) or mock option (M) and Psta for 9 h. Fenpyroximate Description Dashed line represents the fivefold cutoff among weak and sturdy TF-DNA interactions. Data represent median SE of 4 replicates of 15 two seedlings every single. Supply data of Figs. 3a and 3c are supplied as a Source Data file(Supplementary Fig. 5c ), a W-box region that also does not contain any WRKY33-specific motifs. WRKY33 reportedly binds to a region encompassing W1 in response to flg2231,49 and Psta31. These findings recommend that WRKY33 may perhaps use W-box extended motifs or option specificity motifs to target camalexin biosynthetic genes in response to pathogen effectors, or 4OHICN biosynthetic genes in response to MAMPs or fungal pathogens. CYP82C2 underwent regulatory neofunctionalization. CYP82C2 catalyzes the last step in 4OH-ICN biosynthesis, hydroxylating ICN to type 4OH-ICN23, and most likely was the last 4OH-ICN pathway gene to become recruited to the WRKY33 regulon within a. thaliana. To discover the phylogenetic distribution pattern of 4OH-ICN biosynthesis, we profiled ICN and 4OH-ICN metabolites in close and distant relatives of A. thaliana in response to Psta. Even though ICN biosynthesis was observed across numerous close relatives, 4OH-ICN was only detected within a. thaliana (Fig. 4a and Supplementary Fig. 6a). This result suggests that 4OH-ICNmanifests a species-specific diversification of pathogen-inducible Trp-derived metabolism in the mustard loved ones. Within a. thaliana, CYP82C2 resides in a near-tandem cluster with paralogs CYP82C3 and CYP82C4 (Fig. 4b). We performed phylogenetic and syntenic analyses to determine putative CYP82C2 orthologs in a clade inclusive of ICN-synthesizing species. All identified homologs are syntenic to CYP82C2 or CYP.
