Sinolate (4MI3M)19,20, camalexin21,22, and 4-hydroxyindole-3-carbonylnitrile (4OH-ICN)23–are identified to expand innate immunity in Arabidopsis thaliana. Their biosynthetic pathways share an early step, which can be the conversion of Trp to indole-3-acetaldoxime (IAOx) via the genetically redundant P450 monooxygenases Tebufenozide Epigenetic Reader Domain CYP79B2 and CYP79B3236 (Fig. 1a). The AF647-NHS ester Epigenetics camalexin and 4OHICN pathways also share the conversion of IAOx to indole-3-cyanohydrin (ICY) by partially redundant P450s CYP71A12 and CYP71A1323,27,28 (Fig. 1a). CYP71A13 and CYP71B15PAD3 catalyze additional reactions, leading to camalexin production28,29, whereas the flavin-dependent oxidase FOX1AtBBE3 and P450 CYP82C2 convert ICY to 4OH-ICN (Fig. 1a)23. 4M-I3M is widely distributed across the mustard loved ones (Brassicaceae), whereas camalexin is restricted to the Camelineae tribe of Brassicaceae30. The evolutionary conservation of 4OH-ICN has not however been investigated. The TF WRKY33 regulates the pathogen-inducible biosynthesis of camalexin inside a. thaliana31,32 and its orthologs regulate numerous unrelated specialized metabolites in other flowering plant lineages33. The group I class of WRKYs to which WRKY33 belongs is an ancient clade of regulators; orthologs within the green alga Chlamydomonas reinhardtii may perhaps be ancestral to all greater plant WRKYs33,34. While all WRKY TFs bind towards the W-box core sequence [TTGAC(TC)], WRKY33 preferentially binds Wboxes which might be within 500 nt with the WRKY33-specific motif [(TG) TTGAAT])35.aL-Trp CYP79B2 CYP79B3 IAOxbnmol phytoalexin mg DW 150 one hundred 50 0 50 CYP71A12 CYP83B1 CYP71A13 ICY FOX1 ICN CYP71B15 CYP82COHCamalexin d d f ace n.d. b b ef ae abc bc bICN f df df cd ab n.d ce abnmol phytoalexin mg DW30 be ten 0 n.d. ab aANI SURCYP81F2 nmol phytoalexin mg DWOCH3 GIc S1.5 1.4OH-ICN e be bc cdN SON N N H N H OSO0.five 0.0 n.d. n.d. n.d. n.d.k M oc 22 f2 6 ito sa flg elN Hn.dadn.daPtFig. 1 4OH-ICN is synthesized under ETI-like responses. a Schematic of tryptophan (L-Trp)-derived specialized metabolism in a. thaliana. White arrows denote the presence of extra enzymes. ANI, aci-nitro indole; ICY, indole cyanohydrin. b. LC-DAD-FLD-MS analysis of camalexin (best), ICN (middle), and 4OH-ICN (bottom) in seedlings elicited with indicated MAMPs and bacterial strains for 27 h. Information represent mean SE of four replicates of 15 2 seedlings every. Distinctive letters denote statistically important differences (P 0.05, one-factor ANOVA coupled to Tukey’s test). ICN totals consist in the sum of ICN and methanolic degradation product ICA-ME. 4OH-ICN totals consist of the sum of aqueous and methanolic degradation items 4OHICA-ME and 4OH-ICA, respectivelyNATURE COMMUNICATIONS | (2019)ten:3444 | 41467-019-11406-3 | www.nature.comnaturecommunicationsPsch n ito 30 sa 00 n ta (P vr st R pm ) 1 (P st Ps a ta ) vr R Ps pt 2 ta vr R ps 4 Pm Pm a M a two E Pm S43 26 a av rR pt two o D CCamalexin4OH-ICN4M-I3Mm L chmLNATURE COMMUNICATIONS | 41467-019-11406-ARTICLEHere we show that a current, lineage-specific TE exaptation has resulted in the expansion of a core regulon inside the framework of Arabidopsis Trp-derived defense metabolism. Especially, the LINE retrotransposon EPCOT3 has retroduplicated from a WRKY33-TFBS-carrying progenitor and inserted upstream of the newly duplicated gene CYP82C2. Subsequent chromatin remodeling inside a. thaliana has led EPCOT3 to come to be a bona fide enhancer with demonstrated biochemical, regulatory, physiological, and fitness-promoting qualities by way.
