Of WRKY33binding and pathogen-responsive CYP82C2 transcription, 4OHICN biosynthesis, and antibacterial defense. Benefits 4OH-ICN requires ETI-like responses. To identify the major Trp-derived specialized metabolites synthesized in ETI in a. thaliana, we compared host transcriptional and metabolic responses towards the PTI-eliciting bacterial MAMPs flg22, elf26, and fungal MAMP chitosan; the PTIETS-eliciting pathogens Pseudomonas syringae pv. tomato DC3000 (Pto DC3000 or Pst); P. syringae pv. maculicola ES4326 (Pma); plus the ETI-eliciting pathogens Pst avrRpm1 (Psta), Pst avrRpt2, Pst avrRps4, Pma M2, and Pma avrRpt2 under similar conditions as these of earlier studies19,36. Psm M2 is an ETI-eliciting strain from which the avrRpm1 gene was originally isolated37. Each flg22 and Psta induced genes involved in camalexin, 4OH-ICN, and 4MI3M biosynthesis, with camalexin and 4OH-ICN biosynthetic genes obtaining a higher degree of induction than those of 4M-I3M in Psta-inoculated plants36 (Supplementary Table 1). Alternatively, metabolite responses amongst PTI and ETI differed qualitatively. 4M-I3M and its quick precursor 4-hydroxy-I3M (4OH-I3M) have been present in uninfected plants and accumulated to modest levels in the expense of parent metabolite I3M in flg22and Psta-inoculated plants19 (Supplementary Fig. 1a). By comparison, camalexin, ICN, and 4OH-ICN had been absent in uninfected plants and accumulated to higher levels upon inoculation with ETI-inducing pathogens (Fig. 1b and Supplementary Fig. 1b). Additionally, camalexin, ICN, and 4OH-ICN metabolism was greatly diminished, and 4M-I3M, 4OH-I3M, and I3M levels have been mainly unchanged inside the rpm1 mutant (Supplementary Fig. 1), which can be impaired in ETI recognition of Psta40. By contrast, camalexin and ICN have been largely at ��-Aminopropionitrile Technical Information low-to-undetectable levels in plants treated with saturating concentrations with the bacterial MAMPs flg22 and elf2638,39 and PTIETS-eliciting pathogens, with 4OH-ICN not detected in most cases (Fig. 1b). A Histamine dihydrochloride Endogenous Metabolite single exception was the fungal MAMP chitosan. Chitosan (150 g mL) induced high levels of camalexin and detectable levels of ICN (Fig. 1b), consistent with preceding observations of camalexin biosynthetic gene upregulation41. Larger chitosan concentrations ( 200 gmL) have been shown to induce HR-like cell death in Arabidopsis42, a phenomenon usually observed for ETI16. To our surprise, 300 gmL chitosan also induced detectable levels of 4OH-ICN (Fig. 1b). These benefits suggest that 4OH-I3M, 4M-I3M, camalexin, and ICN are synthesized in response to various PTI elicitors, whereas 4OH-ICN biosynthesis is precise to ETI-like responses. WRKY33 is essential to activate 4OH-ICN in response to Psta. 4OH-ICN biosynthetic genes are very co-expressed with each other23 and with camalexin biosynthetic genes (Supplementary Table 2), that are in the WRKY33 regulon31,43. To decide whether 4OH-ICN biosynthetic genes are also within the WRKY33 regulon, we compared camalexin, ICN, and 4OH-ICN levels involving wild-type and a wrky33 loss-of-function mutant that encodes two differently truncated proteins44 (Fig. 2a). Constant using a earlier report31, wrky33 was impaired in camalexinbiosynthesis in response to Psta and Pst avrRps4 (Fig. 2b and Supplementary Fig. 2a). The wrky33 mutant was similarly impaired in 4OH-ICN biosynthesis (Fig. 2b and Supplementary Fig. 2a). These results indicate that WRKY33 is expected for camalexin and 4OH-ICN biosynthesis in response to several ETI elicitors. To confirm.