TRANSCRIPTION ACTIVATOR-LIKE EFFECTOR NUCLEASE-Mediated Generation and Metabolic Analysis of Camalexin-Deficient cyp71a12 cyp71a13 Double Knockout Lines

Abstract

In Arabidopsis (Arabidopsis thaliana), a number of defense-related metabolites are synthesized via indole-3-acetonitrile (IAN), including camalexin and indole-3-carboxylic acid (ICOOH) derivatives. Cytochrome P450 71A13 (CYP71A13) is a key enzyme for camalexin biosynthesis and catalyzes the conversion of indole-3-acetaldoxime (IAOx) to IAN. The CYP71A13 gene is located in tandem with its close homolog CYP71A12, also encoding an IAOx dehydratase. However, for CYP71A12, indole-3-carbaldehyde and cyanide were identified as major reaction products. To clarify CYP71A12 function in vivo and to better understand IAN metabolism, we generated two cyp71a12 cyp71a13 double knockout mutant lines. CYP71A12-specific transcription activator-like effector nucleases were introduced into the cyp71a13 background, and very efficient somatic mutagenesis was achieved. We observed stable transmission of the cyp71a12 mutation to the following generations, which is a major challenge for targeted mutagenesis in Arabidopsis. In contrast to cyp71a13 plants, in which camalexin accumulation is partially reduced, double mutants synthesized only traces of camalexin, demonstrating that CYP71A12 contributes to camalexin biosynthesis in leaf tissue. A major role of CYP71A12 was identified for the inducible biosynthesis of ICOOH. Specifically, the ICOOH methyl ester was reduced to 12% of the wild-type level in AgNO 3 -challenged cyp71a12 leaves. In contrast, indole-3-carbaldehyde derivatives apparently are synthesized via alternative pathways, such as the degradation of indole glucosinolates. Based on these results, we present a model for this surprisingly complex metabolic network with multiple IAN sources and channeling of IAOx-derived IAN into camalexin biosynthesis. In conclusion, transcription activator-like effector nuclease-mediated mutation is a powerful tool for functional analysis of tandem genes in secondary metabolism.

Novel triterpene oxidizing activity of Arabidopsis thaliana CYP716A subfamily enzymes

“…In a previous study, Arabidopsis cyp71a12 cyp71a13 double knockout plants were generated using genome-editing tools. This finding supports that artificial nucleases, such as transcription activator-like effector nuclease (TALEN), will be useful in the generation of multigene knockout lines of linked genes [15]. The generation of double knockout plants using genomeediting tools and analysis of these plants are ongoing, and will be the subject of future research efforts in our lab.…”

The role of CYP71A12 monooxygenase in pathogen‐triggered tryptophan metabolism and Arabidopsis immunity

“…Metabolite profiling of cyp 7 1A12 knockout plants indicates the crucial contribution of CYP71A12 monooxygenase to the AgNO 3 ‐, UV‐ and pathogen‐triggered accumulation of ICAs (Müller et al ., ; Rajniak et al ., ). Analysis of the double cyp71A12/A13 mutant suggested that CYP71A13 provides an additional minor contribution to the UV‐induced accumulation of these compounds (Müller et al ., ). To assess the role of both monooxygenases in the pathogen‐triggered biosynthesis of ICA derivatives, we inoculated leaves of cyp71A12 , cyp7A13 and cyp71A12/A13 mutant lines together with wild‐type (WT) plants (Col‐0 ecotype) with a spore suspension of the host‐adapted pathogenic strain of the necrotrophic fungus Plectosphaerella cucumerina BMM ( PcBMM ), which is able to colonize Col‐0 plants (Ton & Mauch‐Mani, ).…”

“…The TALEN‐generated cyp71A12 cyp71A13 was described by Müller et al . (). The myb34/51/122 triple mutant line was reported by Frerigmann et al .…”

“…The TALEN mutation in the cyp71A12 cyp71A13 double mutant line was confirmed according to Müller et al . (). Mutations in the myb34/51/122 triple mutant line were confirmed according to Frerigmann et al .…”

“…) (Böttcher et al ., ). Accumulation of ICAs in single and double cyp71a12 and cyp71a13 mutant lines suggest that biosynthesis of these compounds by Arabidopsis in response to AgNO 3 toxicity depends predominantly on CYP71A12 monooxygenase, whereas specific ICAs are produced in response to UV treatment, with the contribution of CYP71A13 (Müller et al ., ). The cyp71a12 mutant is also reported to accumulate reduced amounts of ICAs when infected with A. brassicicola or Botrytis cinerea (Rajniak et al ., ).…”