Fig. 2: The complete 12-gene avenacin biosynthetic cluster and full pathway reconstitution by transient expression in Nicotiana benthamiana.

a The region of the A. strigosa genome encompassing the avenacin biosynthetic gene cluster. The genes shown in colour are the nine previously characterised avenacin pathway genes^{4,5,6,7,8,9,10,20,21} along with two previously uncharacterised CYP genes (asterisked), shown in this work to catalyse the two missing pathway steps (see Supplementary Table 7 for more information about all genes in this region). UGT74H7, which encodes a previously characterised sugar transferase related to UGT74H5 but with low activity towards avenacin acyl precursors¹⁰, is also indicated. b The complete pathway for the biosynthesis of avenacin A-1, including the newly validated steps catalysed by CYP94D65 and CYP72A476 (asterisked) (Supplementary Figs. 9 and 10; Supplementary Table 8). c Reconstitution of the avenacin pathway in N. benthamiana by transient expression. Full pathway: co-expression of GoldenGate constructs EC80344 (bAS1/Sad1 + CYP51H10/Sad2 + CYP72A475/Sad6 + CYP94D65 + CYP72A476), EC80345 (AAT1 + UGT91G16 + TG1 + P19), EC80379 (MT1/Sad9 + UGT74H5/Sad10 + SCPL1/Sad7). No acyl group control: co-expression of EC80344 and EC80345 only. Leaves were harvested 5 days after agro-infiltration, freeze-dried, and extracts analysed by high-performance liquid chromatography. Avenacin A-1 has strong autofluorescence under ultra-violet illumination. A peak with the same retention time as the avenacin A-1 standard was detected in extracts from leaves co-expressing all of the pathway genes but not in extracts from no acyl group control leaves. Mass spectra in both positive and negative modes confirmed that this peak had the same mass as avenacin A-1 (Supplementary Fig 11). Source data underlying Fig. 2a are provided as a Source Data file.