Non-random gene organization significantly impacts genome evolution in eukaryotes. This study focuses on the avenacin cluster in oat, responsible for producing avenacins—antifungal metabolites crucial for defending against soil-borne diseases like take-all, a significant threat to wheat yield. Previous research identified approximately 100 avenacin-deficient oat mutants, suggesting gene clustering. Ten avenacin pathway genes have been characterized, five of which reside on a ~300kb BAC contig, while the remaining five are genetically linked but their physical proximity remains unclear. Elucidating the avenacin cluster's organization and evolution is vital for understanding metabolic diversity in grasses and offers potential for engineering enhanced disease resistance in other cereals. Biosynthetic gene clusters for various natural products have been identified across various plant species, raising questions regarding their formation mechanisms and the significance of gene clustering. This research utilizes a genomics-driven approach to study the avenacin cluster's origin and structure, seeking to uncover the mechanisms driving genome architecture and adaptive evolution in plants.

Previous work has established the importance of avenacins in oat disease resistance and hinted at the existence of a gene cluster involved in their biosynthesis. Studies have identified and characterized several genes within this cluster, including those encoding enzymes for triterpene biosynthesis, acylation, and glycosylation. However, the complete extent of the cluster, the exact order of genes, and its evolutionary origin remained unknown. Furthermore, the broader context of biosynthetic gene cluster formation and evolution in plants has been a subject of considerable interest, with ongoing investigations into mechanisms such as gene duplication, horizontal gene transfer, and the role of specific genomic regions.

The researchers began by sequencing the genome of *Avena strigosa* accession S75, using a combination of Oxford Nanopore PromethION and Illumina sequencing technologies, followed by hybrid assembly with optical mapping and Hi-C data. This resulted in a high-quality chromosome-scale assembly. RNA-seq data from various tissues were used for gene annotation. The complete avenacin cluster was identified within the genome assembly, revealing the physical locations and relative order of all 12 genes involved. Two previously uncharacterized cytochrome P450 (CYP) enzymes, CYP94D65 and CYP72A476, were identified as candidates for catalyzing the remaining unknown steps in the pathway. The functions of these enzymes were confirmed through *Agrobacterium*-mediated transient expression in *Nicotiana benthamiana*, where co-expression of the genes with other pathway enzymes led to the production of avenacin A-1. The chromosomal location and organization of the cluster were further investigated through karyotyping and fluorescence in situ hybridization (FISH), using probes for genes at either end of the cluster. The evolutionary origin of the cluster was investigated by comparative genomics analysis with other cereal and grass species, including collinearity and sequence similarity comparisons. The study also utilized plantiSMASH, an algorithm designed to identify biosynthetic gene clusters in plant genomes, to assess the density of clusters in the *A. strigosa* genome and compare it to other cereal and grass species. Finally, they analyzed the avenacin cluster in other oat species (*Avena atlantica* and *Avena eriantha*) to understand the cluster's evolutionary dynamics. Metabolite analysis confirmed the production of avenacins in the leaves of *A. eriantha*, a finding not previously observed in other oat species.

The complete 12-gene avenacin biosynthetic gene cluster was identified in *A. strigosa*. The cluster is located in a subtelomeric region of chromosome 1. The gene order within the cluster shows approximate colinearity with the biosynthetic pathway. The two previously unknown pathway steps were identified and characterized as being catalyzed by CYP94D65 and CYP72A476. The entire avenacin A-1 pathway was successfully reconstituted in *N. benthamiana* through transient expression. Comparative genomics revealed that the avenacin cluster likely formed de novo since the divergence of oats from other cereals and grasses. The subtelomeric location of the cluster appears to be a 'hotspot' for gene cluster formation, with higher cluster density observed in this region compared to other regions of the *A. strigosa* genome and other cereal genomes analyzed. The avenacin cluster is significantly more complex than other triterpene biosynthetic gene clusters identified. *A. eriantha*, unlike other A-genome oat species, produces avenacins in leaves, demonstrating broader roles for avenacins in pathogen defense.

This study provides compelling evidence for the de novo formation of a complex biosynthetic gene cluster in a subtelomeric region of the oat genome. The approximate colinearity of the gene order with the biosynthetic pathway suggests a potential mechanism for regulating sequential gene expression. The subtelomeric location may offer advantages, including mitigation against self-poisoning by toxic pathway intermediates through telomeric deletions. The high cluster density in the subtelomeric region of chromosome 1 supports the notion of these regions as 'hotspots' for gene cluster formation. The evolutionary dynamics observed in other oat species highlight the ongoing evolution and plasticity of the avenacin cluster. The finding that *A. eriantha* produces avenacins in leaves, unlike other A-genome oats, broadens the understanding of the avenacin pathway's function. The findings pave the way for engineering enhanced disease resistance in other cereals like wheat.

This research comprehensively characterized the avenacin biosynthetic gene cluster in oat, identifying the last two missing enzymes and reconstituting the entire pathway. The de novo formation of this cluster in a subtelomeric region highlights the remarkable plasticity of plant genomes and provides insights into the evolution of complex metabolic pathways. Future research could explore the regulatory mechanisms controlling gene expression within this cluster and investigate the potential for transferring this pathway to other cereals to enhance disease resistance.

While the study provides a detailed analysis of the avenacin cluster in *A. strigosa*, the findings may not be directly generalizable to all oat species or other cereals. Further investigation is needed to fully understand the regulatory mechanisms controlling gene expression within the cluster and the precise role of the subtelomeric location in cluster formation. The study primarily focused on avenacin A-1; additional work is required to investigate the biosynthesis of other avenacin variants.