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Operons are clusters of unrelated genes with related functions that are a feature of prokaryotic genomes. Here, we report on an operon-like gene cluster in the plant Arabidopsis thaliana that is required for triterpene synthesis (the thalianol pathway). The clustered genes are coexpressed, as in bacterial operons. However, despite the resemblance to a bacterial operon, this gene cluster has been assembled from plant genes by gene duplication, neofunctionalization, and genome reorganization, rather than by horizontal gene transfer from bacteria. Furthermore, recent assembly of operon-like gene clusters for triterpene synthesis has occurred independently in divergent plant lineages (Arabidopsis and oat). Thus, selection pressure may act during the formation of certain plant metabolic pathways to drive gene clustering.

In bacteria, genes with related functions often are grouped together in operons and are cotranscribed as a single polycistronic mRNA. In eukaryotes, functionally related genes generally are scattered across the genome. Notable exceptions include gene clusters for catabolic pathways in yeast, synthesis of secondary metabolites in filamentous fungi, and the major histocompatibility complex in animals. Until quite recently it was thought that gene clusters in plants were restricted to tandem duplicates (for example, arrays of leucine-rich repeat disease-resistance genes). However, operon-like clusters of coregulated nonhomologous genes are an emerging theme in plant biology, where they may be involved in the synthesis of certain defense compounds. These clusters are unlikely to have arisen by horizontal gene transfer, and the mechanisms behind their formation are poorly understood. Previously in thale cress (Arabidopsis thaliana) we identified an operon-like gene cluster that is required for the synthesis and modification of the triterpene thalianol. Here we characterize a second operon-like triterpene cluster (the marneral cluster) from A. thaliana, compare the features of these two clusters, and investigate the evolutionary events that have led to cluster formation. We conclude that common mechanisms are likely to underlie the assembly and control of operon-like gene clusters in plants.

Genes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and Arabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume Lotus japonicus. In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in Nicotiana benthamiana. The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments. A novel triterpene structure, dihydrolupeol, was produced by AMY2. A new plant cytochrome P450, CYP71D353, which catalyses the formation of 20-hydroxybetulinic acid in a sequential three-step oxidation of 20-hydroxylupeol was characterized. The genes within the cluster are highly co-expressed during root and nodule development, in hormone-treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed. A tightly co-regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development.

Operons (clusters of co-regulated genes with related functions) are common features of bacterial genomes. More recently, functional gene clustering has been reported in eukaryotes, from yeasts to filamentous fungi, plants, and animals. Gene clusters can consist of paralogous genes that have most likely arisen by gene duplication. However, there are now many examples of eukaryotic gene clusters that contain functionally related but non-homologous genes and that represent functional gene organizations with operon-like features (physical clustering and co-regulation). These include gene clusters for use of different carbon and nitrogen sources in yeasts, for production of antibiotics, toxins, and virulence determinants in filamentous fungi, for production of defense compounds in plants, and for innate and adaptive immunity in animals (the major histocompatibility locus). The aim of this article is to review features of functional gene clusters in prokaryotes and eukaryotes and the significance of clustering for effective function.

• Triterpenes are plant secondary metabolites, derived from the cyclization of 2,3-oxidosqualene by oxidosqualene cyclases (OSCs). Here, we investigated the role of lupeol synthase, encoded by OSC3, and its product, lupeol, in developing roots and nodules of the model legume Lotus japonicus. • The expression patterns of OSC3 in different developmental stages of uninfected roots and in roots infected with Mesorhizobium loti were determined. The tissue specificity of OSC3 expression was analysed by in situ hybridization. Functional analysis, in which transgenic L. japonicus roots silenced for OSC3 were generated, was performed. The absence of lupeol in the silenced plant lines was determined by GC-MS. • The expression of ENOD40, a marker gene for nodule primordia initiation, was increased significantly in the OSC3-silenced plant lines, suggesting that lupeol influences nodule formation. Silenced plants also showed a more rapid nodulation phenotype, consistent with this. Exogenous application of lupeol to M. loti-infected wild-type plants provided further evidence for a negative regulatory effect of lupeol on the expression of ENOD40. • The synthesis of lupeol in L. japonicus roots and nodules can be solely attributed to OSC3. Taken together, our data suggest a role for lupeol biosynthesis in nodule formation through the regulation of ENOD40 gene expression.

Plants produce a plethora of natural products, including many drugs. It has recently emerged that the genes encoding different natural product pathways may be organized as biosynthetic gene clusters in plant genomes, with >30 examples reported so far. Despite superficial similarities with microbes, these clusters have not arisen by horizontal gene transfer, but rather by gene duplication, neofunctionalization, and relocation via unknown mechanisms. Previously we reported that two Arabidopsis thaliana biosynthetic gene clusters are located in regions of the genome that are significantly enriched in transposable elements (TEs). Other plant biosynthetic gene clusters also harbor abundant TEs. TEs can mediate genomic rearrangement by providing homologous sequences that enable illegitimate recombination and gene relocation. Thus, TE-mediated recombination may contribute to plant biosynthetic gene cluster formation. TEs may also facilitate establishment of regulons. However, a systematic analysis of the TEs associated with plant biosynthetic gene clusters has not been carried out. Here we investigate the TEs associated with clustered terpene biosynthetic genes in multiple plant genomes and find evidence to suggest a role for miniature inverted-repeat transposable elements in cluster formation in eudicots. Through investigation of the newly sequenced Amborella trichopoda, Aquilegia coerulea, and Kalanchoe fedtschenkoi genomes, we further show that the “block” mechanism of founding of biosynthetic gene clusters through duplication and diversification of pairs of terpene synthase and cytochrome P450 genes that is prevalent in the eudicots arose around 90–130 million years ago, after the appearance of the basal eudicots and before the emergence of the superrosid clade.

Pathogens have evolved different strategies to overcome the various barriers that they encounter during infection of their hosts. The rice blast fungus Magnaporthe grisea causes one of the most damaging diseases of cultivated rice and has emerged as a paradigm system for investigation of foliar pathogenicity. This fungus undergoes a series of well-defined developmental steps during leaf infection, including the formation of elaborate penetration structures (appressoria). This process has been studied in great detail, and over thirty M. grisea genes that condition leaf infection have been identified. Here we show a new facet of the M. grisea life cycle: this fungus can undergo a different (and previously uncharacterized) set of programmed developmental events that are typical of root-infecting pathogens. We also show that root colonization can lead to systemic invasion and the development of classical disease symptoms on the aerial parts of the plant. Gene-for-gene type specific disease resistance that is effective against rice blast in leaves also operates in roots. These findings have significant implications for fungal development, epidemiology, plant breeding and disease control.

Cloning of OSCs required for triterpene synthesis from legume species that are amenable to molecular genetics will provide tools to address the importance of triterpenes and their derivatives during normal plant growth and development and also in interactions with symbionts and pathogens. Here we report the cloning and characterization of a total of three triterpene synthases from the legume species Medicago truncatula and Lotus japonicus. These include a beta-amyrin synthase from M. truncatula (MtAMY1) and a mixed function triterpene synthase from Lotus japonicus (LjAMY2). A partial cDNA predicted to encode a beta-amyrin synthase (LjAMY1) was also isolated from L. japonicus. The expression patterns of MtAMY1, LjAMY1 and LjAMY2 and of additional triterpene synthases previously characterised from M. truncatula and pea differ in different plant tissues and during nodulation, suggesting that these enzymes may have distinct roles in plant physiology and development.

Osbourn discusses recent research that reports that the regulation of synthesis of a host-selective cyclic peptide toxin by the maize pathogen Cochliobolus carbonum is controlled by a novel pathway-specific transcription factor that may be unique to plant pathogenic fungi. She believes that secondary metabolites may play a far more significant role in determining the outcome of plant-pathogen interactions than has previously been anticipated.