Modularity of plant metabolic gene clusters: a trio of linked genes that are collectively required for acylation of triterpenes in oat.

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis.

Induction of chlorosis, ROs generation and cell death by a toxin isolated from Pyricularia oryzae.

The ethyl acetate extract of the conidia germination fluid from an Avena isolate (Br58) of Pyricularia oryzae had chlorosis-inducing activity on oat leaf segments. The same activity was also present in the acetone extract of an oatmeal agar culture of Br58. Fungal cultures were used for a large-scale preparation. A series of acetone and ethyl acetate extraction monitored by chromatography was used to isolate an active fraction. The active principle was purified by HPLC. We show by NMR and LC/MS that the toxin was an oxidized C18 unsaturated fatty acid named Mag-toxin. Mag-toxin induced chlorosis on oat leaf segments incubated in the light but not in the dark. Reactive oxygen species (ROS) and cell death were induced by Mag-toxin in oat cells. The sub-cellular localization of ROS generation induced by the toxin treatment was correlated with the location of mitochondria. Interestingly, the induction of ROS generation and cell death by Mag-toxin was light-independent.

Pyrichalasin H production and pathogenicity of Digitaria-specific isolates of Pyricularia grisea.

SUMMARY Culture filtrates from 72 isolates of Pyricularia, grouped into 13 rDNA types, were analysed via HPLC. Of these isolates, 31 (r9 DNA type) from crabgrass (Digitaria sanguinalis), one (r9 DNA type) from pangolagrass (Digitaria smutsii) and six (r8 DNA type) from Digitaria horizontalis produced 20-280 microg pyrichalasin H per millilitre of culture. These same isolates were pathogenic on five Digitaria species. Interestingly, two isolates, KM-1 and Br 29, which were originally isolated from Digitaria plants, did not produce pyrichalasin H, nor caused blast lesion on Digitaria plants. Because these two isolates were identified as Digitaria pathogens by PCR analysis using Digitaria-specific primers, they are likely to be mutants lacking pyrichalasin H production. Isolates that belonged to the remaining 11 rDNA types did not produce pyrichalasin H and were avirulent to Digitaria plants. Therefore, the virulence of Pyricularia on Digitaria plants correlates with pyrichalasin H production. Pyrichalasin H was also present in spore germination fluid of a crabgrass isolate (IBDS 5-1-1), but not in that of isolates from rice, foxtail millet, finger millet, common millet and wheat. In addition, pyrichalasin H was detected in host leaves infected with IBDS 5-1-1, but not in leaves from other plants infected with compatible Pyricularia isolates. Pretreatment of leaf sheaths of crabgrass with 3 microg/mL pyrichalasin H led to the penetration and colonization by non-host isolates. Overall, these results indicate that production of pyrichalasin H is responsible for the genus-specific pathogenicity of Digitaria isolates.