Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells.

Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol.

The reduced mycorrhizal colonisation (rmc) mutation of tomato disrupts five gene sequences including the CYCLOPS/IPD3 homologue.

Arbuscular mycorrhizal (AM) symbiosis in vascular plant roots is an ancient mutualistic interaction that evolved with land plants. More recently evolved root mutualisms have recruited components of the AM signalling pathway as identified with molecular approaches in model legume research. Earlier we reported that the reduced mycorrhizal colonisation (rmc) mutation of tomato mapped to chromosome 8. Here we report additional functional characterisation of the rmc mutation using genotype grafts and proteomic and transcriptomic analyses. Our results led to identification of the precise genome location of the Rmc locus from which we identified the mutation by sequencing. The rmc phenotype results from a deletion that disrupts five predicted gene sequences, one of which has close sequence match to the CYCLOPS/IPD3 gene identified in legumes as an essential intracellular regulator of both AM and rhizobial symbioses. Identification of two other genes not located at the rmc locus but with altered expression in the rmc genotype is also described. Possible roles of the other four disrupted genes in the deleted region are discussed. Our results support the identification of CYCLOPS/IPD3 in legumes and rice as a key gene required for AM symbiosis. The extensive characterisation of rmc in comparison with its 'parent' 76R, which has a normal mycorrhizal phenotype, has validated these lines as an important comparative model for glasshouse and field studies of AM and non-mycorrhizal plants with respect to plant competition and microbial interactions with vascular plant roots.