Discovery and functional analysis of monoterpenoid indole alkaloid pathways in plants

Numerous difficulties have been associated with forward genetic approaches to identify, and functionally characterize genes involved in the biosynthesis, regulation, and transport of monoterpenoid indole alkaloids (MIAs). While the identification of certain classes of genes associated with MIA pathways has facilitated the use of homology-based approaches to clone other genes catalyzing similar reactions in other parts of the pathway, this has not greatly speeded up the pace of gene discovery for the diversity of reactions involved. Compounding this problem has been the lack of knowledge or even availability of certain MIA intermediates that would be required to establish a novel enzyme reaction to functionally identify a biosynthetic step or the candidate gene product involved. The advent of inexpensive sequencing technologies for transcriptome and genome sequencing, combined with proteomics and metabolomics, is now revolutionizing the pace of gene discovery associated with MIA pathways and their regulation. The discovery process uses large databases of genes, proteins, and metabolites from an ever-expanding list of nonmodel plant species competent to produce and accumulate MIAs. Comparative bioinformatics between species, together with gene expression analysis of particular tissue, cell, and developmental types, is helping to identify target genes that can then be investigated for their possible role in an MIA pathway by virus-induced gene silencing. Successful silencing not only confirms the involvement of the candidate gene but also allows identification of the pathway intermediate involved. In many circumstances, the pathway intermediate can be isolated for use as a substrate in order to confirm gene function in heterologous bacterial, yeast, or plant expression systems.