Investigation on the molecular mechanisms of sesquiterpenoid phytoalexin metabolism in Solanum species

概要

Plants are constantly confronted with a large number of pathogens, such as fungi, oomycetes, bacteria and viruses. In order to cope with these threats, plants have evolved various defense strategies. One such strategy is the hypersensitive response, where plant cells deliberately commit cell-death, in order to deprive invading pathogens of nutrients. While this strategy is effective against biotrophic pathogens, which require living plant cells to feed on, this measure is ineffective against hemibiotrophic and necrotrophic pathogens, which can feed on dead plant tissue. In such cases, the rapid production of phytoalexins, a generic term for a wide range of toxic compounds, can represent an effective defense measure. These phytoalexins accumulate near the site of infection where they ideally kill, or at least slow down an invading pathogen. These toxic compounds vary between species and fall into different chemical classes, such as flavonoids, indoles, terpenoids and others.

Solanaceae, the plant family that encompasses the important nutritional plants potato, tomato, eggplant and pepper, produces a substantial number of phytoalexins that belong to the class of sesquiterpenes, such as capsidiol, rishitin, lubimin, oxylubimin, solavetivone, debneyol and others. Not all of these phytoalexins have been scrutinized extensively, but at least some of these compounds were found to damage the plant's own cells as well. The use of these phytoalexins is therefore a double-edged sword, and in the case of potatoes, the main phytoalexin rishitin is therefore eventually detoxified to a less harmful compound once the pathogen threat has subsided.

Although many solanaceous phytoalexins have already been discovered decades ago, our understanding of how plants produce, regulate and detoxify these compounds lacks far behind. With exception to capsidiol and solavetivone, the genes required for the biosynthesis of phytoalexins have not yet been discovered. Discovery of these genes could be valuable, since this knowledge could eventually be used for efficient breeding of more resilient cultivars of important crops such as potato and tomato. To this end, this thesis aimed to contribute to advancing our knowledge of the Solanaceae phytoalexin pathway.

In order to do so, various potato tuber genes were cloned and tested for their involvement in the metabolic pathway of potatoes’ main phytoalexin rishitin, which is thought to derive via the production of solavetivone, lubimin and oxylubimin. A selection of candidate genes, was evaluated by expression in yeast, E. coli and in leaves of N. benthamiana, which produces different sesquiterpenoid phytoalexins than potato. Using these approaches, a novel gene was identified which was named sesquiterpenoid phytoalexin dihydroxylase (SPH). SPH was found to encode for a protein, which detoxifies rishitin via hydroxylation to 13-hydroxrishitin, also referred to as rishitin-M1. Interestingly, this protein was found to metabolize a wide range of other phytoalexins as well, including solavetivone, lubimin, oxylubimin and even capsidiol-type phytoalexins that do not naturally occur in potato. This flexibility in substrate choices and the conservation of closely related homologs in other Solanaceae indicates that SPH presumably plays an important role in phytoalexin detoxification, even in species that do not produce rishitin.

While the genes responsible for the synthesis of potato phytoalexins were ultimately not discovered, this work has laid the groundwork which will improve further testing of phytoalexin synthesis genes. This includes not only methodological improvements, but also results from a large scale RNAseq analysis of elicited potato tubers, which were analyzed using weighted gene co-expression analysis. These results identified gene clusters of distinct functions that correlate to phytoalexin accumulation and thereby substantially help to narrow down the list of genes potentially involved in phytoalexin synthesis.