The mechanism underlying strigolactone diversification in sorghum and its role in resistance against the parasitic weed Striga hermonthica

Striga hermonthica (Striga) is a parasitic plant that attaches to the roots of a host plant from which it drains nutrients and water to complete its life cycle. Sorghum is one of the host plants that is greatly affected by Striga infestation which can result in up to 70-100% yield losses. In Chapter 1 of this thesis, I discuss the challenges of controlling Striga infestation, the different Striga resistance mechanisms, with the emphasis on Low Germination Stimulant activity (LGS) and factors affecting this trait, strigolactones. Strigolactones are the key player in inducing the germination of Striga seeds by serving as a signaling molecule for host presence when exuded into the rhizosphere by the host plant. The current knowledge on their evolution, biosynthesis and diversification is extensively discussed. Furthermore, their positive role in the rhizosphere, to induce a symbiotic relationship with arbuscular mycorrhizal (AM) fungi, and in regulating plant architecture is addressed. So far, more than 20 strigolactones have been identified from different plant species. Different blends of strigolactones can be produced by a single plant species. The amount and/or type of strigolactones produced/exuded differs from one plant species to the other. Moreover, the blend can also differ between different cultivars of the same species. In Chapter 2, we investigated the correlation of these differences with Striga resistance in sorghum. We showed that not the level but the type of strigolactone, in a stereospecific manner, determines the resistance of sorghum lines. Sorghum lines with high Striga germination stimulant activity predominantly produce 5-deoxystrigol while the low germination stimulant lines produce orobanchol. Since the purpose of sorghum to exude strigolactones into the rhizosphere is to attract AM-fungi, we looked at how the stereospecific difference of strigolactones affects this symbiotic relationship. I showed that the colonization by three AM fungi species was similar in the high- and low- germination stimulant sorghum lines. Furthermore, we provided evidence for the functional loss of an enzyme annotated as a sulfotransferase (Sobic.005G213600, SbSOT4A) and hypothesized it is responsible for the stereospecific difference of strigolactones between low- and high- germination stimulant sorghum lines. In Chapter 3, I further investigated the role of SbSOT4A in the total strigolactone profile of low-and high- germination stimulant lines. We provided evidence on how a sulfotransferase can possibly be involved in strigolactone biosynthesis. We showed that SbSOT4A is localized in the cytosol, suggesting it sulfates small molecules such as hormones. Using protein modeling and substrate docking, we showed the enzyme has good affinity to C18-hydroxycarlactone and proposed a new model on strigolactone biosynthesis in sorghum. In summary, this model proposes that in high germination stimulant lines, SbSOT4A is intact; after sulfation of C18-hydroxycarlactone it is further oxidized at the C19 position to form a carboxy group and upon the loss of the sulfate group ring closure occurs which results in the formation of 5-deoxystrigol. The loss of SbSOT4A function results in the lack of the sulfated intermediate; rather further oxidation of the C18 hydroxyl group results in a carbonyl; upon ring closure orobanchol will be produced which will lead to low germination stimulant activity towards Striga. We further showed that inhibition of sulfotransferases using Triclosan gave a similar phenotype which can be integrated as a tool to control Striga. These findings emphasize the importance of strigolactone diversification. Therefore, in Chapter 4, we further studied the production of sorgomol, a strigolactone produced by sorghum that can induce a higher level of Striga germination than 5-deoxystrigol. Using Recombinant Inbred Lines (RILs) derived from parents contrasting for the presence of sorgomol, we identified the locus that correlates with sorgomol production in sorghum. With further investigation using RNAseq and bulk segregant analysis, we narrowed down the list of candidate genes and presented evidences on the involvement of two priori candidate genes in sorgomol production in sorghum. I proposed the role of Sobic.008G106200, which encodes a cytochrome P450, in catalyzing the conversion of 5-deoxystrigol to sorgomol while Sobic.001G319900 is regulating the level of production. Strigolactone diversification is achieved by different modifications such as hydroxylation, acetylation, demethylation, esterification, decarboxylation, epoxidation and oxidation. The key players in catalyzing these steps are MAX1, CYP711A homologs that belong to the cytochrome P450 super family of enzymes. Sorghum has four MAX1 homologs and in Chapter 5, we characterized their response to phosphate starvation and their expression pattern in different sorghum parts such as root, lower stem, axillary buds and the flower head. Using phylogenetic tree analysis with functionally characterized MAX1 homologs from different plants, I predicted the part of the biosynthesis that are likely to be catalyzed by these MAX1 homologs from sorghum. I also showed their affinity to use carlactone as a substrate using a transient assay in Nicotiana benthamiana. In Chapter 6, I discuss the main highlights of the thesis, the challenges and future perspectives. Based on the fact that the success of Striga infestation is dependent on the type of strigolactones exuded by sorghum plants, I propose possible tools that can be used to eradicate Striga. I also address the concept of integrated Striga management with the emphasis on the cultural aspects of the farmers based on my personal observation during a field trip that gave me an opportunity to hear the farmer’s side of the Striga control measures.