A novel strigolactone receptor antagonist provides insights into the structural inhibition, conditioning, and germination of the crop parasite Striga.

Crop parasites of the Striga genera are a major biological deterrent to food security in Africa and are one of the largest obstacles to poverty alleviation on the continent. Striga seeds germinate by sensing small-molecule hormones, strigolactones (SLs), that emanate from host roots. Although SL receptors (Striga hermonthica HYPOSENSITIVE TO LIGHT [ShHTL]) have been identified, discerning their function has been difficult because these parasites cannot be easily grown under laboratory conditions. Moreover, many Striga species are obligate outcrossers that are not transformable, hence not amenable to genetic analysis. By combining phenotypic screening with ShHTL structural information and hybrid drug discovery methods, we discovered a potent SL perception inhibitor for Striga, dormirazine (DOZ). Structural analysis of this piperazine-based antagonist reveals a novel binding mechanism, distinct from that of known SLs, blocking access of the hormone to its receptor. Furthermore, DOZ reduces the flexibility of protein-protein interaction domains important for receptor signaling to downstream partners. In planta, we show, via temporal additions of DOZ, that SL receptors are required at a specific time during seed conditioning. This conditioning is essential to prime seed germination at the right time; thus, this SL-sensitive stage appears to be critical for adequate receptor signaling. Aside from uncovering a function for ShHTL during seed conditioning, these results suggest that future Ag-Biotech Solutions to Striga infestations will need to carefully time the application of antagonists to exploit receptor availability and outcompete natural SLs, critical elements for successful parasitic plant invasions.

Rational design of Striga hermonthica-specific seed germination inhibitors.

The obligate hemiparasitic weed Striga hermonthica grows on cereal roots and presents a severe threat to global food security by causing enormous yield losses, particularly in sub-Saharan Africa. The rapidly increasing Striga seed bank in infested soils provides a major obstacle in controlling this weed. Striga seeds require host-derived strigolactones (SLs) for germination, and corresponding antagonists could be used as germination inhibitors. Recently, we demonstrated that the common detergent Triton X-100 is a specific inhibitor of Striga seed germination by binding noncovalently to its receptor, S. hermonthica HYPO-SENSITIVE TO LIGHT 7 (ShHTL7), without blocking the rice (Oryza sativa) SL receptor DWARF14 (OsD14). Moreover, triazole ureas, the potent covalently binding antagonists of rice SL perception with much higher activity toward OsD14, showed inhibition of Striga but were less specific. Considering that Triton X-100 is not suitable for field application and by combining structural elements of Triton and triazole urea, we developed two hybrid compounds, KK023-N1 and KK023-N2, as potential Striga-specific germination inhibitors. Both compounds blocked the hydrolysis activity of ShHTL7 but did not affect that of OsD14. Binding of KK023-N1 diminished ShHTL7 interaction with S. hermonthica MORE AXILLARY BRANCHING 2, a major component in SL signal transduction, and increased ShHTL7 thermal specificity. Docking studies indicate that KK023-N1 binding is not covalent but is caused by hydrophobic interactions. Finally, in vitro and greenhouse tests revealed specific inhibition of Striga seed germination, which led to a 38% reduction in Striga infestation in pot experiments. These findings reveal that KK023-N1 is a potential candidate for combating Striga and a promising basis for rational design and development of further Striga-specific herbicides.

Strigolactone Analogues Derived from Dihydroflavonoids as Potent Seed Germinators for the Broomrapes.

The broomrapes (Orobanche and Phelipanche spp.) and witchweeds (Striga spp.) are a class of parasitic weeds, which are distributed widely in the tropical, subtropical, and temperate areas of the globe. Since they have completely consistent lifecycles with the host plants, it is difficult to control them selectively through using the conventional herbicides. Inducing suicidal germination of these weed seeds by small molecular signaling agents proved to be a promising strategy for the management of parasitic weeds. As a class of naturally occurring terpenoid metabolites, strigolactones (SLs) show significant biological activities including stimulation germination of weed seeds, inhibition of shoot-branching, and so on. However, the widespread application of these natural SLs is greatly limited by their extremely low natural abundance and complex molecular structures. Design and synthesis of the simplified analogues as natural SLs alternatives provide a viable avenue for the efficient control of these parasitic weeds. We herein disclose the development of a novel class of SLs analogues derived from dihydroflavonoids as potent seed germinators of parasitic weeds. It was shown that one of them displayed a higher potential toward the seed germination of the broomrapes than the positive control GR24. The structure-activity relationship of these SLs analogues was further validated on the basis of the binding affinity experiment to strigolactone receptor protein HTL7 by using a YLG fluorescent probe method.

Efficient Mimics for Elucidating Zaxinone Biology and Promoting Agricultural Applications.

Zaxinone is an apocarotenoid regulatory metabolite required for normal rice growth and development. In addition, zaxinone has a large application potential in agriculture, due to its growth-promoting activity and capability to alleviate infestation by the root parasitic plant Striga through decreasing strigolactone (SL) production. However, zaxinone is poorly accessible to the scientific community because of its laborious organic synthesis that impedes its further investigation and utilization. In this study, we developed easy-to-synthesize and highly efficient mimics of zaxinone (MiZax). We performed a structure-activity relationship study using a series of apocarotenoids distinguished from zaxinone by different structural features. Using the obtained results, we designed several phenyl-based compounds synthesized with a high-yield through a simple method. Activity tests showed that MiZax3 and MiZax5 exert zaxinone activity in rescuing root growth of a zaxinone-deficient rice mutant, promoting growth, and reducing SL content in roots and root exudates of wild-type plants. Moreover, these compounds were at least as efficient as zaxinone in suppressing transcript level of SL biosynthesis genes and in alleviating Striga infestation under greenhouse conditions, and did not negatively impact mycorrhization. Taken together, MiZax are a promising tool for elucidating zaxinone biology and investigating rice development, and suitable candidates for combating Striga and increasing crop growth.

Molecular basis of strigolactone perception in root-parasitic plants: aiming to control its germination with strigolactone agonists/antagonists.

The genus Striga, also called "witchweed", is a member of the family Orobanchaceae, which is a major family of root-parasitic plants. Striga can lead to the formation of seed stocks in the soil and to explosive expansion with enormous seed production and stability once the crops they parasitize are cultivated. Understanding the molecular mechanism underlying the communication between Striga and their host plants through natural seed germination stimulants, "strigolactones (SLs)", is required to develop the technology for Striga control. This review outlines recent findings on the SL perception mechanism, which have been accumulated in Striga hermonthica by the similarity of the protein components that regulate SL signaling in nonparasitic model plants, including Arabidopsis and rice. HTL/KAI2 homologs were identified as SL receptors in the process of Striga seed germination. Recently, this molecular basis has further promoted the development of various types of SL agonists/antagonists as seed germination stimulants or inhibitors. Such chemical compounds are also useful to elucidate the dynamic behavior of SL receptors and the regulation of SL signaling.

Hybrid-type strigolactone analogues derived from auxins.

BACKGROUND: Strigolactones (SLs) have a vast number of ecological implications because of the broad spectrum of their biological activities. Unfortunately, the limited availability of SLs restricts their applicability for the benefit of humanity and renders synthesis the only option for their production. However, the structural complexity of SLs impedes their economical synthesis, which is unfeasible on a large scale. Synthesis of SL analogues and mimics with a simpler structure, but with retention of bioactivity, is the solution to this problem. RESULTS: Here, we present eight new hybrid-type SL analogues derived from auxin, synthesized via coupling of auxin ester [ethyl 2-(1H-indol-3-yl)acetate] and of ethyl 2-phenylacetate with four D-rings (mono-, two di- and trimethylated). The new hybrid-type SL analogues were bioassayed to assess the germination activity of seeds of the parasitic weeds Striga hermonthica, Orobanche minor and Phelipanche ramosa using the classical method of counting germinated seeds and a colorimetric method. The bioassays revealed that analogues with a natural monomethylated D-ring had appreciable to good activity towards the three species and were the most active derivatives. By contrast, derivatives with the trimethylated D-ring showed no activity. The dimethylated derivatives (2,4-dimethyl and 3,4-dimethyl) were slightly active, especially towards P. ramosa. CONCLUSIONS: New hybrid-type analogues derived from auxins have been prepared. These analogues may be attractive as potential suicidal germination agents for parasitic weed control because of their ease of preparation and relevant bioactivity. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

How does Striga hermonthica Bewitch its hosts?

The common name witchweed synonymous with the Latin name Striga befits the bewitching effects, viz wilting and chlorosis, the parasite inflicts on its hosts long before it emerges and becomes visible above the ground. However, interactions in the rhizosphere between host roots and Striga seedlings are concealed and inscrutable. In vitro experiments revealed that abscisic acid was produced by S. hermonthica seedlings and a considerable portion of the phytohormone was exuded. The phytohormone in the rhizosphere could, at least in part, contribute to the bewitching effects, disrupt host immunity and promote commencement of parasitism.

Aberrant protein phosphatase 2C leads to abscisic acid insensitivity and high transpiration in parasitic Striga.

Striga parasitizes major crops in arid regions, depriving the host crop of nutrients through the transpiration stream and causing vast agricultural damage. Here, we report on the mechanism underlying how Striga maintains high transpiration under drought conditions. We found that Striga did not respond to abscisic acid, the phytohormone responsible for controlling stomatal closure. Protein phosphatase 2C of Striga (ShPP2C1) is not regulated by abscisic acid receptors, and this feature is attributable to specific mutations in its amino acid sequence. Moreover, Arabidopsis transformed with ShPP2C1 showed an abscisic acid-insensitive phenotype, indicating that ShPP2C1 functions as a dominant negative regulator of abscisic acid signal transduction. These findings suggest that ShPP2C1 interrupts abscisic acid signalling in Striga, resulting in high transpiration and subsequent efficient absorption of host nutrients under drought conditions.

A femtomolar-range suicide germination stimulant for the parasitic plant Striga hermonthica.

The parasitic plant Striga hermonthica has been causing devastating damage to the crop production in Africa. Because Striga requires host-generated strigolactones to germinate, the identification of selective and potent strigolactone agonists could help control these noxious weeds. We developed a selective agonist, sphynolactone-7, a hybrid molecule originated from chemical screening, that contains two functional modules derived from a synthetic scaffold and a core component of strigolactones. Cooperative action of these modules in the activation of a high-affinity strigolactone receptor ShHTL7 allows sphynolactone-7 to provoke Striga germination with potency in the femtomolar range. We demonstrate that sphynolactone-7 is effective for reducing Striga parasitism without impinging on host strigolactone-related processes.

Structural basis for specific inhibition of the highly sensitive ShHTL7 receptor.

Striga hermonthica is a root parasitic plant that infests cereals, decimating yields, particularly in sub-Saharan Africa. For germination, Striga seeds require host-released strigolactones that are perceived by the family of HYPOSENSITIVE to LIGHT (ShHTL) receptors. Inhibiting seed germination would thus be a promising approach for combating Striga However, there are currently no strigolactone antagonists that specifically block ShHTLs and do not bind to DWARF14, the homologous strigolactone receptor of the host. Here, we show that the octyl phenol ethoxylate Triton X-100 inhibits S. hermonthica seed germination without affecting host plants. High-resolution X-ray structures reveal that Triton X-100 specifically plugs the catalytic pocket of ShHTL7. ShHTL7-specific inhibition by Triton X-100 demonstrates the dominant role of this particular ShHTL receptor for Striga germination. Our structural analysis provides a rationale for the broad specificity and high sensitivity of ShHTL7, and reveals that strigolactones trigger structural changes in ShHTL7 that are required for downstream signaling. Our findings identify Triton and the related 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]acetic acid as promising lead compounds for the rational design of efficient Striga-specific herbicides.

Methyl phenlactonoates are efficient strigolactone analogs with simple structure.

Strigolactones (SLs) are a new class of phytohormones that also act as germination stimulants for root parasitic plants, such as Striga spp., and as branching factors for symbiotic arbuscular mycorrhizal fungi. Sources for natural SLs are very limited. Hence, efficient and simple SL analogs are needed for elucidating SL-related biological processes as well as for agricultural applications. Based on the structure of the non-canonical SL methyl carlactonoate, we developed a new, easy to synthesize series of analogs, termed methyl phenlactonoates (MPs), evaluated their efficacy in exerting different SL functions, and determined their affinity for SL receptors from rice and Striga hermonthica. Most of the MPs showed considerable activity in regulating plant architecture, triggering leaf senescence, and inducing parasitic seed germination. Moreover, some MPs outperformed GR24, a widely used SL analog with a complex structure, in exerting particular SL functions, such as modulating Arabidopsis roots architecture and inhibiting rice tillering. Thus, MPs will help in elucidating the functions of SLs and are promising candidates for agricultural applications. Moreover, MPs demonstrate that slight structural modifications clearly impact the efficiency in exerting particular SL functions, indicating that structural diversity of natural SLs may mirror a functional specificity.

Redox-mediated quorum sensing in plants.

The rhizosphere, the narrow zone of soil around plant roots, is a complex network of interactions between plants, bacteria, and a variety of other organisms. The absolute dependence on host-derived signals, or xenognosins, to regulate critical developmental checkpoints for host commitment in the obligate parasitic plants provides a window into the rhizosphere's chemical dynamics. These sessile intruders use H2O2 in a process known as semagenesis to chemically modify the mature root surfaces of proximal host plants and generate p-benzoquinones (BQs). The resulting redox-active signaling network regulates the spatial and temporal commitments necessary for host attachment. Recent evidence from non-parasites, including Arabidopsis thaliana, establishes that reactive oxygen species (ROS) production regulates similar redox circuits related to root recognition, broadening xenognosins' role beyond the parasites. Here we compare responses to the xenognosin dimethoxybenzoquinone (DMBQ) between the parasitic plant Striga asiatica and the non-parasitic A. thaliana. Exposure to DMBQ simulates the proximity of a mature root surface, stimulating an increase in cytoplasmic Ca2+ concentration in both plants, but leads to remarkably different phenotypic responses in the parasite and non-parasite. In S. asiatica, DMBQ induces development of the host attachment organ, the haustorium, and decreases ROS production at the root tip, while in A. thaliana, ROS production increases and further growth of the root tip is arrested. Obstruction of Ca2+ channels and the addition of antioxidants both lead to a decrease in the DMBQ response in both parasitic and non-parasitic plants. These results are consistent with Ca2+ regulating the activity of NADPH oxidases, which in turn sustain the autocatalytic production of ROS via an external quinone/hydroquinone redox cycle. Mechanistically, this chemistry is similar to black and white photography with the emerging dynamic reaction-diffusion network laying the foundation for the precise temporal and spatial control underlying rhizosphere architecture.

Zealactones. Novel natural strigolactones from maize.

In the root exudate and root extracts of maize hybrid cv NK Falkone seven putative strigolactones were detected using UPLC-TQ-MS-MS. All seven compounds displayed MS-MS-fragmentation common for strigolactones and particularly the presence of a fragment of m/z 97 Da, which may indicate the presence of the so-called D-ring, suggests they are strigolactones. The levels of all these putative strigolactones increased upon phosphate starvation and decreased upon fluridone (carotenoid biosynthesis inhibitor) treatment, both of which are a common response for strigolactones. All seven compounds were subsequently isolated with prep-HPLC-MS. They all exhibited Striga hermonthica seed germination inducing activity just as the synthetic strigolactone analog GR24. The structure of two of the seven compounds was elucidated by NMR spectroscopy as: methyl (2E,3E)-4-(3,3-dimethyl-5-oxo-2-(prop-1-en-2-yl)tetrahydrofuran-2-yl)-2-(((4-methyl-5-oxo-2,5-dihydrofuran-2-yl)oxy)methylene)but-3-enoate (two diastereomers 1a and 1b). Strigolactones (1a/b) are closely related to the methyl ester of carlactonoic acid (MeCLA) and heliolactone. However, they contain a unique 4,4-dimethyltetrahydrofuran-2-one motif as the "A-ring" instead of the classical (di)methylcyclohexene. Because these compounds were isolated from maize (Zea mays) we called them "zealactone 1a and 1b". The implications of this discovery for our view on strigolactones and their biosynthesis are discussed.

Small-molecule antagonists of germination of the parasitic plant Striga hermonthica.

Striga spp. (witchweed) is an obligate parasitic plant that attaches to host roots to deplete them of nutrients. In Sub-Saharan Africa, the most destructive Striga species, Striga hermonthica, parasitizes major food crops affecting two-thirds of the arable land and over 100 million people. One potential weakness in the Striga infection process is the way it senses the presence of a host crop. Striga only germinates in the presence of the plant hormone strigolactone, which exudes from a host root. Hence small molecules that perturb strigolactone signaling may be useful tools for disrupting the Striga lifecycle. Here we developed a chemical screen to suppress strigolactone signaling in the model plant Arabidopsis. One compound, soporidine, specifically inhibited a S. hermonthica strigolactone receptor and inhibited the parasite's germination. This indicates that strigolactone-based screens using Arabidopsis are useful in identifying lead compounds to combat Striga infestations.

Induction of Haustorium Development by Sphaeropsidones in Radicles of the Parasitic Weeds Striga and Orobanche. A Structure-Activity Relationship Study.

Crop attack by parasitic weeds such as Striga and Orobanche occurs through developmental processes triggered by host chemodetection. Seeds of those weed species remain dormant in the soil until germination is triggered by host root exudates. The development of haustorium, a parasitic plant organ that invades the host to withdraw its nutrients, is also initiated in Orobanchaceae by host molecular cues. The induction of haustorium development by exogenous signals has previously been reported for Striga but not for Orobanche species. In this work, we demonstrate that sphaeropsidone and epi-sphaeropsidone, two phytotoxic cyclohexene oxides isolated from the fungus Diplodia cupressi, a causal agent of cypress canker, induce haustorium development in radicles of the parasitic weeds Striga hermonthica, Orobanche crenata, and Orobanche cumana. This is the first report of chemical stimulation of haustorium development in radicles of Orobanche in the absence of host. In addition, SAR studies were carried out by testing the haustorium-inducing activity of the natural cyclohexene oxides, seven already known and four new hemisynthetic derivatives, in O. cumana, O. crenata, and S. hermonthica, to find a molecular specificity model required for haustorium induction. The results suggested that the haustorium-inducing activity is due to the possibility to convert the natural sphaeropsidone and natural and hemisynthetic derivatives in the corresponding 3-methoxyquinone and that the stereochemistry at C-5 also seems to affect this activity.

Chemical modification of a phenoxyfuranone-type strigolactone mimic for selective effects on rice tillering or Striga hermonthica seed germination.

BACKGROUND: We previously reported that a series of phenoxyfuranone compounds, designated 'debranones', mimic strigolactone (SL) activity. 4-Bromodebranone (4BD) is a functionally selective SL mimic that reduces the number of shoot branches on rice more potently than GR24, a typical synthetic SL analogue, but does not induce seed germination in the root-parasitic plant Striga hermonthica. To enhance the selective activity of debranones in stimulating the seed germination of root-parasitic plants, we prepared several analogues of 4BD in which the chlorine atom was substituted with an H atom at the o-, m- or p-position on the phenyl ring (designated 2-, 3-, or 4-chlorodebranone, respectively) or had a bicyclic group instead of the phenyl ring. We evaluated the biological activities of the compounds with rice tillering assays and S. hermonthica seed germination assays. RESULTS: Both assays showed that the substituent position affected debranone efficiency, and among the monochlorodebranones, 2-chlorodebranone was more effective than the other two isomers in both assays. When the activities of the bicyclic debranones were compared in the same two assays, one was more active than GR24 in the rice tillering assay. This debranone also stimulated the germination of S. hermonthica seeds. Thus, some debranone derivatives induced the germination of S. hermonthica seeds, although their activities were still ∼1/20 that of GR24. CONCLUSION: These results strongly suggest that further and rigorous structure-activity relationship studies of the debranones will identify derivatives that more potently stimulate the suicidal germination of S. hermonthica seeds. © 2016 Society of Chemical Industry.

Practicality of the suicidal germination approach for controlling Striga hermonthica.

BACKGROUND: Purple witchweed (Striga hermonthica), Orobanchaceae, is an obligate root-parasitic weed of important cereal crops. The parasite is a copious seed producer, and a huge seed bank develops soon after the onset of the initial infestation. To germinate, a Striga seed requires a pretreatment in a moist warm environment and a subsequent exposure to an exogenous stimulant. One approach to reduce the seed bank is artificially to induce germination of the seeds in the absence of or away from the host roots. A newly developed germination stimulant for S. hermonthica, designated as T-010, was evaluated for efficacy in greenhouse and field experiments under artificial Striga infestation. RESULTS: T-010 displayed germination-inducing activity in soil. Formulated T-010 applied at 0.1, 1 and 10 kg ai ha-1 to potted soil containing S. hermonthica seeds, previously conditioned by judicious irrigation, reduced Striga emergence by 94-100%. Results of a field trial showed that formulated T-010, at the same rates as for the pot experiment, delayed and reduced Striga emergence by 33% and increased sorghum shoot and head dry weight by 18.7-40.2% and 187-241% respectively. CONCLUSION: These findings demonstrated, for the first time, the technical feasibility of suicidal germination for controlling S. hermonthica. Optimising structure, formulation and application protocol of germination stimulants should be the main goal for further improvement of the technology. © 2016 Society of Chemical Industry.

Isolation and identification of Desmodium root exudates from drought tolerant species used as intercrops against Striga hermonthica.

Plants from the genus Desmodium, in particular D. uncinatum, are used on sub-Saharan small-holder farms as intercrops to inhibit parasitism of cereal crops by Striga hermonthica and Striga asiatica via an allelopathic mechanism. The search for Desmodium species which are adapted to more arid conditions, and which show resilience to increased drought stress, previously identified D. intortum, D. incanum and D. ramosissimum as potential drought tolerant intercrops. Their potential as intercrops was assessed for resource poor areas of rain-fed cereal production where drought conditions can persist through normal meteorological activity, or where drought may have increasing impact through climate change. The chemical composition of the root exudates were characterised and the whole exudate biological activity was shown to be active in pot experiments for inhibition of Striga parasitism on maize. Furthermore, rain fed plot experiments showed the drought tolerant Desmodium intercrops to be effective for Striga inhibition. This work demonstrates the allelopathic nature of the new drought tolerant intercrops through activity of root exudates and the major compounds seen in the exudates are characterised as being C-glycosylflavonoid. In young plants, the exudates show large qualitative differences but as the plants mature, there is a high degree of convergence of the C-glycosylflavonoid exudate chemical profile amongst active Desmodium intercrops that confers biological activity. This defines the material for examining the mechanism for Striga inhibition.

Avenaol, a germination stimulant for root parasitic plants from Avena strigosa.

Root exudates from the allelopathic plant, black oat (Avena strigosa Schreb.), were found to contain at least six different germination stimulants for root parasitic plants, but no known strigolactones (SLs). One of these germination stimulants was purified and named avenaol. Its HR-ESI-TOFMS analysis indicated that the molecular formula of avenaol is C20H24O7, and thus it contains an additional carbon compared with known C19-SLs. Its structure was determined as 5-((E)-(5-(3-hydroxy-1,5,5-trimethyl-2-oxobicyclo[4.1.0]heptan-7-yl)-2-oxodihydrofuran-3(2H)-ylidene)methoxy)-3-methylfuran-2(5H)-one, by 1D and 2D NMR spectroscopy, and ESI- and EI-MS spectrometry. Although avenaol contains the C-D moiety, the common structural feature for all known SLs, it lacks the B ring and has an additional carbon atom between the A and C rings. Avenaol is a potent germination stimulant of Phelipanche ramosa seeds, but only a weak stimulant for seeds of Striga hermonthica and Orobanche minor.

New strigolactone mimics: structure-activity relationship and mode of action as germinating stimulants for parasitic weeds.

Strigolactones (SLs) are new plant hormones with varies important bio-functions. This Letter deals with germination of seeds of parasitic weeds. Natural SLs have a too complex structure for synthesis. Therefore, there is an active search for SL analogues and mimics with a simpler structure with retention of activity. SL analogues all contain the D-ring connected with an enone moiety through an enol ether unit. A new mechanism for the hydrolysis SL analogues involving bidentate bound water and an α,ß-hydrolase with a Ser-His-Asp catalytic triad has been proposed. Newly discovered SL mimics only have the D-ring with an appropriate leaving group at C-5. A mode of action for SL mimics was proposed for which now supporting evidence is provided. As predicted an extra methyl group at C-4 of the D-ring blocks the germination of seeds of parasitic weeds.