Exploration of Strigolactone-Like Molecules in Phytopathogenic
Fungi
Yoshiya SETO
School of Agriculture, Meiji University
Research objective
Strigolactones (SLs) were initially identified as germination-inducing molecules for rootparasitic weeds, which cause significant damage to crop production. By sensing this molecule,root-parasitic weeds recognize the presence of a host plant and germinate to initiate parasitism.
This is a survival strategy for root-parasitic weeds that cannot survive in the absence of a host.
In 2005, it was revealed that SL is a signaling molecule that induces symbiosis with arbuscular
mycorrhizal fungi, which are important symbionts for plants. In 2008, it was discovered thatSLs are a class of new hormone molecules that regulate shoot branching. Therefore, SL plays
an extremely important role in the regulation of plant growth, and root-parasitic weeds exploit
such molecules for their own germination.
Many phytohormone molecules have been found not only as plant metabolites but also asmetabolites of phytopathogenic microorganisms. However, no such case has been reported for
SLs to date. In terms of root parasitic weed control, a suicide germination induction method
was devised in which SL molecules were sprayed on the field before planting crops. However,this method has not been put to practical use because of the difficulty in the quantitative
production of SL. If we can find a microorganism that produces a molecule with the sameactivity as SL, it may be possible to produce a suicidal germination inducer using afermentative method. Therefore, the aim of this study was to search for molecules with SLlike activity, especially in phytopathogenic fungi.
Methods
SLs in plants are biosynthesized from carotenoids. When carotenoids are irradiated withlight to induce non-enzymatic degradation, germination-inducing molecules against rootparasitic weeds are produced (Teramoto et al., 49th Annual Meeting of the Society for
Chemical Regulation of Plants). Since carotenoids are found not only as plant metabolites, butalso as metabolites of various microorganisms, we focused on plant pathogenic fungi that are
known to produce carotenoids. We screened for SL-like molecule-producing fungi via agermination-inducing assay using the root-parasitic plant, Orobanche minor. At the time ofapplication for this project, we had already found phytopathogenic fungi (Fungus A) that

produce SL-like molecules. Therefore, we isolated several more pathogenic fungi of the same
genus as Fungus A and tested their germination-inducing activity using their culture broth.
We then narrowed down to one strain that showed the highest activity and examined the culture
and purification conditions for the active molecule. DWARF14 (D14), the SL receptor inplants, belongs to the α/β-hydrolase family and has been reported to perceive and hydrolyze
SLs. To investigate whether the root parasitic plant germination-inducing molecules from
pathogenic fungi have SL-like chemical structures, we conducted an experiment to determine
whether the active molecules are degraded by the SL receptor, D14.
We speculated that fungi that produce SL-like active molecules may respond to SL.
Therefore, we examined SL responsiveness by growing fungi on medium supplemented with
GR24, a synthetic SL analog.
10090above,seven fungi belonging tothe same genus as Fungus
A were obtained. Ethyl
acetate extracts of theculture filtrate of each
fungus were further
purified using a silica gel
cartridge column to80

germination rate(%)

Results
1. As mentioned

7060
50403020
100×1×10
A×1×10
B×1×10
C×1×10
D×1×10
E×1×10
F×1×10
G×1×10
HFig. 1. Germination assay results of Orobamche minor using ethyl acetate
extract of pathogenic fungus A to H（×1, ×10 indicates the concentration
which is compared with the original culture broth）。

obtain several fractions. Using each fraction, we performed a germination assay with O.
minor seeds and found that almost all fungal metabolites showed germination-inducing
activity (Fig. 1). Therefore, it is possible that the active molecules are metabolites common
to the same family of fungi, or analogous molecules with similar skeletons.
2. Strains with strong activity were selected through screening, as described above, andvarious culture conditions were examined. Interestingly, when the active fraction wasconcentrated, germination-inducing activity did not increase significantly, but radicle
elongation was inhibited. This result suggests that the metabolites may contain not only
germination-inducing substances, but also components that inhibit germination or postradicle growth. Considering these points, we decided that ICI medium, a synthetic medium,was suitable for the production of bioactive molecules. Various purification conditions
were examined prior to mass culture and isolation of active molecules. The crude extract
was fractionated using silica gel and octadecyl-silica reversed-phase high-performanceliquid chromatography columns, and the activity of each fraction was tested. The resultsshowed that in both cases, activity was observed in several fractions, suggesting thepresence of multiple active molecules.
Next, we performed an experiment using the SL receptor, D14. One active fraction
obtained using the silica gel normal-phase column described above was pre-incubated with
D14 and subjected to a germination assay. GR24, a synthetic SL analog, was used as acontrol, and its activity was completely lost upon incubation with D14. In contrast, in thesample after treatment of the active fractions of fungal metabolites with D14, the activity
was predominantly, though not completely, reduced. In the case of the D14 mutant protein,in which a catalytic His residue was replaced with Ala, the activity was not reduced,
indicating that the active molecule was most likely■D14
degraded in a D14-dependent manner.
3. We applied a synthetic SL analog, GR24, to a fungus
that produces this active compound. When GR24 was■D14 HA mutant■control (no protein)

administered at final concentrations of 1 and 10 µM,
colony growth was inhibited in a concentrationdependent manner. Furthermore, abnormalities inmycelial growth were observed, with severe
branching following GR24 treatment.
X1

X10

X100Fig. 2. Germination assay results after
incubation of the active fraction with D14.

Conclusion
The objective of this study was to identify the metabolites with SL-like activity inphytopathogenic fungi. Although we were unable to isolate and identify the active molecules,
we found that several pathogenic strains belonging to one genus produced the active molecules.
Furthermore, it was strongly suggested that the active molecule was degraded by the SL
receptor, D14. This result indicates that the active molecule may have a chemical structure
similar to that of SL. Since we were able to examine the purification conditions of the active
molecule to some extent, we proceeded with its mass culture and purification to identify theactive molecule. Since this strain was observed to respond to SL, it is possible that the active
molecules found in this strain are involved in its growth control. In the future, we aim toinvestigate the biological significance of the production of SL-like molecules in detail.