Identification of an Asexual Reproduction Inducer of Phytopathogenic and Toxigenic Fusarium

Article abstract of DOI:10.1002/anie.201803329

Asexual and sexual reproduction are the most important biological events in the life cycle of phytopathogenic and toxigenic Fusarium and are responsible for disease epidemics. However, the signaling molecules which induce the asexual reproduction of Fusarium are unknown. Herein we describe the structure elucidation, including the absolute configuration, of Fusarium asexual reproduction inducer (FARI), a new sesquiterpene derivative, by spectroscopic analysis, total synthesis, and conidium-inducing assays of synthetic isomers. We have also uncovered the universality of FARI among Fusarium species. Moreover, a mechanism-of-action study suggested that the Gpmk1 and LaeA signaling pathways are required for conidium formation induced by FARI; conversely, the Mgv1 of mitogen-activated protein kinase is not involved in conidium formation. FARI exhibited conidium-inducing activity at an extremely low dose and high stereoselectivity, which may suggest the presence of a stereospecific target.

Full text of DOI:10.1002/anie.201803329

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Identification of an Asexual Reproduction Inducer of
Phytopathogenic and Toxigenic Fusarium
Authors: Jianhua Qi, Lihong Cheng, Yujuan Sun, Yushi Hirata, Naoki
Ushida, Zhonghua Ma, Hiroyuki Osada, Toshio Nishikawa,
and Lan Xiang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201803329
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10.1002/anie.201803329
Angewandte Chemie International Edition
Identification of an Asexual Reproduction Inducer of
Phytopathogenic and Toxigenic Fusarium
Jianhua Qi,* Lihong Cheng+ , Yujuan Sun+, Yushi Hirata, Naoki Ushida, Zhonghua
Ma, Hiroyuki Osada, Toshio Nishikawa* and Lan Xiang*
Prof. J. Qi, L. Cheng,+ Dr. Y. Sun,+ Dr. L. Xiang
College of Pharmaceutical Sciences, Zhejiang University, Yu Hang Tang Road 866,
Hangzhou 310058, People’s Republic of China
E-mail: qijianhua@zju.edu.cn
lxiang@zju.edu.cn
Y. Hirata, N. Ushida, Prof. T. Nishikawa
Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku,
Nagoya 464-8601, Japan
E-mail: nisikawa@agr.nagoya-u.ac.jp
Prof. Z. Ma
Institute of Biotechnology, Zhejiang University, Yu Hang Tang Road 866, Hangzhou
310058, People’s Republic of China
Prof. H. Osada
Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science,
Wako-shi, Saitama 351-0198, Japan
[+]These authors contributed equally to this work.
1
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10.1002/anie.201803329
Angewandte Chemie International Edition
Abstract
Asexual and sexual reproductions are the most important biological events
in the lifecycle of phytopathogenic and toxigenic Fusarium and are responsible
for disease epidemics. However, the signaling molecules which induce the asexual
reproduction of Fusarium are unknown. Here we describe the structure
elucidation including the absolute stereochemistry of Fusarium asexual
reproduction inducer (FARI), a new sesquiterpene derivative, which was
determined by spectroscopic analysis, total synthesis, and conidium–inducing
assays of synthetic isomers. We have also uncovered the universality of FARI
among Fusarium species. Moreover, the results of a mechanism of action study
suggested that the Gpmk1 and LaeA signaling pathways are required for the
conidial formation induced by FARI; conversely, the Mgv1 of mitogen-activated
protein kinase is not involved in conidial formation. Furthermore, FARI
exhibited conidium-inducing activity at an extremely low dose and high
stereo-selection, which may suggest the presence of a stereospecific target.
2
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10.1002/anie.201803329
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The Fusarium genus is among the most important phytopathogenic and toxigenic
filamentous fungus that causes destructive diseases in plants and contaminates food
and feed with mycotoxins. Fusarium species can also directly cause human infections.
The most notorious species, F. graminearum, is responsible for Fusarium head blight
(FHB), which is a global problem in wheat, oat, barley, ear and stalk rot of maize and
leads to severe yield and economic losses in these crops.[1-2] In addition to yield and
quality losses, the fungus produces harmful mycotoxins in infected crops, food, and
feed during the pre-harvest and post-harvest period.[3-4] These mycotoxins, such as
deoxynivalenol, zearalenone, T-2 toxin, and fumonisin B1 are a severe threat to
human and domestic animal health.[5-6] Direct Fusarium infection on humans causes
various diseases such as sinusitis,[7] pneumonia,[8] and skin lesions[9] and is
life-threatening in immunocompromised patients.[10-11] Asexual and sexual
reproductions are key biological events in higher microorganisms. In nature, sexual
and asexual reproductions are critical in the origin of outbreak strains for epidemic
diseases, and the rapid spread of diseases is often through asexual reproduction by
pathogenic fungi.[12] Therefore, discovery of asexual and sexual reproductive inducers
and understanding the mechanism of asexual and sexual reproduction greatly
contribute to the control of pathogenic fungi. For this purpose, we focused on the
characterization of signaling molecules which induce the asexual or sexual
reproduction of microorganisms for a long time. We previously characterized the
mating hormones, 1 and2, in Phytophthora oomycetes,[13-15] which
morphologically resemble fungi but are evolutionarily distinct.
Asexual reproduction is the most important means of fungal reproduction. For
plant pathogens like Fusarium, asexual reproduction can greatly affect epidemic
diseases, while sexual reproduction is important as a source of genetic variation.
3
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Studies have been performed to understand asexual reproduction of fungi for several
decades. Results revealed that asexual spore production of Aspergillus spp. was
affected by polyunsaturated fatty acids.[16] Also, reports indicated that conidiation of
Penicillium cyclopium could be prematurely induced in a nutritionally sufficient
medium by an endogenous diterpene and conidiation of Trichoderma was found to be
influenced by the environment and endogenous biological rhythms.[17-18] Findings for
the understanding of the regulation and signaling transduction on asexual
reproduction also showed that the mitogen-activated protein kinase (MAPK),[19]
velvet complex,[20-21] and AbaA[22] signaling pathways are involved in the asexual
reproduction of Fusarium species. However, the signaling molecule that induces the
asexual reproduction of Fusarium spp. is still unknown. Here, we report the isolation
and characterization of a conidium-inducing signaling molecule, Fusarium asexual
reproduction inducer (FARI (1), Figure 1A), from a Fusarium spp., and its generality
in the genus Fusarium as well as a mechanism of action on conidium formation.
F. graminearum strain PH-1 was used to isolate FARI. In a typical case, the
supernatant (100 L) from the culture broths of F. graminearum was partitioned with
EtOAc in batches. The active organic extracts were combined and chromatographed
on a silica gel open column, followed by reversed-phase column chromatography.
Further purification by repeated high performance liquid chromatography (HPLC)
yielded 0.4 mg of pure FARI. Finally, a total of 0.8 mg of pure FARI was obtained
from 201 L of supernatant culture broths. Conidium-inducing activity was evaluated
in F. graminearum strain PH-1 as described in the Supporting Information (Figure S1)
for all collected fractions from chromatography.
FARI has the molecular formula C15H26O2, as determined by high-resolution
ESI-MS measurement (found 261.1827, calculated for C15H26O2Na [M + Na]+
4
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10.1002/anie.201803329
Angewandte Chemie International Edition
1
261.1825). We analyzed the chemical structure of FARI by H nuclear magnetic
resonance (NMR) (Figure S2, Table 1), 13C NMR (Figure S3, Table 1), and
1
two-dimensional NMR spectroscopy (Figures S4-S6). The H NMR spectrum of
FARI show the presence of three methyl groups at δH 0.86 (3H, d, J = 6.8 Hz), 0.91
(3H, d, J = 6.8 Hz) and 2.10 (3H, s); four olefinic protons at δH 4.91 (1H, br s), 4.92
(1H, br s), 5.46 (1H, dd, J = 9.4, 15.8 Hz), 6.01 (1H, d, J = 15.8 Hz); one
oxymethylene δH 3.58 (2H, t, J = 6.6 Hz); four methylenes at δH 1.51 (1H, m), 1.70
(2H, m), 1.78 (1H, m), 2.28 (2H, dd, J = 6.5, 9.0 Hz ), and 2.42 (2H, m); and two
methines at 1.61 (1H, m) and 1.78 (1H, m) (Table 1). The 13C NMR spectrum of
FARI revealed the presence of 15 carbon signals comprised of one ketone carbon (C
212.2), three methyl groups (C 19.7, 21.2 and 30.0), two double bonds (δC 114.4,
132.9, 134.9 and 147.2), an oxymethylene carbon (δC 62.7), four methylene groups
(δC 27.6, 29.7, 32.6 and 42.6) as well as two methine groups (C 33.6 and 51.0) (Table
1). A detailed analysis of a proton-proton correlation spectroscopy (1H–1H COSY)
spectrum clarified the partial connectivity (in bold bonds in Figure S7). The
heteronuclear multiple-bond connectivity (HMBC) spectrum connected the above
partial structures to give a planar structure (Figure S7) as follows: H–1 to C–2; H–3 to
C–1 and C–2; H–6 to C–8; H–7 to C–8 and C–12; H–9 to C–7 and C–8; H–10 to C–8;
and H–12 to C–8 and C–9. The trans-geometry of the double bond at C6–C7 was
determined from the coupling constant of H-6 and H-7, J6–7 = 15.8 Hz. The optical
rotation of the natural FARI was []D25 +7.0 (c 0.42, benzene).
FARI significantly induced conidia formation (1200 ± 165 conidia) at 1 ng dose
on F. graminearum strain PH-1 mycelia, and the number of total conidia increased in
a dose-dependent manner at the range of 1 to 300 ng (Figure 1B) and peaked at 300
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10.1002/anie.201803329
Angewandte Chemie International Edition
ng (Figure S8). Cross effect to other Fusarium species demonstrated that FARI
induced the conidia formation on the other six tested species: F. equiseti and F.
verticillioides (Figure 1B), and F. proliferatum, F. avenaceum, F. culmorum, and F.
oxysporum (Figure S9). Moreover, the conidium-inducing activity of FARI on other
fungi was examined. The results revealed that FARI was inactive on conidiation of
Botrytis cinerea and Penicillium digitatum (Figure S10). Figure 1C shows a
photomicrograph of conidia obtained in the conidium-inducing assay at a dose of 300
ng.
Since only trace amount of FARI was obtained in this study and the structure of
FARI possesses two possible stereoisomers due to the chiral center at C5, the
chemical synthesis of two enantiomers was necessary for the structure confirmation as
well as the assignment of the absolute configuration at C5 (Figure 2, see the
Supporting Information). Two enantiomers of FARI (1) were synthesized from
commercially available chiral precursors, (R)- and (S)-limonene (2) (see the
Supporting Information, Figures S11-S30). The synthesis of (S)-1 from (R)-2 is shown
in Figure 2. First, site-selective hydrogenation of (R)-limonene (2) with Adams’
catalyst (PtO2) followed by Lemiux-Johnson oxidation (cat. OsO4, NaIO4) afforded
(R)-keto aldehyde 3.[23-25] The resulting aldehyde was then transformed into
(R)-geminal dibromide 4 in two steps by Takeda’s procedure.[26] The hydrazone
prepared by treatment of (R)-3 with hydrazine monohydrate was treated with copper
(II) bromide and triethylamine in the presence of a molecular sieve (MS 4A) to give
(R)-gem-dibromide 4 in 34% yield. Dehydrobromination of (R)-4 with DBU provided
(S)-vinyl bromide 5 in a good yield. Though the yield of 4 was relatively low, this
route provided sufficient amount of 5 without epimerization at C5 for the synthesis of
(S)-1 with high optical purity. (S)-vinyl bromide 5 was coupled with alkenylborane
6
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10.1002/anie.201803329
Angewandte Chemie International Edition
6[27] through the Suzuki-Miyaura coupling[28] to give (S)-7 in a yield of 75%. Finally,
removal of the TBS group with TBAF furnished (S)-1 in a good yield. The
(R)-enantiomer of 1 was also synthesized from (S)-limonene (2) in a similar manner
to that of (S)-1. Optical purities of (S)- and (R)-1 were determined by chiral HPLC to
be 96.8 and 97.4% ee, respectively. The maximum wavelength of absorption (λmax) of
(S)-1 was 231 nm and the molar absorption coefficients (ε) in methanol was 18217.
The synthetic stereoisomers did not show any difference from the natural
1
13
compound as observed by H and C NMR data (Figures S31, S32, Tables S1, S2).
Since assignment of the absolute configurations of natural FARI by spectroscopic
methods is difficult, the conidium-inducing activities of the two synthetic isomers,
(S)-1 and (R)-1, were evaluated in comparison with the natural one. The (S)-1 isomer
induced significant conidia formation (P < 0.001) in F. graminearum at a dose of 1 ng,
and the conidia increased in a dose-dependent manner, which is very similar to the
results of the natural FARI (Figure 3). (R)-1 isomer at a dose of 100 ng did not induce
any remarkable conidia formation (Figure 3). Although a higher dose (300 ng) of
(R)-1 induced conidia formation, this activity might be derived from a small amount
of the (S)-enantiomer (1.3%) containing in (R)-1, which may have arisen from the
epimerization in the synthesis and/or from the starting material containing trace
25
amounts of (R)-2. In addition, the optical rotation of natural FARI (1) [[]D +7.0 (c
24
0.42, benzene)] closely matched that of synthesized (S)-1 [[]D +10.8 (c 1.05,
23
benzene)] and opposite that of (R)-1 [[]D -10.7 (c 1.07, benzene)]. These results
strongly suggested that natural FARI has a S configuration at the C-5 position.
To confirm interspecies universality, quantitative analysis of FARI produced by
Fusarium species was performed using the ultra high performance liquid
chromatography-electrospray ionization tandem quadrupole mass spectrometry
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(UHPLC-ESI-MS/MS) and FARI-d2 ((S)-1-d2) as the internal standard (IS) (See
Supporting Information, Figures S33-S39 and Tables S3-S4). The tested species
produced FARI at 0.10-0.32g L−1 (Table 2), which strongly supports the universality
of FARI among Fusarium species. These results suggest that FARI is not only a
conidium-inducing molecule of F. graminearum but also a universal regulator of
conidia formation among Fusarium species.
To investigate the mechanism of action, the FgMgv1 and FgGpmk1 of MAPK,
FgLaeA of velvet complex, and FgAbaA signaling pathways were examined to
evaluate their effects on conidiation induced by FARI using corresponding mutants in
carboxymethyl cellulose (CMC) liquid medium culture. Two concentrations, 200 and
600 ng mL-1, that practically correspond to 100 and 300 ng doses on the solid medium
assay, were used for all experiments. FARI significantly increased conidia formation
on FgMgv1 at tested concentrations, whereas it did not cause an increase in conidia
number on FgGpmk1 andFgLaeA. These results indicated that the Mgv1 MAPK
pathway did not play a crucial role, but the Gpmk1 MAPK and LaeA velvet pathways
are involved in conidiation induced by FARI (Figure 4). As AbaA is known to be
directly involved in the conidial formation, FgAbaA did not make conidium
regardless the existence of FARI.
In conclusion, to our knowledge, this is the first characterization of a
conidium-inducing molecule in the pathogenic and toxigenic fungus, Fusarium.
Isolation, structure elucidation, total synthesis of enantiomers, biological evaluation as
well as an investigation of the mode of action were achieved. The absolute
configuration of natural FARI was determined as 5S by comparing the optical rotation
and conidium-inducing activities of the two synthetic stereoisomers with those of
natural FARI. Chirality is certainly important in the conidium-inducing activity in
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Fusarium, which strongly implies the existence of a stereospecific target at the
beginning of the signal transduction cascade, leading to conidia formation. Despite
being a genus of filamentous fungi, Fusarium is similar to phytopathogenic
Phytophthora oomycetes, in which the configuration change for each methyl group in
the chemical structure of synthetic mating hormones,1 and 2, could have resulted
in an almost complete loss of hormonal activities.[14-15] These results suggested that
the mating hormones of Phytophthora target stereospecific receptors to initiate the
oospore formation. Therefore, our findings also suggest that FARI is a possible
hormone or hormone-like molecule which regulates the conidia formation in
Fusarium.
A study of the mechanism of action showed that the Gpmk1 MAPK and velvet
complex signaling pathways are involved in conidiation of F. graminearum induced
by FARI. These results are consistent with previous reports by genetic methods in
which conidium production was completely abolished in AbaA deletion mutant,[22]
and the Gpmk1 pathway and LaeA pathway of the velvet complex were involved in
conidiation.[19,21] The Mgv1 pathway is involved in the male fertilization and female
fertilization, but not in asexual conidia formation.[29] These support the idea that FARI
is an important inducer for conidia formation of Fusarium species.
Microorganisms may contain specific receptors for signaling molecules, which
trigger asexual and sexual reproductions. Each endogenous reproductive signaling
molecule targeting a specific receptor is possibly a natural occurrence in the microbial
kingdom. Our findings imply that the formation of asexual spores in filamentous
fungi may be regulated by hormones or hormone-like inducers, and the targets of such
substances could be used for the discovery of specific novel antibiotics and fungicides
against fungal pathogens for human and in agriculture, respectively.
9
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32, 390-402.
Acknowledgements
This work was supported by the National Natural Science Foundation of China [Grant
No. 21661140001 and 21572204] (to J.Q.), a Grant-in-Aid for Scientific Research (B)
(No. 16H04915) (to T.N.) and the JSPS A3 Foresight Program. All authors discussed
the results and commented on the manuscript. The authors thank professors Su Zeng
(Zhejiang University, China) and Zhujun Yao (Nanjing University, China) for
valuable discussions. Dr. Julius Lopez (RIKEN, Japan) gave important comments to
the manuscript. The authors acknowledge Dr. Kin-ichi Oyama (Chemical
Instrumental Facility, Research Center for Material Science, Nagoya University) for
measuring HRMS (EI) of deuterated compounds.
Conflict of interest
The authors declare no conflict of interest.
Keywords: asexual reproduction inducer  Fusarium  sesquiterpene  signal
transduction  total synthesis
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Figures and figure legends:
C
A
30
20
10
0
F. equiseti
80
60
40
20
0
B
25
20
15
10
5
F. verticillioides
***
F. graminearum
***
***
**
**
***
*
**
0
C EA 1
3
10 30 100300
C
1
3
10 30 100300
C
1
3
10 30 100300
Dose (ng)
Dose (ng)
Dose (ng)
Figure 1. Chemical and biological character of natural FARI. A) The chemical
structure of Fusarium asexual reproduction inducer (FARI, 1) isolated from Fusarium
graminearum. B) Dose-dependent increase of asexual spores (conidia) formation in F.
graminearum, F. equiseti and F. verticillioides upon treatment with FARI. The total
number of conidia (vertical axis) produced on a FARI-added well was counted. EA
represents the EtOAc extract corresponding to 5 mL of culture broth; C is the negative
control. Values are expressed in terms of means of six replicates ± SE. *P < 0.05, **P
< 0.01 and ***P < 0.001 indicate significant difference from the corresponding
negative control. The total number of conidia formed on the mycelia of Fusarium
represented the conidium-inducing activity of test samples. C) Photomicrograph of
conidia induced by 300 ng of FARI on F. graminearum. The left one is the
micrograph of fluorescence image. The right one is the micrograph of light image.
Fluorescence image of conidia stained with calcofluor white (CFW) were acquired
13
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10.1002/anie.201803329
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using a Zeiss LSM 780 confocal microscope. Conidia were obtained after filtration of
hyphae and agar. Scale bar, 20 m.
Figure 2. Synthesis of (S)-1. Reagents and conditions: (i) H2, PtO2, EtOH; (ii) OsO4,
NaIO4, pyridine, THF-H2O; (iii) (H2N)2·H2O, CH2Cl2, rt, then Et3N, CuBr2, MS4A,
0 °C to rt, 20 min (34%); (iv) DBU, DMF, 50 °C to reflux, 5 h (67%); (v) Pd(PPh3)4,
Cs2CO3, DMF-H2O, 50 °C, 4 h (75%); (vi) TBAF, THF, rt, 80 min (67%).
Abbreviation: MS4A, molecular sieve 4A; TBS, tert-butyldimethylsilyl; DBU,
1,8-diazabicyclo[5.4.0]undec-7-ene; DMF, N,N-dimethylformamide.
14
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10.1002/anie.201803329
Angewandte Chemie International Edition
8
6
4
2
0
***
***
**
C 1 3 10
C 1 3 10
C 10 30 100 (ng)
Natural FARI
Synthetic (S)-1
Stereoisomer (R)-1
Figure 3. Biological properties of the synthetic FARI stereoisomers. Conidia
formation in F. graminearum induced by two synthesized isomers (S)-1 and (R)-1 at
doses of 1, 3, and 10 ng well−1, and 10, 30, 100 ng well−1, respectively, in comparison
with natural FARI at same doses of (S)-1. C, negative control. Values are expressed as
means of six replicates ± SE. **P < 0.01 and ***P < 0.001 indicate significant
difference from the corresponding negative control.
15
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10.1002/anie.201803329
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Figure 4. Mechanism of action of FARI. Conidium-inducing activity of FARI on
mutants (FgAbaA, FgGpmk1, FgMgv1, and FgLaeA) in comparison with F.
graminearum strain PH-1. Conidia were counted after incubation of the wide type and
o
mutants in CMC liquid culture medium for five days at 25 C, 180 rpm. The
concentrations of 200 and 600 ng mL-1 which practically correspond to the doses of
100 and 300 ng on solid medium assay, respectively, were used in all experiments. C
is the negative control. Values are expressed in terms of means of six replicates ± SE.
***P < 0.001 indicates significant difference from the corresponding negative
control.
16
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10.1002/anie.201803329
Angewandte Chemie International Edition
Tables
Table 1. The 1H and 13C NMR data for natural FARI (in CD3OD, ppm)[a]
Carbon
No.[b]
H
C
30.0
1
2
2.10 s
-
212.2
42.6
3
2.42 m
4
1.78 m 1.51 m
1.78 m
27.6
5
51.0
6
5.46 dd (9.4,15.8)
6.01 d (15.8)
-
132.9
134.9
147.2
29.7
7
8
9
2.28 dd (6.5, 9.0)
1.70 m
10
11
12
13
14
15
32.6
3.58 t (6.6)
4.91 br s 4.92 br s
1.61 m
62.7
114.4
33.6
0.86 d (6.8)
0.91 d (6.8)
19.7
21.2
[a] 500 MHz for 1H NMR and 125 MHz for 13C NMR, coupling constants (J in Hz) are
in parentheses.
[b] Numbering of FARI (see Fig. 1A)
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Table 2. Interspecies universality of FARI in Fusarium species (g L-1, n=3).
Species
Strain[b]
FARI[a]
F. proliferatum
F. avenaceum
F. equiseti
CGMCC3.4759
CGMCC3.6813
CGMCC3.6911
CGMCC3.4595
CGMCC3.6787
CGMCC3.7995
0.18 ± 0.04
0.11 ± 0.01
0.30 ± 0.05
0.32 ± 0.10
0.15 ± 0.01
0.10 ± 0.02
F. culmorum
F. oxysporum
F. verticillioides
[a] The values were determined by UHPLC-ESI-MS/MS analysis of FARI containing
fractions which were obtained from the cartridge separation of EtOAc layer of filtered
culture broths using a synthetic FARI-d2 as the internal standard. The experiments
were repeated three times and the data were presented as means ± SEM.
[b]
The strains were purchased from the China General Microbiological Culture
Collection Center (CGMCC, Beijing, China).
18
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Table of contents
Fusarium asexual reproduction inducer (FARI), a new sesquiterpene derivative,
induced conidial formation on notorious Fusarium graminearum at an extremely low
dose. The universality of FARI among Fusarium species was uncovered. Furthermore,
mechanism of action study revealed that Gpmk1 and LaeA signaling pathways are
required for the conidial formation induced by FARI.
19
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