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Identification of Naturally Occurring Polyamines as Root-Knot Nematode Attractants
Journal Pre-proof Identification of naturally-occurring polyamines as nematode Meloidogyne incognita attractants Morihiro Oota, Allen Yi-Lun Tsai, Dan Aoki, Yasuyuki Matsushita, Syuuto Toyoda, Kazuhiko Fukushima, Kentaro Saeki, Kei Toda, Laetitia Perfus-Barbeoch, Bruno Favery, Hayato Ishikawa, Shinichiro Sawa PII: DOI: Reference:
S1674-2052(19)30407-1 https://doi.org/10.1016/j.molp.2019.12.010 MOLP 870
To appear in: MOLECULAR PLANT Accepted Date: 23 December 2019
Please cite this article as: Oota M., Yi-Lun Tsai A., Aoki D., Matsushita Y., Toyoda S., Fukushima K., Saeki K., Toda K., Perfus-Barbeoch L., Favery B., Ishikawa H., and Sawa S. (2020). Identification of naturally-occurring polyamines as nematode Meloidogyne incognita attractants. Mol. Plant. doi: https:// doi.org/10.1016/j.molp.2019.12.010. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs. © 2019 The Author
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Research Report
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Identification of naturally-occurring polyamines as nematode Meloidogyne
4
incognita attractants
5 6
Morihiro Oota1, Allen Yi-Lun Tsai1, 4, Dan Aoki2, Yasuyuki Matsushita2, Syuuto
7
Toyoda1, Kazuhiko Fukushima2, Kentaro Saeki1, Kei Toda1, Laetitia Perfus-Barbeoch3,
8
Bruno Favery3, Hayato Ishikawa1*, and Shinichiro Sawa1*
9 1
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860-8555, Japan 2
12
13
3
17
INRA, Université Côte d’Azur, CNRS, UMR 1355-7254 Institut Sophia Agrobiotech,
06900 Sophia-Antipolis, France 4
16
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601,
Japan.
14
15
Graduate School of Science and Technology, Kumamoto University, Kumamoto
Present address: Dormancy and Adaptation Research Unit, RIKEN Center for
Sustainable Resource Science, Yokohama 230-0045, Japan.
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Running Title:
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Polyamines as plant attractants of nematodes
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Short Summary:
22
We have Identifid naturally-occurring polyamines, cadaverine, putrescine and
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1,3-diaminopropane,
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cryo-TOF-SIMS/SEM, cadaverine was indeed detected in soybean root cortex cells and
25
the surrounding rhizosphere, establishing a chemical gradient.
as
nematode
Meloidogyne
incognita
attractants.
Using
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*Corresponding authors:
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Shinichiro Sawa
29
Graduate School of Science and Technology, Kumamoto University, Kumamoto
30
Kurokami 2-39-1, Kumamoto, 860-8555, Japan
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Telephone / fax: +81-96-342-3439
32
[email protected]
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Hayato Ishikawa
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Graduate School of Science and Technology, Kumamoto University, Kumamoto
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Kurokami 2-39-1, Kumamoto, 860-8555, Japan
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Telephone / fax: +81-96-342-3397
38
[email protected]
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Abstract
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Root-knot nematodes (RKN, genus Meloidogyne) are a class of plant parasites that seek
42
out and infect roots of many plant species. It is believed that RKN target certain
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signaling molecules derived from plants to locate their hosts, however currently no plant
44
compound has been unambiguously identified as a universal RKN attractant. To address
45
this question, we screened a chemical library of synthetic compound for M. incognita
46
attractants.
47
1,3-diaminopropane, as well as related compounds putrescine and cadaverine were
48
found to attract M. incognita. After examining various polyamines, M. incognita were
49
found to be attracted specifically by natural compounds that possess three to five
50
methylene groups between two terminal amino groups. Using cryo-TOF-SIMS/SEM,
51
cadaverine was indeed detected in soybean root cortex cells and the surrounding
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rhizosphere, establishing a chemical gradient. In addition to cadaverine, putrescine and
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1,3-diaminopropane were also detected in root exudate by HPLC-MS/MS. Furthermore,
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exogenously applied cadaverine is sufficient to enhance M. incognita infection of
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Arabidopsis seedlings. These results suggest M. incognita may indeed target
56
polyamines to locate the appropriate host plants, and these naturally-occurring
Break-down
product
of
aminopropylamino-anthraquinone,
57
polyamines may have viable applications in agriculture to develop protection strategies
58
for crops from RKN infections.
59
60
Introduction
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Plant-parasitic root-knot nematodes (genus Meloidogyne, RKN) are obligate
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parasites that infect the roots of many plant species, including several crop plants
63
causing more than $100 billion USD-worth of agricultural damages annually. As a
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mean to target their hosts, infective RKN J2 larvae are attracted to certain signals
65
derived from plant root tips (Torto et al., 2018; Čepulytė et al., 2018). Chemical and
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physical cues such as carbon dioxide, pH, ions, and temperature in the rhizosphere have
67
been reported to attract or repel RKN (Rasmann et al., 2012). In addition, moisture,
68
carbohydrates, amino acids, phenolic compounds, redox potentials, chelating
69
compounds, and electrical potentials may also regulate RKN behaviors (Perry and
70
Aumann, 1998). Lastly, RKN attractants and repellents may be produced from
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organisms in the rhizosphere, including from the RKN themselves (Manosalva et al.,
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2015). These factors combined make the RKN J2 behavior in soil complex, and the
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identification of chemical cues that guide RKN J2 larvae to their hosts has become a
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significant milestone in RKN biology. However currently very few plant-derived
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chemicals have been unambiguously shown to function as RKN attractants. The
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fractionation of host root exudate is expected to identify the RKN attractants involved in
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RKN behavior regulation (Perry and Aumann, 1998); however, such approach has yet to
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definitively isolate plant-derived RKN chemoattractants (Devine and Jones, 2003). The
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identification of RKN chemoattractants can contribute not only to the development of
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RKN control strategies in agriculture, but also for the basic science of multicellular
81
organism interactions. To this end, we’ve decided to employ a different approach by
82
screening a chemical library to identify compounds with nematode-attracting activities,
83
then determine whether these compounds are indeed synthesized and secreted by plants.
84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
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Results and Discussion
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To identify compounds that attract RKN, we screened the NPDepo pilot chemical
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library which includes 376 synthetic chemical compounds. The library compounds were
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tested at 10 mg/mL on a Pluronic F-127-based media with mobile M. incognita J2
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larvae
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(aminopropylamino-anthraquinone, 1) was identified as a potential M. incognita
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attractant (Fig. 1A,B). To confirm whether 1-mediated RKN-attraction is genuine, in
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vitro-synthesized 1 was used to examine M. incognita response. However, freshly
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synthesized 1 did not show any M. incognita attraction activity (Fig. 1B). We then
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hypothesized that the biologically-active component may in fact be a decomposition
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product derived from 1. To test this hypothesis, freshly prepared 1 was exposed to
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oxygen under fluorescent lamp for 233 h followed by liquid chromatography-mass
111
spectrometry
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1,3-Diaminopropane (2) was found to be produced from 1 via oxidation followed by
113
hydrolysis (Supplemental Fig. 1). We found that both oxygen-exposed 1 and
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commercially-available 2 strongly attracted M. incognita (Fig. 1B,C). From these
(Wang
et
al.,
(LC/MS)
2009a).
and
1-[(3-aminopropyl)amino]-anthracene-9,10-dione
nuclear
magnetic
resonance
(NMR)
analysis.
115
results, alkyl groups tethered with terminal amino moiety such as in 2 appear to be the
116
key functional groups responsible for M. incognita attraction.
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We then examined the RKN-attraction properties of other polyamines, ranging
118
from n-propylamine (3) and ethylenediamine (4) to 1,9-diaminonane (10) with
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backbones of up to nine carbons conjugated with up to two terminal amine groups, as
120
well as spermidine (11) and spermine (12) that contain three and four amino groups,
121
respectively (Fig. 1A,C; Supplemental Fig. 2). We found that not only 2, but also
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putrescine (5) and cadaverine (6) have significant M. incognita attraction activity (Fig.
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1C) with chemotaxis index values of 0.58 to 0.82, respectively. Interestingly, 11 and 12
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also showed modest attraction activities, though not as high as 2, 5 and 6 (Fig. 1C；
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Supplemental Fig. 2). However, none of the terminal diamines with backbone shorter
126
than three carbons or longer than five carbons showed M. incognita attraction activity,
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suggesting the size of the attractant is strictly limited from three to five carbons.
128
Interestingly, 3 did not show M. incognita attraction (Fig. 1C; Supplemental Fig. 2),
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suggesting that two terminal amino groups are required for attraction. Thus, we are
130
confident that structurally limited diamines act selectively as M. incognita attractants. M.
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incognita are known to be attracted by acetic acid gradients of pH 4.5-5.4 (Wang et al.,
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2009b), and as a diamine 6 may alter the rhizosphere pH and non-specifically attract M.
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incognita.
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1,8-diaminooctane (9) nor 10 showed attraction activity despite all having two terminal
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amino groups, indicating diaminoalkyl moieties affecting pH alone is not sufficient to
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regulate M. incognita behaviors. The dosage effects of 2, 5, and 6 in M. incognita
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attraction were also examined. 6 at as low as 5 mM was sufficient to visibly attract M.
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incognita. However discernible colonies could only be observed in the presence of 2
139
and 5 of at least 25 mM (Supplemental Fig. 3), suggesting that 6 is the more robust
140
attractant compared to 2 and 5.
However,
neither
1,6-diaminohexane
(7),
1,7-diaminoheptane
(8),
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To evaluate whether the affinity towards 6 is conserved in other parasitic
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nematodes, the response of two other RKN species M. arenaria and M. enterolobii
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towards 6 were tested. Neither M. arenaria nor M. enterolobii showed any response
144
toward 10 mg/mL 6 (Supplemental Table 1), suggesting this attraction behavior may be
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unique to M. incognita. Despite being closely related species, M. incognita, M. arenaria
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and M. enterolobii each have distinct host preferences (Quénéhervé et al., 2011), which
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may partially be regulated by 6 or other natural diamines.
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Cadaverine (6) is known as an essential diamine for normal development and
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responses to environmental conditions in plants (Gamarnik et al., 1991; Jancewicz et al.,
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2016). We hypothesize that 6 may be one of the molecules responsible for M. incognita
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root attraction. To test this hypothesis, we planned to detect the presence of diamines
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from the root exudates of soybean (Glycine max) and tomato (Solanum lycopersicum),
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two crop plants often targeted by RKNs. 100 roots were dipped to 75 ml water for 20
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hours to prepare root exudates. We showed that soybean and tomato root exudate both
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attract M. incognita (Supplemental Fig. 4). By using diluted root exudates, we found
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that soybean root exudates are more effective in attraction compared to tomato in our
157
assay system (Supplemental Fig. 4).
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Diamine concentrations were quantified using HPLC-MS/MS. 6 was detected
159
from soybean and tomato root exudates at 12.4 nM and 465.5 nM, respectively (Table
160
1), indicating that 6 is indeed secreted from soybean and tomato root tips. The amount
161
of 6 detected in soybean root exudate may be lower than the 5 mM cut-off determined
162
earlier (Supplemental Fig. 3), however since 6 is predicted to form a gradient from the
163
root it will likely be more concentrated closer to the root. Furthermore, M. incognita
164
attraction is likely a concerted effort mediated by multiple compounds, as other
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attractants may also be present in soybean root exudates (Oota and Sawa, unpublished
166
results). Other than 6, 2 and 5 were also detected from soybean and tomato root exudate
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at 38.1 nM and 272.3 nM for 2, and 4.0 nM and 11.6 mM for 5, respectively (Table 1).
168
Interestingly, soybean root exudate appears to contain less diamines compared to
169
tomato, despite being the more potent M. incognita attractant. As root exudate
170
compositions are complex, it is possible that tomato root exudates contain compounds
171
that antagonize nematode attractions. To test for the presence of such attraction
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antagonists, soybean root exudates mixed with increasing proportions of tomato
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exudates were tested for nematode attraction activities. Soybean root exudates mixed
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with tomato root exudate indeed show reduced attraction activities compared to samples
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mixed with water at equivalent ratios, suggesting tomato root exudate indeed contain
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compounds that negatively regulate nematode attraction (Supplemental Fig. 4C). These
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compounds may either specifically counteract diamine-mediated nematode attraction, or
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non-specifically repel nematodes in general.
179
The spatial distribution of 6 within and around the root was determined using
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time-of-flight secondary ion mass spectrometry/scanning electron microscopy
181
(cryo-TOF-SIMS/SEM) analysis, a technique used to visualize the distribution of
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water-soluble compounds in freeze-fixed samples at microscopic resolution level (Aoki
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et al., 2016; Okumura et al., 2017). Here we decided to focus on soybean roots due to its
184
high attraction potency compared to tomato. First, to determine the characteristic
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secondary ion of 6, an agar gel control and an aqueous solution of 6 in 0.1 M KCl were
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frozen and measured using cryo-TOF-SIMS. The peak at m/z 103.12 was assigned to
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the cadaverine-derived [M+H]+ ion and was not detected in the agar standard
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(Supplemental Fig. 5). From these results, we conclude that cadaverine (6) distribution
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can be visualized by detecting its m/z 103.12 ion. Similarly, we examined 2 and 5 by
190
detecting peaks at m/z 75.09 and 89.11, respectively (Supplemental Fig. 6A,B). 2 was
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hardly detected from soybean root, probably in part due to its extreme volatile nature,
192
while 5 was indeed detected in soybean root. The semi-quantitative spatial distribution
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of 2, 5, and 6 in soybean root was then determined using cryo-TOF-SIMS/SEM analysis.
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Soybean roots were grown on agar, then frozen to enable the transverse sectioning of
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the root tip and the surrounding agar to be examined (Supplemental Fig. 7). The images
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generated using cryo-SEM (Fig. 2A) and cryo-TOF-SIMS total ion (Fig. 2B) showed
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total ion accumulation in epidermis, cortex and vascular tissues of the transverse section
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of the soybean root. Potassium ions ([39K]+, m/z 38.96) were detected in the entire
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transverse section of the root, with stronger signals in the epidermal cells (Fig. 2C).
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Some potassium ions were also detected in the agar surrounding the root. The m/z
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184.07 ion represents the [phosphocholine]+ fragment derived from phosphatidylcholine
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([C5H13NO4P][lipids]2) (Khan and Williams, 1977), which is a main component of
203
biological membranes in plant cells (Hussain et al., 2013). The detection of potassium
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and phosphocholine ions confirms that the cells were alive and active at the measured
205
surface before fixation (Fig. 2C, D). Furthermore, the clear boundary between the root
206
and agar indicates that the distribution of water-soluble chemicals were freeze-fixed
207
well and visualized without any alterations.
208
The majority of 6 is distributed in the root cortex and epidermis, with lower levels
209
of 6 also present in the central vasculature (Fig. 2E,F). Thus, it is likely that 6 is
210
synthesized locally in the root cortex, and would therefore not require active transport
211
through the vasculature. We also detected significant ion counts in the agar within 150–
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250 µm from the root (Fig. 2F-J). The ion count intensity from the agar adjacent to the
213
root was roughly one-tenth the level relative to the cortex, decreasing with the distance
214
away from the root. These findings indicate that 6 produced in the root tip is likely
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secreted into the rhizosphere to establish a chemical gradient. Furthermore 6 is also
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volatile, suggesting that it may disperse easily and can indeed function as a positional
217
cue for M. incognita. On the other hand, ion counts of m/z 75.09, corresponding to 2,
218
was uniformly detected as background noise, and we could not detect significant
219
presence of 2 in the rhizosphere and in root tissues using cryo-TOF-SIMS/SEM analysis
220
(Supplemental Fig. 6C). Background noise of 5 is lower and 5 was detectable in the root,
221
although a clear gradient could not be clearly observed in the rhizosphere
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(Supplemental Fig. 6C). The molecular weight of 5 is less than that of 6, which may
223
explain why 5 was unable to maintain a gradient in the rhizosphere.
224
To further characterize this diamine-dependent M. incognita attraction, we
225
assayed nematode infection frequencies to determine whether exogenously-applied
226
diamine is sufficient to influence infection. For this purpose, we dipped Arabidopsis
227
root at five days after germination to 100 mM diamine solution, and examined M.
228
incognita infection rates. We have selected Arabidopsis for this assay because
229
Arabidopsis root does not appear to release nematode attractants, thus nematode
230
attraction and infection rates are generally lower compared to other plants.
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Consequently, the effect of diamine enhancement of nematode infection should be more
232
pronounced in Arabidopsis. After diamine treatments, seedlings were grown on MS
233
media for three days to allow nematode infection. The number of galls in seedlings
234
treated with 2, 5, and 6 increased significantly compared to mock-treated seedlings at
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three days post-inoculation (Fig. 3). Gall numbers in mock-treated seedlings gradually
236
caught up with diamine-treated seedlings by six to nine days post-inoculation, possibly
237
due to the applied diamines dispersing over the media, thus losing attraction potency
238
over time (data not shown). Nevertheless, these results suggest that 2, 5, and
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6-dependent M. incognita attraction likely positively regulates M. incognita infection.
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Here we revealed that several naturally-occurring polyamines possess M.
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incognita attraction activity, with cadaverine (6) being the most effective. M. incognita
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preferentially responded to the naturally-occurring 2, 5 and 6 over other synthetic
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polyamine species. 5 and 6 are best known as bacterial by-products of tissue decay, and
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animals respond both positively and negatively toward these diamines depending on the
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species and context. Zebra fish (Danio rerio) trace amine-associated receptor 13c
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(TAAR13c) indeed functions as a receptor of 5 and 6 and mediates negative chemotaxis
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against 5 and 6 (Hayafune et al., 2014). It would be interesting to identify the structure
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of the M. incognita diamine receptor, and determine whether it shares any similarities
249
with TAAR13c. The M. incognita attractant receptor appears to have a ligand size limit
250
for alkyl chain with three to five carbon atoms. On the other hand, the rice (Oryza
251
sativa) chitin elicitor-binding protein (CEBiP) function as a sandwich-type dimer to
252
perceive a single N-acetylchitooctaose ((GlcNAc)8) as a dimer molecule (Hayafune et al.,
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2014). Due to the receptor pocket size constrain, only (GlcNAc)7 or (GlcNAc)8 can be
254
bound by CEBiP. Similarly, the M. incognita receptor for 6 may also function as a
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sandwich-type receptor dimer in order to impose the size limitation of the ligand.
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Endogenous 6 has been detected in several plant species, including wheat, rice,
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corn, and legumes (Jancewicz et al., 2016). 6 has been shown to be involved in various
258
aspects of plant development, and may act as a signaling molecule during stress from
259
heat, drought, salt, and oxidative stress (Jancewicz et al., 2016; Wang et al., 2019). 6 is
260
also the precursor of quinolizidine alkaloids, which are involved in defense responses
261
against insect herbivory (Bunsupa et al., 2012). Furthermore, plant has been shown to
262
actively take up 6 secreted by rhizosphere bacteria (Jancewicz et al., 2016). Here, 6 was
263
detected in both soybean and tomato root exudate, indicating that 6 is likely secreted
264
from the root tip. 2 and 5 could also be detected from root exudate, thus may also be
265
responsible for nematode attraction. Further, other compounds that function as
266
nematode attractants may also be present in root exudates, acting in conjunction with
267
these polyamines. The combined effects of these attractants, as well as other potential
268
repellents likely dictate the overall nematode behavior.
269
M. incognita may indeed use 2, 5, and 6 as one of the cues to locate potential
270
hosts. Host-finding behavior studies of RKN suggests that plant-derived compounds are
271
predicted to be able to diffuse for least 10 cm while remaining potent to attract RKN
272
(Green, 1971). 2, 5, and 6 is volatile in room temperature, and thus may function as a M.
273
incognita attractant once secreted from the root tip. The fact that 6 is associated with
274
stress suggests M. incognita may favor plants under stress, and implies RKN infection
275
may interact with other stresses as well.
276
Our work demonstrated that cadaverine (6) and other naturally-occurring
277
polyamines can act as M. incognita attractants. The role of these polyamines may be
278
further investigated not only in basic research regarding plant–parasite interactions, but
279
also for practical applications in agriculture. The development of crop varieties
280
defective in the biosynthesis of 6 may potentially be less sensitive to RKN infection.
281
Alternatively, 2, 5 and 6 may be utilized in conjunction with nematode-trapping
282
technology to redirect RKN movements, and eventually reduce RKN in the field. The
283
fact that 2, 5 and 6 are naturally-occurring chemicals makes them ideal for agricultural
284
applications, as their synthesis and extraction should be simple and effect on the
285
environment relatively benign.
286 287 288
Online methods
289
Nematode attraction assay
290
32 % Pluronic F-127 (Sigma-Aldrich) gel media was prepared based on
291
previously published protocol (Wang et al., 2009a). Approximately 7,000 J2 larvae of M.
292
incognita were thoroughly mixed with 1 ml of pluronic gel contained in a 2.5 cm well
293
in a 12-well culture plate. Compounds from the NPDepo pilot chemical library
294
(http://www.cbrg.riken.jp/npd/en/?Itemid=163) were dissolved with dimethyl sulfoxide
295
(DMSO) at 10 mg/ml. 1 µl of the chemicals were applied at the center of the well, and
296
the samples were kept at 26 °C in the dark for 20 hours. Attraction of the J2 nematodes
297
were monitored with an AxioZoom V16 dissecting microscope (Zeiss) mounted with a
298
DP74 digital camera (Olympus).
299
Freshly synthesized aminopropylamino-anthraquinone (1) was made using
300
previously described methods (Barasch et al., 1999), then degraded by exposing to
301
oxygen under fluorescent light for 233 hours.
302
To assay the RKN attraction of the polyamines, the chemicals were dissolved in
303
water (except 11 and 12 were dissolved in 250 mM pH 4 acetate buffer) at 100 mM. 1
304
µl polyamine solutions and respective solvent as negative control were each applied
305
three times to one 60 mm petri dish with 3 ml of the Pluronic gel media as described
306
above, with 20,000 RKN J2 larvae. To quantify the RKN attraction behavior, the
307
chemotaxis index was calculated with formula (1) (Margie et al., 2013):
308
ℎ =
[ # − # ] [ #]
(1)
309
310
Where #attracted and #background refer to the quantity of RKN that have gathered to
311
an attractant and the cognate negative control, respectively and #total is the total amount
312
of RKN used. The quantity of RKN was defined as the number of pixels occupied by
313
RKN in an image of the area where attractant or negative control were applied. Images
314
of the area were converted into binary, with the saturation level adjusted such that as
315
much of the nematode bodies and as little background artefacts as possible were
316
highlighted, and the number of pixels were counted using ImageJ. The values from the
317
three mucilage samples and negative controls from a single petri dish were summed to
318
be considered as one replicate. At least three replicates were performed with averages ±
319
SE shown. Data significance were determined with Tukey’s HSD test, p<0.05.
320
Preparation of soybean and tomato root exudates under sterilized condition
321
Surface-sterilized soybean and tomato seeds were sown on sterilized wet paper towels,
322
and grown for 2 and 6 days at 27°C under dark condition to prepare roots long enough
323
to be submerged. Roots of 100 soybean and tomato seedlings were dipped in distilled
324
water for 20 hours. Root exudates were then freeze-dried then re-suspended 1 ml water.
325
1 µl aliquots of the concentrated exudates were used to examine the dosage effects of
326
root exudates on nematode attraction, and mixed in to test for the presence of attraction
327
antagonists in tomato root exudates (Supplemental Fig. 4).
328
Cadaverine detection from soybean and tomato root leachate by HPLC-MS/MS
329
Soybean root exudate was centrifuged for 20 min at 2000 rpm, and the
330
supernatant was filtrated with 0.2 µm syringe filter. 10 µL of exudate was then injected
331
in the HPLC system (HPLC-8040, Shimadzu) equipped with a HILIC separation
332
column (HILICpak VC-50, Shodex, 2 mm × 150 mm). The polyamines were detected
333
by tandem mass spectrometer with electron spray ionization in positive mode.
334
Cryo-TOF-SIMS/SEM
335
Soybeans were germinated on 0.7% agar. The sample block containing the root
336
tip with surrounding agar was cut from the agar gel and immediately frozen with dry ice.
337
The frozen sample block was fixed on the copper sample holder via ice embedding for
338
the analysis. The transverse surface at the 4 mm distance from the root tip was observed
339
by cryo-TOF-SIMS/SEM. The sample preparation procedure is illustrated in
340
Supplemental Fig. 7. For the standard solution of 6, 0.1 M KCl was added to simulate
341
the chemical environment of the plant containing potassium as the most abundant
342
inorganic cation (Kirkby, 2012; Wang et al., 2013), as different ionization can be
343
induced under different chemical conditions during TOF-SIMS measurements because
344
of the “matrix effect” (Delcorte, 2012).
345
Each cryo-TOF-SIMS image was recorded for 10 minutes under the following
346
conditions: polarity, positive; primary ion, 22 keV Au+; raster size, 300 × 300 µm (256
347
× 256 pixels); mass range, m/z 0.5–1800; temperature, −120 °C. An electron gun (30 eV,
348
pulsed) was used to compensate the surface charge. Then, the soybean root sample was
349
transported to cryo-SEM and the same surface was observed under the following
350
conditions: acceleration voltage, 1.5 kV; working distance, 10 mm; temperature,
351
−120 °C. The details of the manufactured cryo-TOF-SIMS/SEM system were formerly
352
described (Kuroda et al., 2013; Masumi et al., 2014).
353
The obtained TOF-SIMS data were combined without any ion count normalization
354
using WinCadence (Ulvac-Phi Inc., ver. 5.1.2.8), MATLAB (MathWorks, Inc., ver.
355
R2014a), and PLS Toolbox (Eigenvector Research, Inc., ver. 7.5.2). Using ImageJ (The
356
National Institutes of Health, USA, ver. 1.50i), the color scale of the combined image
357
was modified. The SEM images were connected using Photoshop (CS5Ex, Adobe
358
Systems, Inc.).
359
Nematode infection test after polyamine treatment using Arabidopsis
360
Surface-sterilized and vernalized Arabidopsis (Col-0) seeds were germinated on
361
MS media containing 0.5% sucrose and 0.6% gellan gum (pH6.4) under continuous
362
light at 23°C for five days. Seedling roots were dipped in sterile water or 100mM 2, 5,
363
or 6 solutions briefly for 1~2 seconds, then incubated with M. incognita J2 larvae
364
(approximately 80 larvae per seedling) on MS media under the short-day condition (8h
365
light/ 16h dark) at 25°C. 3 mock-treated and diamine-treated seedlings are placed
366
together on one petri dish. The number of galls were counted at three days
367
post-incubation. The sum of gall numbers of 3 similarly treated seedlings from 1 petri
368
dish is considered 1 replicate, 9 technical replicates (using in total 27 seedlings) were
369
performed per experiment, 3 biological replicates were performed with similar results.
370
371
Acknowledgements
372
We thank T. Akita for help with cryo-TOF-SIMS/SEM system operation. NPDepo pilot
373
library was provided by the RIKEN BRDC through the National Bio-resource Project of
374
the MEXT, Japan. This work was supported by JSPS KAKENHI Grant Numbers
375
(25252032 and 15H01230 to KF; 16H04168 to KT; 24114009, 24370024, 16K14757,
376
17H03967, and 18H05487 to S.S.). L, P.-B. and B. F. are supported by INRA and the
377
French Government (National Research Agency, ANR) through the "Investments for
378
the Future" LABEX SIGNALIFE : program reference # ANR-11-LABX-0028-01.
379
380
Author contributions
381
M.O. and S.S. initiated the study and directed the project. S.T. and A.Y.-L.T. performed
382
nematode attraction tests. K.S. and K.T. performed LC-MS/MS to detect cadaverine
383
from root exudate. D.A., Y.M., and K.F. identified spatial cadaverine distribution by
384
cryo-TOF-SIMS/SEM. L,P.-B. and B.F. examined different nematode attraction. H.I.
385
hydrolyzed aminopropylamino-anthraquinone of chemical library and performed NMR.
386
D.A., H.I., A.Y.-L.T., and S.S. prepared the manuscript, which was revised and
387
approved by all authors.
388
389
Competing interests
390
The authors declare no competing interests.
391
392
Correspondence and requests for materials should be addressed to S. S.
393 394
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499 500 501
Figure legends
502
Figure 1. Naturally-occurring polyamines attract M. incognita. (A) Chemical
503
structures of polyamines tested in this study. (B) representative images of M. incognita
504
behavior in the presence of DMSO, 10 mg/ml aminopropylamino-anthraquinone (1)
505
from the NPDepo pilot chemical library, 10 mg/ml freshly-synthesized 1 and 10 mg/ml
506
1 exposed to oxygen. Bar = 2 mm. (C) Chemotaxis indexes of various polyamines,
507
averages N = 3 ± SE are shown, letters denote statistically significant differences
508
(P<0.05 Tukey’s HSD test). Experiment was repeated twice with similar results.
509
510
Figure 2. Cadaverine can be detected within and around soybean root using
511
cryo-TOF-SIMS/SEM. cryo-SEM image (A) and the corresponding distribution
512
patterns of total ions (B), potassium (C), phosphocholine (D) and cadaverine (6) (E) in
513
soybean root cross-section. (F) distribution of cadaverine (6) m/z 103.12 ion intensities
514
across the soybean root section as seen in (E), where x’s indicate regions outside of the
515
root where cadaverine (6) could be detected. (G-J) MS spectra of regions α, β, γ and δ
516
from (F), arrowheads denote the m/z 103.12 peak and numbers denote the magnitude of
517
the cadaverine (6) signal.
518
519
Figure 3. Diamine-dependent RKN attraction promote M. incognita infection.
520
Numbers of galls per three Col-0 seedlings that have been mock-treated or treated with
521
100 mM 1,3-diaminopropane (2), putrescine (5) or cadaverine (6) at 3 days-post
522
inoculation from M. incognita infection. Averages of n = 9 ± SD are shown, 3
523
biological replicates were performed with similar results. **P ≤ 0.01, ***P ≤ 0.001
524
compared to mock-treated seedlings.
525
526
Table 1. Amounts of polyamines detected from soybean and tomato root exudates.
527
528
Supplemental Figure 1. 1,3-diaminopropane (2) can be detected as a breakdown
529
product of aminopropylamino-anthraquinone (1). (A) HPLC elution profile of
530
aminopropylamino-anthraquinone (1) exposed to oxygen with the hypothetical
531
aminopropylamino-anthraquinone (1) degradation pathway to form 1,3-diaminopropane
532
(2);
533
aminopropylamino-anthraquinone (1) and 1,3-diaminopropane (2). (B) MS ion
dashed
arrowheads
indicate
the
peaks
representing
534
spectrum at 3.9min elution time from (A), red circle denotes the ion peak representing
535
1,3-diaminopropane (2). (C) MS ion spectrum at 15.8min elution time from (A), red
536
circle denotes the ion peak representing aminopropylamino-anthraquinone (1).
537
538
Supplemental Figure 2. Representative M. incognita behavior toward polyamines.
539
Representative images of M. incognita behavior in the presence of DMSO or 10mg/ml
540
of polyamines shown in Fig. 1C. Bar = 2 mm.
541
542
Supplemental Figure 3. Diamines attract M. incognita in a dose-dependent fashion.
543
(A) Chemotaxis index of 1,3-diaminopropane (2), putrescine (5) and cadaverine (6) at
544
100, 50, 25, 10, 5, and 1 mM. Averages of n = 3 ± SE are shown. (B) Representative
545
images of M. incognita behavior for each diamine at the indicated concentration. Bar =
546
2 mm.
547
548
Supplemental Figure 4. M. incognita attraction strengths of soybean and tomato
549
root exudates.
550
(A) Chemotaxis indexes of soybean and tomato root exudates diluted at 1/1, 1/5, 1/10,
551
1/20, 1/50, and 1/100. Averages of n = 3 ± SE are shown, **P ≤ 0.01, ***P ≤ 0.001
552
compared to tomato root exudate. (B) Representative images of M. incognita behavior
553
in the presence of root exudate samples. Bar = 2 mm. (C) Chemotaxis indexes of
554
soybean root exudates mixed with increasing amounts of tomato root exudate or water
555
added. Averages of n =7 ± SE are shown, **P ≤ 0.01 compared to control treatment.
556
557
Supplemental Figure 5. Cadaverine can be detected with the m/z 103.12 ion.
558
(A) MS spectrum of cadaverine (6) standard in KCl solution. (B-E) close-up of the m/z
559
103.12 ion peak from the MS spectra of (B) cadaverine (6) standard as shown in (A),
560
(C) agar standard, (D) soybean root, and (E) agar from the same section as the soybean
561
root from (D) not in contact with the root. Arrowheads denote the position of the m/z
562
103.12 ion peak, solid arrowheads denote the presence of (6), while dashed arrowhead
563
denote the absence of (6).
564
565
Supplemental Figure 6. Detection of 1,3-diaminopropane (2) and putrescine (5)
566
from soybean root
567
(A, B) the m/z 75.09 1,3-diaminopropane (2) ion peak (A) and the m/z 89.11 putrescine
568
(5) ion peak (B) from the MS spectra of soybean root (upper graphs), and agar from the
569
same section as the soybean root from (A) not in contact with the root (lower graphs).
570
Arrowheads denote the position of the relevant diamine ion peaks, solid arrowheads
571
denote the presence of diamine, while dashed arrowhead denote the absence of diamine.
572
(C) Detection of radial distribution of m/z 75.09 and 89.11 ion counts corresponding to
573
1,3-diaminopropane (2) (upper graph) and putrescine (5) (lower graph) in soybean root.
574
Dashed lines denote the boundaries between the root and adjacent agar.
575
576
Supplemental
577
cryo-TOF-SIMS/SEM. Images denoting the region of the soybean root tip chosen for
578
cryo-TOF-SIMS/SEM analysis in Fig. 2.
579
Figure
7.
Region
of
soybean
root
analyzed
with
580
Supplemental Table 1. Frequencies of attraction occurrence with 10mg/ml
581
cadaverine solution for M. incognita, M. enterolobii and M. arenaria
A 1,3-diaminopropane (2)
1,7-diaminoheptane (8)
1,6-diaminohexane (7)
1,8-diaminooctane (9)
spermidine (11)
1,9-diaminononane (10)
ethylenediamine (4)
cadaverine (6)
putrescine (5) 1-[(3-aminopropyl)amino]anthracene-9,10-dione (1)
propylamine (3)
spermine (12)
B
1-[(3-aminopropyl)amino]Freshly synthesized 1-[(3-aminopropyl)amino]- 1,3-diaminopropane (2) anthracene-9,10-dione (1) 1-[(3-aminopropyl)amino]- anthracene-9,10-dione (1) anthracene-9,10-dione (1) exposed to oxygen
DMSO
C Chemotaxis index
1 A
0.8
A
A 0.6 0.4 0.2 0
B
BCD CD
CD
BC
BCD BCD D
-0.2
Figure 1. Naturally-occurring polyamines attract M. incognita
Ion count 0
B
Total ion
C
[39K]+, m/z 38.96
D
[Phosphocholine]+, m/z 184.07
Max count 8/pixel
E
Cadaverine, m/z 103.12 (binary image)
Max count 4/pixel
Max count 439/pixel
Max count 37/pixel
α
100 Radial distribution of m/z 103.12 ion count X 10
β
γ
δ
X
1
300
500
1000 1500 2000 Distance from the left end (µm)
6122 cts
Region α
H
200 100
120
103
Region γ
m/z
103.2
312 cts
80
J
103
m/z
103.2
103.4
120
2500
Region β
3000
621 cts
80 40 0 102.8
103.4
40 0 102.8
Ion Counts
Ion Counts Ion Counts
Agar gel
Cryo-SEM
0 102.8
I
Vascular tissues
A
0
G
Cortex
Ion Counts
F
Ion Counts
Epidermis
100 µm
Max
120
103
Region δ
m/z
103.2
103.4
284 cts
80 40 0 102.8
103
m/z
103.2
103.4
Figure 2. Cadaverine can be detected within and around soybean root using cryo-TOF-SIMS/SEM
# of galls per 3 seedlings
7 6
Mock-treated Diamine-treated ***
5
**
** 4 3 2 1 0 Diaminopropane (2)
Putrescene (5)
Cadaverine (6)
Figure 3. Diamine-dependent RKN attraction enhances M. incognita infection.
Table 1. Amounts of polyamines detected from soybean and tomato root exudates Concentration (nM) Diaminopropane (2)
Putrescine (5)
Cadaverine (6)
Soybean
38.1±2.7
4.0±0.6
12.4±1.2
Tomato
272.3±84.4
11619.3±2323.9 465.5±149.0
2,
NH2
A
O
N
hν
O
HN
O2 HN NH
OH22,Ohν oxidation
hydrolysis
1,3-diaminopropane (2) O
O
NH
NH2
N
O2, H hν O 2
H2N
O
O
NH2
H2O
O
NH2
NH2
H2N
O
O
N
O
O
O2, hν
O
1-[(3-aminopropyl)amino]anthracene-9,10-dione (1)
B H2N
C
NH2
MS spectrum at 3.9 min
MS spectrum at 15.8 min
O HN
NH2
O
Supplemental Figure 1. 1,3-diaminopropane (2) can be detected as a breakdown product of aminopropylaminoanthraquinone (1)
propylamine (3)
ethylenediamine (4)
putrescine (5)
1,7-diaminoheptane (8) 1,8-diaminooctane (9) 1,9-diaminononane(10)
cadaverine (6)
1,6-diaminohexane (7)
spermidine (11)
spermine (12)
Supplemental Figure 2. Representative M. incognita behavior toward polyamines
A
1
Chemotaxis index
0.8
0.6
0.4 Cadaverine (6) Putrescine (5)
0.2
1-3diaminopropane (2) 0 0
20
40
80
100
120
Concentration (mM)
-0.2
B
60
1mM
5mM
10mM
25mM
50mM
100mM
(2)
(5)
(6)
Supplemental Figure 3. Diamines attract M. incognita in a dosage-dependent fashion.
A Chemotaxis index
1.2
***
1
**
0.8 0.6
Soybean
0.4
Tomato
0.2 0 0
0.2
0.4
0.6
0.8
1
1.2
Dilution factor
B
1/1
1/5
1/10
1/20
1/50
1/100
Soybean
C
Chemotaxis index
Tomato
1 0.8
**
0.6
**
**
0.4
**
**
Soybean+Water 0.2
Soybean+Tomato
0 10:0
8:2
6:4
5:5
4:6
2:8
0:10
Soybean dilution ratio Supplemental Figure 4. M. incognita attraction strengths of soybean and tomato root exudates
Cadaverine standard from (A)
102.8
Ion Counts
D
0
20
40
60 m/z
C [M+H]+
103.12
103.0 103.2 m/z
250 Soybean root 200 150 100 50 0 102.8 103.0 103.2 m/z
Ion Counts
200 150 100 50 0
[M+H]+ 103.12
103.4
E Ion Counts
Ion Counts
B
Cadaverine standard in KCl solution
10 8 6 4 2 0
Ion Counts/10
2
A
103.4
150
80
100
Agar standard 103.03
100 50 0 102.8
150
103.0 103.2 m/z
103.4
103.0 103.2 m/z
103.4
Agar
100 50 0 102.8
Supplemental Figure 5. Cadaverine can be detected with the m/z 103.12 ion
B
Ion counts
Soybean root
Soybean root
Ion counts
A 1,3-diaminopropane (2) [M+H]+ 75.0922
m/z
m/z Agar
Ion counts
Ion counts
Agar
m/z
Ion Counts
100
Ion Counts
m/z
C
100
Agar
Soybean root
m/z 75.09 1,3-diaminopropane (2)
10
Putrescine (5) [M+H]+ 89.1078
Agar
1 0
500
1000
1500
2000
2500
3000
2500
3000
m/z 89.11 putrescine (5)
10 1 0
500
1000 1500 2000 Distance from the left end (µm)
Supplemental Figure 6. Detection of 1,3-diaminopropane (2) and putrescine (5) from soybean root
Measured area
Cut Agar gel
≈ 4mm Enlarge
Supplemental Figure 7. Region of soybean root analyzed with cryo-TOF-SIMS/SEM
Supplemental Table 1. Frequencies of attraction occurrence with 10mg/ml cadaverine solution for M. incognita, M. enterolobii and M. arenaria RKN species
Number of times attraction observed
Total number of trials attempted
M. incognita
18
18
M. enterolobii
0
7
M. arenaria
0
7
S1674-2052(19)30407-1 https://doi.org/10.1016/j.molp.2019.12.010 MOLP 870
To appear in: MOLECULAR PLANT Accepted Date: 23 December 2019
Please cite this article as: Oota M., Yi-Lun Tsai A., Aoki D., Matsushita Y., Toyoda S., Fukushima K., Saeki K., Toda K., Perfus-Barbeoch L., Favery B., Ishikawa H., and Sawa S. (2020). Identification of naturally-occurring polyamines as nematode Meloidogyne incognita attractants. Mol. Plant. doi: https:// doi.org/10.1016/j.molp.2019.12.010. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. All studies published in MOLECULAR PLANT are embargoed until 3PM ET of the day they are published as corrected proofs on-line. Studies cannot be publicized as accepted manuscripts or uncorrected proofs. © 2019 The Author
1
Research Report
2
3
Identification of naturally-occurring polyamines as nematode Meloidogyne
4
incognita attractants
5 6
Morihiro Oota1, Allen Yi-Lun Tsai1, 4, Dan Aoki2, Yasuyuki Matsushita2, Syuuto
7
Toyoda1, Kazuhiko Fukushima2, Kentaro Saeki1, Kei Toda1, Laetitia Perfus-Barbeoch3,
8
Bruno Favery3, Hayato Ishikawa1*, and Shinichiro Sawa1*
9 1
10
11
860-8555, Japan 2
12
13
3
17
INRA, Université Côte d’Azur, CNRS, UMR 1355-7254 Institut Sophia Agrobiotech,
06900 Sophia-Antipolis, France 4
16
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601,
Japan.
14
15
Graduate School of Science and Technology, Kumamoto University, Kumamoto
Present address: Dormancy and Adaptation Research Unit, RIKEN Center for
Sustainable Resource Science, Yokohama 230-0045, Japan.
18
Running Title:
19
Polyamines as plant attractants of nematodes
20
21
Short Summary:
22
We have Identifid naturally-occurring polyamines, cadaverine, putrescine and
23
1,3-diaminopropane,
24
cryo-TOF-SIMS/SEM, cadaverine was indeed detected in soybean root cortex cells and
25
the surrounding rhizosphere, establishing a chemical gradient.
as
nematode
Meloidogyne
incognita
attractants.
Using
26
27
*Corresponding authors:
28
Shinichiro Sawa
29
Graduate School of Science and Technology, Kumamoto University, Kumamoto
30
Kurokami 2-39-1, Kumamoto, 860-8555, Japan
31
Telephone / fax: +81-96-342-3439
32
[email protected]
33
34
Hayato Ishikawa
35
Graduate School of Science and Technology, Kumamoto University, Kumamoto
36
Kurokami 2-39-1, Kumamoto, 860-8555, Japan
37
Telephone / fax: +81-96-342-3397
38
[email protected]
39
40
Abstract
41
Root-knot nematodes (RKN, genus Meloidogyne) are a class of plant parasites that seek
42
out and infect roots of many plant species. It is believed that RKN target certain
43
signaling molecules derived from plants to locate their hosts, however currently no plant
44
compound has been unambiguously identified as a universal RKN attractant. To address
45
this question, we screened a chemical library of synthetic compound for M. incognita
46
attractants.
47
1,3-diaminopropane, as well as related compounds putrescine and cadaverine were
48
found to attract M. incognita. After examining various polyamines, M. incognita were
49
found to be attracted specifically by natural compounds that possess three to five
50
methylene groups between two terminal amino groups. Using cryo-TOF-SIMS/SEM,
51
cadaverine was indeed detected in soybean root cortex cells and the surrounding
52
rhizosphere, establishing a chemical gradient. In addition to cadaverine, putrescine and
53
1,3-diaminopropane were also detected in root exudate by HPLC-MS/MS. Furthermore,
54
exogenously applied cadaverine is sufficient to enhance M. incognita infection of
55
Arabidopsis seedlings. These results suggest M. incognita may indeed target
56
polyamines to locate the appropriate host plants, and these naturally-occurring
Break-down
product
of
aminopropylamino-anthraquinone,
57
polyamines may have viable applications in agriculture to develop protection strategies
58
for crops from RKN infections.
59
60
Introduction
61
Plant-parasitic root-knot nematodes (genus Meloidogyne, RKN) are obligate
62
parasites that infect the roots of many plant species, including several crop plants
63
causing more than $100 billion USD-worth of agricultural damages annually. As a
64
mean to target their hosts, infective RKN J2 larvae are attracted to certain signals
65
derived from plant root tips (Torto et al., 2018; Čepulytė et al., 2018). Chemical and
66
physical cues such as carbon dioxide, pH, ions, and temperature in the rhizosphere have
67
been reported to attract or repel RKN (Rasmann et al., 2012). In addition, moisture,
68
carbohydrates, amino acids, phenolic compounds, redox potentials, chelating
69
compounds, and electrical potentials may also regulate RKN behaviors (Perry and
70
Aumann, 1998). Lastly, RKN attractants and repellents may be produced from
71
organisms in the rhizosphere, including from the RKN themselves (Manosalva et al.,
72
2015). These factors combined make the RKN J2 behavior in soil complex, and the
73
identification of chemical cues that guide RKN J2 larvae to their hosts has become a
74
significant milestone in RKN biology. However currently very few plant-derived
75
chemicals have been unambiguously shown to function as RKN attractants. The
76
fractionation of host root exudate is expected to identify the RKN attractants involved in
77
RKN behavior regulation (Perry and Aumann, 1998); however, such approach has yet to
78
definitively isolate plant-derived RKN chemoattractants (Devine and Jones, 2003). The
79
identification of RKN chemoattractants can contribute not only to the development of
80
RKN control strategies in agriculture, but also for the basic science of multicellular
81
organism interactions. To this end, we’ve decided to employ a different approach by
82
screening a chemical library to identify compounds with nematode-attracting activities,
83
then determine whether these compounds are indeed synthesized and secreted by plants.
84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
99
Results and Discussion
100
To identify compounds that attract RKN, we screened the NPDepo pilot chemical
101
library which includes 376 synthetic chemical compounds. The library compounds were
102
tested at 10 mg/mL on a Pluronic F-127-based media with mobile M. incognita J2
103
larvae
104
(aminopropylamino-anthraquinone, 1) was identified as a potential M. incognita
105
attractant (Fig. 1A,B). To confirm whether 1-mediated RKN-attraction is genuine, in
106
vitro-synthesized 1 was used to examine M. incognita response. However, freshly
107
synthesized 1 did not show any M. incognita attraction activity (Fig. 1B). We then
108
hypothesized that the biologically-active component may in fact be a decomposition
109
product derived from 1. To test this hypothesis, freshly prepared 1 was exposed to
110
oxygen under fluorescent lamp for 233 h followed by liquid chromatography-mass
111
spectrometry
112
1,3-Diaminopropane (2) was found to be produced from 1 via oxidation followed by
113
hydrolysis (Supplemental Fig. 1). We found that both oxygen-exposed 1 and
114
commercially-available 2 strongly attracted M. incognita (Fig. 1B,C). From these
(Wang
et
al.,
(LC/MS)
2009a).
and
1-[(3-aminopropyl)amino]-anthracene-9,10-dione
nuclear
magnetic
resonance
(NMR)
analysis.
115
results, alkyl groups tethered with terminal amino moiety such as in 2 appear to be the
116
key functional groups responsible for M. incognita attraction.
117
We then examined the RKN-attraction properties of other polyamines, ranging
118
from n-propylamine (3) and ethylenediamine (4) to 1,9-diaminonane (10) with
119
backbones of up to nine carbons conjugated with up to two terminal amine groups, as
120
well as spermidine (11) and spermine (12) that contain three and four amino groups,
121
respectively (Fig. 1A,C; Supplemental Fig. 2). We found that not only 2, but also
122
putrescine (5) and cadaverine (6) have significant M. incognita attraction activity (Fig.
123
1C) with chemotaxis index values of 0.58 to 0.82, respectively. Interestingly, 11 and 12
124
also showed modest attraction activities, though not as high as 2, 5 and 6 (Fig. 1C；
125
Supplemental Fig. 2). However, none of the terminal diamines with backbone shorter
126
than three carbons or longer than five carbons showed M. incognita attraction activity,
127
suggesting the size of the attractant is strictly limited from three to five carbons.
128
Interestingly, 3 did not show M. incognita attraction (Fig. 1C; Supplemental Fig. 2),
129
suggesting that two terminal amino groups are required for attraction. Thus, we are
130
confident that structurally limited diamines act selectively as M. incognita attractants. M.
131
incognita are known to be attracted by acetic acid gradients of pH 4.5-5.4 (Wang et al.,
132
2009b), and as a diamine 6 may alter the rhizosphere pH and non-specifically attract M.
133
incognita.
134
1,8-diaminooctane (9) nor 10 showed attraction activity despite all having two terminal
135
amino groups, indicating diaminoalkyl moieties affecting pH alone is not sufficient to
136
regulate M. incognita behaviors. The dosage effects of 2, 5, and 6 in M. incognita
137
attraction were also examined. 6 at as low as 5 mM was sufficient to visibly attract M.
138
incognita. However discernible colonies could only be observed in the presence of 2
139
and 5 of at least 25 mM (Supplemental Fig. 3), suggesting that 6 is the more robust
140
attractant compared to 2 and 5.
However,
neither
1,6-diaminohexane
(7),
1,7-diaminoheptane
(8),
141
To evaluate whether the affinity towards 6 is conserved in other parasitic
142
nematodes, the response of two other RKN species M. arenaria and M. enterolobii
143
towards 6 were tested. Neither M. arenaria nor M. enterolobii showed any response
144
toward 10 mg/mL 6 (Supplemental Table 1), suggesting this attraction behavior may be
145
unique to M. incognita. Despite being closely related species, M. incognita, M. arenaria
146
and M. enterolobii each have distinct host preferences (Quénéhervé et al., 2011), which
147
may partially be regulated by 6 or other natural diamines.
148
Cadaverine (6) is known as an essential diamine for normal development and
149
responses to environmental conditions in plants (Gamarnik et al., 1991; Jancewicz et al.,
150
2016). We hypothesize that 6 may be one of the molecules responsible for M. incognita
151
root attraction. To test this hypothesis, we planned to detect the presence of diamines
152
from the root exudates of soybean (Glycine max) and tomato (Solanum lycopersicum),
153
two crop plants often targeted by RKNs. 100 roots were dipped to 75 ml water for 20
154
hours to prepare root exudates. We showed that soybean and tomato root exudate both
155
attract M. incognita (Supplemental Fig. 4). By using diluted root exudates, we found
156
that soybean root exudates are more effective in attraction compared to tomato in our
157
assay system (Supplemental Fig. 4).
158
Diamine concentrations were quantified using HPLC-MS/MS. 6 was detected
159
from soybean and tomato root exudates at 12.4 nM and 465.5 nM, respectively (Table
160
1), indicating that 6 is indeed secreted from soybean and tomato root tips. The amount
161
of 6 detected in soybean root exudate may be lower than the 5 mM cut-off determined
162
earlier (Supplemental Fig. 3), however since 6 is predicted to form a gradient from the
163
root it will likely be more concentrated closer to the root. Furthermore, M. incognita
164
attraction is likely a concerted effort mediated by multiple compounds, as other
165
attractants may also be present in soybean root exudates (Oota and Sawa, unpublished
166
results). Other than 6, 2 and 5 were also detected from soybean and tomato root exudate
167
at 38.1 nM and 272.3 nM for 2, and 4.0 nM and 11.6 mM for 5, respectively (Table 1).
168
Interestingly, soybean root exudate appears to contain less diamines compared to
169
tomato, despite being the more potent M. incognita attractant. As root exudate
170
compositions are complex, it is possible that tomato root exudates contain compounds
171
that antagonize nematode attractions. To test for the presence of such attraction
172
antagonists, soybean root exudates mixed with increasing proportions of tomato
173
exudates were tested for nematode attraction activities. Soybean root exudates mixed
174
with tomato root exudate indeed show reduced attraction activities compared to samples
175
mixed with water at equivalent ratios, suggesting tomato root exudate indeed contain
176
compounds that negatively regulate nematode attraction (Supplemental Fig. 4C). These
177
compounds may either specifically counteract diamine-mediated nematode attraction, or
178
non-specifically repel nematodes in general.
179
The spatial distribution of 6 within and around the root was determined using
180
time-of-flight secondary ion mass spectrometry/scanning electron microscopy
181
(cryo-TOF-SIMS/SEM) analysis, a technique used to visualize the distribution of
182
water-soluble compounds in freeze-fixed samples at microscopic resolution level (Aoki
183
et al., 2016; Okumura et al., 2017). Here we decided to focus on soybean roots due to its
184
high attraction potency compared to tomato. First, to determine the characteristic
185
secondary ion of 6, an agar gel control and an aqueous solution of 6 in 0.1 M KCl were
186
frozen and measured using cryo-TOF-SIMS. The peak at m/z 103.12 was assigned to
187
the cadaverine-derived [M+H]+ ion and was not detected in the agar standard
188
(Supplemental Fig. 5). From these results, we conclude that cadaverine (6) distribution
189
can be visualized by detecting its m/z 103.12 ion. Similarly, we examined 2 and 5 by
190
detecting peaks at m/z 75.09 and 89.11, respectively (Supplemental Fig. 6A,B). 2 was
191
hardly detected from soybean root, probably in part due to its extreme volatile nature,
192
while 5 was indeed detected in soybean root. The semi-quantitative spatial distribution
193
of 2, 5, and 6 in soybean root was then determined using cryo-TOF-SIMS/SEM analysis.
194
Soybean roots were grown on agar, then frozen to enable the transverse sectioning of
195
the root tip and the surrounding agar to be examined (Supplemental Fig. 7). The images
196
generated using cryo-SEM (Fig. 2A) and cryo-TOF-SIMS total ion (Fig. 2B) showed
197
total ion accumulation in epidermis, cortex and vascular tissues of the transverse section
198
of the soybean root. Potassium ions ([39K]+, m/z 38.96) were detected in the entire
199
transverse section of the root, with stronger signals in the epidermal cells (Fig. 2C).
200
Some potassium ions were also detected in the agar surrounding the root. The m/z
201
184.07 ion represents the [phosphocholine]+ fragment derived from phosphatidylcholine
202
([C5H13NO4P][lipids]2) (Khan and Williams, 1977), which is a main component of
203
biological membranes in plant cells (Hussain et al., 2013). The detection of potassium
204
and phosphocholine ions confirms that the cells were alive and active at the measured
205
surface before fixation (Fig. 2C, D). Furthermore, the clear boundary between the root
206
and agar indicates that the distribution of water-soluble chemicals were freeze-fixed
207
well and visualized without any alterations.
208
The majority of 6 is distributed in the root cortex and epidermis, with lower levels
209
of 6 also present in the central vasculature (Fig. 2E,F). Thus, it is likely that 6 is
210
synthesized locally in the root cortex, and would therefore not require active transport
211
through the vasculature. We also detected significant ion counts in the agar within 150–
212
250 µm from the root (Fig. 2F-J). The ion count intensity from the agar adjacent to the
213
root was roughly one-tenth the level relative to the cortex, decreasing with the distance
214
away from the root. These findings indicate that 6 produced in the root tip is likely
215
secreted into the rhizosphere to establish a chemical gradient. Furthermore 6 is also
216
volatile, suggesting that it may disperse easily and can indeed function as a positional
217
cue for M. incognita. On the other hand, ion counts of m/z 75.09, corresponding to 2,
218
was uniformly detected as background noise, and we could not detect significant
219
presence of 2 in the rhizosphere and in root tissues using cryo-TOF-SIMS/SEM analysis
220
(Supplemental Fig. 6C). Background noise of 5 is lower and 5 was detectable in the root,
221
although a clear gradient could not be clearly observed in the rhizosphere
222
(Supplemental Fig. 6C). The molecular weight of 5 is less than that of 6, which may
223
explain why 5 was unable to maintain a gradient in the rhizosphere.
224
To further characterize this diamine-dependent M. incognita attraction, we
225
assayed nematode infection frequencies to determine whether exogenously-applied
226
diamine is sufficient to influence infection. For this purpose, we dipped Arabidopsis
227
root at five days after germination to 100 mM diamine solution, and examined M.
228
incognita infection rates. We have selected Arabidopsis for this assay because
229
Arabidopsis root does not appear to release nematode attractants, thus nematode
230
attraction and infection rates are generally lower compared to other plants.
231
Consequently, the effect of diamine enhancement of nematode infection should be more
232
pronounced in Arabidopsis. After diamine treatments, seedlings were grown on MS
233
media for three days to allow nematode infection. The number of galls in seedlings
234
treated with 2, 5, and 6 increased significantly compared to mock-treated seedlings at
235
three days post-inoculation (Fig. 3). Gall numbers in mock-treated seedlings gradually
236
caught up with diamine-treated seedlings by six to nine days post-inoculation, possibly
237
due to the applied diamines dispersing over the media, thus losing attraction potency
238
over time (data not shown). Nevertheless, these results suggest that 2, 5, and
239
6-dependent M. incognita attraction likely positively regulates M. incognita infection.
240
Here we revealed that several naturally-occurring polyamines possess M.
241
incognita attraction activity, with cadaverine (6) being the most effective. M. incognita
242
preferentially responded to the naturally-occurring 2, 5 and 6 over other synthetic
243
polyamine species. 5 and 6 are best known as bacterial by-products of tissue decay, and
244
animals respond both positively and negatively toward these diamines depending on the
245
species and context. Zebra fish (Danio rerio) trace amine-associated receptor 13c
246
(TAAR13c) indeed functions as a receptor of 5 and 6 and mediates negative chemotaxis
247
against 5 and 6 (Hayafune et al., 2014). It would be interesting to identify the structure
248
of the M. incognita diamine receptor, and determine whether it shares any similarities
249
with TAAR13c. The M. incognita attractant receptor appears to have a ligand size limit
250
for alkyl chain with three to five carbon atoms. On the other hand, the rice (Oryza
251
sativa) chitin elicitor-binding protein (CEBiP) function as a sandwich-type dimer to
252
perceive a single N-acetylchitooctaose ((GlcNAc)8) as a dimer molecule (Hayafune et al.,
253
2014). Due to the receptor pocket size constrain, only (GlcNAc)7 or (GlcNAc)8 can be
254
bound by CEBiP. Similarly, the M. incognita receptor for 6 may also function as a
255
sandwich-type receptor dimer in order to impose the size limitation of the ligand.
256
Endogenous 6 has been detected in several plant species, including wheat, rice,
257
corn, and legumes (Jancewicz et al., 2016). 6 has been shown to be involved in various
258
aspects of plant development, and may act as a signaling molecule during stress from
259
heat, drought, salt, and oxidative stress (Jancewicz et al., 2016; Wang et al., 2019). 6 is
260
also the precursor of quinolizidine alkaloids, which are involved in defense responses
261
against insect herbivory (Bunsupa et al., 2012). Furthermore, plant has been shown to
262
actively take up 6 secreted by rhizosphere bacteria (Jancewicz et al., 2016). Here, 6 was
263
detected in both soybean and tomato root exudate, indicating that 6 is likely secreted
264
from the root tip. 2 and 5 could also be detected from root exudate, thus may also be
265
responsible for nematode attraction. Further, other compounds that function as
266
nematode attractants may also be present in root exudates, acting in conjunction with
267
these polyamines. The combined effects of these attractants, as well as other potential
268
repellents likely dictate the overall nematode behavior.
269
M. incognita may indeed use 2, 5, and 6 as one of the cues to locate potential
270
hosts. Host-finding behavior studies of RKN suggests that plant-derived compounds are
271
predicted to be able to diffuse for least 10 cm while remaining potent to attract RKN
272
(Green, 1971). 2, 5, and 6 is volatile in room temperature, and thus may function as a M.
273
incognita attractant once secreted from the root tip. The fact that 6 is associated with
274
stress suggests M. incognita may favor plants under stress, and implies RKN infection
275
may interact with other stresses as well.
276
Our work demonstrated that cadaverine (6) and other naturally-occurring
277
polyamines can act as M. incognita attractants. The role of these polyamines may be
278
further investigated not only in basic research regarding plant–parasite interactions, but
279
also for practical applications in agriculture. The development of crop varieties
280
defective in the biosynthesis of 6 may potentially be less sensitive to RKN infection.
281
Alternatively, 2, 5 and 6 may be utilized in conjunction with nematode-trapping
282
technology to redirect RKN movements, and eventually reduce RKN in the field. The
283
fact that 2, 5 and 6 are naturally-occurring chemicals makes them ideal for agricultural
284
applications, as their synthesis and extraction should be simple and effect on the
285
environment relatively benign.
286 287 288
Online methods
289
Nematode attraction assay
290
32 % Pluronic F-127 (Sigma-Aldrich) gel media was prepared based on
291
previously published protocol (Wang et al., 2009a). Approximately 7,000 J2 larvae of M.
292
incognita were thoroughly mixed with 1 ml of pluronic gel contained in a 2.5 cm well
293
in a 12-well culture plate. Compounds from the NPDepo pilot chemical library
294
(http://www.cbrg.riken.jp/npd/en/?Itemid=163) were dissolved with dimethyl sulfoxide
295
(DMSO) at 10 mg/ml. 1 µl of the chemicals were applied at the center of the well, and
296
the samples were kept at 26 °C in the dark for 20 hours. Attraction of the J2 nematodes
297
were monitored with an AxioZoom V16 dissecting microscope (Zeiss) mounted with a
298
DP74 digital camera (Olympus).
299
Freshly synthesized aminopropylamino-anthraquinone (1) was made using
300
previously described methods (Barasch et al., 1999), then degraded by exposing to
301
oxygen under fluorescent light for 233 hours.
302
To assay the RKN attraction of the polyamines, the chemicals were dissolved in
303
water (except 11 and 12 were dissolved in 250 mM pH 4 acetate buffer) at 100 mM. 1
304
µl polyamine solutions and respective solvent as negative control were each applied
305
three times to one 60 mm petri dish with 3 ml of the Pluronic gel media as described
306
above, with 20,000 RKN J2 larvae. To quantify the RKN attraction behavior, the
307
chemotaxis index was calculated with formula (1) (Margie et al., 2013):
308
ℎ =
[ # − # ] [ #]
(1)
309
310
Where #attracted and #background refer to the quantity of RKN that have gathered to
311
an attractant and the cognate negative control, respectively and #total is the total amount
312
of RKN used. The quantity of RKN was defined as the number of pixels occupied by
313
RKN in an image of the area where attractant or negative control were applied. Images
314
of the area were converted into binary, with the saturation level adjusted such that as
315
much of the nematode bodies and as little background artefacts as possible were
316
highlighted, and the number of pixels were counted using ImageJ. The values from the
317
three mucilage samples and negative controls from a single petri dish were summed to
318
be considered as one replicate. At least three replicates were performed with averages ±
319
SE shown. Data significance were determined with Tukey’s HSD test, p<0.05.
320
Preparation of soybean and tomato root exudates under sterilized condition
321
Surface-sterilized soybean and tomato seeds were sown on sterilized wet paper towels,
322
and grown for 2 and 6 days at 27°C under dark condition to prepare roots long enough
323
to be submerged. Roots of 100 soybean and tomato seedlings were dipped in distilled
324
water for 20 hours. Root exudates were then freeze-dried then re-suspended 1 ml water.
325
1 µl aliquots of the concentrated exudates were used to examine the dosage effects of
326
root exudates on nematode attraction, and mixed in to test for the presence of attraction
327
antagonists in tomato root exudates (Supplemental Fig. 4).
328
Cadaverine detection from soybean and tomato root leachate by HPLC-MS/MS
329
Soybean root exudate was centrifuged for 20 min at 2000 rpm, and the
330
supernatant was filtrated with 0.2 µm syringe filter. 10 µL of exudate was then injected
331
in the HPLC system (HPLC-8040, Shimadzu) equipped with a HILIC separation
332
column (HILICpak VC-50, Shodex, 2 mm × 150 mm). The polyamines were detected
333
by tandem mass spectrometer with electron spray ionization in positive mode.
334
Cryo-TOF-SIMS/SEM
335
Soybeans were germinated on 0.7% agar. The sample block containing the root
336
tip with surrounding agar was cut from the agar gel and immediately frozen with dry ice.
337
The frozen sample block was fixed on the copper sample holder via ice embedding for
338
the analysis. The transverse surface at the 4 mm distance from the root tip was observed
339
by cryo-TOF-SIMS/SEM. The sample preparation procedure is illustrated in
340
Supplemental Fig. 7. For the standard solution of 6, 0.1 M KCl was added to simulate
341
the chemical environment of the plant containing potassium as the most abundant
342
inorganic cation (Kirkby, 2012; Wang et al., 2013), as different ionization can be
343
induced under different chemical conditions during TOF-SIMS measurements because
344
of the “matrix effect” (Delcorte, 2012).
345
Each cryo-TOF-SIMS image was recorded for 10 minutes under the following
346
conditions: polarity, positive; primary ion, 22 keV Au+; raster size, 300 × 300 µm (256
347
× 256 pixels); mass range, m/z 0.5–1800; temperature, −120 °C. An electron gun (30 eV,
348
pulsed) was used to compensate the surface charge. Then, the soybean root sample was
349
transported to cryo-SEM and the same surface was observed under the following
350
conditions: acceleration voltage, 1.5 kV; working distance, 10 mm; temperature,
351
−120 °C. The details of the manufactured cryo-TOF-SIMS/SEM system were formerly
352
described (Kuroda et al., 2013; Masumi et al., 2014).
353
The obtained TOF-SIMS data were combined without any ion count normalization
354
using WinCadence (Ulvac-Phi Inc., ver. 5.1.2.8), MATLAB (MathWorks, Inc., ver.
355
R2014a), and PLS Toolbox (Eigenvector Research, Inc., ver. 7.5.2). Using ImageJ (The
356
National Institutes of Health, USA, ver. 1.50i), the color scale of the combined image
357
was modified. The SEM images were connected using Photoshop (CS5Ex, Adobe
358
Systems, Inc.).
359
Nematode infection test after polyamine treatment using Arabidopsis
360
Surface-sterilized and vernalized Arabidopsis (Col-0) seeds were germinated on
361
MS media containing 0.5% sucrose and 0.6% gellan gum (pH6.4) under continuous
362
light at 23°C for five days. Seedling roots were dipped in sterile water or 100mM 2, 5,
363
or 6 solutions briefly for 1~2 seconds, then incubated with M. incognita J2 larvae
364
(approximately 80 larvae per seedling) on MS media under the short-day condition (8h
365
light/ 16h dark) at 25°C. 3 mock-treated and diamine-treated seedlings are placed
366
together on one petri dish. The number of galls were counted at three days
367
post-incubation. The sum of gall numbers of 3 similarly treated seedlings from 1 petri
368
dish is considered 1 replicate, 9 technical replicates (using in total 27 seedlings) were
369
performed per experiment, 3 biological replicates were performed with similar results.
370
371
Acknowledgements
372
We thank T. Akita for help with cryo-TOF-SIMS/SEM system operation. NPDepo pilot
373
library was provided by the RIKEN BRDC through the National Bio-resource Project of
374
the MEXT, Japan. This work was supported by JSPS KAKENHI Grant Numbers
375
(25252032 and 15H01230 to KF; 16H04168 to KT; 24114009, 24370024, 16K14757,
376
17H03967, and 18H05487 to S.S.). L, P.-B. and B. F. are supported by INRA and the
377
French Government (National Research Agency, ANR) through the "Investments for
378
the Future" LABEX SIGNALIFE : program reference # ANR-11-LABX-0028-01.
379
380
Author contributions
381
M.O. and S.S. initiated the study and directed the project. S.T. and A.Y.-L.T. performed
382
nematode attraction tests. K.S. and K.T. performed LC-MS/MS to detect cadaverine
383
from root exudate. D.A., Y.M., and K.F. identified spatial cadaverine distribution by
384
cryo-TOF-SIMS/SEM. L,P.-B. and B.F. examined different nematode attraction. H.I.
385
hydrolyzed aminopropylamino-anthraquinone of chemical library and performed NMR.
386
D.A., H.I., A.Y.-L.T., and S.S. prepared the manuscript, which was revised and
387
approved by all authors.
388
389
Competing interests
390
The authors declare no competing interests.
391
392
Correspondence and requests for materials should be addressed to S. S.
393 394
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499 500 501
Figure legends
502
Figure 1. Naturally-occurring polyamines attract M. incognita. (A) Chemical
503
structures of polyamines tested in this study. (B) representative images of M. incognita
504
behavior in the presence of DMSO, 10 mg/ml aminopropylamino-anthraquinone (1)
505
from the NPDepo pilot chemical library, 10 mg/ml freshly-synthesized 1 and 10 mg/ml
506
1 exposed to oxygen. Bar = 2 mm. (C) Chemotaxis indexes of various polyamines,
507
averages N = 3 ± SE are shown, letters denote statistically significant differences
508
(P<0.05 Tukey’s HSD test). Experiment was repeated twice with similar results.
509
510
Figure 2. Cadaverine can be detected within and around soybean root using
511
cryo-TOF-SIMS/SEM. cryo-SEM image (A) and the corresponding distribution
512
patterns of total ions (B), potassium (C), phosphocholine (D) and cadaverine (6) (E) in
513
soybean root cross-section. (F) distribution of cadaverine (6) m/z 103.12 ion intensities
514
across the soybean root section as seen in (E), where x’s indicate regions outside of the
515
root where cadaverine (6) could be detected. (G-J) MS spectra of regions α, β, γ and δ
516
from (F), arrowheads denote the m/z 103.12 peak and numbers denote the magnitude of
517
the cadaverine (6) signal.
518
519
Figure 3. Diamine-dependent RKN attraction promote M. incognita infection.
520
Numbers of galls per three Col-0 seedlings that have been mock-treated or treated with
521
100 mM 1,3-diaminopropane (2), putrescine (5) or cadaverine (6) at 3 days-post
522
inoculation from M. incognita infection. Averages of n = 9 ± SD are shown, 3
523
biological replicates were performed with similar results. **P ≤ 0.01, ***P ≤ 0.001
524
compared to mock-treated seedlings.
525
526
Table 1. Amounts of polyamines detected from soybean and tomato root exudates.
527
528
Supplemental Figure 1. 1,3-diaminopropane (2) can be detected as a breakdown
529
product of aminopropylamino-anthraquinone (1). (A) HPLC elution profile of
530
aminopropylamino-anthraquinone (1) exposed to oxygen with the hypothetical
531
aminopropylamino-anthraquinone (1) degradation pathway to form 1,3-diaminopropane
532
(2);
533
aminopropylamino-anthraquinone (1) and 1,3-diaminopropane (2). (B) MS ion
dashed
arrowheads
indicate
the
peaks
representing
534
spectrum at 3.9min elution time from (A), red circle denotes the ion peak representing
535
1,3-diaminopropane (2). (C) MS ion spectrum at 15.8min elution time from (A), red
536
circle denotes the ion peak representing aminopropylamino-anthraquinone (1).
537
538
Supplemental Figure 2. Representative M. incognita behavior toward polyamines.
539
Representative images of M. incognita behavior in the presence of DMSO or 10mg/ml
540
of polyamines shown in Fig. 1C. Bar = 2 mm.
541
542
Supplemental Figure 3. Diamines attract M. incognita in a dose-dependent fashion.
543
(A) Chemotaxis index of 1,3-diaminopropane (2), putrescine (5) and cadaverine (6) at
544
100, 50, 25, 10, 5, and 1 mM. Averages of n = 3 ± SE are shown. (B) Representative
545
images of M. incognita behavior for each diamine at the indicated concentration. Bar =
546
2 mm.
547
548
Supplemental Figure 4. M. incognita attraction strengths of soybean and tomato
549
root exudates.
550
(A) Chemotaxis indexes of soybean and tomato root exudates diluted at 1/1, 1/5, 1/10,
551
1/20, 1/50, and 1/100. Averages of n = 3 ± SE are shown, **P ≤ 0.01, ***P ≤ 0.001
552
compared to tomato root exudate. (B) Representative images of M. incognita behavior
553
in the presence of root exudate samples. Bar = 2 mm. (C) Chemotaxis indexes of
554
soybean root exudates mixed with increasing amounts of tomato root exudate or water
555
added. Averages of n =7 ± SE are shown, **P ≤ 0.01 compared to control treatment.
556
557
Supplemental Figure 5. Cadaverine can be detected with the m/z 103.12 ion.
558
(A) MS spectrum of cadaverine (6) standard in KCl solution. (B-E) close-up of the m/z
559
103.12 ion peak from the MS spectra of (B) cadaverine (6) standard as shown in (A),
560
(C) agar standard, (D) soybean root, and (E) agar from the same section as the soybean
561
root from (D) not in contact with the root. Arrowheads denote the position of the m/z
562
103.12 ion peak, solid arrowheads denote the presence of (6), while dashed arrowhead
563
denote the absence of (6).
564
565
Supplemental Figure 6. Detection of 1,3-diaminopropane (2) and putrescine (5)
566
from soybean root
567
(A, B) the m/z 75.09 1,3-diaminopropane (2) ion peak (A) and the m/z 89.11 putrescine
568
(5) ion peak (B) from the MS spectra of soybean root (upper graphs), and agar from the
569
same section as the soybean root from (A) not in contact with the root (lower graphs).
570
Arrowheads denote the position of the relevant diamine ion peaks, solid arrowheads
571
denote the presence of diamine, while dashed arrowhead denote the absence of diamine.
572
(C) Detection of radial distribution of m/z 75.09 and 89.11 ion counts corresponding to
573
1,3-diaminopropane (2) (upper graph) and putrescine (5) (lower graph) in soybean root.
574
Dashed lines denote the boundaries between the root and adjacent agar.
575
576
Supplemental
577
cryo-TOF-SIMS/SEM. Images denoting the region of the soybean root tip chosen for
578
cryo-TOF-SIMS/SEM analysis in Fig. 2.
579
Figure
7.
Region
of
soybean
root
analyzed
with
580
Supplemental Table 1. Frequencies of attraction occurrence with 10mg/ml
581
cadaverine solution for M. incognita, M. enterolobii and M. arenaria
A 1,3-diaminopropane (2)
1,7-diaminoheptane (8)
1,6-diaminohexane (7)
1,8-diaminooctane (9)
spermidine (11)
1,9-diaminononane (10)
ethylenediamine (4)
cadaverine (6)
putrescine (5) 1-[(3-aminopropyl)amino]anthracene-9,10-dione (1)
propylamine (3)
spermine (12)
B
1-[(3-aminopropyl)amino]Freshly synthesized 1-[(3-aminopropyl)amino]- 1,3-diaminopropane (2) anthracene-9,10-dione (1) 1-[(3-aminopropyl)amino]- anthracene-9,10-dione (1) anthracene-9,10-dione (1) exposed to oxygen
DMSO
C Chemotaxis index
1 A
0.8
A
A 0.6 0.4 0.2 0
B
BCD CD
CD
BC
BCD BCD D
-0.2
Figure 1. Naturally-occurring polyamines attract M. incognita
Ion count 0
B
Total ion
C
[39K]+, m/z 38.96
D
[Phosphocholine]+, m/z 184.07
Max count 8/pixel
E
Cadaverine, m/z 103.12 (binary image)
Max count 4/pixel
Max count 439/pixel
Max count 37/pixel
α
100 Radial distribution of m/z 103.12 ion count X 10
β
γ
δ
X
1
300
500
1000 1500 2000 Distance from the left end (µm)
6122 cts
Region α
H
200 100
120
103
Region γ
m/z
103.2
312 cts
80
J
103
m/z
103.2
103.4
120
2500
Region β
3000
621 cts
80 40 0 102.8
103.4
40 0 102.8
Ion Counts
Ion Counts Ion Counts
Agar gel
Cryo-SEM
0 102.8
I
Vascular tissues
A
0
G
Cortex
Ion Counts
F
Ion Counts
Epidermis
100 µm
Max
120
103
Region δ
m/z
103.2
103.4
284 cts
80 40 0 102.8
103
m/z
103.2
103.4
Figure 2. Cadaverine can be detected within and around soybean root using cryo-TOF-SIMS/SEM
# of galls per 3 seedlings
7 6
Mock-treated Diamine-treated ***
5
**
** 4 3 2 1 0 Diaminopropane (2)
Putrescene (5)
Cadaverine (6)
Figure 3. Diamine-dependent RKN attraction enhances M. incognita infection.
Table 1. Amounts of polyamines detected from soybean and tomato root exudates Concentration (nM) Diaminopropane (2)
Putrescine (5)
Cadaverine (6)
Soybean
38.1±2.7
4.0±0.6
12.4±1.2
Tomato
272.3±84.4
11619.3±2323.9 465.5±149.0
2,
NH2
A
O
N
hν
O
HN
O2 HN NH
OH22,Ohν oxidation
hydrolysis
1,3-diaminopropane (2) O
O
NH
NH2
N
O2, H hν O 2
H2N
O
O
NH2
H2O
O
NH2
NH2
H2N
O
O
N
O
O
O2, hν
O
1-[(3-aminopropyl)amino]anthracene-9,10-dione (1)
B H2N
C
NH2
MS spectrum at 3.9 min
MS spectrum at 15.8 min
O HN
NH2
O
Supplemental Figure 1. 1,3-diaminopropane (2) can be detected as a breakdown product of aminopropylaminoanthraquinone (1)
propylamine (3)
ethylenediamine (4)
putrescine (5)
1,7-diaminoheptane (8) 1,8-diaminooctane (9) 1,9-diaminononane(10)
cadaverine (6)
1,6-diaminohexane (7)
spermidine (11)
spermine (12)
Supplemental Figure 2. Representative M. incognita behavior toward polyamines
A
1
Chemotaxis index
0.8
0.6
0.4 Cadaverine (6) Putrescine (5)
0.2
1-3diaminopropane (2) 0 0
20
40
80
100
120
Concentration (mM)
-0.2
B
60
1mM
5mM
10mM
25mM
50mM
100mM
(2)
(5)
(6)
Supplemental Figure 3. Diamines attract M. incognita in a dosage-dependent fashion.
A Chemotaxis index
1.2
***
1
**
0.8 0.6
Soybean
0.4
Tomato
0.2 0 0
0.2
0.4
0.6
0.8
1
1.2
Dilution factor
B
1/1
1/5
1/10
1/20
1/50
1/100
Soybean
C
Chemotaxis index
Tomato
1 0.8
**
0.6
**
**
0.4
**
**
Soybean+Water 0.2
Soybean+Tomato
0 10:0
8:2
6:4
5:5
4:6
2:8
0:10
Soybean dilution ratio Supplemental Figure 4. M. incognita attraction strengths of soybean and tomato root exudates
Cadaverine standard from (A)
102.8
Ion Counts
D
0
20
40
60 m/z
C [M+H]+
103.12
103.0 103.2 m/z
250 Soybean root 200 150 100 50 0 102.8 103.0 103.2 m/z
Ion Counts
200 150 100 50 0
[M+H]+ 103.12
103.4
E Ion Counts
Ion Counts
B
Cadaverine standard in KCl solution
10 8 6 4 2 0
Ion Counts/10
2
A
103.4
150
80
100
Agar standard 103.03
100 50 0 102.8
150
103.0 103.2 m/z
103.4
103.0 103.2 m/z
103.4
Agar
100 50 0 102.8
Supplemental Figure 5. Cadaverine can be detected with the m/z 103.12 ion
B
Ion counts
Soybean root
Soybean root
Ion counts
A 1,3-diaminopropane (2) [M+H]+ 75.0922
m/z
m/z Agar
Ion counts
Ion counts
Agar
m/z
Ion Counts
100
Ion Counts
m/z
C
100
Agar
Soybean root
m/z 75.09 1,3-diaminopropane (2)
10
Putrescine (5) [M+H]+ 89.1078
Agar
1 0
500
1000
1500
2000
2500
3000
2500
3000
m/z 89.11 putrescine (5)
10 1 0
500
1000 1500 2000 Distance from the left end (µm)
Supplemental Figure 6. Detection of 1,3-diaminopropane (2) and putrescine (5) from soybean root
Measured area
Cut Agar gel
≈ 4mm Enlarge
Supplemental Figure 7. Region of soybean root analyzed with cryo-TOF-SIMS/SEM
Supplemental Table 1. Frequencies of attraction occurrence with 10mg/ml cadaverine solution for M. incognita, M. enterolobii and M. arenaria RKN species
Number of times attraction observed
Total number of trials attempted
M. incognita
18
18
M. enterolobii
0
7
M. arenaria
0
7