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. 2020 Jun 29;15(6):e0235568. doi: 10.1371/journal.pone.0235568

Undefeated—Changing the phenamacril scaffold is not enough to beat resistant Fusarium

Rasmus D Wollenberg 1, Søren S Donau 1, Manuel H Taft 2, Zoltan Balázs 1, Sven Giese 2, Claudia Thiel 2, Jens L Sørensen 3, Thorbjørn T Nielsen 1, Henriette Giese 1, Dietmar J Manstein 2, Reinhard Wimmer 1, Teis E Sondergaard 1,*
Editor: Andreas Hofmann4
PMCID: PMC7323951  PMID: 32598376

Abstract

Filamentous fungi belonging to the genus Fusarium are notorious plant-pathogens that infect, damage and contaminate a wide variety of important crops. Phenamacril is the first member of a novel class of single-site acting cyanoacrylate fungicides which has proven highly effective against important members of the genus Fusarium. However, the recent emergence of field-resistant strains exhibiting qualitative resistance poses a major obstacle for the continued use of phenamacril. In this study, we synthesized novel cyanoacrylate compounds based on the phenamacril-scaffold to test their growth-inhibitory potential against wild-type Fusarium and phenamacril-resistant strains. Our findings show that most chemical modifications to the phenamacril-scaffold are associated with almost complete loss of fungicidal activity and in vitro inhibition of myosin motor domain ATPase activity.

Introduction

Phenamacril (formerly known by the experimental code JS399-19) belongs to a novel group of single-site acting cyanoacrylate fungicides, which exerts its growth-inhibitory effect against certain members of the much-dreaded genus Fusarium [1–5]. Most notably, it is highly effective against F. graminearum [1,6], the primary cause of Fusarium head-blight (FHB), a devastating disease of cereals with profound socio-economic consequences [7,8]. Informally classified as a Fusarium-specific and an environmentally benign fungicide [9], phenamacril targets Fusarium class I myosin [2,10], a member of the ubiquitous eukaryotic myosin superfamily of molecular motor proteins that couple hydrolysis of ATP to the production of force and movement thereby facilitating such diverse and essential actin-associated processes as vesicle- and particle-transport [11,12], phagocytosis [13], septation [14] and cytokinesis [15]. In F. graminearum, the phenamacril-mediated inhibition results in mycelial growth defects and aberration of vesicle-transport [2]. This affects both essential cellular processes and more specialized tasks such as the regulation of deoxynivalenol (DON) biosynthesis and toxisome formation [12].

At the molecular level, phenamacril inhibits the ATPase activity associated with the generic myosin motor domain of F.spp. class I myosins [2,10]. While the nucleotide-binding motif is common to all P-loop nucleotide triphosphatases [16,17], four allosteric sites also constitute known binding sites for allosteric effectors of myosin ATPase activity [18–24]. We have recently shown that phenamacril is an allosteric effector and non-competitive inhibitor of the class I myosin motors from F. graminearum, F. avenaceum, and F. solani myosin-1 [25].

Since the establishment of a baseline sensitivity [1,6], several UV-induced phenamacrylic-resistant laboratory and more recently resistant field strains of Fusarium and the underlying individual amino acid mutations associated with the resistant phenotypes have been characterized [2,5,10,26,27]. In F. graminearum, the mutations confer either low (A135T, V151M, P204S, I434M, A577T, R580G/H and I581F), moderate (S418R, I424R and A577G) or high (K216R/E, S217P/L and E420K/G/D) levels of resistance [28] and mostly involve amino acid residues that are clustered deep within the actin-binding cleft, in immediate vicinity of an allosteric communication pathway responsible for the coupling of the actin and nucleotide binding sites [19,20,29]. While the qualitative resistance mechanism and the emergence of field-resistant strains [5] may render the use of phenamacril short-lived, novel cyanoacrylate derivatives based on the phenamacril-scaffold promise to provide a useful strategy and rational approach to overcome resistant genotypes and preserve the usefulness of this new class of fungicides and inhibitors of myosin ATPase activity.

In this study, we synthesized a small library of 18 cyanoacrylate compounds based on the phenamacril-scaffold and evaluated their growth-inhibitory potential against F. avenaceum, F. solani, F. oxysporum, F. verticilliodes, F. graminearum PH-1 and nine phenamacril-resistant mutants (K216E/R, S217L/P, S418R, E420K/G/D and A577G), which were generated by introducing point-mutations into the F. graminearum Myo5 locus, which encodes FgMyo1. We subsequently showed a correlation of in vivo growth inhibition with an in vitro inhibition of the ATPase activity of the purified class I myosin motor domain construct from F. graminearum PH-1, thereby demonstrating that cyanoacrylate-compounds based on the phenamacril-scaffold do de facto target Fusarium class I myosin.

While our findings demonstrate that most substitutions are associated with a significant loss of fungicidal activity, both subtle and large substitutions can sustain or slightly increase the inhibitory potency. While these structure-activity data provide novel insight into the nature of the binding-pocket, they more importantly suggest that overcoming phenamacril-resistance through iterative chemical synthesis of derivatives based on the phenamacril-scaffold is not straightforward.

Material and methods

Species and strains

Fusarium graminearum PH-1 (NRRL 31084) and Fusarium avenaceum 05001 were acquired from the Agricultural Research Service Culture Collection (ARS Culture Collection, USA) and the IBT culture collection at the Danish Technical University, respectively. Fusarium solani f. sp. pisi 77-14-4 (FGSC 9596), F. verticillioides CS7600 (FGSG_7600) and F. oxysporum f. sp. lycopersici (FGSC 9935) were retrieved from the Fungal Genetics Stock Center (FGSC, USA) [30].

Vector construction

A mutagenesis vector (pFgMyo1::hph) for targeted double-homologous recombination in the genomic Myo5 locus (FGSG_01410) was constructed through the four-fragment USER cloning procedure described in Frandsen et. al (2008) [31]. Briefly, to allow for the incorporation of the hph-marker cassette downstream of the myosin5 gene, left and right borders were amplified with uracil-containing primers and Phusion U Hot Start DNA Polymerase (Thermo Fisher Scientific, USA). The shuttle-vector pRF-HU2 was linearized with PacI (New England Biolabs, USA) and single-stranded overhangs generated by Nt.BbvCI (New England Biolabs, USA). Following gel-purification with the Qiagen gel-extraction kit (Qiagen, Germany) equimolar quantities of vector and border regions were incubated in an uracil-specific excisions reagent mix (USER-mix) (New England Biolabs, USA). Single- and di-nucleotide mutations were subsequently introduced into purified pFgMyo1::hph by the QuickChange II XL site-directed mutagenesis kit (Agilent, USA) according to manufacturer’s recommendations and using the primers listed in S1 Table in S1 File. Mutations were subsequently verified by Sanger sequencing (Eurofins Genomics, Germany).

Agrobacterium tumefaciens mediated transformation

Myosin mutagenesis vectors were electroporated into electrocompetent Agrobacterium tumefaciens LBA4404 (Thermo Fisher Scientific, USA) using a quartz cuvette (Bio-Rad, USA) and a Gene Pulser II electroporation chamber (Bio-Rad, USA) operated at 2.5 kV, 25 μF and 200 Ω. Following selection and sub-culturing on Lennox Luria-Broth agar with 20 μg·mL-1 Rifampicin and 25 μg·mL-1 Kanamycin, positive clones were inoculated into liquid IMAS media (0.18% (v/v) glucose, 0.5% (v/v) glycerol and 40% (v/v) 2.5x salt solution (3.625 g·L-1 KH2PO4, 5.125 g·L-1 K2HPO4, 0.375 g·L-1 NaCl, 1.250 g·L-1 MgSO4•7H2O, 0.165 g·L-1 CaCl2•2H2O, 0.0062 g·L-1 FeSO4•7H2O, (NH4)2SO4), 200 μM Acetosyringone (Sigma-Aldrich, USA) and 40 mM MES) and kept at 28°C and 150 RPM. At OD600 • 0.6, they were mixed 1:1 (v/v) with 1 mL 2.5·106 F. graminearum PH-1 macroconidia and distributed onto 10 IMAS agar plates with sterile 80 mm AGF220 filters. At the appearance of an even layer of mycelia (• 2–3 days), filters were transferred onto defined Fusarium agar medium (DFM) (pH 6.0 ± 0.1, 1.25% glucose, 10 mM Asparagin, 2.1 mM MgSO4, 11.2 mM KH2PO4, 7 mM KCl, 40 μg·mL-1 Na2B4O7•7H2O, 0.4 mg·mL-1 CuSO4•7H2O, 1.2 mg·mL-1 FeSO4•7H2O, 0.7 mg·mL-1 MnSO4•1H2O, 0.8 mg·mL-1 NaMoO2•2H2O and 10 mg·mL-1 ZnSO4•7H2O) with 150 μg·mL-1 Hygromycin B (Invivogen, USA), 0.3 mg·mL-1 Cefoxitin (Sigma-Aldrich, USA) with or without 10 μM Phenamacril (Aalborg University, DK). After 3–5 days of incubation at 25°C, filters were transferred onto fresh DFM plates with 150 μg·mL-1 hygromycin B and 10 μM Phenamacril, from which single-colony progeny were obtained.

Validation of chromosomal integration

Single chromosomal integration of the myosin mutagenesis-cassettes into the Myo5 locus was verified by long-read whole-genome sequencing (Oxford Nanopore Technology, Oxford, UK). Briefly, the library was prepared from ~1000–2000 ng high-molecular weight (HMW) DNA according to the Ligation Sequencing Kit (SQK-LSK108 or SQK-LSK109 for the Minion and Promethion, respectively) and the Native Barcoding Kit (EXP-NBD103) (Oxford Nanopore Technologies, UK). The libraries were loaded onto primed Flo-min6 (R9.4 chemistry) or FLO-PRO002 (R9.4.1 chemistry) flow-cells and sequenced on the MinION or Promethion (Alfa/Beta) DNA sequencers (Oxford Nanopore Technologies, UK). Reads were base-called by Albacore v.2.0.1 and then trimmed and demultiplexed in Porechop v.0.2.3. The incorporation of the individual mutations was further verified by Sanger Sequencing (Eurofins Genomics, Germany) of PCR products spanning the relevant codons. All primers are listed in S1 Table in S1 File.

NADH-coupled steady-state ATPase assay

FgMyo1 (residues 1–711) has been heterologously produced and purified in the baculovirus/Sf9 insect cell system (Thermo Fisher Scientific, USA) [25]. ATPase assays were conducted in accordance with this study and that of Furch et al. (1998) [32]. The actin-activated ATPase rates were normalized to that of the controls, which had been amended with either 0.5% ethanol or 0.5% DMSO.

Synthesis of phenamacril-derivatives

Synthesis of phenamacril and phenamacril-derivatives are thoroughly described in S1 Methods in S1 File. NMR data were recorded by dissolving each compound in CDCl3 (550 μL), with the only exception being compound 9, which for structure validation was dissolved in DMSO-d6 (500 μL). Reference spectra of Phenamacril, 1, was also recorded in DMSO-d6. For all compounds a complete set of 1H, 13C, HSQC and HMBC NMR spectra was measured at 298 K for samples in CDCl3 and 308 K for samples in DMSO-d6. All spectra were recorded on a BRUKER AVIII-600 MHz NMR spectrometer equipped with a 5 mm CPP-TCI probe.

Cyanoacrylate stocks

All derivatives were dissolved in either 96% ethanol (VWR Chemicals, DK) or DMSO (Sigma-Aldrich, USA) as 20 mM stocks.

Amended agar assays

All amended agar assays were conducted on YPG agar media pH 6.5 ± 0.1 (10 g·L-1 yeast extract, 20 g·L-1 peptone, 2.5% (w/v) D-(+)-glucose and 20 g·L-1 agar) according to Wollenberg et. al. [4]. Briefly, D-(+)-glucose, ethanol, phenamacril and phenamacril-derivatives were added aseptically to autoclaved, cooled media and poured into 5.5 cm vented petri-dishes. These were inoculated with mycelia plugs (5 mm) from actively growing cultures. The inoculated plates were grown in dark as three replicates at 25 °C in an INCU-Line incubator (VWR, Denmark) with natural convection. Growth was monitored until the controls reached the edge of the plates (2–5 days depending on the species).

In silico modeling

The crystal structure of D. discoideum myosin-1E (PDB ID 1LKX) [33] was used as template for homology modelling of the F. graminearum class I myosin motor domains in the pre-powerstroke state. The C chain of 1LKX with ADP-vanadate was energy-minimized and refined in YASARA Structure and WHAT IF ver. 17.8.15 (Yasara2 forcefield, 25 °C, TIP3P water model, 1000 ps simulation, 40 snapshots) [34]. The lowest-energy conformer was subsequently used as a template in SWISS-MODEL [35]. Following re-positioning of the ADP-vanadate, the model was refined using the above parameters.

Statistical analysis

All plate and ATPase assays were done in six replicates. Error bars denote mean ± S.D. Student’s two-tailed t test was used to compare sample means, with statistical significance p ≤ 0.05.

Results and discussion

We synthesized a total of 18 derivatives based on the phenamacril-scaffold (Fig 1). Seven of those (2–8) vary only by the alcohol group esterified to the carboxylate. Five compounds (9–13) are modified on the aromatic system, five more (14–18) feature changes to the amino group, while compound 19 has its cyano-group replaced by another ethyl ester.

Fig 1. Chemical structures of phenamacril (1) and its derivatives synthesized.

Fig 1

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Compound 16 is in rapid z/e equilibrium and occurs at 47.6% z and 52.4% e. Compounds 18a and 18b are not exchanging rapidly, but the mixture obtained after synthesis and purification was used as obtained and contained 69% 18a and 31% 18b.

It is difficult to distinguish between fungicidal and fungistatic activity working with fungicides. In the case Phenamacril, we have previous demonstrated that Phenamacril predominately works in a fungistatic way in concentrations lower than 100μM inhibiting elongation of hyphae [4].

Our results show that fungicidal activity depends critically on the alcohol moiety esterified to the carboxylic acid. (Fig 2 and S2 Fig in S1 File). Specifically, while shortening the alcohol (2) resulted in reduction in the ability to inhibit growth of wild-type F. graminearum, linearly extending the aliphatic chain (derivatives 3, 5) either slightly increased or retained most of the fungicidal activity. Longer aliphatic extensions (7 and 8) or branching (4, 6) of the aliphatic chain reduced the fungicidal potency. These observations collectively suggest that the aliphatic moiety is involved in essential and highly specific interactions of importance to the mechano-chemical mode of inhibition and that it interacts with a part of the binding-pocket unable to accommodate excessively extended or bulky (aliphatic) substituents. The effect of aromatic substitutions varied significantly. While p-Fluor substitution (10) sustained the fungicidal activity, introducing an electron-withdrawing group such as in the p-nitro derivative (9) or substituting the phenyl-moiety for a pyridine-ring (11) resulted in significant loss of fungicidal activity. Derivatives 12 and 13 further serve to illustrate that aromaticity (planarity, hydrophobicity and delocalized electro-distribution) is vital to the fungicidal activity. Compared to phenamacril, the non-potent cyclohexyl-derivative has a non-planar ring system and no π-electrons. Conversely, the large, planar, naphthyl-derivative (12) maintains an electronic and stereochemical configuration similar to that of phenamacril. It has a growth-inhibitory effect against F. graminearum, which indicates that the aromatic-moiety occupies a more exposed part of the binding pocket, in which such a relatively large substitution can be accommodated.

Fig 2.

Fig 2

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(A) The growth-inhibitory effect of phenamacril (1) and its derivatives (2–18) on F. graminearum PH-1. Plates were amended with 0–100 μM. Controls were amended with 0.5% ethanol (1–7, 10–19) or 0.5% DMSO (8 and 9). * Derivatives 8 and 9 were assayed at 0, 1, 10 and 20 μM due to low solubility. ** both (E) and (Z)-configurations are present. (B) Normalized actin-activated steady-state ATPase activity of FgMyo1. All values are normalized to the respective 0.5% ethanol (compounds 1–7, 10–19) or DMSO (8 and 9) controls. Error bars represent the standard deviations around the mean (n = 6).

N-methylation (15) and N,N-di-methylation (16) abolish the fungicidal effect but surprisingly the N-acetylated derivative (14) retains most of the growth-inhibitor effect. Donau et al. (2017) recently showed that despite an overrepresentation of phenamacril in either the (E)-configuration in the newer literature [1,3,6,9,10,36,37] or as a mixture of both the (E)- and (Z)-configuration [5], phenamacril only exists in its (Z)-configuration [38]. In the absence of the intramolecular hydrogen-bond, the electronic structure of phenamacril allows for rotation around the central “double-bond”. For derivatives 16 and 18, this resulted in the presence of both the (E)- and (Z)-configuration, thus making interpretation difficult. For the N,N-di-methylated derivative (16), the (Z)-isomer likely has an effect similar to the N-methylated version (15). The deaminated derivative (17) is similarly ineffective in inhibiting the growth of F. graminearum, which is in line with the proposed importance of the hydrogen-bond mediated intra-molecular stabilization. Derivative 18 was the only compound in which the nitrile-group of phenamacril was substituted. Replacing the nitrile group with another ethyl-ester group resulted in a complete loss of fungicidal potency. Whether this is caused by the removal of the nitrile-group, which might act as a hydrogen-bond acceptor, or is the result of introducing a bulkier substituent (cf. derivative 4 and 6) remains to be resolved. However, Wollenberg et al. [25] recently suggested that the nitrile-group occupies a pivotal tight-binding position in the proposed binding pocket, from where it also facilitates inter-molecular hydrogen-bond interactions.

The halogenated pseudilins have demonstrated that subtle chemical modifications to a myosin inhibitor can alter the myosin-class specificity of the inhibitor [19,20,39]. We therefore measured the steady-state ATPase activity of the class I myosin motor domain from F. graminearum (FgMyo1) in the presence 0–10 μM cyanoacrylate. Our results showed a general correlation between in vitro and in vivo inhibition, overall demonstrating that inhibitors based on the phenamacril-scaffold target FgMyo1 (Fig 2). Compounds (3) and (12) were slightly more potent than phenamacril (1) (p ≤ 0.05) (Fig 2B). Compared to the results from the amended agar plate assays (Fig 2A), the N-acetylated derivative (15) seemed to have less inhibitory effect against FgMyo1. Although speculative, it is possible that this compound also has some inhibitory effect against one of the myosin class II, V and XVII members present in F. graminearum.

Once we had tested the growth-inhibiting effect of F. graminearum, we then proceeded with the testing of a subset of the derivatives directed against the phenamacril-resistant F. solani and other relevant crop-pathogenic Fusarium spp. but found that the general observations made for F. graminearum are also applicable to other phenamacril-susceptible species such as F. oxysporum, F. avenaceum and F. verticilliodes (Fig 3). Although slightly affected by compounds 3 and 5, F. solani remained resistant to all compounds based on the phenamacril-scaffold. The congruence of the responses of the different Fusarium species to the inhibitors suggests an identical mechanism of inhibition.

Fig 3. The effect of a representative subset of cyanoacrylate-derivatives on the growth of the Phenamacril-resistant (A) F. solani and (B) Fusarium spp. with differential susceptibility to Phenamacril.

Fig 3

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Plates were either amended with 0.5% ethanol or DMSO (control) or 1–100 μM cyanoacrylate. * Derivatives 8 and 9 were assayed at 0, 1, 10 and 20 μM due to low solubility. ** both (E) and (Z)-configurations are present.

Resistant Fusarium strains

To assess the potential of our phenamacril-derivatives to bypass phenamacril-resistant genotypes, we used Agrobacterium tumefaciens mediated transformation to introduce point-mutations into the Myo5 locus (FGSG_01410) of the F. graminearum PH-1 wild-type genome (S1 Fig in S1 File). We chose a subset of moderate- and high-resistance conferring mutations [2,10,26], which included the S217L and S217P mutations observed in phenamacril-resistant field strains of F. fujikuroi [5]. Interestingly, except maybe for 3, none of the derivatives tested had any significant effect on the resistant strains (Fig 4). Mutants K216R and S217L were found to be more susceptible to phenamacril than previously reported [2,10,28], with EC50 values below the previously characterized A577G and S418R moderate-resistance conferring-mutations [28]. While the reason for this discrepancy remains elusive, Fig 4 more importantly shows that (our) derivatives based on the Phenamacril-scaffold are not able to tackle the resistant genotypes. The literature mentions some isolated, low-resistance mutations in the motor domain (A135T, V151M and P204S) [28], the presence of which seems to suggest that global or transmitted structural changes can influence the resistance-mechanism. However, they are presumably of less relevance as the recently reported resistant isolates carried the equivalent of the S217L and S217P mutations [5].

Fig 4.

Fig 4

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(A) The effect of phenamacril (1) and derivatives 1, 3, 4, 6, 8 and 10 on the growth of phenamacril-resistant strains of F. graminearum. Mutants are named for the resistant-conferring mutation (E420D, E420K, E420G, K216E, K216R, S217L, S418R and A577G). Wthph++ reflects a non-resistant transformant with the hph (Hygromycin B phosphotransferase) cassette inserted into the Myo5 locus (FGSG_01410). (B) Relative position of the resistance-conferring mutations in a homology model of the F. graminearum PH-1 class I myosin motor domain.

Currently, no phenamacril-FgMyo1 structure derived from X-ray diffraction crystallography is available to aid the interpretation of our findings. From one perspective, residues such as K216 in FgMyo1 (one of the amino acids associated with resistance) is known to constitute part of an allosteric communication pathway which transmits information between the nucleotide- and the actin-binding site [19,20,29,39]. These mechano-chemical events involve larger structural changes such as cleft-opening or closure and rotation of the lever arm. This could potentially affect more distal binding-sites. From another perspective, the actin-binding cleft is also known to harbor allosteric binding sites [18–21,24] and the halogenated pseudilins mediate their inhibitory effect through direct interactions with a conserved lysine residue (K216 in F. graminearum) [19,20,29,39]. This therefore speaks for a binding of phenamacril in the vicinity of the residues associated with resistance. If this is the case and in light of the loss of effect from subtle modifications to the phenamacril-scaffold (e.g. derivative 2), it is highly likely that chemical derivatization of phenamacril holds little potential to overcome phenamacril-resistance. This is consistent with our previous findings, in which phenamacril docked in the immediate vicinity of the high- and moderate resistance-conferring amino acid residues in a homology model of FgMyo1 [25]. It is however well-known that proper conclusions require screening of a substantial number of derivatives [40].

Conclusion

We successfully synthesized 18 cyanoacrylate derivatives based on the phenamacril scaffold and while this allowed us to deduce the importance of the different functional groups on phenamacril for the fungicidal potency, we found that only subtle changes could be accommodated without loss of effect. Similarly, none of the derivatives were effective against phenamacril-resistant strains of Fusarium to any significant degree, which seems to indicate that derivatization of phenamacril might not be a viable strategy for combatting these emerging resistant genotypes.

Supporting information

S1 File

(PDF)

Click here for additional data file. (1.2MB, pdf)

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

Funding was provided by The Danish Ministry of Higher Education and Science (grant no. 4005-00204B). M.H.T. was supported by the Volkswagen Stiftung, Niedersächsisches Vorab, Joint Lower Saxony-Israeli Research Projects (Grant VWZN3012) and D.J.M. by DFG grant MA1081/22.1.

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Decision Letter 0

Andreas Hofmann

27 Apr 2020

PONE-D-20-09031

Undefeated – Changing the phenamacril scaffold is not enough to beat resistant Fusarium

PLOS ONE

Dear Prof Søndergaard,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript has been reviewed by two experts in the field; please find their comments appended at the end of this email. Reviewer #2 in particular identified a number of issues. Based on the reviewers’ comments as well as my own reading of the manuscript I have decided for ‘Major Revision’ as some experiments, statistics, and other analyses are not performed at the required high technical standard and not described in sufficient detail, respectively.

Specifically, the following points needs to be addressed in a revised version of this manuscript:

1. The agar assay description in Materials and Methods needs to be improved to include all relevant environmental conditions and experimental parameters.

2. Materials and Methods as well as individual experimental section need to be updated to include appropriate description of the statistical methodologies and analyses used. The description and presentation of results needs to include relevant statistical data and critical appraisal based on those data.

3. In the Supplementary Information, the description of all chemical compounds synthesised needs to include experimental yields and purity data as well as NMR, mass spectrometric (MS) and, if available, HPLC and/or any other physico-chemical characterisation data. NMR spectral data need to be assigned, and MS data need to explained.

4. The manuscript needs to be thoroughly edited such as to provide valid referencing throughout.

5. In addition to the above points, all issues raised by the reviewers need to be addressed.

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To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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We look forward to receiving your revised manuscript.

Kind regards,

Andreas Hofmann

Academic Editor

PLOS ONE

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Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript is very well described, with clear objectives and an appropriate methodology. The results of the analysis of the new molecules show us that structural changes do not always cause improvements in biological activity / toxicity. It is very difficult to find a chemical analog with an activity superior to the base compound, however planned and rational the synthesis process may be. Some less adjustments are needed:

- Results and discussion: lines 182/183/231/233, correct: (Error! Reference source not found.).

- Line 197: lots of space.

- Lines 253/256: Missed mentioning the figure number.

- The quality of the chemical structures in figures 3 and 4 is poor.

Reviewer #2: I found the manuscript very intriguing in its hypotheses, and I’m very supportive with researches aimed at demonstrating using indirect strategies or studies. Unfortunately, I have many conceptual concerns about the present manuscript, reported as follows:

Mat&Met: The section about the agar assay lacks of the culture incubation conditions (temperature, light/dark, approximate number of incubation days). It is a key information that must be provided. Additionally, no statistical analysis of growth-inhibition data seems to have been performed, since neither information about analysis software is described nor p values are indicated in charts or their legends, while something suddenly appears in Line 232 (“were slightly more potent than phenamacril (1) (P < 0.05).”)

The chemical part is very wanting. The characterisation of the compounds has been made only by NMR and no reaction yields are reported.

Results & Discussion

In this paragraph there are at least 5 references missing (Error! Reference source not found)

An evaluation of inhibitory effect of compounds was described. Although visual data were shown as pictures of strains cultures, no measurement was conducted to numerically estimate differences in growth rate…thus, I wonder how Authors could discuss and then correlate such differences to the biological activity of compounds (for example in Line 192: “branching of the aliphatic chain reduced the fungicidal potency by ≈100-fold”).

Another consideration I would like to raise is about the “fungicide” potential of compounds: standing to a simple radial growth assay, as reported in the present work, it is not possible to distinguish a fungicidal activity from a fungistatic one. In fact, as frequently demonstrated, inhibitory effect of bioactives on fungi often rely on a strong delay of hyphae elongation, To be able to assess the difference, viability assay and biomass accumulation measurement should be done.

Minor issues:

Line 135: “w/wo” is this abbreviation allowed by the Journal?

Line 182, 188, 231, 233, 241: reference error highlighted.

Line 253, 256: figure number is missing

Figure 4: the relative position of the resistance-conferring mutations in the homology model is shown, but it is not clear if this docking analysis was performed by Author specifically for this work (if this is the case, methodological information in the proper section is missing) or if it is reported for a reader’s better comprehension only.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Daiane Flores Dalla Lana

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Jun 29;15(6):e0235568. doi: 10.1371/journal.pone.0235568.r002

Author response to Decision Letter 0

10 Jun 2020

We would like to thanks the reviewers for the comments.

Reviewer #1: The manuscript is very well described, with clear objectives and an appropriate methodology. The results of the analysis of the new molecules show us that structural changes do not always cause improvements in biological activity / toxicity. It is very difficult to find a chemical analog with an activity superior to the base compound, however planned and rational the synthesis process may be. Some less adjustments are needed:

- Results and discussion: lines 182/183/231/233, correct: (Error! Reference source not found.). Thanks, corrected.

- Line 197: lots of space. Corrected.

- Lines 253/256: Missed mentioning the figure number. Corrected

- The quality of the chemical structures in figures 3 and 4 is poor. We have made new figures 3 and 4 in better quality.

Reviewer #2: I found the manuscript very intriguing in its hypotheses, and I’m very supportive with researches aimed at demonstrating using indirect strategies or studies. Unfortunately, I have many conceptual concerns about the present manuscript, reported as follows:

Mat&Met: The section about the agar assay lacks of the culture incubation conditions (temperature, light/dark, approximate number of incubation days). It is a key information that must be provided. Additionally, no statistical analysis of growth-inhibition data seems to have been performed, since neither information about analysis software is described nor p values are indicated in charts or their legends, while something suddenly appears in Line 232 (“were slightly more potent than phenamacril (1) (P < 0.05).”)

We have added the following: These were inoculated with mycelia plugs (5 mm) from actively growing cultures. The inoculated plates were grown in dark as three replicates at 25°C in an INCU-Line incubator (VWR, Denmark) with natural convection. Growth was monitored until the controls reached the edge of the plates (2-5 days depending on the species).

And added a new paragraph: “Statistical analysis

All plate and ATPase assays were done in triplicates. Error bars denote mean ± S.D. Student's two-tailed t test was used to compare sample means, with statistical significance p > 0.05.”

The chemical part is very wanting. The characterisation of the compounds has been made only by NMR and no reaction yields are reported.

We have added all NMR spectra in supplemental materials and added the yield to the table in supplemental methods.

Results & Discussion

In this paragraph there are at least 5 references missing (Error! Reference source not found)

Corrected.

An evaluation of inhibitory effect of compounds was described. Although visual data were shown as pictures of strains cultures, no measurement was conducted to numerically estimate differences in growth rate…thus, I wonder how Authors could discuss and then correlate such differences to the biological activity of compounds (for example in Line 192: “branching of the aliphatic chain reduced the fungicidal potency by ≈100-fold”).

We agree that 100 fold is an unsuccessful term to use. It was meant that the inhibitory effect changed from 1uM to 100 uM. We have canceled the numbers.

Another consideration I would like to raise is about the “fungicide” potential of compounds: standing to a simple radial growth assay, as reported in the present work, it is not possible to distinguish a fungicidal activity from a fungistatic one. In fact, as frequently demonstrated, inhibitory effect of bioactives on fungi often rely on a strong delay of hyphae elongation, To be able to assess the difference, viability assay and biomass accumulation measurement should be done.

We completely agree and we have previous (ref. 4) been concerned about that fact (4. Wollenberg RD, Donau SS, Nielsen TT, Sorensen JL, Giese H, et al. (2016) Real-time imaging of the growth-inhibitory effect of JS399-19 on Fusarium. Pestic Biochem Physiol 134: 24-30. Based on our observation and combined with the literature, we find that Phenamacril possess both fungicidal and fungistatic effects dependent by concentration.

Minor issues:

Line 135: “w/wo” is this abbreviation allowed by the Journal? Changed

Line 182, 188, 231, 233, 241: reference error highlighted. It refers to figures because of automated track not recognized in PlosOnes system. Changed.

Line 253, 256: figure number is missing Changed

Figure 4: the relative position of the resistance-conferring mutations in the homology model is shown, but it is not clear if this docking analysis was performed by Author specifically for this work (if this is the case, methodological information in the proper section is missing) or if it is reported for a reader’s better comprehension only.

Methodological information has been added

In silico modeling

The crystal structure of D. discoideum myosin-1E (PDB ID 1LKX) (Kollmar et al., 2002) was used as template for homology modelling of the F. graminearum class I myosin motor domains in the pre-powerstroke state. The C chain of 1LKX with ADP-vanadate was energy-minimized and refined in YASARA Structure and WHAT IF ver. 17.8.15 (Yasara2 forcefield, 25�C, TIP3P water model, 1000 ps simulation, 40 snapshots) (Krieger and Vriend, 2014). The lowest-energy conformer was subsequently used as a template in SWISS-MODEL (Bordoli et al., 2009). Following re-positioning of the ADP-vanadate, the model was refined using the above parameters.

Three refs from this section have been added to the literature list.

Attachment

Submitted filename: Rev comments.docx

Click here for additional data file. (23.2KB, docx)

Decision Letter 1

Andreas Hofmann

15 Jun 2020

PONE-D-20-09031R1

Undefeated – Changing the phenamacril scaffold is not enough to beat resistant Fusarium

PLOS ONE

Dear Dr. Søndergaard,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The revised version of this manuscript has been sent to both reviewers of the original submission and reviewer #1 is satisfied with the present version. However, based on my own reading of the mansucript, I have decided for 'Major Revision', as one issue raised in the previous reviews has not been considred in the revised version of the manuscript. Additionally, there are inconsistencies regarding the statitical analyses which need to be addressed.

A revised version of this manuscript should address the following points:

1. Statistical analyses: Line 190 says n = 3, but in line 427 it says n = 6.

2. Fungicidal vs. fungistatic activity: I appreicate your response to the issue raised by reviewer #2. However, I can't see that this has point has been taken into account in the revised manuscript. If you believe that the reviewer's comment can be addressed by previous results/studies, then this needs to be discussed in the main text with appropriate literature being referenced.

3. Line 191: "Student's two-tailed t test was used to compare sample means, with statistical significance p > 0.05."

Presumably, this should be p <= 0.05.

4. Line 424: "Derivatives 8 and 9 were due low solubility assayed at 0, 1, 10 and 20 μM."

Please amend to: Derivatives 8 and 9 were assayed at 0, 1, 10 and 20 μM due low solubility.

5. Supplementary methods, compounds table: yield should be given with only one significant decimal after the floating point or as integer values.

6. Throughout (manuscript and supplemental information):

a. Please use consistent upper or, preferrably, lower case 'p' to denote the p-value.

b. Consistently add space between the number and '°C' when giving temperatures(e.g. '0 °C' instead of '0°C').

c. check spelling, in particular the name of chemicals (e.g. dichloromethane instead of Dichloromethane).

d. Variables should be set in italics, e.g. <i>t</i>-test, <i>p</i>-value, etc.

Please submit your revised manuscript by Jul 30 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Andreas Hofmann

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Jun 29;15(6):e0235568. doi: 10.1371/journal.pone.0235568.r004

Author response to Decision Letter 1

16 Jun 2020

A revised version of this manuscript should address the following points:

1. Statistical analyses: Line 190 says n = 3, but in line 427 it says n = 6. Corrected, it was six replicates

2. Fungicidal vs. fungistatic activity: I appreicate your response to the issue raised by reviewer #2. However, I can't see that this has point has been taken into account in the revised manuscript. If you believe that the reviewer's comment can be addressed by previous results/studies, then this needs to be discussed in the main text with appropriate literature being referenced.

We have added the following to results and discussion:

It is difficult to distinguish between fungicidal and fungistatic activity working with fungicides. In the case Phenamacril, we have previous demonstrated that Phenamacril predominately works in a fungistatic way in concentrations lower than 100µM inhibiting elongation of hyphae [4].

3. Line 191: "Student's two-tailed t test was used to compare sample means, with statistical significance p > 0.05."

Presumably, this should be p <= 0.05. Corrected

4. Line 424: "Derivatives 8 and 9 were due low solubility assayed at 0, 1, 10 and 20 μM."

Please amend to: Derivatives 8 and 9 were assayed at 0, 1, 10 and 20 μM due low solubility. Changed

5. Supplementary methods, compounds table: yield should be given with only one significant decimal after the floating point or as integer values. Corrected

6. Throughout (manuscript and supplemental information):

a. Please use consistent upper or, preferrably, lower case 'p' to denote the p-value. Corrected

b. Consistently add space between the number and '°C' when giving temperatures(e.g. '0 °C' instead of '0°C'). Corrected

c. check spelling, in particular the name of chemicals (e.g. dichloromethane instead of Dichloromethane). Corrected

d. Variables should be set in italics, e.g. t-test, p-value, etc. Corrected

Attachment

Submitted filename: rev comments2.docx

Click here for additional data file. (21.7KB, docx)

Decision Letter 2

Andreas Hofmann

18 Jun 2020

Undefeated – Changing the phenamacril scaffold is not enough to beat resistant Fusarium

PONE-D-20-09031R2

Dear Dr. Søndergaard,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Andreas Hofmann

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Andreas Hofmann

19 Jun 2020

PONE-D-20-09031R2

Undefeated – Changing the phenamacril scaffold is not enough to beat resistant Fusarium

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