https://orcid.org/0000-0001-5930-5763
Figures
Abstract
Plant pathogenic fungi provoke devastating agricultural losses and are difficult to control. How these organisms acquire micronutrients during growth in the host environment remains poorly understood. Here we show that efficient regulation of copper acquisition mechanisms is crucial for plant colonization and virulence in the soilborne ascomycete Fusarium oxysporum, the causal agent of vascular wilt disease in more than 150 different crops. Using a combination of RNA-seq and ChIP-seq, we establish a direct role of the transcriptional regulator Mac1 in activation of copper deficiency response genes, many of which are induced during plant infection. Loss of Mac1 impaired growth of F. oxysporum under low copper conditions and abolishes pathogenicity on tomato plants and on the invertebrate animal host Galleria mellonella. Importantly, overexpression of two Mac1 target genes encoding a copper reductase and a copper transporter was sufficient to restore virulence in the mac1 mutant background. Our results establish a previously unrecognized role of copper reduction and uptake in fungal infection of plants and reveal new ways to protect crops from phytopathogens.
Author summary
Fusarium oxysporum is the causal agent of vascular wilt disease and an opportunistic pathogen of humans. How fungal pathogens obtain micronutrients, such as metal ions, during growth inside the host remains unclear. The role of copper acquisition during fungal infection has been investigated in human pathogenic fungi but so far never in a plant pathogen. Here, we show that efficient regulation of copper uptake mechanisms, including copper reduction in the extracellular space and its internalization into the cytoplasm, is critical for effective plant colonization and virulence in F. oxysporum. Genes encoding metalloreductases and high-affinity copper transporters, whose activation is directly mediated by the copper-responsive transcription factor Mac1 and is required for growth under copper-limiting conditions, are transcriptionally induced during colonization of plant roots. Inactivation of Mac1 reduces growth of F. oxysporum during copper limitation and abolishes pathogenicity on tomato plants. Importantly, we provide evidence that the loss-of-virulence phenotype of the mac1 null mutant is strictly linked with its inability to acquire copper. Since copper is found primarily in its oxidized form in the xylem vessels of tomato plants, our work suggests that targeting copper acquisition mechanisms may represent a promising strategy for controlling diseases caused by phytopathogenic fungi.
Citation: Palos-Fernández R, Aguilar-Pontes MV, Puebla-Planas G, Berger H, Studt-Reinhold L, Strauss J, et al. (2024) Copper acquisition is essential for plant colonization and virulence in a root-infecting vascular wilt fungus. PLoS Pathog 20(11): e1012671. https://doi.org/10.1371/journal.ppat.1012671
Editor: Richard A. Wilson, University of Nebraska-Lincoln, UNITED STATES OF AMERICA
Received: June 5, 2024; Accepted: October 15, 2024; Published: November 4, 2024
Copyright: © 2024 Palos-Fernández et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All raw quantitative data and statistical analyses are provided in the file Source Data. Uncropped PCR and Southern and Western blot images are provided in the file Raw Images. RNA-seq and ChIP-seq data have been deposited in the Gene Expression Omnibus database [79] (Accession No. GSE243247 and GSE244102, respectively).
Funding: This work was supported by grants from Junta de Andalucía (ProyExcel_00488) to MSLB; from the Spanish Ministry of Science and Innovation (MICINN, grant PID2022-140187OB-I00) to MSLB and ADP and (MICINN, grants PLEC2021-007777, TED2021-130262B-I00 and PDC2022-133749-I00) to ADP; and from the Austrian Science Fund (FWF, grant P32790-B) to JS and (FWF, grant T 1266) to LSR. RPF was supported by Ph.D. fellowship FPU18/00028 from the Spanish Ministry of Universities and by the EMBO Scientific Exchange Grant 9756. MVAP was supported by the María Zambrano program to attract international talent 2021 from the Spanish Ministry of Universities and by an UCOLIDERA grant from University of Córdoba. GPP was supported by Ph.D. fellowship FPI PRE2020-092679 from MICINN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Vascular wilt fungi constitute a particularly destructive group attacking almost every crop except cereals and are extremely difficult to control [1]. The Fusarium oxysporum (Fo) species complex provokes devastating losses in global agriculture [2]. Its destructive potential is exemplified by an aggressive clone named tropical race 4 (TR4) that is currently threatening the world’s most important staple crop banana [3]. Fo infection initiates in the soil, when the fungus senses chemical signals released by roots that trigger directed hyphal growth towards the plant [4]. After entering the plant, Fo initially grows intercellularly in the root cortex and subsequently enters and colonizes the xylem vessels, causing characteristic wilt symptoms and plant death [2]. Besides provoking wilt disease in plants, Fo is also an opportunistic pathogen of humans causing symptoms ranging from superficial skin and cornea infections to lethal systemic fusariosis [5].
During the infection process, fungal pathogens compete with the host for limited nutrients and microelements. The latter include transition elements such as iron, copper or zinc, which act as essential cofactors in many cellular processes like electron transfer. All fungi have evolved mechanisms to ensure the efficient uptake and use of these metals under limiting conditions [6]. For example, adaptation to iron limitation was previously shown to be critical for fungal virulence on both plant and animal hosts [7,8].
Similar to iron, copper exists in two relevant oxidation states, Cu+ and Cu2+, acting as a cofactor for enzymes due to its potential to either accept or donate an electron while switching between the two states [9]. Moreover, copper can bind to certain proteins thereby stabilizing their conformation [10]. In the model fungus Saccharomyces cerevisiae, adaptation to copper limitation is mediated by the transcription factor Mac1 [11,12], which is conserved in most filamentous fungi [13,14]. Under conditions of copper deficiency (-Cu), Mac1 directly binds to and transcriptionally activates copper uptake genes such as those encoding metalloreductases that convert Cu2+ into Cu+, or high-affinity copper transporters that internalize Cu+ [9,13]. Mac1-mediated adaptation to copper limiting conditions was previously shown to be important for virulence in human fungal pathogens such as Aspergillus fumigatus, Cryptococcus neoformans, Histoplasma capsulatum or Candida albicans, which face copper limitation in certain host tissues such as kidney or brain [13,15–19].
The role of copper uptake during fungal infection of plant hosts has not been explored so far. Here we show that -Cu response genes such as metalloreductases and high-affinity copper transporters are markedly upregulated in Fo during colonization of tomato roots and that this upregulation is directly mediated by Mac1. We further demonstrate that reduction of Cu2+ to Cu+ and its subsequent uptake by the fungal cell are essential for Fo virulence. Our results reveal a previously unknown role of copper uptake in fungal pathogenicity on plants and suggest novel ways to control plant disease.
Results
F. oxysporum Mac1 is essential for adaptation to copper limiting conditions
A BLASTp search of the genome database of Fusarium oxysporum f. sp. lycopersici 4287 (Fol4287) using the Mac1 amino acid sequences of S. cerevisiae (YMR021C) [20] and A. fumigatus (Afu1g13190) [21] identified a single putative Mac1 ortholog, FOXG_03227, with a predicted CDS starting at position 1,808,878 of chromosome 8 (NC_030993.1). Initial inspection revealed that the protein predicted in the database was significantly shorter than the homologs from S. cerevisiae and A. fumigatus and lacked the copper-fist DNA-binding domain. Manual inspection of the DNA sequence surrounding FOXG_03227 identified an additional putative ATG start codon at position 1,808,411, giving rise to a CDS of 1,607 bp and an intron between position 33 and 103. The predicted protein has a length of 511 aa, shows 26.92% and 30.02% identity with S. cerevisiae and A. fumigatus Mac1 proteins, respectively, and contains the conserved Mac1 copper-fist DNA-binding and Cu-binding domains (S1A and S1B Fig). We therefore concluded that the newly annotated FOXG_03227 ORF corresponds to the correct mac1 gene of Fol4287. Phylogenetic analysis with characterized Mac1 proteins from different fungal species revealed that A. fumigatus Mac1 is the closest ortholog and that the orthologs of C. neoformans and Schizosaccharomyces pombe are closer to Fo Mac1 than those of C. albicans and S. cerevisiae (S1C Fig). A mac1Δ mutant was generated by replacing the complete mac1 ORF in Fol4287 with the HygR resistance cassette (S2A and S2B Fig). The mac1Δ strain was subsequently complemented in locus with a DNA construct containing the wild-type mac1 ORF fused at the 3’ end to the S-tag oligopeptide (mac1Stag) (S2C and S2D Fig).
To determine the role of Mac1 in adaptation of Fo to -Cu conditions, growth rate on solid medium and biomass production in liquid media of the mac1Δ mutant were compared to those of the wild-type and the mac1Stag complemented strain. Growth of the mac1Δ mutant was drastically impaired on -Cu solid media but was similar to that of the wild-type strain at CuSO4 concentrations starting at 10 μM, including the toxic concentration of 2 mM CuSO4. Importantly, the mac1Δ mutant was more resistant than the wild-type strain to high copper concentrations (5 mM CuSO4) (Fig 1A). The complemented mac1Stag strain displayed the same phenotype as the wild-type suggesting that the C-terminal S-tag fusion of Mac1 is fully functional. Biomass production of the mac1Δ mutant in -Cu was significantly reduced compared to the wild-type and the complemented strain. Unexpectedly, mac1Δ produced significantly more fungal biomass than the wild-type or mac1Stag strains when grown in the presence of 10 μM CuSO4 (Fig 1B).
(A) Mac1 is required for growth under copper-limiting conditions. Colony phenotypes of the wild type (wt), the deletion mutant (mac1Δ) and the in locus complemented strain (mac1Stag) after growth for the specified time on minimal medium containing 20 mM L-glutamine, pH 6.5 and trace elements lacking copper (MM+TE-Cu), supplemented with the indicated concentrations of CuSO4. Scale bar, 1 cm. (B) Fungal biomass (dry weight) obtained from the indicated strains after 16 h gemination in MM+TE-Cu supplemented (+Cu) or not (-Cu) with 10 μM CuSO4. Bars represent standard deviations (n = 3, biological replicates). p-values: ns>0.05, **<0.01, ***<0.001 versus wt under the same condition according to two-tailed unpaired Student’s t test. (C) Fold change (FC) of transcript levels of the indicated genes in the wt (left column) and the mac1Δ strain (right column) under -Cu versus +Cu conditions was measured by RNA-seq. Strains germinated 15 h at 28°C in Potato Dextrose Broth were transferred for 6 additional h to MM+TE-Cu with (+Cu) or without (-Cu) 100 μM CuSO4. Differentially expressed genes are ordered according to FC in the wt. p-value: *≤0.05 within each comparison. Data were calculated from three independent biological replicates. (D, E) Abundance of RNA-seq transcript reads of the wt (dark blue) or the mac1Δ strain (red) under -Cu conditions (RNA-seq, upper graphs); or of gDNA reads from ChIP-seq analysis in the mac1Stag strain under -Cu (grey) or +Cu (light blue) conditions (ChIP-seq, lower graphs). Data are represented as base-level coverage to two Fol4287 gene clusters, harboring the ctr3, fre9 and FOXG_07771 genes (D) or the crmC, FOXG_09770, crmA, FOXG_09772, and crmB genes (E). Genes are indicated as red boxes and putative Mac1 binding sites on each strand by black triangles.
Mac1 directly activates expression of copper limitation response genes
We next determined the role of Mac1 in the transcriptional response of Fo to -Cu conditions. RNA-seq analysis of the wild-type strain grown in the absence or presence of 100 μM CuSO4 (-Cu vs +Cu) identified 25 differentially expressed genes |Log2 Fold change| ≥ 2, p ≤ 0.05), 17 of which were upregulated and 8 of which were downregulated in -Cu conditions (Fig 1C and Table 1). Importantly, 16 of the 17 genes upregulated in -Cu failed to display a significant change of transcript levels in the mac1Δ mutant, indicating that Mac1 acts as a positive regulator of genes upregulated during copper limitation. Conversely, 5 of the 8 genes downregulated in the wild-type in -Cu were also significantly downregulated in mac1Δ (Fig 1C).
The closest orthologs in S. cerevisiae and/or A. fumigatus are indicated, together with a description of the protein encoded by each gene.
The 16 Fo genes induced under copper limitation in a Mac1-dependent manner encode known proteins involved in copper acquisition, including 3 Ctr high-affinity copper transporters (FOXG_03101, FOXG_07770, FOXG_13082) and 4 Fre metalloreductases (FOXG_07769, FOXG_18310, FOXG_11474, FOXG_13081). A BLASTp search with the predicted gene products in the genome databases of A. fumigatus, Neurospora crassa, Pyricularia oryzae and S. cerevisiae revealed that FOXG_03101 and FOXG_07770 are direct orthologs of the S. cerevisiae high-affinity copper transporters Ctr1 and Ctr3, respectively. Interestingly, FOXG_13082 encodes an additional predicted Ctr transporter with high similarity to FOXG_03101, but a shorter protein length which was named Ctr1b (S3 Fig). The three predicted Ctr copper transporters found in Fo are evolutionarily related with the Ctr1 and Ctr3 orthologs in other fungal species (S4A Fig). Interestingly, none of the four Fre metalloreductases upregulated in Fo under -Cu conditions are direct orthologs of the 8 Fre proteins reported in S. cerevisiae or of the FreB reductases previously annotated in P. oryzae and N. crassa (S4B Fig). We therefore named these proteins Fre9, 10, 11 and 12, with numbers ranked according to their respective transcript levels in -Cu. Among these newly annotated metalloreductases, only Fre12 is phylogenetically close to one of the previously annotated Fre proteins from S. cerevisiae (Fre7), whereas the others appear to be unique to filamentous fungi. Fre9 has one ortholog in A. fumigatus and N. crassa and two orthologs in P. oryzae, respectively, while Fre10 has a predicted ortholog in A. fumigatus, N. crassa and P. oryzae (S4B Fig). For Fre11 and Fre12, only one ortholog was found in P. oryzae and A. fumigatus, respectively. In addition to the high-affinity copper transporters and metalloreductases, the Fo genes induced under copper limitation in a Mac1-dependent manner include sod3 encoding a Mn-dependent cytosolic superoxide dismutase, crmB encoding an alcohol-O-acetyltransferase, and crmC encoding a siderophore iron transporter (Fig 1C and Table 1).
To identify the Mac1 binding sites in the Fo genome, chromatin immunoprecipitation coupled with Next-Generation sequencing (ChIP-seq) was performed by growing the mac1Stag strain under the same -Cu and +Cu conditions employed in the RNA-seq experiment. Using a monoclonal anti-S-tag antibody for ChIP, we identified 12 putative Mac1-binding regions in -Cu conditions whereas no Mac1-DNA interaction was detected in +Cu conditions. A comparison of these identified Mac1 binding sites defined the putative consensus DNA binding sequence of Fo Mac1 as 5’-DHNTGCTCANNN-3’ (D = A, G, or T; H = A, C, or T; N = any nucleotide) (S5A Fig), similar to the sequence 5’-TTTGCTCA-3’ identified in other fungi such as S. cerevisiae and C. albicans [22,23].
The 12 Mac1-binding sites identified in the Fo genome map to the promoter regions of 16 genes, most of which are part of 5 gene clusters: 1) a cluster composed of the two divergently transcribed genes ctr3, fre9 and the gene FOXG_07771, with at least one predicted Mac1-binding site in each promoter region (Fig 1D); 2) a predicted biosynthetic gene cluster composed of 5 genes: crmA encoding an isocyanide synthase-non ribosomal peptide synthase (ICS-NRPS)-like enzyme, crmB, crmC, FOXG_09770 encoding a transferase family protein and FOXG_09772 encoding a hydrolase, showing up to three putative Mac1-binding sites in each promoter except for the shared promoter of the divergently transcribed genes FOXG_09772 and crmB (Fig 1E); 3) and 4) two clusters, each composed of two divergently transcribed genes sharing a common promoter region with multiple Mac1-binding sites (fre10/FOXG_02393 and fre12/ctr1b) (S5B and S5C Fig); and 5) a gene cluster composed of FOXG_18820 and sod3, which are transcribed in the same direction, containing a single Mac1-binding site between the two genes located on the opposite DNA strand (S5D Fig). Additionally, ChIP-seq identified 3 unclustered genes that are significantly upregulated in -Cu: ctr1a, FOXG_01130, and fre11, all of which contain Mac1-binding sites in their promoter regions (S5E–S5G Fig). Importantly, all the genes identified by ChIP-seq to be bound by Mac1 were also found by RNA-seq to be upregulated during -Cu, including FOXG_09770, crmA, FOXG_09772 and FOXG_18820 which were excluded from the main list due to their high variability between the experimental repeats (Fig 1C and Table 1).
Mac1 is highly stable and localizes to the nucleus independently of copper status
In S. cerevisiae, Mac1 is rapidly degraded upon a shift from copper limitation to sufficiency [24]. To follow mac1 expression and Mac1 protein stability in Fo, the mac1Stag strain was transferred from -Cu to +Cu conditions and total RNA and protein extracts were subjected to real-time RT-qPCR and to immunoblotting with a monoclonal anti-S-tag antibody, respectively. While transcript levels were approximately halfway down 1 h after the shift, Mac1 protein levels remained unchanged even 3 h after copper addition (Fig 2A and 2B). Furthermore, protein levels in -Cu conditions remained stable after addition of the translation inhibitor cycloheximide (chx) [25] during the entire duration of the experiment, suggesting that Mac1 turnover in Fo is much lower than in S. cerevisiae [24] (Fig 2C). Collectively, these results suggest that Fo Mac1 is a highly stable protein whose activity is not primarily controlled by protein degradation.
(A) Transcript levels of mac1 in the mac1Stag strain were measured by real-time RT-qPCR before and at the indicated time points after adding 20 μM CuSO4 (sCu) to a culture in MM-TE-Cu (-Cu) medium and expressed relative to those in -Cu. Bars represent standard deviations (n = 3, biological replicates). p-values: *<0.05, **<0.01 versus -Cu according to two-tailed unpaired Student’s t test. (B, C) Protein samples obtained from the mac1Stag strain grown as described in (A) were subjected to SDS-PAGE and immunoblot analysis with anti-S-tag antibody (S-tag). Anti-α-Tubulin (α-Tub) was used as loading control. Left panels: Representative immunoblot showing Mac1 protein levels. Right panels: Densitometric quantification of Mac1 protein levels normalized to those of α-Tubulin and expressed relative to the -Cu condition. In (C), -Cu cultures were supplemented with 50 μg/ml cycloheximide (chx), instead CuSO4, to determine Mac1 turnover rate. Bars represent standard deviations (n = 3, biological replicates). p-values: ns>0.05 versus -Cu according to two-tailed unpaired Student’s t test. (D) Subcellular localization of Mac1clover was monitored after germinating the mac1clover strain for 16 h at 28°C in MM-TE-Cu supplemented either with 100 μM (+Cu) or 2 μM CuSO4 (-Cu). For the copper shift experiment (sCu), 20 μM CuSO4 was added to the -Cu samples 10 min before imaging. Fungal nuclei were stained with Hoechst 33342. Hyphae were imaged using differential interference contrast (DIC), green fluorescence (mClover3) or blue fluorescence filters (Hoechst). The three images were merged using ImageJ v1.8. Scale bar, 25 μm.
In A. fumigatus and S. pombe, Mac1 was reported to be translocated outside of the nucleus under +Cu conditions [26,27]. To follow the subcellular localization of Mac1 in Fo, the mac1Δ mutant was transformed with a DNA construct carrying the mac1 gene with a C-terminal fusion to the green fluorophore mClover3, driven by the constitutive Aspergillus nidulans gpdA promoter (S6A and S6B Fig). Colony growth phenotypes in -Cu and transcriptional induction of the ctr3 and fre9 genes of the mac1clover strain were similar to those of the wild-type suggesting that the fluorescent tag does not interfere with Mac1 function (S6C and S6D Fig). Next, we performed fluorescence microscopy studies with the mac1clover strain germinated either in -Cu or +Cu conditions or submitted to a shift from -Cu to +Cu (sCu). In contrast to previous reports in A. fumigatus and S. pombe, Fo Mac1clover colocalized with the nuclear stain Hoechst 33342 indicating that Fo Mac1 is continuously present in the nucleus independent of the copper status (Fig 2D).
Copper limitation response genes are upregulated during plant infection in a Mac1-dependent manner
To study the role of Mac1 in regulation of copper response genes during plant infection, we performed RNA-seq of tomato roots inoculated with the wild-type strain or the mac1Δ mutant, either at 2 or 6 days post inoculation (dpi). We first compared transcript levels of the wild-type strain during root infection to those in axenic culture under +Cu conditions and found that several copper limitation response genes were significantly upregulated during tomato plant infection (Figs 3A and S7A). These include FOXG_17215 and fre11 which were upregulated at 2 dpi, FOXG_02393 which was upregulated at 6 dpi, and fre10 and ctr1a which were upregulated at both infection time points. We also identified three genes that were significantly downregulated in planta, crmB, FOXG_07771 and FOXG_06143. Importantly, none of the genes induced in planta in the wild-type were upregulated in the mac1Δ mutant except FOXG_17215 (Fig 3B). Furthermore, 6 additional -Cu response genes were downregulated in the mac1Δ mutant compared to the wild-type in planta: ctr1b, ctr3, FOXG_01130, FOXG_07771, FOXG_08010 and fre9 (Fig 3B). Taken together, these results suggest that Fo faces copper limitation conditions in tomato roots and establish a key role of Mac1 in transcriptional activation of copper deficiency response genes during plant infection.
(A and B) Fold change (FC) of transcript levels of the indicated genes during infection of tomato roots by the wt at 2 (left column) or 6 (right column) days post inoculation (dpi) versus wt in axenic culture under +Cu conditions (A); or during infection of tomato roots by the mac1Δ strain at 2 (left column) or 6 (right column) dpi versus the wt at the same time points (B) was measured by RNA-seq. Differentially expressed genes are in the same order as in Fig 1C. Genes upregulated both under copper limitation and plant infection conditions are in bold. p-value: *≤0.05 within each comparison. Data were calculated from three independent biological replicates. (C, D) Kaplan-Meier plot showing survival of groups of 10 tomato plants (cv. Moneymaker) inoculated by dipping the roots into a suspension of 5x106 microconidia/ml of the indicated fungal strain or water (Mock), planted in minipots and irrigated either with water (C) or with a 10 μM CuSO4 solution (D). Data shown are from one representative experiment. Experiments were performed at least three times with similar results. p-values: ***<0.001, ****<0.0001 versus the wt according to Log-rank (Mantel-Cox) test.
Mac1 is essential for virulence of Fusarium oxysporum on tomato plants
To determine the role of Fo Mac1 during plant infection, roots of tomato plants were dip-inoculated with microconidia of the different fungal strains, planted in minipots, and supplied either with water or with a solution of 10 μM CuSO4. Under both irrigation regimes, the plants inoculated with the wild-type or the complemented strain showed mortality rates close to 100% at 25 dpi whereas those inoculated with the mac1Δ mutant showed no visible disease symptoms (Fig 3C and 3D). Using a plate invasion assay [28], we found that the mac1Δ mutant was still able to penetrate across a cellophane membrane (S7B Fig). We conclude that Mac1 plays a key role in pathogenicity of Fo on tomato plants which is independent of invasive growth and cannot be rescued by exogenous application of copper to the roots.
Overexpression of a copper transporter and a metalloreductase in the mac1Δ mutant rescues growth under copper limitation and virulence on tomato plants
We hypothesized that the essential role of Mac1 during plant infection could be related to the acquisition of copper during growth of Fo in the tomato root cortex and xylem. To test this idea, we first generated single and double deletion mutants in the high-affinity copper transporters Ctr1a and/or Ctr3 (S8A–S8E Fig). In S. cerevisiae and Aspergillus, Ctr1 and Ctr3 are functionally redundant but double deletion mutants have a severe growth phenotype in -Cu conditions [15,29–31]. Here we found that growth of the Fo ctr3Δ and ctr1aΔ single mutants and of the ctr3Δctr1aΔ double mutant under -Cu conditions was indistinguishable from that of the wild-type strain (S8F Fig). In line with this result, no reduction in virulence on tomato plants was observed in the single or the double mutants (S8G Fig). These results suggest the occurrence of functional redundancy in the high-affinity Ctr copper transporters of Fo, possibly due to the presence of the additional Ctr1 paralog Ctr1b (S3 and S4 Figs).
We next asked whether Mac1-independent expression of copper deficiency response genes in the mac1Δ mutant could rescue growth under -Cu conditions. This strategy is based on previous work in Aspergillus, where overexpression of the high-affinity copper transporters CtrA2 or CtrC (orthologs of S. cerevisiae Ctr1 and Ctr3, respectively) in a mac1Δ background partially restored growth during copper limitation [15,29]. To test this idea, we generated transformants in the mac1Δ mutant background overexpressing either the copper transporter ctr3 alone (mac1Δctr3OE) or both ctr3 and the metalloreductase fre9 (mac1Δctr3OEfre9OE) from the constitutive A. nidulans gpdA promoter (Figs 4A and S9A–S9C). The ctr3 and fre9 genes were chosen for co-expression because they are divergently transcribed from a promoter containing 3 predicted Mac1 binding sites and are both upregulated in -Cu conditions and during plant infection in a Mac1-dependent manner (Figs 1C, 1D, 3A and 3B). Successful overexpression of ctr3 and fre9 in the mac1Δ background was confirmed in two independent mac1Δctr3OE and mac1Δctr3OEfre9OE transformants, respectively. RT-qPCR showed that transformants mac1Δctr3OE #4 and mac1Δctr3OEfre9OE #2 exhibited markedly increased ctr3 transcript levels in -Cu conditions compared to mac1Δ, which were similar or even higher than those of the wild-type strain (S9D Fig). Furthermore, fre9 transcript levels in the mac1Δctr3OEfre9OE #2 and #4 transformants were similar to those of the wild-type strain and significantly higher than those of the mac1Δ mutant (S9E Fig). We next tested growth of these overexpressing strains on plates containing different levels of copper. While overexpression of ctr3 alone was not sufficient to rescue growth of the mac1Δ mutant under -Cu conditions, simultaneous overexpression of ctr3 and fre9 restored growth in -Cu close to wild-type levels, particularly in the mac1Δctr3OEfre9OE #2 transformant. In addition, all the transformants, and particularly those overexpressing both genes, were more sensitive to toxic copper levels than the wild-type strain (Fig 4B). This result strongly suggest that reduction of Cu2+ to Cu+ by plasma membrane metalloreductases such as Fre9 is essential for efficient copper acquisition by Fo. In line with this, root infection assays revealed a direct correlation between the growth phenotype of the different strains in -Cu conditions and their ability to cause mortality on tomato plants. While the mac1Δctr3OE transformants were only slightly more virulent than the mac1Δ mutant, the mac1Δctr3OEfre9OE strains were as virulent as the wild-type (Fig 4C). Furthermore, fungal biomass at 10 dpi in stems of tomato plants inoculated with the mac1Δctr3OEfre9OE #4 strain was not significantly different from that of the wild-type or the complemented mac1Stag strain and significantly higher compared to plants inoculated with the mac1Δ mutant (Fig 4D). Similar to the wild-type strain, at 4 dpi the mac1Δctr3OEfre9OE #4 strain labelled with the green protein mClover3 (S10 Fig) was abundantly growing in the root cortex and reached the vascular bundles of the xylem, in contrast to the mac1Δ mutant whose growth was more sparse and remained restricted to the outermost layers of the root cortex (Figs 4E and S11). Collectively, these findings confirm that Mac1-dependent reduction and uptake of extracellular copper is essential for the progression of Fo from the root cortex to the vascular system and for virulence on tomato plants and demonstrate that the loss-of-virulence phenotype of the mac1Δ mutant is strictly associated with the inability to acquire copper.
(A) Schematic representation of the strategy used for overexpression of the high affinity copper transporter ctr3 alone or in combination with the metalloreductase fre9 in the mac1Δ mutant driven by the constitutive Aspergillus nidulans gpdA promoter. (B) Colony phenotypes of the indicated strains after growth (2 or 7 d) on MM+TE-Cu supplemented with the specified concentrations of CuSO4. The colonies of the wt and mac1Δ are the same as those shown in Fig 1A and are repeated here for clarity. Scale bar, 1 cm. (C) Kaplan-Meier plot showing survival of groups of 10 tomato plants (cv. Momotaro) inoculated by dipping the roots into a suspension of 5x106 microconidia/ml of the indicated fungal strain or water (Mock). Data shown are from one representative experiment. Experiments were performed at least two times with similar results. p-values: ***<0.001, ****<0.0001 versus the wt according to Log-rank (Mantel-Cox) test. (D) Fungal burden in tomato plants inoculated with the indicated strains was measured by qPCR of the Fol4287-specific six1 gene using total DNA extracted from roots (left panel) or stems (right panel) of plants at 4 or 10 dpi. Fungal burden was calculated using the 2-ΔΔCt method and normalized to the tomato gapdh gene. Bars represent standard deviations (n = 3, biological replicates). p-value: ns>0.05, *<0.05 versus the wt in the same condition according to two-tailed unpaired Student’s t test. (E) Micrographs showing fungal colonization of tomato roots (cv. Momotaro) dip-inoculated with the indicated F. oxysporum strains expressing 3XFo-mClover3 or water (Mock) at 4 dpi. Fungal fluorescence (mClover3, green) is overlaid with propidium iodide staining of the plant cell wall (PI, magenta). The two images were merged using ImageJ v1.8. The images shown are representative of at least three lateral secondary roots from six different tomato plants. Scale bar, 50 μm.
Mac1-mediated copper uptake is required for virulence of F. oxysporum on an animal host
In the human fungal pathogens A. fumigatus, C. albicans or C. neoformans, loss of Mac1 leads to decreased virulence on animal hosts [15,17,18]. Because Fo can also cause disseminated infections in humans [5], we tested the virulence phenotype of the mac1Δ mutant on the wax moth Galleria mellonella, an invertebrate model that is widely used to study microbial pathogens of humans including Fol4287 [32,33]. All larvae inoculated with the wild-type or the mac1Stag strain were dead at 2 dpi while most larvae inoculated with mac1Δ remained alive at 5 dpi (S12A Fig). Importantly, the ability to cause mortality on G. mellonella was rescued to wild-type levels in the two mac1Δctr3OEfre9OE transformants (S12B Fig), demonstrating that Mac1-dependent copper acquisition is required for full virulence of F. oxysporum on this animal host.
Discussion
Copper is a crucial micronutrient for all living organisms, including plants and microbes, and plays a key role in a variety of physiological processes [9]. However, high copper concentrations are toxic, and copper is widely used as a fungicide. Copper-based fungicides, such as the well-known Bordeaux mixture, have been used for more than a century in agriculture, although concerns about its toxicity have been raised almost from the beginning [34]. Moreover, the presence of high concentrations of this metal ion in the environment could lead to the selection of organisms evolved for copper resistance [35,36]. In this context, and considering that targeting metal ion acquisition mechanisms has been proposed as a useful method to control fungal pathogens [8,37–39], we have investigated, for the first time, the relevance of the transcriptional response to copper limitation in the virulence of a fungal plant pathogen.
The copper-sensing transcription factor Mac1 was identified more than thirty years ago in S. cerevisiae [20], in which copper homeostasis has been studied in detail. Mac1 activates the transcription of genes encoding metalloreductases and high-affinity copper transporters which are required for efficient copper acquisition under copper limitation, and its loss provokes a severe phenotype in -Cu conditions [9]. While a role of Mac1 in fungal virulence on animal hosts has been demonstrated [13,15–19]; its role during plant infection has not been studied before.
Here we functionally characterized Mac1 in Fo, an important fungal phytopathogen. The structural organization of Fo Mac1 is similar to its orthologs in other fungi, with an N-terminal copper fist DNA-binding domain containing the characteristic C, RGHR and GRP residues, and two C-terminal Cys-rich motifs. Furthermore, the inability of the Fo mac1Δ mutant to grow in -Cu conditions confirms that the function of Mac1 is highly conserved across different fungi, which is in line with a previous study showing that the mac1 gene from A. fumigatus can functionally complement a mac1Δ mutant of S. cerevisiae [21]. By contrast, we found that the post-translational regulation of Mac1 activity in Fo differs from that reported in other fungi where inactivation under copper sufficiency occurs mainly by protein degradation, analogous to what has been reported for the iron-responsive transcription factor HapX [40], and/or cytoplasmic retention. In S. cerevisiae, Mac1 is stable at low copper concentrations but rapidly degraded at concentrations above 10 μM CuSO4 [24]. Furthermore, Mac1 in A. fumigatus and S. pombe localizes to the nucleus in -Cu but is present in the cytoplasm during copper sufficiency [26,27]. Here we found that Fo Mac1 has a low turnover rate, is highly stable and remains predominantly localized in the nucleus during +Cu, suggesting that its activity is not regulated by protein degradation or cytoplasmic retention. The most likely explanation for the high Mac1 DNA binding specificity under -Cu conditions is a mechanism previously proposed in S. pombe [27], where copper induces a conformational change in the protein that promotes physical interaction between the N-terminal DNA-binding domain and a Cys-rich motif thereby preventing binding of the transcription factor to its target sites in the genome.
Using a combination of RNA-seq and ChIP-seq, we detected direct DNA binding of Fo Mac1 only under -Cu conditions and found that it transcriptionally activates a group of genes involved in copper acquisition (Ctr copper transporters and Fre reductases), reactive oxygen species (ROS) detoxification (Sod3), and production of isocyanine metabolites (Crm gene cluster). In other fungi, Mac1 also was shown to activate copper acquisition genes, including the orthologs of S. cerevisiae ctr1 and ctr3 [9,13]. In addition, Fo has a third high affinity copper transporter, ctr1b, which is also present in the rice blast fungus P. oryzae and is transcriptionally induced by Mac1 during -Cu conditions. Our finding that the ctr1/ctr3 double mutant is still able to grow under -Cu conditions strongly suggests that Ctr1b is functionally redundant with Ctr1a and Ctr3.
In S. cerevisiae, the two copper reductases Fre1 and Fre7 are transcriptionally induced during -Cu in a Mac1-dependent manner [41]. Interestingly, the direct fre1 and fre7 orthologs were not upregulated in Fo during -Cu. Instead, we found 4 Fre reductases that were direct targets of Fo Mac1, which have no close orthologs in S. cerevisiae and appear to be specific for filamentous fungi. Two of these genes, fre9 and fre12, are clustered and divergently transcribed with the high-affinity copper transporters ctr3 and ctr1b, respectively, suggesting that they are functionally related. Apart from the genes involved in copper acquisition, we found that Fo Mac1 directly activates sod3 encoding a cytosolic Mn-dependent superoxide dismutase. In C. albicans, Sod3 was shown to be important for the maintenance of ROS homeostasis under -Cu conditions, due to the reduced activity of the most abundant cytosolic superoxide dismutase Sod1, which requires copper as cofactor [18]. Moreover, in Aspergillus sod1 expression is downregulated under -Cu conditions [42]. In line with this, our RNA-seq data show significantly reduced sod1 transcript levels in -Cu conditions. Furthermore, several genes in the crm biosynthetic gene cluster of Fo were identified as direct Mac1 targets in Fo. The crm cluster was recently shown to be induced under copper starvation in A. fumigatus and is conserved in a wide range of pathogenic and non-pathogenic fungi. It encodes, among others, an isocyanide synthase that contributes to two distinct biosynthetic pathways whose final products have antibacterial properties [43,44]. Importantly, several of the direct Mac1 targets in Fo were previously reported to contribute to virulence in human fungal pathogens [13,43].
Our results demonstrate for the first time an essential role of Mac1-mediated copper uptake during plant infection by a fungal pathogen. Based on the finding that exogenous addition of 10 μM CuSO4, a concentration sufficient to rescue growth of the mac1Δ mutant on plates, failed to restore virulence on tomato plants, we initially hypothesized that Fo Mac1 could directly or indirectly regulate virulence-related genes other than those related to copper limitation. However, RNA-seq analysis during tomato root infection demonstrated that the transcripts downregulated in mac1Δ versus wild-type in planta all correspond to genes induced under copper starvation, most of which are directly related to copper acquisition. Together with the finding that simultaneous overexpression of the high-affinity copper transporter Ctr3 and the metalloreductase Fre9, but not of Ctr3 alone, rescues growth in -Cu conditions and virulence of the mac1Δ mutant, our results strongly indicate that both extracellular copper reduction as well as copper uptake are essential during these processes, a conclusion that is further supported by the direct correlation observed between the ability of the different Fo strains to grow in -Cu conditions and to cause wilt disease on tomato plants. Fungal burden experiments and fluorescence microscopy studies revealed that in planta growth of the mac1Δ mutant was largely restricted to the outermost layers of the cortex, in stark contrast to the wild-type or the mac1Δctr3OEfre9OE strains who were able to progress towards the innermost root layers and reach the vascular system. Interestingly, the acquisition mechanism of copper by tomato roots has been proposed to function similar to that of fungi, whereby Cu2+ is initially reduced to Cu+ which is subsequently taken up by the root [45]. However, for loading of copper into the xylem re-oxidation of Cu+ to Cu2+ is required, followed by complexation with the mugineic acid-derived metal chelate nicotianamine for further translocation [45]. This redox-selective process implies that Cu+ is available in the root cortex but largely absent during later stages of fungal infection in the xylem vessels due to its oxidation to Cu2+, explaining the residual growth of the mac1Δ mutant in the outer root layers and its failure to colonize the xylem and progress to the plant stems and to cause wilt disease (Fig 5). Interestingly, we found that efficient copper reduction and uptake are also essential for virulence of Fo on G. mellonella. This establishes a conserved role of copper acquisition in fungal pathogenicity on hosts from different kingdoms of life and highlights that general mechanisms such as copper uptake could be promising targets for new broad-spectrum antifungal strategies.
Copper (Cu2+) is reduced and then taken up by tomato roots as Cu+. However, Cu+ is re-oxidized to Cu2+ for xylem loading and complexed with the metal chelate nicotianamine (NA) for translocation. Successful root infection and vascular colonization of F. oxysporum therefore depends on in planta copper uptake, which in turn requires upregulation of copper reductase and transporter genes by direct binding of the transcription factor Mac1.
Materials and methods
Fusarium oxysporum strains
Fusarium oxysporum f. sp. lycopersici 4287 (Fol4287) was used in all experiments. All the strains for this study were generated in this genetic background and are listed in S1 Table. Primers and plasmids used in this work are listed in S2 and S3 Tables. Targeted deletion of the mac1 (FOXG_03227), ctr1a (FOXG_03101), and ctr3 (FOXG_07770) genes was performed by homologous gene replacement with the hygromycin B (mac1 and ctr3) or the neomycin (ctr1a) resistant cassettes using the split marker method [46] (S2A, S9A and S9C Figs). Additionally, ctr1a was deleted in a ctr3Δ background. Briefly, two PCR fragments encompassing 1.5 kb of the 5’- and 3’-flanking regions were amplified by PCR with primer pairs Mac1-5’-F + Mac1-5’-R and Mac1-3’-F + Mac1-3’-R, Ctr3-5’-F + Ctr3-5’-R and Ctr3-3’-F + Ctr3-3’-R, and Ctr1a-5’-F + Ctr1a-5’-R and Ctr1a-3’-F + Ctr1a-3’-R, for mac1, ctr3 and ctr1a, respectively. The amplified fragments were then fused to the hygromycin/neomycin resistance cassettes, previously amplified from pAN-7.1 [47] or pGEMT-Neo [48] plasmids, with primer pairs Mac1-hph-F + Mac1-hph-R, Ctr3-hph-F + Ctr3-hph-R or Ctr1a-neo-F + Ctr1a-neo-R, using the fusion primer combinations Mac1-5’-Fn + HygY and Mac1-3’-Rn + HygG, Ctr3-5’-Fn + HygY and Ctr3-3’-Rn + HygG or Ctr1a-5’-Fn + NeoY, and Ctr1a-3’-Rn + NeoG. The two resulting DNA constructs for each target gene were used to co-transform freshly prepared F. oxysporum protoplasts. The obtained transformants were purified by two rounds of monoconidial isolation as described [49]. Hygromycin/geneticin-resistant transformants were analyzed by Southern blot analysis with gene-specific probes (S2B, S8B, S8D and S8E Figs).
For complementation of the mac1Δ mutant #1, a 3,938 bp DNA fragment containing the wild-type mac1 ORF fused at the 3’ end with a DNA sequence encoding for the S-tag oligopeptide (mac1Stag) was used (S2C Fig). To this aim, a 72 bp sequence encoding a 4X-GA linker and the oligopeptide S-tag was amplified from plasmid Stag::pyrG [50] with the primer pair Mac1-Stag-F + Mac1-Stag-R. Next, two fragments containing the mac1 ORF without the Stop codon preceded by 1,167 bp of its 5’ region, and 1,095 bp of its 3’ region were amplified from Fol4287 genomic DNA with the primers Mac1-5’-F + Mac1-ORF-STOP-R and Mac1-TER-F + Mac1-3’-R, respectively. The three obtained DNA fragments were fused by PCR using the primer pair Mac1-5’-Fn + Mac1-3’-Rn. Taking advantage of the inability of mac1Δ to grow under -Cu conditions (Fig 1A), no selection marker was used. Transformants that grew in the absence of copper (MM+TE-Cu) were inoculated in plates with and without hygromycin. Among all the transformants capable of growing in MM+TE-Cu, one had lost hygromycin resistance indicating in locus integration of the complementation construct. This strain (hereafter called mac1Stag) was further analyzed by PCR with locus-specific primers (S2D Fig).
The mac1clover strain was generated by the co-transformation of mac1Δ protoplasts with the PhleoR cassette, amplified from plasmid pAN8-1 [51] using the primer pair Gpda15B + TrpC8B, and the mac1clover allele (S6A Fig). To generate this DNA construct, four DNA fragments were obtained by PCR: the A. nidulans gpdA promoter and the 1XFo-mClover3 gene were both amplified from plasmid pUC57-1XFomClover3 [52] with primer pairs Gpda15B + Gpda-Mac1-R and Mac1-Clover-F + Clover-Mac1-Ter-R, respectively. The mac1 ORF without the Stop codon and a 1,124 bp fragment of the 3’ flanking region of mac1 were amplified from Fol4287 genomic DNA using primer pairs Mac1-ATG-F + Mac1-ORF-STOP-R and Mac1-TER-F + Mac1-3’-R, respectively. The four obtained DNA fragments were fused by PCR using the primer pair Gpda15Bnest + Mac1-3’-Rn. PCR analysis with the primers Mac1-qPCR-F and EYFPrev identified four independent transformants showing a PCR amplification product with the expected size of 1,418 bp (S6B Fig).
To achieve constitutive expression of ctr3 and fre9 on the mac1Δ background, the CDSs followed by approximately 1.3 kb of the terminator regions were amplified using primer pairs Gpda-Ctr3 + Ctr3-3’-R or Gpda-Fre9 + Fre9-3’-R, respectively. Then the gpdA promoter of A. nidulans was amplified from plasmid pAN7-1 [47] with the primer pair GpdA15B + GpdA9 and fused to the 5’ ends of the ctr3 or the fre9 fragments using the primers Gpda15nest + Ctr3-3’-Rn or Fre9-3’-Rn, to generate the DNA constructs ctr3OE and fre9OE, respectively (S9A Fig). Next, mac1Δ protoplast were co-transformed with the NatR cassette, amplified from plasmid pDNat [53] with the primer pair M13-F + M13-R, together with the ctr3OE DNA construct (for generating mac1Δctr3OE) or with both ctr3OE and fre9OE DNA fragments (for generating mac1Δctr3OEfre9OE). PCR analysis of two independent nourseothricin-resistant transformants from each transformation experiment, with specific the primers Gpda4 + Ctr3-FOXG_07770-R or Fre9-3’-Rn, confirmed the presence of the ctr3OE or the fre9OE DNA constructs in these strains (S8B and S8C Fig).
For fluorescence microscopy assays under infection conditions, we generated mac1Δ or mac1Δctr3OEfre9OE #4 strains expressing 3 copies of Fo-mClover3 by co-transformation with the PhleoR cassette, amplified from plasmid pAN8-1 [51] using the primers Gpda15B + TrpC8B, and the Fo-mClover3 expression cassette, amplified from plasmid pUC57-3XFomClover3 [52] with primer pair Gpda15B + SV40rev (S10A Fig). PCR analysis with the gene-specific primers Gpda4 + 3XFLAGrev revealed the presence of the Fo-3XmClover3 expression cassette in four mac1Δ-3XmClover transformants and in two mac1Δctr3OEfre9OE-3XmClover transformants (S10B and S10C Fig). mac1Δ-3XmClover #11 and mac1Δctr3OEfre9OE-3XmClover #1 were selected for microscopy studies because they presented the highest fluorescence intensity.
Culture conditions
Fungal strains were stored at -80°C as microconidial suspensions in 30% glycerol (v/v). For microconidia production and DNA extraction, strains were grown for 3–4 d in liquid potato dextrose broth (PDB) at 28°C and 170 rpm. When needed, appropriate antibiotics (hygromycin B at 20 μg/ml, geneticin at 10 μg/ml, phleomycin at 4 μg/ml, and nourseothricin at 2.5 μg/ml) were added to the culture medium.
For phenotypic analysis of colony growth, 5 μl drops of 107 microconidia/ml in water were spotted onto 20 mM L-glutamine minimal medium with -Cu trace elements (MM+TE-Cu) solid plates supplemented with 0 to 2 mM CuSO4. To determine the fungal biomass production, 2.5x106 microconidia/ml were germinated in liquid MM+TE-Cu supplemented, or not, with 10 μM CuSO4 for 16 h at 28°C and 170 rpm. The obtained mycelium was lyophilized and weighed.
Axenic liquid cultures at specific copper concentrations were grown as previously described [54]. 2.5x106/ml freshly obtained microconidia were inoculated in PDB and cultured for 15 h at 28°C and 170 rpm. Germlings were filtered using a Monodur filter membrane, washed three times with sterile Milli-Q water, collected with a sterile spatula, divided in two flasks containing pH 6.5 MM+TE-Cu with the desired CuSO4 concentration and incubated for 6 h at 28°C and 170 rpm. In some experiments, a shift from -Cu to +Cu was carried out by the addition of CuSO4 after 6 h of incubation in MM+TE-Cu. Copper concentrations used in each experiment are indicated in the figures. When needed, protein biosynthesis was blocked using 50 μg/ml of the translation inhibitor cycloheximide (chx) (Millipore). All glassware used was pre-washed with 3.5% HCl for 30 min and rinsed five times with distilled water to remove any traces of metal adhering to the glass.
Nucleic acid manipulation and quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) analysis
Genomic DNA was extracted as previously reported [55]. DNA was quantified in a Nanodrop ND1000 spectrophotometer at 260 nm and 280 nm wavelengths. The quality of the DNA was monitored by electrophoresis in 0.7% agarose gels (w/v). PCR amplification reactions were performed using different thermostable Taq DNA polymerases depending on the experiment and the expected fragment size. The enzymes Expand High Fidelity PCR System (Roche) or Phusion High-Fidelity DNA Polymerase (New England Biolabs) were used for reactions where high fidelity PCR amplification was required. For routine PCRs and Southern blot probes, the thermostable BioTaq DNA Polymerase (Meridian Bioscience) was used. The amplifications were performed according to the manufacturer’s instructions in a T100 Thermal Cycler (Bio-Rad).
To measure transcript levels of the desired genes, total RNA was isolated from snap frozen tissue of three biological replicates and used for reverse transcription quantitative PCR (RT-qPCR) analysis as described [8,54]. Briefly, RNA was extracted using the Tripure Reagent and treated with DNase (both from Roche). The resulting RNA was reverse transcribed with the iScript cDNA Synthesis Kit (Bio-Rad) to synthesize the cDNA, and qPCR was carried out using the FastStart Essential DNA Green Master (Roche) in a CFX Connect Real-Time System (Bio-Rad) according to the manufacturer´s instruction. Data were analyzed using the double delta Ct method [56,57] by calculating the relative transcript level normalized to the act1 gene (FOXG_01569).
RNA sequencing analysis
RNA-seq analysis was carried out using RNA isolated from samples obtained from axenic cultures (supplemented or not with 100 μM CuSO4) and from infected tomato roots. For this, roots of 2-week-old tomato plants were inoculated with the indicated F. oxysporum strains and harvested either at 2 or 6 days post inoculation (dpi). Groups of three roots were sampled together and considered as one biological replicate. Once collected, samples (either mycelium or tomato roots) were frozen in liquid nitrogen and lyophilized. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) as described [52] and treated with DNase I using the Turbo DNA-Free Kit (Invitrogen) according to the manufacturer’s instructions. RNA sequencing was performed by Novogene, UK. For library preparation mRNA was captured through poly-A enrichment on the total RNA, and a TruSeq RNA Library Preparation Kit (Illumina, USA) was used to build the libraries according to the manufacturer’s protocol. Libraries were sequenced on a NovaSeq6000 sequencing platform (Illumina). Paired-end 150 bp reads were obtained for each RNA-seq library. Raw reads were produced from the original image data by base calling. Reads containing adaptors, highly ‘N’ containing reads (>10% of unknown bases) and low-quality reads (more than 50% bases with quality value of <5%) were removed. After data filtering, on average, ~99.3% clean reads remained. Transcript quantification was performed with Salmon v1.6.0 [58]. RNA-seq paired-end read data sets were quasimapped against the reference transcriptome of Fusarium oxysporum f. sp. lycopersici 4287 (GCF_000149955.1_ASM14995v2_rna.fna, obtained from NCBI RefSeq). Raw gene counts were used to evaluate the level of correlation between biological replicates using Pearson’s correlation matrix with corrplot R package v0.92 [59] (S13 Fig).
Differential gene expression analysis at transcript level was analyzed using DESeq2 R package v1.40.2 [60]. Transformed raw counts (vst function) were used for Principal Component Analysis (PCA) using prcomp function from stats R package [61] (S14 Fig). A cut-off of absolute FC |Log2 Fold change| ≥ 2 and adjusted p-value ≤ 0.05 by Benjamini and Hochberg method were used to identify differentially expressed genes (DEGs). Genes with less than 1 transcript per million (TPM) across all samples were considered lowly expressed and ignored in the analysis. Intersection between DEGs in the wild-type strain in -Cu and root samples at 2 and 6 dpi, against +Cu as control were calculated and visualized with ComplexUpset R package v1.3.3 [62,63]. Genes were hierarchically clustered based on FC using the Heatmap function from the ComplexHeatmap package R package v2.16.0 [64]. The R statistical language and environment v4.3.0 was used for RNA-seq data analysis and visualization [61]. Scripts used are available at https://github.com/mvapontes/palosfernandez_et_al_plant_2023.
Chromatin immunoprecipitation-coupled sequencing analysis (ChIP)
ChIP-seq analysis of fungal cells grown in submerged liquid cultures followed the procedures described [65]. Briefly, DNA was crosslinked to proteins by adding 1% formaldehyde (v/v) to the axenic cultures and incubating them for 15 min at 28°C and 170 rpm. Crosslinking was stopped by the addition of 125 mM glycine (final concentration) and 5 min incubation with shaking. The mycelium was collected by filtration through a Monodur nylon filter and flash-frozen in liquid nitrogen. Mycelium was ground in liquid nitrogen with a mortar and a pestle. ChIP was carried out as described [66] with minor modifications. The monoclonal anti-S-tag antibody (SAB2702227, Sigma-Aldrich) was used. Precipitation of the protein-antibody conjugate was performed with Dynabeads Protein G (10003D, Thermo Fisher Scientific™). Chromatin-bead complexes were washed three times with Low-salt buffer followed by one wash with 1 ml High-salt buffer and eluted in TES buffer (50 mM TRIS-HCl pH 8, 10 mM EDTA pH 8, 1% SDS). Chromatin was treated with Proteinase K (MBI) and DNA purification was done using the PCR and DNA Cleanup Kit (Monarch). All experiments were performed in biological triplicates.
The obtained DNA was sent for sequencing at the Vienna BioCenter Core Facilities (Vienna, Austria). Paired-end sequencing was performed using a NextSeq550 PE75 Illumina sequencer. Obtained sequences were de-multiplexed, quality controlled, filtered using trimmomatic 0.36 [67] and mapped on the already available Fusarium oxysporum f. sp. lycopersici 4287 genome assembly (GCF_000149955.1_ASM14995v2_genomic.fna from NCBI RefSeq). Mapping was performed using BWA [68] and further processing was done using samtools 1.7 and bedtools v2.27.1 to obtain normalized genome coverage tracks.
For identification of Mac1 binding site coverage, tracks were loaded into R and peaks were identified using R function locate_peak_height (https://github.com/symbiocyte/MNase). Peaks identified in -Cu and +Cu were visually selected using IGB v9.1.10 (https://www.bioviz.org/) resulting in 12 Mac1 specific peaks. Genomic sequences around these peak locations (500 bp) were exported and submitted to NCBI-BLAST homology searches against nt database for the identification of conserved regions. Within these regions the consensus sequence TGCTCA could be identified. A search of the motif was conducted using FIMO [69] online tool at MEM suite with default parameters.
ChIP-seq and RNA-seq coverage plots of Mac1 selected genomic regions were created using kpPlotBAMCoverage function from KaryoplotR [70] Bioconductor package version 1.26.0 in R statistical language [61]. The employed script is available at https://github.com/mvapontes/palosfernandez_et_al_plant_2023.
Sequence search and phylogenetic analysis
In silico gene and protein searches of Fol4287 and related fungal species was performed using the BLAST algorithm [71] from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov), Fungal and Oomycete genome database (FungiDB; https://fungidb.org/fungidb/app) and Saccharomyces Genome Database (SGD; https://www.yeastgenome.org). Protein domain prediction was done using the Prosite database (ExPASy; https://prosite.expasy.org), Pfam (http://pfam.xfam.org), InterPro (https://www.ebi.ac.uk/interpro/) and NCBI Conserved Domain Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Protein alignments were done using the BioEdit software v7.7.1.
For phylogenetic analysis, genome mining of Fol4287 against selected proteins was performed using BLASTp. Results were manually curated based on percentage of identity and e-value. MAFFT v7.453 [72] with default parameters was used to align protein sequences. Manually curated alignments were used to generate phylogenetic trees using MEGA v11 [73] with Maximum Likelihood method and JTT matrix-based model. The bootstrap consensus phylogenetic tree was inferred from 1000 replicates.
Fluorescence microscopy
For studying the subcellular localization of Mac1, 2.5x106 freshly obtained microconidia/ml of the mac1clover strain were germinated for 16 h at 28°C and 170 rpm in 20 mM L-glutamine MM+TE-Cu with 100 μM CuSO4 (+Cu) or 2 μM CuSO4 (-Cu). In some experiments, -Cu cultures were shifted to +Cu (20 μM CuSO4) during 10 min before imaging. Fungal nuclei were stained 5 min before imaging with 2 μg/ml Hoechst 33342 (Invitrogen™) in water in the dark. Wide-field fluorescence imaging was performed with a Zeiss Axio Imager M2 microscope equipped with a Photometrics Evolve EMCCD camera, using the 40X oil objective. Fo-mClover and Hoechst 33342 fluorescence were visualized at an excitation of 459 and 352 nm, and emission detected at 519 and 461 nm, respectively.
For microscopic observations of F. oxysporum during tomato plant infection, roots of 2-week-old tomato seedlings inoculated with the F. oxysporum strains of interest were collected at 4 dpi and secondary lateral roots were sampled. To visualize plant cell walls, samples were stained with 2 mg/ml propidium iodide (PI) (Sigma-Aldrich) in water in the dark for 15 min before imaging. Wide-field fluorescence imaging was performed with a Zeiss Axio Imager M2 microscope equipped with a Photometrics Evolve EMCCD camera, using the 40X oil objective. Fo-mClover and PI fluorescence were visualized at an excitation of 459 and 587 nm, and emission detected at 519 and 610 nm, respectively.
Western blot analysis
Proteins were extracted using a reported procedure [74, 75] involving solubilization from lyophilized mycelial biomass with NaOH, followed by precipitation with trichloroacetic acid (TCA). Aliquots were resolved in 10% SDS-polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes with a Trans-Blot Turbo Transfer System (Bio-Rad) for blotting. Western blots were reacted with monoclonal anti-S-tag (1:5,000; SAB2702227, Sigma-Aldrich) as primary antibody and with polyclonal anti-mouse IgG peroxidase (1:5,000; #7076, Cell Signalling Technology) as secondary antibody. Tubulin, used as loading control, was detected with monoclonal anti-α-Tub (1:5,000; T9026, Sigma-Aldrich) as primary antibody and with polyclonal anti-mouse IgG peroxidase (1:5,000; #7076, Cell Signalling Technology) as secondary antibody. Proteins were detected by chemiluminescence using ECL Select Western blotting Detection reagent (GE Healthcare, Amersham) and a Fujifilm LAS-3000 camera.
Cellophane penetration assay
The cellophane penetration assay was performed as previously described [28, 76]. Briefly, cellophane membranes were cut the same size of a Petri dish, autoclaved in deionized water, and placed on top of PDA plates. 5 μl drops of in 2x107 microconidia/ml in water were spot-inoculated at the center of the plate and plates were incubated at 28°C for 3 d. After this time, the cellophane membrane with the fungal colony was carefully removed and the plates were incubated for another 24 h at 28°C to visualize the mycelium that had penetrated through the cellophane. Plates were imaged before and after cellophane removal. All experiments were performed in triplicate.
Plant infection assay
Tomato seeds (Solanum lycopersicum cv. Moneymaker from EELM-CSIC, or cv. Momotaro from Takii Seed Co., Ltd.; susceptible to F. oxysporum f. sp. lycopersici race 2) were surface-sterilized by immersion in 20% bleach (v/v) for 30 min and sown in moist vermiculite. Seedlings were grown in a growth chamber under the following conditions: 28°C, 40–70% relative humidity and a photoperiod of 14 h of 36 W white light and 10 h of darkness.
Tomato plant infection assays were performed as described [49]. Briefly, two-week-old seedlings were inoculated with the different fungal strains by immersing the roots in a suspension of 5x106 microconidia/ml. Depending on the experiment, plants were irrigated with tap water or with a 10 μM CuSO4 solution. Disease symptoms and the survival rate were analyzed during 30–40 days [77]. Death of the infected plants was diagnosed as a complete collapse of the stem, without any green parts left accompanied by visible proliferation of the fungal mycelium on the dead tissue. The Kaplan-Meier test was used to assess statistical significance of differences in survival among groups using the log-rank test with the software GraphPad Prims version v8.0.1 [8]. All infection experiments were performed at least three times.
Determination of in planta fungal burden
Fungal burden in tomato plants inoculated with F. oxysporum was measured by qPCR as described previously [78] using total DNA extracted from tomato roots and stems at 4 or 10 dpi. Relative fungal burden was calculated using the 2-ΔΔCt method, with primers of the Fol4287 six1 gene (FOXG_16418) and normalized to the tomato gapdh gene.
Infection assays in Galleria mellonella
G. mellonella infection assays were performed as described [32,33]. G. mellonella larvae (CASA REINA SA, Bilbao, Spain) were maintained in plastic boxes for 2–3 d before the infection. Fifteen larvae were used for each treatment. An automicroapplicator (0.1–10 μl; Burkard Manufacturing Co. Ltd) with a 1 ml syringe (Terumo Medical Corporation) was used to inject 8 μl of a 1.6x105 microconidial suspension into the haemocoel of each larva. After injection, larvae were incubated in glass containers at 30°C. Survival was recorded daily for 5 dpi. Data were analyzed with the software GraphPad Prims version v8.0.1 [8].
Supporting information
S1 Fig. Fusarium oxysporum Mac1.
(A) Schematic diagram of the domain structure of F. oxysporum Mac1. The copper fist DNA-binding domain containing the conserved RGHR and a GRP residues and the copper-binding domain containing two cysteine-rich motifs are located at the N- and C-terminus, respectively. (B) Alignment of Mac1 homologs from different fungi. Identical amino acid residues are highlighted. (C) Phylogenetic tree of Mac1 homologs from different fungi generated using the Maximum Likelihood method and JTT matrix-based model. Bootstrap consensus tree inferred from 1000 replicates. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown at the base of each clade. The transcription factor Pro1 from F. oxysporum was used as outgroup for the analysis. Protein sequences were aligned with MAFFT and evolutionary analyses were conducted in MEGA.
https://doi.org/10.1371/journal.ppat.1012671.s001
(PDF)
S2 Fig. Generation of mac1 knockout and complemented strains.
(A) Physical map of the F. oxysporum mac1 locus in the wt and the mac1Δ strains. Relative positions of restriction sites, PCR primers and the probe used in the Southern blot analysis are indicated. HygR, hygromycin resistance gene. (B) Southern blot analysis of putative mac1Δ deletion mutants. Genomic DNA of the wt strain and twelve independent hygromycin resistant transformants was treated with BamHI, separated on a 0.7% agarose gel, transferred to a nylon membrane, and hybridized with the DNA probe corresponding to the 5’ flanking region of mac1 indicated in (A). Molecular sizes of the hybridizing bands are indicated on the left. Transformants #1, #7, #10 and #11 show hybridizing bands consistent with homologous replacement of the mac1 gene with the hygromycin resistance cassette. (C) Physical map of the F. oxysporum mac1 locus in the mac1Δ mutant and the complemented mac1Stag strain. Relative positions of PCR primers are indicated. (D) Agarose gel electrophoresis of PCR products obtained using the primer pair Mac1-5’-F and Mac1-Stag-R with genomic DNA extracted from the indicated strains. M, molecular size markers.The presence of the 2916 bp amplification band is consistent with insertion of the mac1Stag allele at the native mac1 locus.
https://doi.org/10.1371/journal.ppat.1012671.s002
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S3 Fig. Alignment of the two F. oxysporum isoforms of the high affinity copper transporter Ctr1.
Alignment of the F. oxysporum Ctr1a and Ctr1b amino acid sequences with Ctr1 homologs from other fungi. Ctr1 regions absent in F. oxysporum Ctr1b are indicated with red boxes.
https://doi.org/10.1371/journal.ppat.1012671.s003
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S4 Fig. Phylogenetic analysis of Ctr high affinity copper transporters and Fre metalloreductases in fungi.
(A, B) Phylogenetic trees of Ctr and Fre homologs from different fungi generated using the Maximum Likelihood method and JTT matrix-based model. Bootstrap consensus tree inferred from 1000 replicates. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown at the base of each clade. The Cu2+-transporting P-type ATPase Ccc2 (A) and the soluble fumarate reductase FRD1 (B) from S. cerevisiae were used as outgroups for the analysis. Protein sequences were aligned with MAFFT and evolutionary analyses were conducted in MEGA. Proteins encoded by F. oxysporum genes induced under copper limiting conditions are in bold.
https://doi.org/10.1371/journal.ppat.1012671.s004
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S5 Fig. F. oxysporum Mac1 directly activates transcription of copper deficiency response genes.
(A) Putative consensus DNA sequence bound by F. oxysporum Mac1, identified by exporting the genomic sequences around the peak locations (500 bp) of Mac1 binding sites obtained by ChIP-seq and submitting them to BLASTn analysis. Within these regions the consensus sequence TGCTCA was identified. (B-G). Abundance of RNA-seq transcript reads of the wt (dark blue) or the mac1Δ strain (red) under -Cu conditions (RNA-seq, upper graphs); or of gDNA reads from ChIP-seq analysis in the mac1Stag strain under -Cu (grey) or +Cu (light blue) conditions (ChIP-seq, lower graphs). Data are represented as base-level coverage to three Fol4287 gene clusters (B-D) or genes (E-G). Genes are indicated as red boxes and putative Mac1 binding sites on each strand by black triangles.
https://doi.org/10.1371/journal.ppat.1012671.s005
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S6 Fig. Generation of a Fusarium oxysporum mac1clover strain.
(A) Physical map of the F. oxysporum mac1clover DNA construct. Relative positions of PCR primers are indicated. (B) Agarose gel electrophoresis of PCR products obtained using primer pairs Mac1-qPCR-F and EYFPrev with genomic DNA extracted from the indicated transformants. M, molecular size markers. (C) Colony phenotypes of the indicated strains grown for 2 d at 28°C on MM+TE-Cu (20 mM NaNO3) without any copper supply. Scale bar, 0.5 cm. (D) Transcript levels of the indicated genes in the indicated strains transferred for 6 h to MM-TE-Cu with (+Cu) or without (-Cu) 100 μM CuSO4 were measured by real-time RT-qPCR and expressed relative to those of the wt strain in -Cu. Bars represent standard deviations (n = 3, biological replicates). p-vaules: ns>0.05, *<0.05, **<0.01 versus -Cu, in each strain, according to two-tailed unpaired Student’s t test.
https://doi.org/10.1371/journal.ppat.1012671.s006
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S7 Fig. Comparative transcriptional analysis in -Cu and plant infection versus +Cu conditions and invasive growth assay on cellophane-covered plates.
(A) Comparative analysis of differentially expressed genes from RNA-seq analysis of the wt strain in axenic culture in MM+TE-Cu (-Cu) or during infection of tomato plants at 2 or 6 dpi. Upregulated (yellow) or downregulated (purple) sets of genes showing significant differential expression in the indicated condition versus axenic culture under copper sufficiency (MM+TE-Cu + 100 μM CuSO4). Vertical bars show the intersection between sets and numbers refer to genes unique for each intersection. Genes upregulated both in copper limitation and under infection conditions are highlighted in red with gene names indicated above. Data were visualized using ComplexUpSet. (B) The indicated strains were spot-inoculated on top of a cellophane membrane on PDA plates with (+Cu) or without 5 μM CuSO4, grown for 3 d at 28°C and imaged (Before). The cellophane with the fungal colony was removed and plates were incubated for an additional day to determine the presence of mycelial growth on the plate, indicating penetration of the cellophane (After). Scale bar, 2 cm.
https://doi.org/10.1371/journal.ppat.1012671.s007
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S8 Fig. Targeted deletion of the high-affinity copper transporters Ctr3 and Ctr1a in F. oxysporum.
(A, C) Physical maps of the F. oxysporum ctr3 and ctr1a loci in the wt and in the ctr3Δ (A) and ctr1aΔ (C) strains, respectively. Relative positions of restriction sites, PCR primers and the probes used in the Southern blots are indicated. HygR, hygromycin resistance gene. NeoR, neomycin resistance gene. (B, D, E) Southern blot analysis of putative ctr3Δ (B), ctr1aΔ (D) and ctr3Δctr1aΔ (E) deletion mutants. Genomic DNA of the wt strain and independent hygromycin or neomycin resistant transformants was treated with EcoRI (B) or BamHI (D, E), separated on 0.7% agarose gels, transferred to nylon membranes and hybridized with a DNA probe corresponding to the 5’ flanking region of ctr3 (B, indicated in A) or to the 3’ flanking region of ctr1a (D, E indicated in C). Molecular sizes of the hybridizing bands are indicated on the left. (F) Colony phenotypes of the indicated strains after 2 d growth on MM+TE-Cu, supplemented with the indicated concentrations of CuSO4. Scale bar, 1 cm. The colonies of wt and mac1Δ are the same as those shown in Fig 1A and are repeated here for clarity. Scale bar, 1 cm. (G) Kaplan-Meier plot showing survival of groups of 10 tomato plants (cv. Momotaro) inoculated by dipping the roots into a suspension of 5x106 microconidia/ml of the indicated fungal strains or water (Mock). Data shown are from one representative experiment. Experiments were performed at least two times with similar results. p-value: ***<0.001 versus the wt according to Log-rank (Mantel-Cox) test.
https://doi.org/10.1371/journal.ppat.1012671.s008
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S9 Fig. Generation of F. oxysporum strains overexpressing ctr3 and/or fre9 in a mac1Δ background.
(A) Physical maps of the DNA constructs used for over-expression of ctr3 and fre9 in mac1Δ. Relative positions of PCR primers are indicated. (B, C) Agarose gel electrophoresis of PCR products obtained using primer pairs Gpda4 and Ctr3-FOXG_07770-R (B), and Gpda4 and Fre9-3’-Rn (C) using genomic DNA extracted from the indicated nourseothricin-resistant transformants or from mac1Δ as a negative control. M, molecular size markers. (D, E) Real-time RT-qPCR analysis was performed in the indicated strains grown in the absence (-Cu) or presence (+Cu) of 100 μM CuSO4. Transcript levels of the indicated genes are expressed relative to those of the wt strain in -Cu conditions. Bars represent standard deviations (n = 3, biological replicates). p-values: ns>0.05, *<0.05, **<0.01 versus mac1Δ, under the same condition, according to two-tailed unpaired Student’s t test.
https://doi.org/10.1371/journal.ppat.1012671.s009
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S10 Fig. Generation of fluorescent mac1Δ- and mac1Δctr3OEfre9OE strains expressing 3XmClover3.
(A) Physical map of the Fo-mClover3 expression cassette used to transform mac1Δ or mac1Δctr3OEfre9OE #4 strains. Relative positions of the primers used for diagnostic PCR are indicated. (B, C) Agarose gel electrophoresis of PCR products obtained using the primer pair Gpda4 and 3XFLAGrev with genomic DNA extracted from the indicated phleomycin-resistant transformants or mac1Δ or mac1Δctr3OEfre9OE #4 as negative controls. M, molecular size markers.
https://doi.org/10.1371/journal.ppat.1012671.s010
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S11 Fig. Overexpression of the high affinity copper transporter ctr3 and the metalloreductase fre9 rescues vascular colonization of tomato roots in the mac1Δ mutant.
Tomato root colonization of the indicated F. oxysporum strains expressing 3XFo-mClover3 at 4 dpi. Fungal fluorescence (mClover3, green) is overlaid with propidium iodide staining of the plant cell wall (PI, magenta). The two images were merged using ImageJ v1.8. The images shown are representative of at least three lateral secondary roots from six different tomato plants. Scale bar, 50 μm.
https://doi.org/10.1371/journal.ppat.1012671.s011
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S12 Fig. Overexpression of the high affinity copper transporter ctr3 and the metalloreductase fre9 rescues restores virulence of the mac1Δ mutant on the animal host Galleria mellonella.
(A, B) Kaplan-Meier plot showing survival of groups of 15 G. mellonella larvae after injection into the hemocoel of 1.6x105 microconidia of the indicated F. oxysporum strains or phosphate-buffered saline (PBS) as a negative control. Insects were maintained at 30°C. p-value: ****<0.0001 versus the wt according to Log-rank (Mantel-Cox) test. Data shown are from one representative experiment. Experiments were performed at least two times with similar results.
https://doi.org/10.1371/journal.ppat.1012671.s012
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S13 Fig. Pearson’s correlation matrix of Fol4287 transcriptomes.
Raw gene counts used to evaluate the level of correlation between biological replicates using Pearson’s correlation. Pearson‘s correlation matrix were performed in R (v4.3.0) statistical language and environment, the core function from the stats base package and the corrplot package v0.92 were used for the analysis.
https://doi.org/10.1371/journal.ppat.1012671.s013
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S14 Fig. Principal Component Analysis (PCA) of the RNA-seq data demonstrating the reproducibility of the three biological replicates.
Transformed raw counts (vst function from DESeq2 R package) per gene were used as variable for prcomp function from stats R package. PCA shows the samples in the 2D plane spanned by their first two principal components. Sample color is coded according to the experimental condition.
https://doi.org/10.1371/journal.ppat.1012671.s014
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S1 Table. Fungal strains and tomato cultivars used in this study.
https://doi.org/10.1371/journal.ppat.1012671.s015
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S2 Table. List of primers used in this study.
Lowercase nucleotides do not belong to the original sequence and were introduced to generate overlapping ends for fusion PCR reactions.
https://doi.org/10.1371/journal.ppat.1012671.s016
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Acknowledgments
We are grateful to Mariló Alcaide Caballero and Florian Kastner for valuable technical assistance and to Rafael Fernández Muñoz, IHSM “La Mayora”, UMA-CSIC, Malaga, Spain, and Tsutomu Arie, Tokyo University of Agriculture and Technology, TUAT, Tokyo, Japan, for kindly providing seeds of tomato cultivars Moneymaker and Momotaro, respectively.
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