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Assessment of Ustilago maydis as a fungal model for root infection studies
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Assessment of Ustilago maydis as a fungal model for root infection studies Mahta MAZAHERI-NAEINIa, Seyed Kazem SABBAGHb,*, Yves MARTINEZc, d,e Nathalie SEJALON-DELMAS , Christophe ROUXd,e a
Department of Medical Genetic, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, BP634, F8916978477 Yazd, Iran b Department of Plant Protection and Institute of Plant Biotechnology, University of Zabol, BP98615-58, F9861335856 Zabol, Iran c CNRS, FR3450, BP42617, F-31326 Castanet-Tolosan CEDEX, France d Universite de Toulouse, UPS, UMR5546, Laboratoire de Recherche en Sciences Vegetales, Castanet-Tolosan CEDEX 31326, France e CNRS, UMR5546, 31326 Castanet-Tolosan CEDEX, France
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abstract
Article history:
Ustilago maydis is a fungus infecting aerial parts of maize to form smutted galls. Due to its
Received 21 June 2014
interest as a genetic tool in plant pathology, we evaluated its ability to penetrate into plant
Received in revised form
roots. The fungus can penetrate between epidermic root cells, forming inter and intracel-
25 November 2014
lular pseudohyphae. Root infection didn’t provoke gall formation on the maize lines tested,
Accepted 1 December 2014
and targeted PCR detection showed that U. maydis, unlike the other maize smut fungus
Available online 17 December 2014
Sporisorium reilianum, has a weak aptitude to grow from the roots up to the aerial part of
Corresponding Editor:
maize. We also observed that U. maydis can infect Medicago truncatula hairy roots as an al-
Pieter van West
ternative host. This plant species is a model host to study root symbiosis, and this pathosystem can provide new insights on rootemicrobe interactions. Considering that U. maydis
Keywords:
could be a soil fungus, we tested its responsiveness to GR24, a strigolactone analogue.
alternative host
Strigolactones are root exuded molecules which activate mitochondrial metabolism of
cell respiration
arbuscular mycorrhizal (AM) fungi. Physiologic and molecular analysis revealed that
soil fungus
GR24 also increases cell respiration of U. maydis. This result points out that strigolactones
strigolactone
could have an incidence on several rhizospheric microbes. These data provide evidences
Ustilaginaceae
that the biotrophic pathogen U. maydis has to be considered for studying root infection. ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Among plant parasites, biotrophic and hemibiotrophic pathogenic fungi are described as highly adapted to their hosts, showing a tissue-specific infection site and a limited host spectrum: Blumeria graminis f. sp. hordei infects leaves of
barley, Colletotrichum lindemuthianum is pathogen on leaves of bean. One limitation of this restricted description is that the tissue specificity and the host range of a pathogen are usually defined by observation of the symptoms. Symptom formation and severity are not always strictly correlated to the level of invasion of a plant by a fungus, particularly in the case of
* Corresponding author. Tel.: +98 5431232100; fax: +98 5432240696. E-mail address: [email protected] (S. K. Sabbagh). http://dx.doi.org/10.1016/j.funbio.2014.12.002 1878-6146/ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
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biotrophic fungi which can interact with a host cell without causing major damage (Koeck et al. 2011). Ustilago maydis for instance is a biotrophic fungus causing corn smut disease, described in mycological flora as a strict pathogen of Zea genus nky 1994). Recent works showed that a number of monocot (Va and dicot species, like papaya or Arabidopsis thaliana, were susndezceptible to inoculation (Leon-Ramirez et al. 2004; Me n et al. 2005). Although U. maydis didn’t sporulate in these Mora alternative hosts, these observations underline that this biotrophic fungus has an extended capacity to infect plants and point out the interest to revisit its biology. Ustilago maydis is a major fungal model to study biotrophic plant parasitism (Dean et al. 2012). It is a dimorphic fungus: compatible saprotrophic haploid yeasts mate and form pathogenic dikaryotic pseudohyphae (Nadal et al. 2008). A great interest in this model is that sexual cycle, morphology and pathogenicity are interconnected processes (Kahmann & Kamper 2004). For these reasons its genome was sequenced € mper et al. 2006) and lot of mutants were obtained. Based (Ka on symptom formation e e.g. gall formation e U. maydis is considered as infecting maize via different aerial part of maize (Christensen 1963; references therein; Wenzler & Meins 1987). Several works described with accuracy the penetration process of dikaryotic pseudohyphae issuing from fusion of compatible yeast strains on stem, young and aged leaves 1981; Snetselaar & Mims 1992, 1993, and silks (Mills & Kotze 1994; Snetselaar 1993). We investigated here whether this pathogen could efficiently infect maize plantlets via roots. Different inoculum forms were tested: teliospores, a mix of compatible haploid yeasts, a solopathogenic strain, and a haploid yeast strain as control. Solopathogenic strains are diploid strains of the fungus which can infect plants in absence of mating with a compatible one (Holliday 1974). The efficiency of infection was analysed by using a set of complementary microscopic techniques and PCR detection. To further evaluate the potentialities of U. maydis as a rhizospheric pathogen, we also tested its ability to infect alternative host roots and to perceive root exuded molecules. Inoculations were carried out on Medicago truncatula hairy roots. This plant species is a model plant to investigate root biotrophic symbiosis. As the main root pathosystems available on barrel medic involve necrotrophic fungi (Tivoli et al. 2006) or hemibiotrophic ones (Rey et al. 2013), it was interesting to test on this plant the infectious potential of the biotrophic fungus U. maydis. The second evaluation consisted in monitoring the response of U. maydis to root exuded molecules. Many soilborne organisms were described to perceive molecules exuded by plant roots. This is the case for Sporisorium reilianum f. sp. zeae, a root infecting fungus phylogenetically related to U. maydis, causing maize head smut, which was described to be sensitive to a lipophilic fraction of maize root exudates (Martinez et al. 2001). We particularly tested the response of U. maydis to GR24, a synthetic chemical analogue of strigolactones. These molecules, present in root exudates of a broad spectrum of plants, have been described as important signals occurring in previous steps of interactions of plants with parasitic weeds (Cook et al. 1966, 1972) and with arbuscular mycorrhizal (AM) fungi (Akiyama et al. 2005). On AM fungi, strigolactones boost mitochondrial activity, causing a putative metabolic switch (Besserer et al. 2006).
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We tested the effect of GR24 on cell respiration of U. maydis and S. reilianum, this last species being considered as a natural root infecting pathogen of maize. The aim of these investigations is to evaluate U. maydis as a pathogenic fungal model to study root infection.
Materials and methods Fungal strains and culture condition Haploid strains FB6a and FB6b (Banuett & Herskowitz 1989), teliospores obtained from artificial contamination of maize with the strains FB6aFB6b, and a solopathogenic strain FBD11/pOTEF expressing the green fluorescent protein (GFP) (Spellig et al. 1996) were used in this study. Haploid (CRM) and solopathogenic (CRS1) strains of Sporisorium reilianum were isolated in our laboratory. All yeast strains were cultivated in liquid potato dextrose broth medium (PDB) at 24 C under shaking 100 rpm.
Plants and culture conditions A maize line susceptible to Ustilago maydis (LMZ66-France) was used as natural host. Corn seeds free of microbial contamination were cultivated in Magenta boxes on solidified M medium or in pot on sterilized peat (1 h at 120 C) as previously described (Martinez et al. 2002). In Magenta boxes, inoculations on one-week-old maize plantlets were carried out by adding in the vicinity of roots 500 mL of cell suspension at 107 cells mL1. In pot, inoculations were carried out by pouring directly on roots 1 mL of cell suspension at 107 cells mL1. For these experiments, the plants were grown in a night/day temperature of 18/24 5 C for three to five weeks, except for symptom observation where cultivation was performed in greenhouse up to floral formation. Infection of hairy roots of Medicago truncatula cv. Jemalong grown on M medium were performed by adding droplets of a culture of FBD11/pOTEF on root tips, followed by a two-week cocultivation before observation.
Microscopic observations Observations of infection and development of Ustilago maydis in roots were performed by macroscopic and microscopic observation. For light microscopy, samples of root were embedded in agarose before cutting sections of 150 mm width. The sections were stained with lactophenol-cotton blue and rinsed in lactophenol for 10 min. All the specimens were mounted on a glass slide and observed under bright-field optics or differential interference contrast (DIC). Observations under bright field, DIC, or epifluorescence were carried out in a Leica DMIRB Microscope (Leica, Wetzlar, Germany). Images were acquired using a colour video camera (Hyper Had, Japan). For transmission electron microscopy (TEM) observations, we used the method described by Martinez et al. 1999. Ultrathin sections (50e60 nm in thickness) were stained with uranyl-acetate or PATAg (periodic acidethiocarbohyrazideesilver) and examined using a Philips 301 transmission electron microscope at 80 kV (Philips, Eindhoven, The
Assessment of Ustilago maydis
Netherlands). For confocal microscopy, roots inoculated with a solopathogenic strain of U. maydis expressing the green fluorescence protein (FBD11-pOTEF) were observed by a spectral confocal laser scanning system (SP2 SE, Leica, Germany) equipped with an upright microscope (DM 6000, Leica, Germany). An Argon laser emitted at 488 nm was used to detect GFP fluorescence in the range between 500 and 530 nm. The ray line of the HeeNe laser emitted at 543 nm was able to excite the cell wall coloured by a solution of blue evans 0.01 % for 30 s and the fluorescence was collected in the range 590e710 nm. The images were acquired in the dual mode. The overlay images were then computed by projection of 20e30 plan-confocal images acquired in Z dimension with 0.8 mm increments between two focal planes.
PCR detection Plants used for these experiments were cultivated in pots for five weeks after addition of different inoculum sources on root plantlets. A set of ten plants was used for each inoculum, and the experiments were triplicated. Aerial parts were then collected and caulinar apices were isolated to avoid risk of contamination by the inoculum on plant surface. DNA extractions from 50 mg of plant material were performed using the CTAB procedure. We designed the specific primer pUM1 (50 -CGGTCGGTCTGTCGAAACC-30 ) on the ribosomic internal transcribed spacer one region of Ustilago maydis. PCR reactions (5 min 94 Ce35 cycles 1 min 54 C 1 min 72 C 1 min 94 Ce10 min 72 C) using pUM1epITS4 primers were followed by electrophoresis of amplicons on agarose 1.5 %. The presence of U. maydis in samples is assessed by the presence of a 680 bp band.
Effect of GR24 on cell respiration Respiratory measurements were performed by polarography and by a fluorescent assay 1 h after addition of GR24, a strigolactone analogue. For all strains (FB6b and FBD11/pOTEF for Ustilago maydis, CRM and CRS1 for Sporisorium reilianum), young cultures of ten-hour-old were used for these assays. Cell density was measured in assay and control at the end of the experiment and compared to the starting respective concentration. Polarography was performed by using a Clark electrode according to (Besserer et al. 2006). A volume of 10 mL of cells (106 cells mL1) were incubated during 1 h with GR24 or with the corresponding solvent (0.01 % acetone/water) for control before measuring oxygen consumption. For fluorescent assays, the protocol used is adapted from the recommendation of the manufacturer. A volume of 20 ml of Cell TiterBlue reagent (CTBR Promega, Madison, USA) was added to 100 ml of cell suspension at 107 cell mL1. The blue dye (resazurin) could be reduced in resorufin, a fluorescent compound, by the cell redox potential. According to provider technical protocol, CTB reagent was previously used for respiration assays (https://france.promega.com/resources/product-guidesand-selectors/protocols-and-applications-guide/cell-viability/). As a validation, we used specific inhibitors of fungal cell nol 100 mM): fluoresrespiration (carboxin 300 mM, di-nitro-phe cence formation on cell cultures was totally inhibited (data not shown), indicating that the Redox potential formed and
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then the resulting fluorescence emitted is essentially issuing from mitochondrial activity. Cell respiration was measured on 96-well-optical reaction plates at 25 C by recording fluorescence with an excitation wavelength of 560 nm and an emission wavelength of 590 nm using a Fluoroskan FL600 (Bio-Tek, Vermont, USA).
RNA extraction and RT-PCR Control (acetone 0.01 %) and GR24-stimulated cells (FBD11) were harvested by centrifugation after elicitation for 1 h and conserved at 80 C for RNA extraction. Total RNAs were isolated using a standard phenolechloroform procedure, purified using RNA purification Kit (promega) and treated by the RNase-Free DNase Set (Promega) according to the manufacturer’s recommendations. Contamination of residual genomic DNA in all RNA samples was verified by conventional PCR amplification on total RNA using the designed primers. Total RNA yield and concentration were measured with Nanodrop ND1000 spectrophotometer and the concentration of each sample was adjusted to 100 ng mL1. Quality of RNA was assessed by 1 % agarose gel electrophoresis stained by ethidium bromide and evaluated with Agilent 2100 Bioanalyzer (Agilent Technologies). For reverse transcription step, ImPromII enzyme (Promega) was used according to the manufacturer’s recommendations. First strands of cDNA were purified and concentration of final working solution was adjusted according to ND-1000 spectrophotometer quantification.
Primer design and qRT-PCR Oligonucleotide primers used in this study were designed using Primer3 site (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Ras1 (GenBank accession number CAA51689, fp 50 -TACCATTGAGGACTCTTACC-30 and rp 50 -CGGCAGTATCCAACACATC-30 ), (Muller et al. 2003), Rop1 (GenBank accession number CAA51689, fp 50 -CAGAGGTTTGGGACAAGCTC-30 and rp 50 -TAGGACGAGGAAGAGGACGA-30 ), (Brefort et al. 2005) and mig2-2 (fp 50 -AACCCAAAGCAGCCGTACCT-3’and rp 50 GCTCTTCCACCAACGAATCG-30 ), (Basse et al. 2002) were used as marker genes involved in pathogenicity. Cytochrome c1 Oxydase e Cytc1 (GenBank accession number ABQ96919, fp 50 CTATGGCTGAGGAGGTCGAG-3’and rp 50 -TAAGGTCGGGGCATGTAGTC-30 ) is a gene marker of cell respiration, hypothetical 60S ribosomal protein (GenBank accession number UM02518.1 fp 50 -GCAGGTGACTTTCGAGATCC-30 and rp 50 GGTCCGTGTGAATTTTGGTC-30 ) is as gene marker of cell translation. Elongation factor-EF1 (GenBank accession number XP_757071, fp 50 -TGGTAAGACCCTCCTTGACG-30 and rp 30 -ACCGCCGATCTTGTAGACAT-50 ) and glycine N-acyltransferase GNAT (GenBank accession no EDN60858, fp 50 TGCCATCCTCGACTTTATCC-30 and rp 30 -TCAGCAATCAACACCTCTGC-50 ) were used as housekeeping genes deduced from a preliminary transcriptomic analysis (not shown). Realtime PCR was performed on cDNA templates using a lightcycler ABI PRISM 7900 HT sequence detection system (Perkin Elmer/ Applied Biosystem, Foster City, USA) and PCR Master Mix for Syber Green Assays (Applied Biosystems, Foster City, USA) according to the manufacturer’s protocol. Real-time PCR was carried out in optical 384-well plates using the ABI PRISM
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7900 HT sequence detection system (Applied Biosystems) using the following concentration: 8 ml of SYBR Green PCR Master Mix (Applied Biosystems), 10 mM of each oligonucleotide primer (final concentration) and 2 ng of cDNA template in 10 ml reaction volume. Each gene amplification was prepared in triplicates. Two biological repetitions were carried out. Triplicates were validated with technical error under 0.5 CT. The amplification condition was: 50 C for 2 min, 95 C for 10 min, 40 cycles at 95 C for 15 sec, 60 C for 1 min. Melting curves analysis was performed after each reaction, to exclude nonspecific amplifications, with the thermal cycle at 95 C for 15sec, 60 C for 15 sec, and 95 C for 15 sec. The optimal baseline and threshold values were determined using automatic CT function available with the SDS 2.2 software (Applied Biosystems). The equation of the line that best fits the data was determined by minimizing error for regression analysis. The R2 value was calculated to estimate the accuracy of the real-time RT-PCR as a quantification method. The slope of the standard curve was used to calculate the efficiency of the real-time QRT-PCR according to the formula E ¼ 101/slope (Ptaffi 2001; Wen et al. 2005). The calibration curves were created using series of cDNA dilutions from 10 to 100 ng. EF1 gene was used for normalization experimental design. All data exported from SDS 2.2 software were calculated with Microsoft Excel software.
Results Ustilago maydis infects maize roots The ability of Ustilago maydis to penetrate and proliferate in maize roots, then to systemically grow up to aerial parts was respectively investigated by using microscopy techniques and PCR detection. Mock plants and also plants inoculated with the nonpathogenic haploid strain Fb6b were used as controls. After three weeks of culture in Magenta box, roots of maize inoculated with the pathogenic inoculums forms of U. maydis (FB6aFB6b mix; teliospores; FBD11/pOTEF) were covered with a hyphal net (not shown). Transverse sections stained with cotton blue reveal that the fungus penetrates the roots by growing at the junction of epidermic cells, then breaches the cell wall and forms hyphae in host cells (Fig 1A). Fungal growth is mainly intracellular, the fungus growing from cell to cell through the host cell wall in the inner part of the roots (Fig 1B). In larger roots, the observation was performed in confocal microscopy with a fluorescent solopathogenic strain (FBD11 solo-pOTEF) expressing GFP. The fungus can invade all root tissues except xylem (Fig 1C). The density of fungal cells in roots increased between three and five weeks. In general, we observed that U. maydis was less abundant in secondary roots and in the central pith. Although the fungus is mainly intracellular, growth in intercellular space is often observed in root epidermis (Fig 1D). In TEM observations, the fungus appeared as rounded cells or part of pseudohyphae depending on the angle of cutting (Fig 1E,F). The fungus hydrolyses the outer cell wall of root cells, penetrates by growing in the middle lamella of cell wall, then penetrates root cells and grows intracellularly from cell to cell. Same observations were obtained whatever the inoculum source, except with the nonpathogenic haploid FB6b strain (not shown).
M. Mazaheri-Naeini et al.
Maize root infection by Ustilago maydis didn’t cause symptoms Microscopic observations on the aerial part of the maize, three weeks after root inoculation, didn’t reveal the presence of fungal structure either by light or fluorescence microscopy. To define if the fungus could migrate systemically from roots to stem on more lignified five-week-old maize, we designed a specific primer to detect the presence of the fungus by using PCR. The primer pUM1 was designed on the ribosomal ITS1 region after alignment of several ITS sequences of Ustilaginales. Its specificity was assessed by amplification of a 680 bp sequence on DNA of Ustilago maydis, but not with DNA from Sporisorium reilianum, Ustilago hordei or maize (not shown). We used the primers pUM1epITS4 to the fungus in stems of five-week-old infected maize. To prevent DNA contamination by fungal cells outside plant tissues, we extracted DNA on isolated caulinary apices. As reported in Fig 2, the fungus could be detected in aerial part of the maize after root infection, whatever the pathogenic inoculum tested (FB6aFB6b, teliospores, FBD11/pOTEF). However, among all inoculations performed, only 16 % of inoculated plants showed a PCR signature, indicating that systemic growth of U. maydis from roots to aerial parts is weak. No significant difference was noticed according to fungal inoculum. In a last series of experimentation, three independent series of ten maize plants were inoculated on roots with teliospores and cultivated for 12 weeks in greenhouse. No symptom appeared on the aerial part of the maize line used, and overall we never observed any difference of growth during our assays in magenta boxes or in pot between infected, mock and control (FB6b inoculation) plants.
Ustilago maydis can infect Medicago roots Hairy roots of Medicago truncatula cv. Jemalong cultivated on Petri dishes were inoculated by FBD11-pOTEF. The inoculum was applied at root tips for a two-week coculture, until formation of fuzzy colonies at the tip of the roots (Fig 3A). Some aerial roots, not in contact with the solidified medium, appeared swollen with a visible white hyphal development at the surface (Fig 3B). Green fluorescence allowed to assess that this mycelium corresponded to the FBD11-pOTEF strain (Fig 3C). Fluorescence is not limited to external hyphae and could be observed into root tissue (Fig 3C double arrow). Confocal observations confirm the presence of typical pseudohyphae of Ustilago maydis inside root tissues (Fig 3D). Observation of root slides after staining with lactophenol-cotton blue revealed that the mycelium is more abundant in epidermal root cells than in the cortical zone (not shown). When observed by TEM (Fig 3E), the fungus could be observed inside tissues, sometimes forming grouped hyphae inside root cells. These results indicate that U. maydis is able to penetrate the roots of M. truncatula in absence of wounding.
A strigolactone analogue stimulates Ustilago maydis cell respiration The effect of GR24 on cell respiration of Sporisorium reilianum and Ustilago maydis was evaluated. Results in Table 1
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Fig 1 e Observation of Ustilago maydis in maize roots. (AeB) Transverse section of two-week-old roots stained by cotton blue. (A) The fungus penetrates essentially into the junction of epidermis cells (double arrows) and then develops intercellular and intracellular pseudohyphae (arrows). (B) The fungus invades the epidermis and the root cortex, passing from cells to cells through host cell wall (arrows). (CeD) Confocal microscopy of five-week-old roots infected by a solopathogenic strain expressing GFP (FBD11-pOTEF). (C) The use of a bi-photon apparatus allows revealing the different root cell tissues and the fungus. The fungus is present in all inner tissues of roots, except xylem. (D) In cortical cells, the fungus is visible in intercellular space (white double arrow) and inside host cells, forming hyphae crossing host cell wall (white arrows). (EeF) TEM of infected roots, stained with PATAg. (E) External fungal cells (*) are tightly stuck to the epidermal cell wall by peeling the outer root matrix (m). The fungus develops wedge cells which penetrate between two epidermal host cells (white arrow) before being intracellular (black arrow). (F) Perforation of the host cell wall (arrow) by the fungus. Magnification bars: (AeC) 30 mm; (D) 10 mm; (EeF) 5 mm.
indicate that GR24 have a positive effect on cell respiration of S. reilianum and U. maydis whereas the density of cells remained unchanged on control and assay during the experiment. This induction was observed on the two fungi either
on haploid or solopathogenic strains, and whatever the technique used (polarography or Cell Titer Blue). Using the Cell Titer Blue method, the optimal concentration to induce cell respiration was reached at 1011 M whereas a toxic effect
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Fig 2 e Electrophoresis of amplicons after PCR amplification of ITS regions using the primers pUM1epITS4. Lane 1: maize inoculated with a haploid strain of U. maydis FB6b. Lane 2: noninoculated maize. Lane 3: U. maydis FB6b strain. Lanes 4 and 10: 100 bp DNA ladder. Lanes 5 and 6: maize inoculated with (FB6a3FB6b) strains. Lanes 7: maize inoculated with FBD11pOTEF strain. Lanes 8 and 9: maize inoculated with teliospores. The arrow represents the size of amplicon (680 bp).
was observed at 105 M (Fig 4). In order to have a better understanding of such a response, we analysed the effect of GR24 on the expression of some genes involved in cell respiration (cytochrome C oxidase), translation (ribosomal
protein), and genes identified in their role in the previous (ras1, rop1) and later step (mig2.2) of pathogenicity. Data acquired on the solopathogenic strain by qRT-PCR showed that 1 h post addition of GR24, transcription of genes Cytc1
Fig 3 e Infection of hairy roots of Medicago truncatula with Ustilago maydis FBD11-pOTEF strain. (A) Spots of Ustilago in the vicinity of roots on surface of gelified medium, forming white filamentous colonies (arrows). (BeC) Observation of an aerial root with a stereomicroscope under visible and epifluorescence light. (B) Parts of roots appear swollen (white arrow), and white mycelium is sometime visible outside the root (black arrow). (C) Under fluorescent light, green fluorescence is visible in the areas where fungal structures are present (simple arrows), but also inside the root tissues (double arrow). (D) Fluorescent hyphae are observed inside Medicago root tissue using confocal microscopy (arrow). The hyphae cells are passing from cell to cell through host cell wall (white arrow). (E) TEM of infected Medicago roots, stained with PATAg. The fungus is observed inside nonhost root cell. Some cells are heavily infected. Magnification bars: (A) 1 cm; (BeC) 1 mm; (D) 20 mm; (E) 5 mm.
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Table 1 e Effect of GR24 10L8 M on cell respiration of Ustilago maydis and Sporisorium reilianum.
U. maydis haploid strain U. maydis solopathogenic strain S. reilianum haploid strain S. reilianum solopathogenic strain
O2 consumption
Redox potential
þ11.9 % ND þ13.1 % ND
þ14.1 % þ9.2 % þ11.6 % þ11.8 %
Oxygen consumption was measured by using polarographic method. Values correspond to the ratio of slopes (100 (sGR sCt)/sCt) obtained on cells elicited or not with GR24 during 1 h. Global cell redox potential, mainly resulting from cell respiration (see Material and methods) was measured by using Cell Titer Blue reagent. Values correspond to the ratio of fluorescence measured 1 h post addition of GR24 and in control condition (100 (fGR fCt)/fCt). No significant difference of cell density was noticed on assays and controls before and after 1 h for the different experiments. ND: Not Determined (values obtained were biased due to sensitivity of mycelial colonies to stirring in polarographic chamber). Values correspond to one of the three independent experiments.
(2.7 fold) and prib60S (3.24 fold) was up-regulated, but not the other genes.
Discussion Our experiments show that Ustilago maydis is able to infect maize roots. All the pathogenic forms of the fungus tested (teliospores, mix of compatible sporidia, solopathogenic strains) were infective, whereas the nonpathogen haploid strain didn’t penetrate roots. Penetration occurred by pseudohyphae which degrade epidermic root cell surface, form an infection wedge between two epidermic cells and then penetrate into epidermic or cortical cells. A similar mechanism of penetration between two adjacent epidermic cells was observed when U. maydis penetrates maize stigmas (Snetselaar & Mims
Fig 4 e Dose response effect of GR24 on cell respiration of Ustilago maydis. Respiration was measured using the Cell Titer Blue reaction. The relative cell respiration (Rc) was calculated from the respiration rate measured 1 h post addition of a range of concentration of GR24 (RGR24) and the corresponding control (RCtrol) (Rc [ 100 3 (RGR24 L RCtrol)/ RCtrol).
1993). This behaviour is in accordance with the fact that U. maydis possesses only discrete appressorium-like structures, showing slightly swollen cells (Snetselaar & Mims 1993; Snetselaar et al. 2001; Freitag et al. 2011) and doesn’t have the capacity to penetrate plant tissue by mechanical force. After penetration, hyphae grew intracellularly and intercellularly and invaded all parts of maize roots. Ustilago maydis was unable to form galls in roots. Based on transcriptomic approaches, Skibbe and co-authors (2010) proposed that the induction of sporogenesis in U. maydis is dependent of host organ specific gene expression. As a carbon sink, the fungus seems unable to recruit enough photosynthetic carbon as in leaves to fuel tumour metabolism (Horst et al. 2010). Maize is a host of another smut fungus, Sporisorium reilianum f. sp. zeae, causing maize head smut disease. Typical symptom corresponds to a black sorus full of teliospores instead of ear or tassel. Sporisorium reilianum infects maize only via roots of plantlets and sporulates only if it reaches apical meristem (Martinez et al. 2000). In our assays with U. maydis, PCR detection revealed that only 16 % of maize stems were infected five weeks after root infection and we were unable to observe hyphae in aerial tissues. The systemic growth of the fungus from root to stem seems to be insufficient to allow sporulation and gall formation. Based on the observation of gall and sorus formation, it was supposed that these two smut fungi have different strategies of penetration in maize e U. maydis on aerial parts, S. reilianum on roots. It is likely that their difference seems to be related more to their ability to growth systemically in planta than to their potentiality to infect roots. Infection of aerial parts of alternative hosts by U. maydis has been already observed (Leon-Ramirez et al. 2004; ndez-Mora n et al. 2005). We observed here that U. maydis Me could parasite roots of Medicago truncatula. The fungus penetrated in the absence of wounding on the apical zone of roots, and then grew on all parts of hairy roots. Although obtained in controlled condition, such observation has to be considered in the light of metagenomic data that showed that plant root hosted microbiome is more diversified than expected (Lundberg et al. 2012). Altogether, these observations suggest that the concept of host specificity can be larger for biotrophic fungi than previously identified based on symptom formation. Medicago truncatula is a model legume to study root biotrophic symbiosis by rhizobia and AM fungi. Several comparative transcriptomic works have been developed to find shared signalling and metabolic pathways involved in these two biotrophic symbiotic interactions (Journet et al. 2002; Manthey et al. 2004). Such comparative analyses between symbiotic and pathogenic interactions have already shown their interest to identify conserved mechanism in plant microbe interactions: comparison of the rice root transcriptomes in presence of AM fungi and Magnaporthe grisea revealed common traits (Guimil et al. 2005). Additionally, the observation that U. maydis is hosted by M. truncatula opens the way to investigate the incidence in biotrophic pathogenesis of the common signalling pathway required for rhizobial and mycorrhizal symbiosis, recently described as involved in the perception of Aphanomyces euteiches (Rey et al. 2013). The ability of U. maydis to infect alternative host is intriguing. Although U. maydis is one of the best known fungal models to study biotrophic interaction, its epidemiology is
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poorly documented: teliospores formed at the end of life cycle are widespread in soil and overwinter, but the primo infective inoculum in field is not clearly identified. In alternative hosts, the fungus doesn’t form teliospores but external hyphae were observed on different parts of Medicago roots not in contact with the inoculum. Aerial mycelium was also observed on different alternative hosts (Mendes-Giannini 2005). These discrete fungal structures were only observed in controlled conditions but it seems necessary to evaluate the incidence of infected weeds as a source of inoculum in field, discarding airborne pathogenic structures. It will be of interest to investigate the epidemiology incidence of these nonconventional propagative forms compared to teliospore dispersal. In a last series of experiments to argue that U. maydis can be a soilborne pathogenic microorganism, we tested its ability to perceive root exuded molecules. We tested the effect of GR24, a chemical analogue of strigolactones. This chemical family is involved in plant-parasitic weeds interaction (see Bouwmeester et al. 2003 for review) and in AM symbiosis (Akiyama et al. 2005; Gomez-Roldan et al. 2007). On AM fungi, GR24 elicits cell respiration prior hyphal branching (Besserer et al. 2006). Based on hyphal branching tests, contradictory conclusions were reported about perception of GR24 by soil filamentous fungi at 106 M: from no effect (Steinkellner et al. 2007) to branching activation (Dor et al. 2011). This last study also reports an inhibitory effect of colony growth at 105 M as we observed on U. maydis. Using respiratory direct (polarography) and indirect (Cell Titer Blue) assays, we were able to demonstrate that GR24 rapidly stimulates cell respiration of U. maydis and S. reilianum at concentration as lower as 1013 M, in accordance with the most active concentrations already observed on AM fungi (Besserer et al. 2006). The increase of cell respiration is confirmed by the observed induction of cytc1 gene transcription 1 h post addition of GR24. At this time of induction, we didn’t observe induction of genes involved in early (ras1, rop1) or late (mig2.2) pathogenic events of U. maydis. More information about the biological incidence of the perception of strigolactones in U. maydis and S. reilianum pathogenesis would need the use of maize mutant lines repressed in strigolactones biosynthesis. To date, such mutants are still not available, but this result underlines that strigolactones could have a larger spectrum of targeted organisms than considered and could be root signalling components involved in the establishment of the rhizospheric flora. Incidently, this investigation supports that U. maydis doesn’t differ from the root infecting pathogen S. reilianum about its ability to perceive root exuded molecules and argued to the fact that it could be considered as a rhizospheric fungus. This new insight into the ability of U. maydis to penetrate roots of maize and barrel medic and to perceive strigolactones makes it a powerful tool to better understand the underground interactions.
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journal homepage: www.elsevier.com/locate/funbio
Assessment of Ustilago maydis as a fungal model for root infection studies Mahta MAZAHERI-NAEINIa, Seyed Kazem SABBAGHb,*, Yves MARTINEZc, d,e Nathalie SEJALON-DELMAS , Christophe ROUXd,e a
Department of Medical Genetic, Faculty of Medicine, Shahid Sadoughi University of Medical Sciences, BP634, F8916978477 Yazd, Iran b Department of Plant Protection and Institute of Plant Biotechnology, University of Zabol, BP98615-58, F9861335856 Zabol, Iran c CNRS, FR3450, BP42617, F-31326 Castanet-Tolosan CEDEX, France d Universite de Toulouse, UPS, UMR5546, Laboratoire de Recherche en Sciences Vegetales, Castanet-Tolosan CEDEX 31326, France e CNRS, UMR5546, 31326 Castanet-Tolosan CEDEX, France
article info
abstract
Article history:
Ustilago maydis is a fungus infecting aerial parts of maize to form smutted galls. Due to its
Received 21 June 2014
interest as a genetic tool in plant pathology, we evaluated its ability to penetrate into plant
Received in revised form
roots. The fungus can penetrate between epidermic root cells, forming inter and intracel-
25 November 2014
lular pseudohyphae. Root infection didn’t provoke gall formation on the maize lines tested,
Accepted 1 December 2014
and targeted PCR detection showed that U. maydis, unlike the other maize smut fungus
Available online 17 December 2014
Sporisorium reilianum, has a weak aptitude to grow from the roots up to the aerial part of
Corresponding Editor:
maize. We also observed that U. maydis can infect Medicago truncatula hairy roots as an al-
Pieter van West
ternative host. This plant species is a model host to study root symbiosis, and this pathosystem can provide new insights on rootemicrobe interactions. Considering that U. maydis
Keywords:
could be a soil fungus, we tested its responsiveness to GR24, a strigolactone analogue.
alternative host
Strigolactones are root exuded molecules which activate mitochondrial metabolism of
cell respiration
arbuscular mycorrhizal (AM) fungi. Physiologic and molecular analysis revealed that
soil fungus
GR24 also increases cell respiration of U. maydis. This result points out that strigolactones
strigolactone
could have an incidence on several rhizospheric microbes. These data provide evidences
Ustilaginaceae
that the biotrophic pathogen U. maydis has to be considered for studying root infection. ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Introduction Among plant parasites, biotrophic and hemibiotrophic pathogenic fungi are described as highly adapted to their hosts, showing a tissue-specific infection site and a limited host spectrum: Blumeria graminis f. sp. hordei infects leaves of
barley, Colletotrichum lindemuthianum is pathogen on leaves of bean. One limitation of this restricted description is that the tissue specificity and the host range of a pathogen are usually defined by observation of the symptoms. Symptom formation and severity are not always strictly correlated to the level of invasion of a plant by a fungus, particularly in the case of
* Corresponding author. Tel.: +98 5431232100; fax: +98 5432240696. E-mail address: [email protected] (S. K. Sabbagh). http://dx.doi.org/10.1016/j.funbio.2014.12.002 1878-6146/ª 2014 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
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biotrophic fungi which can interact with a host cell without causing major damage (Koeck et al. 2011). Ustilago maydis for instance is a biotrophic fungus causing corn smut disease, described in mycological flora as a strict pathogen of Zea genus nky 1994). Recent works showed that a number of monocot (Va and dicot species, like papaya or Arabidopsis thaliana, were susndezceptible to inoculation (Leon-Ramirez et al. 2004; Me n et al. 2005). Although U. maydis didn’t sporulate in these Mora alternative hosts, these observations underline that this biotrophic fungus has an extended capacity to infect plants and point out the interest to revisit its biology. Ustilago maydis is a major fungal model to study biotrophic plant parasitism (Dean et al. 2012). It is a dimorphic fungus: compatible saprotrophic haploid yeasts mate and form pathogenic dikaryotic pseudohyphae (Nadal et al. 2008). A great interest in this model is that sexual cycle, morphology and pathogenicity are interconnected processes (Kahmann & Kamper 2004). For these reasons its genome was sequenced € mper et al. 2006) and lot of mutants were obtained. Based (Ka on symptom formation e e.g. gall formation e U. maydis is considered as infecting maize via different aerial part of maize (Christensen 1963; references therein; Wenzler & Meins 1987). Several works described with accuracy the penetration process of dikaryotic pseudohyphae issuing from fusion of compatible yeast strains on stem, young and aged leaves 1981; Snetselaar & Mims 1992, 1993, and silks (Mills & Kotze 1994; Snetselaar 1993). We investigated here whether this pathogen could efficiently infect maize plantlets via roots. Different inoculum forms were tested: teliospores, a mix of compatible haploid yeasts, a solopathogenic strain, and a haploid yeast strain as control. Solopathogenic strains are diploid strains of the fungus which can infect plants in absence of mating with a compatible one (Holliday 1974). The efficiency of infection was analysed by using a set of complementary microscopic techniques and PCR detection. To further evaluate the potentialities of U. maydis as a rhizospheric pathogen, we also tested its ability to infect alternative host roots and to perceive root exuded molecules. Inoculations were carried out on Medicago truncatula hairy roots. This plant species is a model plant to investigate root biotrophic symbiosis. As the main root pathosystems available on barrel medic involve necrotrophic fungi (Tivoli et al. 2006) or hemibiotrophic ones (Rey et al. 2013), it was interesting to test on this plant the infectious potential of the biotrophic fungus U. maydis. The second evaluation consisted in monitoring the response of U. maydis to root exuded molecules. Many soilborne organisms were described to perceive molecules exuded by plant roots. This is the case for Sporisorium reilianum f. sp. zeae, a root infecting fungus phylogenetically related to U. maydis, causing maize head smut, which was described to be sensitive to a lipophilic fraction of maize root exudates (Martinez et al. 2001). We particularly tested the response of U. maydis to GR24, a synthetic chemical analogue of strigolactones. These molecules, present in root exudates of a broad spectrum of plants, have been described as important signals occurring in previous steps of interactions of plants with parasitic weeds (Cook et al. 1966, 1972) and with arbuscular mycorrhizal (AM) fungi (Akiyama et al. 2005). On AM fungi, strigolactones boost mitochondrial activity, causing a putative metabolic switch (Besserer et al. 2006).
M. Mazaheri-Naeini et al.
We tested the effect of GR24 on cell respiration of U. maydis and S. reilianum, this last species being considered as a natural root infecting pathogen of maize. The aim of these investigations is to evaluate U. maydis as a pathogenic fungal model to study root infection.
Materials and methods Fungal strains and culture condition Haploid strains FB6a and FB6b (Banuett & Herskowitz 1989), teliospores obtained from artificial contamination of maize with the strains FB6aFB6b, and a solopathogenic strain FBD11/pOTEF expressing the green fluorescent protein (GFP) (Spellig et al. 1996) were used in this study. Haploid (CRM) and solopathogenic (CRS1) strains of Sporisorium reilianum were isolated in our laboratory. All yeast strains were cultivated in liquid potato dextrose broth medium (PDB) at 24 C under shaking 100 rpm.
Plants and culture conditions A maize line susceptible to Ustilago maydis (LMZ66-France) was used as natural host. Corn seeds free of microbial contamination were cultivated in Magenta boxes on solidified M medium or in pot on sterilized peat (1 h at 120 C) as previously described (Martinez et al. 2002). In Magenta boxes, inoculations on one-week-old maize plantlets were carried out by adding in the vicinity of roots 500 mL of cell suspension at 107 cells mL1. In pot, inoculations were carried out by pouring directly on roots 1 mL of cell suspension at 107 cells mL1. For these experiments, the plants were grown in a night/day temperature of 18/24 5 C for three to five weeks, except for symptom observation where cultivation was performed in greenhouse up to floral formation. Infection of hairy roots of Medicago truncatula cv. Jemalong grown on M medium were performed by adding droplets of a culture of FBD11/pOTEF on root tips, followed by a two-week cocultivation before observation.
Microscopic observations Observations of infection and development of Ustilago maydis in roots were performed by macroscopic and microscopic observation. For light microscopy, samples of root were embedded in agarose before cutting sections of 150 mm width. The sections were stained with lactophenol-cotton blue and rinsed in lactophenol for 10 min. All the specimens were mounted on a glass slide and observed under bright-field optics or differential interference contrast (DIC). Observations under bright field, DIC, or epifluorescence were carried out in a Leica DMIRB Microscope (Leica, Wetzlar, Germany). Images were acquired using a colour video camera (Hyper Had, Japan). For transmission electron microscopy (TEM) observations, we used the method described by Martinez et al. 1999. Ultrathin sections (50e60 nm in thickness) were stained with uranyl-acetate or PATAg (periodic acidethiocarbohyrazideesilver) and examined using a Philips 301 transmission electron microscope at 80 kV (Philips, Eindhoven, The
Assessment of Ustilago maydis
Netherlands). For confocal microscopy, roots inoculated with a solopathogenic strain of U. maydis expressing the green fluorescence protein (FBD11-pOTEF) were observed by a spectral confocal laser scanning system (SP2 SE, Leica, Germany) equipped with an upright microscope (DM 6000, Leica, Germany). An Argon laser emitted at 488 nm was used to detect GFP fluorescence in the range between 500 and 530 nm. The ray line of the HeeNe laser emitted at 543 nm was able to excite the cell wall coloured by a solution of blue evans 0.01 % for 30 s and the fluorescence was collected in the range 590e710 nm. The images were acquired in the dual mode. The overlay images were then computed by projection of 20e30 plan-confocal images acquired in Z dimension with 0.8 mm increments between two focal planes.
PCR detection Plants used for these experiments were cultivated in pots for five weeks after addition of different inoculum sources on root plantlets. A set of ten plants was used for each inoculum, and the experiments were triplicated. Aerial parts were then collected and caulinar apices were isolated to avoid risk of contamination by the inoculum on plant surface. DNA extractions from 50 mg of plant material were performed using the CTAB procedure. We designed the specific primer pUM1 (50 -CGGTCGGTCTGTCGAAACC-30 ) on the ribosomic internal transcribed spacer one region of Ustilago maydis. PCR reactions (5 min 94 Ce35 cycles 1 min 54 C 1 min 72 C 1 min 94 Ce10 min 72 C) using pUM1epITS4 primers were followed by electrophoresis of amplicons on agarose 1.5 %. The presence of U. maydis in samples is assessed by the presence of a 680 bp band.
Effect of GR24 on cell respiration Respiratory measurements were performed by polarography and by a fluorescent assay 1 h after addition of GR24, a strigolactone analogue. For all strains (FB6b and FBD11/pOTEF for Ustilago maydis, CRM and CRS1 for Sporisorium reilianum), young cultures of ten-hour-old were used for these assays. Cell density was measured in assay and control at the end of the experiment and compared to the starting respective concentration. Polarography was performed by using a Clark electrode according to (Besserer et al. 2006). A volume of 10 mL of cells (106 cells mL1) were incubated during 1 h with GR24 or with the corresponding solvent (0.01 % acetone/water) for control before measuring oxygen consumption. For fluorescent assays, the protocol used is adapted from the recommendation of the manufacturer. A volume of 20 ml of Cell TiterBlue reagent (CTBR Promega, Madison, USA) was added to 100 ml of cell suspension at 107 cell mL1. The blue dye (resazurin) could be reduced in resorufin, a fluorescent compound, by the cell redox potential. According to provider technical protocol, CTB reagent was previously used for respiration assays (https://france.promega.com/resources/product-guidesand-selectors/protocols-and-applications-guide/cell-viability/). As a validation, we used specific inhibitors of fungal cell nol 100 mM): fluoresrespiration (carboxin 300 mM, di-nitro-phe cence formation on cell cultures was totally inhibited (data not shown), indicating that the Redox potential formed and
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then the resulting fluorescence emitted is essentially issuing from mitochondrial activity. Cell respiration was measured on 96-well-optical reaction plates at 25 C by recording fluorescence with an excitation wavelength of 560 nm and an emission wavelength of 590 nm using a Fluoroskan FL600 (Bio-Tek, Vermont, USA).
RNA extraction and RT-PCR Control (acetone 0.01 %) and GR24-stimulated cells (FBD11) were harvested by centrifugation after elicitation for 1 h and conserved at 80 C for RNA extraction. Total RNAs were isolated using a standard phenolechloroform procedure, purified using RNA purification Kit (promega) and treated by the RNase-Free DNase Set (Promega) according to the manufacturer’s recommendations. Contamination of residual genomic DNA in all RNA samples was verified by conventional PCR amplification on total RNA using the designed primers. Total RNA yield and concentration were measured with Nanodrop ND1000 spectrophotometer and the concentration of each sample was adjusted to 100 ng mL1. Quality of RNA was assessed by 1 % agarose gel electrophoresis stained by ethidium bromide and evaluated with Agilent 2100 Bioanalyzer (Agilent Technologies). For reverse transcription step, ImPromII enzyme (Promega) was used according to the manufacturer’s recommendations. First strands of cDNA were purified and concentration of final working solution was adjusted according to ND-1000 spectrophotometer quantification.
Primer design and qRT-PCR Oligonucleotide primers used in this study were designed using Primer3 site (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Ras1 (GenBank accession number CAA51689, fp 50 -TACCATTGAGGACTCTTACC-30 and rp 50 -CGGCAGTATCCAACACATC-30 ), (Muller et al. 2003), Rop1 (GenBank accession number CAA51689, fp 50 -CAGAGGTTTGGGACAAGCTC-30 and rp 50 -TAGGACGAGGAAGAGGACGA-30 ), (Brefort et al. 2005) and mig2-2 (fp 50 -AACCCAAAGCAGCCGTACCT-3’and rp 50 GCTCTTCCACCAACGAATCG-30 ), (Basse et al. 2002) were used as marker genes involved in pathogenicity. Cytochrome c1 Oxydase e Cytc1 (GenBank accession number ABQ96919, fp 50 CTATGGCTGAGGAGGTCGAG-3’and rp 50 -TAAGGTCGGGGCATGTAGTC-30 ) is a gene marker of cell respiration, hypothetical 60S ribosomal protein (GenBank accession number UM02518.1 fp 50 -GCAGGTGACTTTCGAGATCC-30 and rp 50 GGTCCGTGTGAATTTTGGTC-30 ) is as gene marker of cell translation. Elongation factor-EF1 (GenBank accession number XP_757071, fp 50 -TGGTAAGACCCTCCTTGACG-30 and rp 30 -ACCGCCGATCTTGTAGACAT-50 ) and glycine N-acyltransferase GNAT (GenBank accession no EDN60858, fp 50 TGCCATCCTCGACTTTATCC-30 and rp 30 -TCAGCAATCAACACCTCTGC-50 ) were used as housekeeping genes deduced from a preliminary transcriptomic analysis (not shown). Realtime PCR was performed on cDNA templates using a lightcycler ABI PRISM 7900 HT sequence detection system (Perkin Elmer/ Applied Biosystem, Foster City, USA) and PCR Master Mix for Syber Green Assays (Applied Biosystems, Foster City, USA) according to the manufacturer’s protocol. Real-time PCR was carried out in optical 384-well plates using the ABI PRISM
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7900 HT sequence detection system (Applied Biosystems) using the following concentration: 8 ml of SYBR Green PCR Master Mix (Applied Biosystems), 10 mM of each oligonucleotide primer (final concentration) and 2 ng of cDNA template in 10 ml reaction volume. Each gene amplification was prepared in triplicates. Two biological repetitions were carried out. Triplicates were validated with technical error under 0.5 CT. The amplification condition was: 50 C for 2 min, 95 C for 10 min, 40 cycles at 95 C for 15 sec, 60 C for 1 min. Melting curves analysis was performed after each reaction, to exclude nonspecific amplifications, with the thermal cycle at 95 C for 15sec, 60 C for 15 sec, and 95 C for 15 sec. The optimal baseline and threshold values were determined using automatic CT function available with the SDS 2.2 software (Applied Biosystems). The equation of the line that best fits the data was determined by minimizing error for regression analysis. The R2 value was calculated to estimate the accuracy of the real-time RT-PCR as a quantification method. The slope of the standard curve was used to calculate the efficiency of the real-time QRT-PCR according to the formula E ¼ 101/slope (Ptaffi 2001; Wen et al. 2005). The calibration curves were created using series of cDNA dilutions from 10 to 100 ng. EF1 gene was used for normalization experimental design. All data exported from SDS 2.2 software were calculated with Microsoft Excel software.
Results Ustilago maydis infects maize roots The ability of Ustilago maydis to penetrate and proliferate in maize roots, then to systemically grow up to aerial parts was respectively investigated by using microscopy techniques and PCR detection. Mock plants and also plants inoculated with the nonpathogenic haploid strain Fb6b were used as controls. After three weeks of culture in Magenta box, roots of maize inoculated with the pathogenic inoculums forms of U. maydis (FB6aFB6b mix; teliospores; FBD11/pOTEF) were covered with a hyphal net (not shown). Transverse sections stained with cotton blue reveal that the fungus penetrates the roots by growing at the junction of epidermic cells, then breaches the cell wall and forms hyphae in host cells (Fig 1A). Fungal growth is mainly intracellular, the fungus growing from cell to cell through the host cell wall in the inner part of the roots (Fig 1B). In larger roots, the observation was performed in confocal microscopy with a fluorescent solopathogenic strain (FBD11 solo-pOTEF) expressing GFP. The fungus can invade all root tissues except xylem (Fig 1C). The density of fungal cells in roots increased between three and five weeks. In general, we observed that U. maydis was less abundant in secondary roots and in the central pith. Although the fungus is mainly intracellular, growth in intercellular space is often observed in root epidermis (Fig 1D). In TEM observations, the fungus appeared as rounded cells or part of pseudohyphae depending on the angle of cutting (Fig 1E,F). The fungus hydrolyses the outer cell wall of root cells, penetrates by growing in the middle lamella of cell wall, then penetrates root cells and grows intracellularly from cell to cell. Same observations were obtained whatever the inoculum source, except with the nonpathogenic haploid FB6b strain (not shown).
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Maize root infection by Ustilago maydis didn’t cause symptoms Microscopic observations on the aerial part of the maize, three weeks after root inoculation, didn’t reveal the presence of fungal structure either by light or fluorescence microscopy. To define if the fungus could migrate systemically from roots to stem on more lignified five-week-old maize, we designed a specific primer to detect the presence of the fungus by using PCR. The primer pUM1 was designed on the ribosomal ITS1 region after alignment of several ITS sequences of Ustilaginales. Its specificity was assessed by amplification of a 680 bp sequence on DNA of Ustilago maydis, but not with DNA from Sporisorium reilianum, Ustilago hordei or maize (not shown). We used the primers pUM1epITS4 to the fungus in stems of five-week-old infected maize. To prevent DNA contamination by fungal cells outside plant tissues, we extracted DNA on isolated caulinary apices. As reported in Fig 2, the fungus could be detected in aerial part of the maize after root infection, whatever the pathogenic inoculum tested (FB6aFB6b, teliospores, FBD11/pOTEF). However, among all inoculations performed, only 16 % of inoculated plants showed a PCR signature, indicating that systemic growth of U. maydis from roots to aerial parts is weak. No significant difference was noticed according to fungal inoculum. In a last series of experimentation, three independent series of ten maize plants were inoculated on roots with teliospores and cultivated for 12 weeks in greenhouse. No symptom appeared on the aerial part of the maize line used, and overall we never observed any difference of growth during our assays in magenta boxes or in pot between infected, mock and control (FB6b inoculation) plants.
Ustilago maydis can infect Medicago roots Hairy roots of Medicago truncatula cv. Jemalong cultivated on Petri dishes were inoculated by FBD11-pOTEF. The inoculum was applied at root tips for a two-week coculture, until formation of fuzzy colonies at the tip of the roots (Fig 3A). Some aerial roots, not in contact with the solidified medium, appeared swollen with a visible white hyphal development at the surface (Fig 3B). Green fluorescence allowed to assess that this mycelium corresponded to the FBD11-pOTEF strain (Fig 3C). Fluorescence is not limited to external hyphae and could be observed into root tissue (Fig 3C double arrow). Confocal observations confirm the presence of typical pseudohyphae of Ustilago maydis inside root tissues (Fig 3D). Observation of root slides after staining with lactophenol-cotton blue revealed that the mycelium is more abundant in epidermal root cells than in the cortical zone (not shown). When observed by TEM (Fig 3E), the fungus could be observed inside tissues, sometimes forming grouped hyphae inside root cells. These results indicate that U. maydis is able to penetrate the roots of M. truncatula in absence of wounding.
A strigolactone analogue stimulates Ustilago maydis cell respiration The effect of GR24 on cell respiration of Sporisorium reilianum and Ustilago maydis was evaluated. Results in Table 1
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Fig 1 e Observation of Ustilago maydis in maize roots. (AeB) Transverse section of two-week-old roots stained by cotton blue. (A) The fungus penetrates essentially into the junction of epidermis cells (double arrows) and then develops intercellular and intracellular pseudohyphae (arrows). (B) The fungus invades the epidermis and the root cortex, passing from cells to cells through host cell wall (arrows). (CeD) Confocal microscopy of five-week-old roots infected by a solopathogenic strain expressing GFP (FBD11-pOTEF). (C) The use of a bi-photon apparatus allows revealing the different root cell tissues and the fungus. The fungus is present in all inner tissues of roots, except xylem. (D) In cortical cells, the fungus is visible in intercellular space (white double arrow) and inside host cells, forming hyphae crossing host cell wall (white arrows). (EeF) TEM of infected roots, stained with PATAg. (E) External fungal cells (*) are tightly stuck to the epidermal cell wall by peeling the outer root matrix (m). The fungus develops wedge cells which penetrate between two epidermal host cells (white arrow) before being intracellular (black arrow). (F) Perforation of the host cell wall (arrow) by the fungus. Magnification bars: (AeC) 30 mm; (D) 10 mm; (EeF) 5 mm.
indicate that GR24 have a positive effect on cell respiration of S. reilianum and U. maydis whereas the density of cells remained unchanged on control and assay during the experiment. This induction was observed on the two fungi either
on haploid or solopathogenic strains, and whatever the technique used (polarography or Cell Titer Blue). Using the Cell Titer Blue method, the optimal concentration to induce cell respiration was reached at 1011 M whereas a toxic effect
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Fig 2 e Electrophoresis of amplicons after PCR amplification of ITS regions using the primers pUM1epITS4. Lane 1: maize inoculated with a haploid strain of U. maydis FB6b. Lane 2: noninoculated maize. Lane 3: U. maydis FB6b strain. Lanes 4 and 10: 100 bp DNA ladder. Lanes 5 and 6: maize inoculated with (FB6a3FB6b) strains. Lanes 7: maize inoculated with FBD11pOTEF strain. Lanes 8 and 9: maize inoculated with teliospores. The arrow represents the size of amplicon (680 bp).
was observed at 105 M (Fig 4). In order to have a better understanding of such a response, we analysed the effect of GR24 on the expression of some genes involved in cell respiration (cytochrome C oxidase), translation (ribosomal
protein), and genes identified in their role in the previous (ras1, rop1) and later step (mig2.2) of pathogenicity. Data acquired on the solopathogenic strain by qRT-PCR showed that 1 h post addition of GR24, transcription of genes Cytc1
Fig 3 e Infection of hairy roots of Medicago truncatula with Ustilago maydis FBD11-pOTEF strain. (A) Spots of Ustilago in the vicinity of roots on surface of gelified medium, forming white filamentous colonies (arrows). (BeC) Observation of an aerial root with a stereomicroscope under visible and epifluorescence light. (B) Parts of roots appear swollen (white arrow), and white mycelium is sometime visible outside the root (black arrow). (C) Under fluorescent light, green fluorescence is visible in the areas where fungal structures are present (simple arrows), but also inside the root tissues (double arrow). (D) Fluorescent hyphae are observed inside Medicago root tissue using confocal microscopy (arrow). The hyphae cells are passing from cell to cell through host cell wall (white arrow). (E) TEM of infected Medicago roots, stained with PATAg. The fungus is observed inside nonhost root cell. Some cells are heavily infected. Magnification bars: (A) 1 cm; (BeC) 1 mm; (D) 20 mm; (E) 5 mm.
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Table 1 e Effect of GR24 10L8 M on cell respiration of Ustilago maydis and Sporisorium reilianum.
U. maydis haploid strain U. maydis solopathogenic strain S. reilianum haploid strain S. reilianum solopathogenic strain
O2 consumption
Redox potential
þ11.9 % ND þ13.1 % ND
þ14.1 % þ9.2 % þ11.6 % þ11.8 %
Oxygen consumption was measured by using polarographic method. Values correspond to the ratio of slopes (100 (sGR sCt)/sCt) obtained on cells elicited or not with GR24 during 1 h. Global cell redox potential, mainly resulting from cell respiration (see Material and methods) was measured by using Cell Titer Blue reagent. Values correspond to the ratio of fluorescence measured 1 h post addition of GR24 and in control condition (100 (fGR fCt)/fCt). No significant difference of cell density was noticed on assays and controls before and after 1 h for the different experiments. ND: Not Determined (values obtained were biased due to sensitivity of mycelial colonies to stirring in polarographic chamber). Values correspond to one of the three independent experiments.
(2.7 fold) and prib60S (3.24 fold) was up-regulated, but not the other genes.
Discussion Our experiments show that Ustilago maydis is able to infect maize roots. All the pathogenic forms of the fungus tested (teliospores, mix of compatible sporidia, solopathogenic strains) were infective, whereas the nonpathogen haploid strain didn’t penetrate roots. Penetration occurred by pseudohyphae which degrade epidermic root cell surface, form an infection wedge between two epidermic cells and then penetrate into epidermic or cortical cells. A similar mechanism of penetration between two adjacent epidermic cells was observed when U. maydis penetrates maize stigmas (Snetselaar & Mims
Fig 4 e Dose response effect of GR24 on cell respiration of Ustilago maydis. Respiration was measured using the Cell Titer Blue reaction. The relative cell respiration (Rc) was calculated from the respiration rate measured 1 h post addition of a range of concentration of GR24 (RGR24) and the corresponding control (RCtrol) (Rc [ 100 3 (RGR24 L RCtrol)/ RCtrol).
1993). This behaviour is in accordance with the fact that U. maydis possesses only discrete appressorium-like structures, showing slightly swollen cells (Snetselaar & Mims 1993; Snetselaar et al. 2001; Freitag et al. 2011) and doesn’t have the capacity to penetrate plant tissue by mechanical force. After penetration, hyphae grew intracellularly and intercellularly and invaded all parts of maize roots. Ustilago maydis was unable to form galls in roots. Based on transcriptomic approaches, Skibbe and co-authors (2010) proposed that the induction of sporogenesis in U. maydis is dependent of host organ specific gene expression. As a carbon sink, the fungus seems unable to recruit enough photosynthetic carbon as in leaves to fuel tumour metabolism (Horst et al. 2010). Maize is a host of another smut fungus, Sporisorium reilianum f. sp. zeae, causing maize head smut disease. Typical symptom corresponds to a black sorus full of teliospores instead of ear or tassel. Sporisorium reilianum infects maize only via roots of plantlets and sporulates only if it reaches apical meristem (Martinez et al. 2000). In our assays with U. maydis, PCR detection revealed that only 16 % of maize stems were infected five weeks after root infection and we were unable to observe hyphae in aerial tissues. The systemic growth of the fungus from root to stem seems to be insufficient to allow sporulation and gall formation. Based on the observation of gall and sorus formation, it was supposed that these two smut fungi have different strategies of penetration in maize e U. maydis on aerial parts, S. reilianum on roots. It is likely that their difference seems to be related more to their ability to growth systemically in planta than to their potentiality to infect roots. Infection of aerial parts of alternative hosts by U. maydis has been already observed (Leon-Ramirez et al. 2004; ndez-Mora n et al. 2005). We observed here that U. maydis Me could parasite roots of Medicago truncatula. The fungus penetrated in the absence of wounding on the apical zone of roots, and then grew on all parts of hairy roots. Although obtained in controlled condition, such observation has to be considered in the light of metagenomic data that showed that plant root hosted microbiome is more diversified than expected (Lundberg et al. 2012). Altogether, these observations suggest that the concept of host specificity can be larger for biotrophic fungi than previously identified based on symptom formation. Medicago truncatula is a model legume to study root biotrophic symbiosis by rhizobia and AM fungi. Several comparative transcriptomic works have been developed to find shared signalling and metabolic pathways involved in these two biotrophic symbiotic interactions (Journet et al. 2002; Manthey et al. 2004). Such comparative analyses between symbiotic and pathogenic interactions have already shown their interest to identify conserved mechanism in plant microbe interactions: comparison of the rice root transcriptomes in presence of AM fungi and Magnaporthe grisea revealed common traits (Guimil et al. 2005). Additionally, the observation that U. maydis is hosted by M. truncatula opens the way to investigate the incidence in biotrophic pathogenesis of the common signalling pathway required for rhizobial and mycorrhizal symbiosis, recently described as involved in the perception of Aphanomyces euteiches (Rey et al. 2013). The ability of U. maydis to infect alternative host is intriguing. Although U. maydis is one of the best known fungal models to study biotrophic interaction, its epidemiology is
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poorly documented: teliospores formed at the end of life cycle are widespread in soil and overwinter, but the primo infective inoculum in field is not clearly identified. In alternative hosts, the fungus doesn’t form teliospores but external hyphae were observed on different parts of Medicago roots not in contact with the inoculum. Aerial mycelium was also observed on different alternative hosts (Mendes-Giannini 2005). These discrete fungal structures were only observed in controlled conditions but it seems necessary to evaluate the incidence of infected weeds as a source of inoculum in field, discarding airborne pathogenic structures. It will be of interest to investigate the epidemiology incidence of these nonconventional propagative forms compared to teliospore dispersal. In a last series of experiments to argue that U. maydis can be a soilborne pathogenic microorganism, we tested its ability to perceive root exuded molecules. We tested the effect of GR24, a chemical analogue of strigolactones. This chemical family is involved in plant-parasitic weeds interaction (see Bouwmeester et al. 2003 for review) and in AM symbiosis (Akiyama et al. 2005; Gomez-Roldan et al. 2007). On AM fungi, GR24 elicits cell respiration prior hyphal branching (Besserer et al. 2006). Based on hyphal branching tests, contradictory conclusions were reported about perception of GR24 by soil filamentous fungi at 106 M: from no effect (Steinkellner et al. 2007) to branching activation (Dor et al. 2011). This last study also reports an inhibitory effect of colony growth at 105 M as we observed on U. maydis. Using respiratory direct (polarography) and indirect (Cell Titer Blue) assays, we were able to demonstrate that GR24 rapidly stimulates cell respiration of U. maydis and S. reilianum at concentration as lower as 1013 M, in accordance with the most active concentrations already observed on AM fungi (Besserer et al. 2006). The increase of cell respiration is confirmed by the observed induction of cytc1 gene transcription 1 h post addition of GR24. At this time of induction, we didn’t observe induction of genes involved in early (ras1, rop1) or late (mig2.2) pathogenic events of U. maydis. More information about the biological incidence of the perception of strigolactones in U. maydis and S. reilianum pathogenesis would need the use of maize mutant lines repressed in strigolactones biosynthesis. To date, such mutants are still not available, but this result underlines that strigolactones could have a larger spectrum of targeted organisms than considered and could be root signalling components involved in the establishment of the rhizospheric flora. Incidently, this investigation supports that U. maydis doesn’t differ from the root infecting pathogen S. reilianum about its ability to perceive root exuded molecules and argued to the fact that it could be considered as a rhizospheric fungus. This new insight into the ability of U. maydis to penetrate roots of maize and barrel medic and to perceive strigolactones makes it a powerful tool to better understand the underground interactions.
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