Chemotropic Assay for Testing Fungal Response to Strigolactones and Strigolactone-Like Compounds

Depósito de Investigación de la Universidad de Sevilla https://idus.us.es/ This is an Accepted Manuscript of an article published by Springer Nature in Strigolactones in May 2021, available at: https://doi.org/10.1007/978-1-0716-1429-7_9 © 2021 Springer Science+Business Media, LLC, part of Springer Nature Chapter 9 1 Chemotropic assay for testing fungal response to strigolactones and 2 strigolactones-like compounds 3 Rocío Pineda-Martos1, Antonio Di Pietro2, David Turrà3,* 4 1University of Seville, School of Agricultural Engineering, Area of Agroforestry 5 Engineering, Seville, Spain; [email protected] 6 2University of Córdoba, Department of Genetics, Córdoba, Spain; [email protected] 7 3,*University of Naples Federico II, Department of Agricultural Sciences, Naples, Italy; 8 [email protected] 9 10 Running Head: Fungal chemotropic sensing of SLs 11 12 Abstract 13 Current knowledge on the mechanism of strigolactones (SLs) as signalling 14 molecules during specific interactions in the rhizosphere is mainly related to the 15 control of germination of parasitic weed seeds and hyphal branching of arbuscular 16 mycorrhizal fungi. Thus, the role of plant secreted SLs in regulating the growth and 17 development of root-colonizing fungi still remains controversial. Fusarium oxysporum 18 can sense and respond to extracellular signals through oriented germ tube 19 emergence and redirectioning of hyphal growth towards gradients of nutrients, sex 20 pheromones or plant root exudates. However, chemoattractant activity of SLs 21 against microorganisms living in the soil has not been tested so far. Here we 22 propose a quantitative chemotropic assay to understand if and how soil fungi could 23 sense gradients of SLs and SLs-like sources. In the example case of F. oxysporum, 24 hyphae of fungal representative mutants preferentially grow towards the synthetic SL 25 analogue GR24; and this chemotropic response requires conserved elements of the 26 fungal invasive growth mitogen-activated protein kinase (MAPK) cascade. 27 28 Key Words 29 Chemotropism, fungal signalling, Fusarium oxysporum, GR24, root-colonizing fungi 30 31 1. Introduction 32 Although phytohormones and growth regulators are normally produced in plants at 33 low concentrations to modulate self-growth and physiology, there are several 34 examples in nature of plant-interacting microbes (i.e. fungi and bacteria) that either 35 produce the same or very similar chemical compounds to modulate plant growth or 36 responses to their own benefit (1-2). In recent years, a growing body of evidence has 37 shown that phytohormones might function as signalling messengers, used by plants 38 for underground or aboveground recruitment of beneficial organisms or by 39 pathogens to locate the plant host (3-8). One such example are strigolactones (SLs), 40 a group of carotenoid-derived compounds exhibiting regulatory activity on plant 41 growth and physiology, that also trigger germination of parasitic plants and symbiosis 42 of arbuscular mycorrhizal fungi (3,4,6,9) (see also Chapters 6 and 7). However, the 43 potential role of plant secreted SLs or their synthetic analogues in regulating growth 44 and development of other soil-inhabiting fungi, including plant pathogens, has 45 remained controversial (10-16). 46 The Fusarium oxysporum species complex comprises a large group of cosmopolitan 47 isolates that live in the soil as saprophytes by thriving on dead or decaying organic 48 matter. Some representatives behave as endophytes and as biocontrol agents by 49 inducing plant resistance and/or enhancing soil suppressiveness, while others are 50 detrimental plant pathogens causing vascular wilt disease in more than one hundred 51 different crop species (17). Previous studies revealed that F. oxysporum, like other 52 filamentous fungi, can sense and reorient hyphal growth towards a variety of 53 chemical signals including nutrients, sex pheromones and class III peroxidases, a 54 major chemoattractant excreted from host plant roots (18,19). This sense-and-55 respond mechanism – common to both unicellular and multicellular fungi – is 56 regulated by complex signalling circuits which rely on the activity of three-tiered 57 protein kinase modules termed mitogen-activated protein kinase (MAPK) cascades 58 (18,20). 59 Interestingly, directed growth of F. oxysporum germ tubes towards nutrient sources 60 such as sugars and amino acids is governed by the invasive growth MAPK Fmk1, 61 while chemosensing of fungal pheromones and plant root peroxidases occurs via the 62 functionally distinct cell wall integrity (CWI) MAPK Mpk1 (18,19). Preceding studies 63 have established that fungi with different ecological functions, including F. 64 oxysporum, can detect and degrade different types of SLs (11,16,21). Moreover, a 65 quantitative assay on agar plates (see Fig. 1) was applied to understand if and how 66 different mutants of F. oxysporum sense and track SL gradients, measuring 67 chemotropism of germ tubes towards GR24 (16). In this case, F. oxysporum was 68 able to reorient its growth towards gradients of GR24, a process that required 69 conserved elements of the invasive growth MAPK cascade, but not of the CWI 70 MAPK cascade (16). 71 In the present Chapter, the chemotropic assay – previously tested in F. oxysporum 72 MAPK mutants – is described for its relevance on potential procedures to: (i) screen 73 fungal mutants to work out the SL perception, signalling and response pathway in 74 fungi inhabiting in the rhizosphere; and (ii) screen SL-like molecules and analogues 75 for differential chemoattraction activity. 76 77 2. Materials 78 All solutions are prepared by using sterile distilled ultrapure water (ddH2O) and 79 stored at room temperature (~ 25°C) unless otherwise indicated. 80 2.1 Fungal cultures 81 1. Fungal strains based on a collection of mutants and their wild type isolate. 82 2. Potato dextrose broth (PDB) prepared according to manufacturer’s 83 instructions. 84 3. Flasks and magnetic stirrer for preparation of solutions. 85 4. Autoclave. 86 5. Thermoregulated orbital shaking incubator. 87 6. Autoclavable funnels. 88 7. Cheesecloth (10 µm mesh). 89 8. Refrigerated centrifuge and 50 ml centrifuge conical tubes. 90 9. Sterile ultrapure water. Sterilize by autoclaving. 91 10. Microconidia storage solution: 30% v/v glycerol in water. Mix 30 ml of glycerol 92 with 70 ml of water, transfer to a 250 ml screw-cap glass bottle and sterilize 93 by autoclaving. 94 2.2 Fungal chemotropism 95 1. Water Agar medium (WA): 0.5% (w/v) Bacteriological Agar in ddH2O. 96 Adjusted to pH 7 and sterilize by autoclaving in a screw-cap glass bottle. 97 Store at room temperature or 4°C. 98 2. pH meter. 99 3. Microwave. 100 4. Water bath. 101 5. Round (90 mm diameter) Petri dishes. For chemotropic assays, prepare the 102 Petri dishes by drawing three parallel lines with a 1.0 mm blue indelible 103 marker at the centre of the bottom plate, each at 0.5 cm from the other (see 104 Note 1). Left, control line; centre, scoring line; right, chemoattractant line (Fig. 105 1). 106 6. Neubauer chamber for cell counting. 107 7. Optical microscope to count fungal conidia (400× magnification) or to score 108 the direction of fungal hyphae (200× magnification). 109 8. Microbiological laminar flow hood. 110 9. SL stock solution: 30 mM GR24 (synthetic analogue of SLs) in acetone. A 30 111 µM working solution of GR24 is obtained by diluting the stock solution in 112 water. A 0.1% solution of acetone is used as solvent control. Both SL working 113 solution and solvent may be stored at −20°C until use, but it is recommended 114 to prepare the working solution fresh on the day of the experiment. 115 10. Flat-bottom plastic boxes for chemotropic plate incubation. 116 11. Fungal growth incubator. 117 118 3. Methods 119 Carry out all procedures at room temperature (~ 25°C) unless otherwise specified. 120 3.1 Fungal isolates, culture conditions and microconidia collection 121 1. To obtain fresh fungal microconidia to be used in chemotropic assays, start 122 liquid fungal cultures by inoculating, under a sterile hood, 25 µl of 123 microconidia suspension (from a stock stored at −80°C; see point 4 below and 124 3.2.1) into 250 ml Erlenmeyer flasks containing 25 ml of autoclaved PDB. 125 2. Incubate fungal cultures at 150 rpm and 28°C for 4-5 days in an orbital 126 shaking incubator. 127 3. To collect microconidia, filter the fungal culture through four sterile layers of 128 cheesecloth in a sterile funnel on top of a 50 ml centrifuge tube. Once the 129 conidia have passed through the filter meshes, remove the funnel and 130 centrifuge the precipitate at 6000 x g for 10 min. Carefully remove the 131 supernatant and resuspend the pellet in 1 ml of sterile ddH2O. 132 4. Store the freshly collected fungal microconidia at −80°C in storage solution or 133 use directly for chemotropic assays. 134 3.2 Quantification of fungal chemotropism 135 1. Conidia concentration in the freshly obtained microconidia solution is 136 estimated by the use of a Neubauer chamber and adjusted to 2.5×108 137 conidia∙ml−1 with sterile ddH2O (22,23). 138 2. Fungal conidia are embedded in 4 ml WA (see Note 2) at a final concentration 139 of 2.5×106 conidia∙ml−1. To do this, add 10 µl of the 2.5×108 conidia∙ml−1 140 solution, prepared in step 1, to every 4 ml of WA to obtain the desired fungal 141 concentration (see Notes 3,4); and pour it into pre-drawn chemotropic Petri 142 dishes (see Note 5). 143 3. With a 1000-µl pipette tip cut two parallel wells – respectively, control and 144 chemoattractant well – into the WA layer on top of the control and 145 chemoattractant lines (see Fig. 1 for design). 146 4. Pipette 40 μl of the test compound solution (e.g. GR24) uniformly into the 147 chemoattractant well; and, 40 μl of the solvent (0.1% solution of acetone) into 148 the control well. 149 5. Place the Petri dishes inside a flat bottom plastic box and incubate at 28°C in 150 the dark for 13 hours (see Notes 6-9). 151 6. Use a binocular microscope (200× magnification) to count the number of germ 152 tube tips on the central scoring line pointing, respectively, towards the test 153 compound or the solvent control. For each chemotropic plate, a total of 500 154 hyphal tips are scored from 5 or more different regions of the scoring line. For 155 each test compound, experiments are repeated in two or more independent 156 occasions. 157 7. Use the following mathematical formula to calculate the chemotropic index: 158 [(Htest − Hsolv) / Htotal × 100] where, Htest is the number of hyphae growing towards the test compound; Hsolv 159 is the number of hyphae growing towards the solvent control and; Htotal is the 160 total number of hyphae counted. 161 162 4. Notes 163 1. Avoid the use of a dark-coloured marker (i.e. black) to draw the central 164 scoring line on the bottom of the plate, as opaque lines will prevent light 165 diffusion through the sample and efficient visualization of fungal germ tubes 166 under the microscope. As an example, Staedtler Lumocolor® permanent pen 167 317 M, works fine. 168 2. A stock of autoclaved WA can be stored at room temperature for several 169 months and used when needed. To use solidified WA for chemotropic assays, 170 melt the medium (in a loosened screw-cap bottle) in the microwave and keep 171 the bottle in a water bath set at 45°C for at least 1 h before pouring it. 172 3. To prevent death of fungal propagules, rapidly mix conidia and the WA 173 solution in a 50 ml tube by using a 5000-µl pipette. 174 4. Avoid preparing more than 30 ml of WA plus conidia mixture (equivalent to ~ 175 6-7 chemotropic plates) at a time, as this can result in pipette clogging due to 176 agar solidification and uneven spreading of the medium on the surface of the 177 plate. 178 5. Rapidly spread the mixture of WA plus conidia on the plate by repeatedly 179 rotating the plate horizontally and vertically to prevent quick agar solidification 180 and uneven spreading of the medium on the surface of the plate. 181 6. Be careful to place the Petri dishes inside a flat bottom box, making sure that 182 they are parallel to the base of the box, to prevent spilling of the 183 chemoattractant solutions. 184 7. Wrap the plastic box with aluminium foil when testing light-sensitive 185 chemoattractant compounds 186 8. Do not move the box containing the chemotropic plates during the incubation 187 to prevent spilling of chemoattractant solutions. 188 9. When assaying chemoattractant compounds that exert a stimulatory activity 189 on conidial germination or germ tube growth, plate incubation can be 190 performed at lower (room) temperature, or for shorter time periods to avoid 191 overgrowth. 192 193 Acknowledgements 194