Fungal Mineral Weathering Mechanisms Revealed Through Direct Molecular Visualization

Soil fungi facilitate the translocation of inorganic nutrients from soil minerals to other microorganisms and plants. This ability is particularly advantageous in impoverished soils, because fungal mycelial networks can bridge otherwise spatially disconnected and inaccessible nutrient hotspots. However, the molecular mechanisms underlying fungal mineral weathering and transport through soil remains poorly understood. Here, we addressed this knowledge gap by directly visualizing nutrient acquisition and transport through fungal hyphae in a mineral doped soil micromodel using a multimodal imaging approach. We observed that Fusarium sp. DS 682, a representative of common saprotrophic soil fungi, exhibited a mechanosensory response (thigmotropism) around obstacles and through pore spaces (∼12 µm) in the presence of minerals. The fungus incorporated and translocated potassium (K) from K-rich mineral interfaces, as evidenced by visualization of mineral derived nutrient transport and unique K chemical moieties following fungal induced mineral weathering. Specific membrane transport proteins were expressed in the presence of minerals, including those involved in oxidative phosphorylation pathways and transmembrane transport of small molecular weight organic acids. This study establishes the significance of fungal biology and nutrient translocation mechanisms in maintaining fungal growth under water and nutrient limitations in a soil-like microenvironment.


Introduction
Biotic mineral weathering is critical in impoverished soils where nutrients can be bound 37 to minerals and unavailable to plants and other organisms. Some soil fungi are adept at extraction 38 of micronutrients from soil minerals by weathering rock material to mineralize elemental 39 potassium (K), iron (Fe), manganese (Mn), calcium (Ca), and other inorganic nutrients [1][2][3] . These  The minerals embedded in the surface of the microchannel in the soil micromodel also provided 97 a direct contact interface between the fungus and embedded minerals ( Fig. 1c and S2). 98 The soil micromodel was inoculated with Fusarium sp. DS 682, a saprotrophic fungus 99 that is common in grassland soils 26 . The fungus was inoculated at one end with a potato dextrose 100 agar (PDA) plug (Fig. 1a, location C1), while a second axenic PDA patch (Fig. 1a, location C2) 101 was provided at the other end of the device. The mineral grains embedded in the micromodel 102 were the only source of nutrient, such as K, Na, Ca (Fig. 1a) between the PDA plugs (C1 and 103 C2) at either end of the device, creating a nutrient impoverished condition. Fungal driven 104 weathering of the minerals was thus required for making the mineral nutrients bioavailable. A 105 water limitation stress was simulated in the soil micromodel by creating an unsaturated channel 106 to keep the PDA sources apart, preventing diffusion of nutrients between PDA and mineral 107 nutrient (Fig. 1a) through liquid media in the microchannel. Therefore, fungal contact with 108 minerals was required to induced weathering of mineral surfaces for extraction of nutrients. 109 These stresses generated an environment for studying fungal degradation and transport of 110 mineral derived nutrients. Mineral availability regulates fungal growth in nutrient limited environments 113 The micromodel system provided an unprecedented view of fungal mineral weathering 114 mechanisms using several spatially resolved characterization techniques, such as optical 115 microscopy, electron microscopy, secondary ion mass spectrometry, and X-ray 116 spectromicroscopy. The PDMS-glass configuration of the micromodels was reversibly bonded 117 4 such that the PDMS mineral micromodel side could be separated from the glass coverslip for 118 characterizing fungal mineral weathering using advanced imaging techniques. 119 We used optical microscopy to image the movement of the fungal hyphae from the 120 inoculation site (C1) through the porous environment (12-150 µm pore spaces) to access the 121 axenic nutrient (PDA) pool (Fig. 1a, C2). We observed that fungal mycelia grew extensively in 122 the presence of minerals and exhibited thigmotrophic movement around micro-and macro-pore 123 spaces that mimicked natural soil pores (Fig. 1d). On the contrary, the absence of soil minerals in 124 the microenvironment significantly inhibited fungal growth (Fig. 1d), where fungi grew only 125 around the nutrient plug at the inoculation site (C1). Further tests suggest that this increase in 126 hyphal density in microporous environments mimicking soil were specifically regulated by the 127 fungal-mineral interactions. Notably, the porosity of the micromodels without minerals did not 128 significantly alter hyphal growth, and fungal hyphal biomass was observed in micromodels with 129 larger pore spaces (600 µm). However, growth ceased when the pore size decreased to 5 µm 130 ( Fig. S4a), demonstrating that thigmotrophic movement was at least partly dependent on pore 131 size. Additionally, thigmotrophic movement could not be attributed to the surface chemistry of 132 the PDMS, as micromodels with either hydrophilic or hydrophobic surfaces resulted in fungal 133 growth comparable to micromodels without minerals (Fig. S4b) 139 We observed an increase in the relative concentrations of K + and Na + in fungal hyphae 140 grown in mineral doped soil micromodels compared to mineral free micromodel controls by 141 time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging (Fig. 2a). The micromodels 142 with and without minerals had the same amount of PDA, which contained minimal amounts of K 143 and Na. Therefore, any increase in relative concentrations of K + and Na + in the fungal hyphae in 144 the mineral doped micromodels was attributed to uptake from the K-feldspar and mica minerals. 145 We did not observe an increase in other mineral nutrients detected within mineral grains shown 146 in Fig. 1b and c (e.g., Ca, Mg) into the fungal hyphae. 147 We hypothesized that enhanced fungal growth in the presence of minerals was due to 148 uptake of nutrients by hyphae from minerals in the micromodel through mineral weathering by 149 the release of fungal organic acids. Consequently, we expected to observe differences in K 150 chemistry in mineral interfaces and K + transported in fungal hyphae from K minerals. Using 151 multi-energy micro-X-ray fluorescence imaging (µ-XRF) around the potassium K-edge, 152 combined with potassium X-ray Absorption Near Edge Structure (XANES) spectroscopy we 153 found evidence of K + transport from minerals through the fungal hyphae ( Fig. 2b and c). 154 Specifically, we identified two distinct K chemistries in the mineral doped soil micromodel as a 155 result of fungal growth: (i) inorganic mineral-bound K (referred to as 'inorganic K'), and (ii) 156 unidentified organic, possibly hyphal adsorbed, K (referred to 'organic K') located along distinct that are indicative of fungal hyphal biomass (Fig. S6). The organic K was observed at the fungal 159 inoculation point and in fungal hyphae grown with and without minerals, suggesting that this 160 form of K is highly present in fungal hyphae.

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The extensive fungal induced K speciation of the minerals was probed using spatially 162 defined XANES spectra within the soil micromodel (Fig. 2c). Here, mineral K showed the 163 distinct pre-and post-edge features (Fig. 2c, spectra 7-11), which were absent in the organic K 164 spectra collected from fungal biomass growing in the same region (Fig. 2c, spectra 1-6). 165 Interestingly, we observed peak shifts in energy of the spectral features within inorganic K 166 spectra (Fig. 2c, 7-11), while the peaks for organic K spectra (Fig. 2c, 1-6) did not exhibit energy 167 shifts. Mineral weathering by different fungal derived organic acids resulting in distinctive 168 mineral K bonding environments could contribute to these shifts in energy observed for the pre-169 and post-edge features in inorganic K spectra ( Fig. 2c and S8). In addition, a linear combination 170 fitting of end-member organic K and mineral XANES spectra (Fig. S7) to the experimental 171 micromodel K XANES spectra indicates discrepancies in the fits of spectra 7-11, which alludes 172 to a change in the mineral surface chemistry (Fig. S8).

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Furthermore, hyphal trails that specifically tracked with the location of mineral grains 174 rich in K were revealed by imaging the micromodel surface after fungal biomass was removed 175 (Fig. S9). This imaging was made possible because areas where the hyphae attached to the 176 surface of the micromodel were enriched in carbon and directly observable by scanning electron 177 microscopy (SEM) imaging. These images suggest the potential that fungi can sense and extract 178 mineral derived nutrients based on their requirement of a specific nutrient, K in this case. We did 179 not detect formation of previously reported micro-tunnels, cavities, or micropores on the mineral 180 grain surfaces, as observed during direct mineral weathering by ectomycorrhizal fungi 4,29 .

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Together, our results showing transport of K and Na through hyphae, K speciation in 182 micromodel minerals after fungal growth, and the absence of micro-tunnels on micromodel 183 mineral grains suggest that the mineral derived nutrient extraction by Fusarium hyphae is via an 184 indirect weathering mechanism. are associated with increased transport of K and Na into fungal hyphae 33,34 . Therefore, we 212 attribute the unique expression of the P-type ATPase in +M treatment group to increased 213 transport of K and Na into the fungal hyphae, as observed in our imaging data (Fig. 2a).

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In addition to the unique expression of P-type ATPase for Na and K transport described   . K is an essential macronutrient required for fungal growth, while Na uptake by fungi can 234 occur simultaneously or as a substitute for K 36 . Nevertheless, fungal uptake of both Na and K 235 greatly enhances hyphal growth, where hyphal uptake of both nutrients occurs through P-type 236 ATPases 33,34 , as was observed in our proteomics analysis. Here, we observed fungal hyphal 237 bridging between two nutrient plugs (C1 and C2, Fig. 1a and d) only in presence of minerals.

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Therefore, we propose that the increase in K + and Na + transport promotes hyphal growth to  We also observed changes in the K chemistry as K was transported from minerals into 242 the fungal hyphae, where two distinct types of K chemistries, inorganic K and organic K, were 243 observed. XANES spectra of the natural kaolinite contained pre-and post-edge features, which 244 are low intensity peaks before and after the highest intensity peaks, which are spectroscopic 245 features consistently seen in other K minerals 37 ( Fig. S7; 'Powdered mineral' and 'Etched 246 mineral'). We observed that the organic K spectra in fungal hyphae do not contain pre-and/or        The instrument has a 32-element multichannel detection system. The X-ray beam is incident    The micromodels with fungal growth were disassembled by removing the PDMS membrane 442 covers and PDMS coated glass coverslips. The micromodel surface with fungi was then exposed 443 to 4% paraformaldehyde (PFA) vapors by placing opened micromodels with a PFA-soaked filter 444 in a petri dish for 24 h at room temperature, which arrested fungal growth. Micromodels that 445 contained no fungi, as well as a natural kaolinite powder, were used as controls and these 446 samples were not exposed to the PFA protocol. Micro-X-ray fluorescence (µ-XRF) imaging and 447 X-ray absorption near edge structure (XANES) spectroscopy were conducted using the tender X- the processed XANES spectra from each mapped region was used to identify the spectra that are 476 the most dissimilar to one another (i.e., most different K chemistries). A least-squares fitting of 477 these spectra to the ME maps was generated to a second set of spatial abundance maps of the same 478 region in order to further show the distinct K chemistries 47-49 . Prior to this experiment the 479 differences between XANES spectra of inorganic vs. organic complexed K was unknown. In these that each ME map appears very similar, and a least-squares XANES fitting was required to tease 483 apart the locations of organic complexed K from mineral-bound K.    abundance values for 2/3 across proteomic sample replicates within a given treatment group.

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To explore sequence data match quality statistics supplement to this proteogenomic method 611 analysis, see publication data DOI: 10 640 3b and S10).  oxidative phosphorylation pathway (Fig. S12). The P-type ATPase for Ca + /K + /Na + transport was 898 observed in the +M treatment group alone.