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Hyphae of the fungus Aspergillus nidulans demonstrate chemotropism to nutrients and pH [1]

['Riho Yamamoto', 'Microbiology Research Center For Sustainability', 'Mics', 'Faculty Of Life', 'Environmental Sciences', 'University Of Tsukuba', 'Tsukuba', 'Hinata Miki', 'Ayaka Itani', 'Norio Takeshita']

Date: 2024-08

The importance of fungi in ecological systems and pathogenicity hinges on their ability to search for nutrients, substrates, and hosts. Despite this, the question of whether fungal hyphae exhibit chemotropism toward them remains largely unresolved and requires close examination at the cellular level. Here, we designed a microfluidic device to assess hyphal chemotropism of Aspergillus nidulans in response to carbon and nitrogen sources, as well as pH. Within this device, hyphae could determine their growth direction in a two-layer flow with distinct compositions that were adjacent but non-mixing. Under conditions with and without a carbon source, hyphae changed growth direction to remain in the presence of a carbon source, but it was still difficult to distinguish between differences in growth and chemotropism. Although nitrogen sources such as ammonia and nitrate are important for growth, the hyphae indicated negative chemotropism to avoid them depending on the specific transporters. This fungus grows equally well at the colony level in the pH range of 4 to 9, but the hyphae exhibited chemotropism to acidic pH. The proton pump PmaA is vital for the chemotropism to acid pH, while the master regulatory for pH adaptation PacC is not involved, suggesting that chemotropism and adaptive growth via gene expression regulation are distinct regulatory mechanisms. Despite various plasma membrane transporters are distributed across membranes except at the hyphal tip, the control of growth direction occurs at the tip. Finally, we explored the mechanisms linking these two phenomena, tip growth and chemotropism.

Microfluidic devices provide precise regulation of environmental conditions within purposely designed spaces and, when integrated with live imaging, unveil novel aspects of mycelial growth [ 18 – 20 ]. In this study, we have established an assay system using microfluidic control technology to determine the chemotropism of hyphae in response to nutrients and pH at the cellular level. Within this device, hyphae exhibit the capability to choose their growth direction in a two-layer flow with distinct compositions, positioned adjacent to each other but remaining unmixed.

When fungal colonies alter their shape in response to beneficial or harmful chemicals, distinguishing whether the growth at that site was affected or if the hyphae exhibited chemotropism becomes challenging. This underscores the importance of analyzing hyphal chemotropism at the single-cell level for accurate monitoring. Within the phytopathogenic fungus Fusarium oxysporum, microconidia extend germtubes in the direction of gradients involving diverse nitrogen and carbon sources on agar medium, such as glutamate, aspartate, and glucose [ 7 , 14 ]. A comparable assessment in the fungal parasitic mold Trichoderma atroviride reveals a chemotropism in germtube elongation toward various nitrogen and carbon sources [ 15 ]. Moreover, F. oxysporum demonstrates the ability to discern the presence of host plants by detecting peroxidase released from the host roots, guiding spore germination towards the host [ 16 , 17 ]. In these experiments, the microscopic observation of spore germination direction is conducted. Nevertheless, the mechanism by which hyphae regulate the growth direction is still largely unknown.

Microorganisms in the environment adapt to their changing surroundings by moving toward or away from beneficial or toxic chemicals. Bacterial chemotaxis is the movement of bacteria towards or away from a particular chemical stimulus in the environment [ 5 ]. Fungal chemotropism has been analyzed well regarding sexual and vegetative hyphal fusion with mating pheromones [ 6 , 7 ]; nevertheless, there is limited knowledge regarding chemotropism to nutrients or hosts. Most fungi grow by extending their hyphae and branching, which make up the mycelial network [ 8 ]. Fungal hyphae grow by apical extension, where the tips of the hyphae secrete enzymes that break down surrounding organic matter, allowing the fungus to absorb nutrients [ 8 – 10 ]. Fungi engage in interactions and perform functions with a diverse array of targets, encompassing human and plant pathogens, decomposers of plant biomass, and symbiotic partners with plant roots [ 11 – 13 ].

Tropism refers to the directional growth or movement of an organism or a part of an organism in response to a particular stimulus, such as light, gravity, touch, or chemicals. Tropism is a fundamental process in the behavior of many living organisms, and it allows them to adapt and respond to changes in their environment. For example, the giant sporangiophores (fruiting body) of the fungus Phycomyces bend toward blue light (phototropism) via blue-light receptor [ 1 , 2 ] and display negative gravitropism [ 3 ]. Thigmotropism (contact) and galvanotropism (electrical currents) have also been observed in some fungi [ 4 ].

Results

Role of proton pump and transcription factor PacC on pH chemotropism Hyphae of A. nidulans maintain intracellular pH by expelling protons outside the cell through the plasma membrane proton pump, PmaA [25]. Pma1, an ortholog of the proton pump, is an essential gene in the yeast [26]. To investigate the role of PmaA, we utilized a knockdown strain where the expression of the pmaA gene was suppressed through carbon catabolite repression of the alcA promoter. This strain exhibited severe growth defects on a glucose medium plate but normal growth on a plate with glycerol as the carbon source (S4A Fig). The spores were harvested from the glycerol plate and introduced into the device under conditions of pH 4 and 6.5 with a glucose medium. The pmaA knockdown strain exhibited slow growth and lacked the chemotropism response to pH 4 (Fig 4A–4C). In contrast, wild-type hyphae selectively grew in the pH 4 medium (Fig 4A). Wild-type hyphae in the pH 4 medium changed their growth direction at the boundary with the pH 6.5 medium, repeatedly returning to the pH 4 layer. However, pmaA knockdown hyphae did not exhibit a clear preference for the 2 layers (Fig 4B and 4C). In the pmaA de-repressed condition with glycerol as carbon source, the chemotropism response to pH 4 was restored (Fig 4A). These results suggest that the chemotropism response to acid pH is PmaA dependent, although the repression of pmaA renders the cell unable to tolerate acidic pH, making it difficult to distinguish between lack of chemotropism and growth defects. Repression of pmaA was also implicated in negative chemotropism to nitrate (S4B Fig). Since repression of pmaA affects plasma membrane integrity and MAPK signaling as well as growth inhibition [27], it may also exert indirect effects, such as altering the localization of nitrate transporters. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 4. Molecular mechanism involved in chemotropism. (A) Difference in the percent of hyphal tips between pH 4 and pH6.5 in the wild type, pmaA knockdown strain (repressed in glucose, de-repressed in glycerol), acidity-mimicking and alkalinity-mimicking pacC mutants. The wild-type strain in identical 2 layers of pH 6.5 with glucose or pH 6.5 with glycerol as the control conditions. Error bars represent standard deviations (SDs). n = 3; 40~50 hyphae in 3 independent experiments. ***, p ≤ 0.001. (B) Hyphal growth in the condition with pH 4 (upper) and pH 6.5 (lower) in the pmaA knockdown strain repressed in glucose. The positions of each hyphal tip are marked with red circles. The border of 2 layers is indicated by the dot line. Scale bars: 100 μm. (C) Merged plots of the position of the hyphal tips in the channel with the change in color over time from (B). (D, E) Hyphal growth in the condition with pH 4 (upper) and pH 6.5 (lower) in the acidity-mimicking pacC mutant (D) and in the alkalinity-mimicking pacC mutant (E). The positions of each hyphal tip are marked with red circles. The border of 2 layer is indicated by the dot line. Scale bars: 100 μm. (F) DIC and GFP images of PmaA-GFP on the plasma membrane except the hyphal apex. The arrows indicate the apex of the hypha. Scale bar: 10 μm. (G) Hyphal growth in the condition with pH 4 (upper) and pH 6.5 (lower) in the ΔteaA and ΔteaR strain. Scale bars: 100 μm. (H) Fluorescence image of GFP-labeled microtubules and calmodulin CaM-mRFP. Scale bar: 5 μm. (I) Time course of signal intensity of GFP-microtubules in arbitrary unit (a.u.) in the upper and lower region shown in (H). Error bars represent SDs. n = 6 ROI (region of interest). (J) Image sequence of change in the hyphal growth direction and fluorescence image of GFP-microtubules and CaM-mRFP. The elapsed time is given in minutes. The arrows indicate the growth direction of the hypha. Scale bar: 5 μm. (K) Enlarged images of GFP-microtubules and CaM-mRFP at the hyphal tip of (J). The elapsed time is given in minutes. Scale bar: 2 μm. The data underlying this figure can be found in S1 Data. https://doi.org/10.1371/journal.pbio.3002726.g004 Numerous fungi exhibit growth across a broad pH spectrum and their gene expression is adapted to the prevailing environmental pH. In A. nidulans, the transcription factor PacC serves as an activator for genes expressed under alkaline conditions and a repressor for those expressed under acidic conditions [28–32]. Under acidic conditions, the full-length form of PacC predominates but, under neutral-to-alkaline conditions, PacC undergoes sequential proteolytic cleavages. The processed PacC leave the zinc-finger region intact and acts as a repressor for genes expressed in acidic conditions and as an activator for genes expressed in alkaline conditions. To explore the relevance of PacC on chemotropism response to pH, we employed acidity-mimicking and alkalinity-mimicking pacC mutants, respectively, under the conditions pH 4 and 6.5 [33]. The acidity-mimicking pacC mutant still exhibited selective growth in the pH 4 medium (Fig 4A and 4D). Similarly, the alkalinity-mimicking pacC mutant selectively grew in the pH 4 medium (Fig 4A and 4E). These outcomes suggest that PacC does not play a role in the chemotropism response to acidity. The acidity-mimicking pacC mutant indicated growth defect on the plate with pH 6.5 and 9, whereas the alkalinity-mimicking pacC mutant did not (S4C Fig).

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002726

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