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E, Barski A, et al. Microbiology Pt 10 : — J Biol Chem — F, Schulz W. L, Davis D. PLoS One 5: e TeaC requires microtubules, but not KipA, for its localisation at hyphal tips and at septa [ 53 ]. Mutants in TeaC also exhibited zig-zag growth and were affected in septation. Therefore, the TeaA and TeaC proteins couple the microtubule and actin-based vesicle delivery systems involved in hypha orientation.
However, under these conditions zig-zag growth did not occur, suggesting that disruption of the apical sterol-rich domain had multiple effects on hyphal polarity. The control of directionality in hyphae, therefore, implicates both microtubules in delivering vesicles and localising parts of the actin-dependent vesicle docking system and localised calcium ion uptake and signalling via Ras-type GTPase modules that form part of the polarisome complex within a sterol-rich apical domain Figure 4.
Components of the hyphal apex involved in orientation of the growth axis. Directionality involves calcium signalling through the Mid1—Cch1 channel complex, GTP—GDP cycling of the Ras-GTPase, Rsr1, delivery of specific cargo by kinesin motor proteins and proteins that tether microtubule plus ends to complexes in the apical sterol-rich domain. Vesicles finally are delivered to the membrane in the apex via actinomyosin.
The spatial localisation of the KipA kinesin and Tea cell-end proteins in A. The ability to orient the axis of growth of hyphae is a vital aspect of the physiology of fungal cells.
Recent work has demonstrated that the molecular machinery that regulates hypha orientation can be distinguished from that inducing polarised growth since mutants and growth conditions can be generated, in which the trajectory of fungal hyphae is influenced without blocking the ability of hyphae to undergo apical growth. This orientation apparatus apparently involves calcium signalling, GTPase signalling modules and protein complexes that orchestrate sites of actin recruitment and microtubule-tethering at the hyphal apex.
These components orchestrate the growth, morphogenesis and various lifestyles of filamentous fungi. Papers of particular interest, published within the period of review, have been highlighted as:. National Center for Biotechnology Information , U. Sponsored Document from. Current Opinion in Microbiology. Curr Opin Microbiol.
Author information Copyright and License information Disclaimer. Alexandra Brand: ku. This article has been cited by other articles in PMC.
Abstract Hypha orientation is an essential aspect of polarised growth and the morphogenesis, spatial ecology and pathogenesis of fungi. Introduction Most fungi are sessile filamentous organisms that grow by extending the tips of hyphae to form an expanding mycelial network. Tropisms in plant pathogens and saprophytes Tropic alignment of hyphae plays both general and specific roles in the growth of mycelial fungi. Open in a separate window. Figure 1.
Tropisms in human pathogens Tropic growth of hyphae of fungal pathogens has been reported for mating interactions for those few species that have recognised sexual cycles Figure 2 [ 16,17 ]. Figure 2. Molecular mechanisms Whilst the response to environmental cues is likely to be fungus-specific, tip re-orientation may be achieved by the modulation of the conserved machinery that sustains polarised hyphal growth.
Candida albicans Germ tube growth of C. Figure 3. Figure 4. Conclusions The ability to orient the axis of growth of hyphae is a vital aspect of the physiology of fungal cells. References 1. Gow N. Fungal morphogenesis and host invasion. Virag A. Mycol Res. Steinberg G. Eukaryot Cell. Machesky L. Curr Opin Cell Biol. Lipschutz J. Exocytosis: the many masters of the exocyst. Curr Biol. Sudbery P. Polarised growth in fungi. In: Howard R. Springer-Verlag; Martin S.
Lipid raft polarization contributes to hyphal growth in Candida albicans. Pearson C. MesA, a novel fungal protein required for the stabilization of polarity axes in Aspergillus nidulans. Mol Biol Cell. Alvarez J. Sterol-rich plasma membrane domains in fungi. Irazoqui J. Cdc42p, GTP hydrolysis, and the cell's sense of direction.
Cell Cycle. Malassezia yeasts are thought to be taken up by keratinocytes and exist as facultative intracellular parasites by actively suppressing the inflammatory response [ 26 ]. Malassezia hyphae are not well studied because they are slow growing in vitro and require specialised growth media that contains a source of lipid.
In vivo , hyphae are observed only in individuals with hyperactive sebaceous-gland activity, where the presence of excess sebum appears to be the inducer of morphogenesis [ 27 ].
The hyphae are short but penetrate keratinised skin cells to gain access to deeper cavities below, where growth reverts to yeast, and new colonies are formed Figure 1 a [ 13 , 14 ]. One of the roles of M. In lipophilic Malassezia spp. Interestingly, the C.
The specific involvement of hyphae in the generation of immunostimulatory molecules by Malassezia has yet to be addressed. The keratinolytic dermatophytes, T. Dermatophytes are visible as arthroconidia and hyphae within the epidermal layers and surrounding hair shafts, although the in situ signals that induce morphogenesis are not known. In addition to hyphae, T.
The deeper layers of the epidermis are penetrated by hyphae that meander and produce branches that extend parallel to the predominant cell layers Figure 1 b. It is thought that further penetration is inhibited by limited iron availability due to the activity of host ferritin in the underlying layer of the dermis [ 34 ].
No direct penetration of the keratinised cells by dermatophytes has been reported, but fungal cells flatten themselves against the substratum, a further possible aid to adhesion [ 28 ]. In contrast, C.
Hyphae were entangled and less likely to stratify within the epidermal layers than dermatophytes or Malassezia species [ 14 ]. Additionally, many hyphae grew in a helical or spiral growth trajectory, a contact-dependent and low-nutrient response in this fungus Figure 1 c [ 35 , 36 ]. Detection of an epidermal infection by the host leads to the proliferation of epidermal cells to increase shedding of the stratum corneum and the microbes contained within it.
The physical penetration by hyphae and their accompanying enzymatic activity, therefore, contribute to the thickened and chaotic appearance of the skin and nail bed that is a marked feature of epidermal infection.
The formation of hyphae is a key feature in the development of the 3-dimensional structure of fungal biofilms, which present specific clinical problems due to their relative resistance to treatment with antifungal drugs and their potential to release infective cells. Biofilms formed by C. Surprisingly little is known about the activation of morphogenesis by C.
In in vitro models of epithelial infection, the germination of hyphae commences as soon as yeast cells are introduced into the system [ 44 ].
This begs the question as to whether this process is actively suppressed during commensalism, or whether hyphae are constantly germinating in vivo but immediately being cleared by the immune system. Contaminated medical plastics, such as catheters and prostheses, also offer stable substrates for biofilm formation Figure 2 a i , and recent studies of aspergilloma suggest that these enclosed foci of matrix-bound A.
In vitro studies of the temporal process of biofilm formation by C. Hypha formation is not essential for biofilm establishment or maintenance, but biofilms formed by yeast alone are thin and more easily removed from surfaces by mechanical disruption, suggesting that the tangle of hyphal filaments serves to strengthen the structure [ 48 ]. Studies of C.
Hyphae in mature biofilms show a strong propensity to invade the underlying substrate, even when there is little nutrient value Figure 2 a. In the mucosa, intercalation of hyphae disrupts the epithelial layer, which activates a localised inflammatory response. Remarkably, the hyphae of C. Hyphal infiltration causes the silicone to expand and stiffen, thus, compromising the function of the device Figure 2 a ii [ 7 , 8 ].
Mucosal biofilms form at sites occupied by multiple species of microbes [ 49 ]. The presence of bacteria within C. These oral bacteria can attenuate or promote hyphal growth, respectively, through physical interactions and chemical signalling via the production of quorum-sensing molecules [ 50 , 51 ].
The role of hyphae in mucosal infection by C. The first method is through the passive uptake of fungal particles by host cells, and the second is by a method that is unique to filamentous fungi-active penetration of host cell membranes by the hyphal tip [ 55 ]. Passive uptake occurs either during receptor-mediated engulfment of the fungus by phagocytic cells with the aim of killing the microbe or by nonphagocytic endothelial or epithelial cells where molecules on the fungal surface stimulate their own endocytosis Figure 2 b [ 40 , 56 , 57 ].
It has been observed that germinating C. This could be the result of evolutionary pressure on the fungus because the immune system is activated by the presence of hyphae, making escape into a safer environment a matter of some urgency for the fungus. This is of particular importance in the bloodstream because exposure to serum is a strong inducer of morphogenesis in C.
The molecular interactions that stimulate the uptake of C. These proteins have alternative cellular functions Als3 is an amyloid-like, hypha-specific adhesion, and Ssa1 is an intracellular heat-shock protein , but Ssa1 interacts with N-cadherin on endothelial cells and both interact with E-cadherin on epithelial cells [ 33 , 57 , 59 ].
In vitro assays show that A. Escape from the intracellular host environment requires morphogenesis followed by sustained polarised growth. As growth reverted to yeast, the mutant was unable to punch its way out of the host cell, and hence, dissemination into the underlying cell layers did not occur. Sustained hyphal growth is, therefore, important for the endocytic route of tissue invasion in C.
This finding underscores a fundamental difference between the mechanisms used by pathogens that undergo morphogenesis and those that infect solely as yeast, such as Cryptococcus neoformans. The dissemination of C. Unlike C. Induced uptake appears to be a common strategy for the first step of translocation within the host, but pathogenic fungi have alternative mechanisms for breaking free from the host vehicle.
In hypha-generating fungi, the second stage of invasion is one of morphogenesis followed by the active penetration of host cell membranes by hyphal tips Figure 2 c. This enables the fungus to establish fungal masses in the underlying matrix of solid organ tissue where, compared to the bloodstream, infiltrates of host immune cells find it relatively difficult to access the invading fungus Figures 2 c i and 2 c ii. Reversible morphogenesis offers fungi a choice between two lifestyles within the host.
When cells divide as yeast, a mother cell and her daughters remain confined at a single site and must compete with each other for nutrients. If nutrients are scarce, the formation of hyphae allows new cells to be produced sequentially by expansion at the tip. The bulk of the mother-cell cytosol, which contains most of the elements required to generate new cells, is pushed forward by turgor pressure coupled with expansion of the vacuoles positioned sub-apically [ 64 ].
Thus, the tip cell actively extends while the sub-apical cells lie dormant until new nutrients are assimilated. This is more than enough to escape from a phagocyte, anchor within a cell layer, or penetrate endothelia and reach the solid organs below.
Once a more favourable environment is reached, the fungus can choose to revert once again to growth as yeast. Morphogenesis from yeast or conidia is stimulated by a perceived change in the environment. For A. For Malassezia , it is thought to be the sensing of lipids. It requires the integration of multiple signalling pathways involved in the sensing of ambient conditions, such as pH, temperature, and nitrogen availability see reviews by [ 18 , 19 ].
The complexity of these inputs reflects the unusually wide variety of host environments this fungus is capable of colonising. Signals act on the master regulators of morphogenesis, activators Cph1 and Efg1, and suppressors, Tup1 and Nrg1.
Early comparative expression studies revealed that several HSGs encode surface proteins that are involved in adhesion or host interactions and are essential for full virulence: Als3 is an adhesin and invasin, Hyr1 is involved in interaction with neutrophils, and Hwp1 delivers strong adhesion properties because it is a substrate for crosslinking to extracellular matrix by host transglutaminase [ 65 , 66 ]. These include Ume6, a master regulator of hypha-specific genes, Czf1 embedded growth , Bcr1 biofilm maturation , Eed1 escape after endocytosis , and Hgc1, which suppresses cell separation and is expressed at the hyphal tip only [ 39 , 67 — 72 ].
Analyses of temporal and spatial gene expression during infection, coupled with studies of physical changes induced by the environment in other fungi, suggest that a combination of site-specific and hypha-specific gene expression is likely to produce hyphae with subtly different properties [ 73 , 74 ].
It is possible that some of the HSGs of unknown function are involved in modulating the structural status of hyphae. For example, C. Morphogenesis has a significant consequence for the fungus because it exposes surface molecules that alert the immune system to its presence.
The primary mechanism is through the detection of pathogen-associated molecular patterns PAMPs , microbe-derived molecules that are recognised as nonhost by phagocytes. PAMPs that are derived from fungi include cell wall polysaccharides galactomannan and galactofuranose in A.
During epithelial colonisation by C. Although taken up and cleared by macrophages, the inflammatory response is not activated unless conidia swell and germinate, when RodA is degraded and the cell wall polysaccharides are exposed [ 78 , 79 ]. Mucosal defence at most body sites is mediated by epithelial cells and macrophages, which specifically recognise hyphae [ 80 — 82 ].
On detection of hyphal PAMPs via Dectin-1 and other receptors, cytokine signalling activates a group of proteins called the inflammasome, which is expressed within mucosal macrophages and dendritic cells [ 83 ].
The hyphal tip is of particular importance because it controls the direction of new growth in response to the environment, steering the hypha around obstacles or towards nutrients [ 87 ]. In plants, the direction of polarised cell growth is determined by specific cues from the environment, such as light or gravity, which elicit pre-programmed directional responses, or tropisms.
In fungi, contact-dependent growth behaviour, or thigmotropism, has been studied in some detail in plant pathogens. Growing hyphal tips are able to detect defined topographical features on the host leaf, enabling them to locate host penetration sites. For example, the hyphae of Cymodothea trifolii follow depressions at the cell boundaries of its host, white clover, because the stomatal pores are located at cell junctions [ 88 ].
A different growth strategy has evolved in Uromyces appendiculatus , a rust fungus whose host bean plant arranges its stomata transversely across the leaf. Here the hyphae grow perpendicularly to leaf depressions to maximise the chance of finding a penetration site. These responses can be induced on inert surfaces that topographically mimic the host, demonstrating that hyphae can sense surface topography through thigmotropism [ 89 ].
Are fungal tropic responses important for disease progression in humans? However, it is not clear whether they are actively responding to chemical or topographical clues and how this tropism is relieved when the fungus exits the bloodstream.
The growth behaviour of T. Other topical fungi, such as T. Studies of tropisms in C. However, no chemotropic growth has been observed in response to cAMP or retinoic acid, which can mediate growth behaviour in mammalian cells, although polarised mating projections do respond chemotropically to mating pheromone Brand, unpublished data [ 96 ]. Instead, pathogenic fungi seem to be hard-wired to penetrate any substrate they are in contact with, since hyphae in biofilms formed on inert silicones invade the abiotic material despite the apparent lack of extrinisic biological signals [ 7 ].
Galvanotropism is also calcium dependent in C. These tropisms are potentially relevant to hyphal guidance within the host but are difficult to isolate in the complex in vivo environment. Helical growth is also observed in Aspergillus spp. While further tropic responses to in vivo stimuli have yet to be identified, normal regulation of hyphal tip directionality does seem to be involved in tissue penetration.
In the C. A refinement of this study confirmed that Rsr1 is required for damage to an epithelial monolayer but is not required for damage in deeper cell layers [ 44 ]. This suggests that a hyphal tip must orient correctly against the outer host surface to achieve initial cell penetration, but, once embedded, directional control is not required within a tissue matrix.
This and other studies demonstrate that hyphal steering can be uncoupled from polarised growth, but there is no straightforward correlation between the loss of response to tropic stimuli that generate responses in vitro and avirulence in vivo. Directional growth has to be coupled with mechanical force if hyphae are to push the surrounding matrix out of the way or to penetrate physical barriers.
The hyphae of A. More is known about the penetration of leaf cuticles by plant fungal pathogens. The waxy coating of the leaf is sufficiently tough to require the formation of specialised structures called appressoria, from which a penetration peg emerges to pierce the host leaf below. Two conditions must be in place for this to be successful. First, the appressorium must generate a high internal turgor pressure, which can be up to 5.
Secondly, the appressorium must become sufficiently anchored to the host leaf so that the penetration peg enters the plant rather than pushing the appressorium away from it. Thus, adequate adhesion to the host is required before the necessary pressure can be exerted by the fungus. This observation may explain why hyphae need to be so much stickier than yeast and why many of the genes upregulated during morphogenesis in C.
Although yeast cells adhere to the host perfectly well, their complement of adhesins may simply not be able to deliver the anchorage required during the application of hyphal tip pressure to an obstacle. The generation of sufficient adhesion by emerging hyphae may be a time-dependent process, where the number of crosslinks formed with the host increases with hypha length. This in turn could be influenced by whether the hypha is growing on a two-dimensional surface low surface area of contact or in a three-dimensional matrix hypha surrounded by contact points.
The need to establish multiple adhesion sites may be why active penetration of the host is not seen in the early stages of tissue invasion [ 44 ]. A further aid to applying force is turgor pressure, which must be accompanied by wall loosening at the tip so that the turgor pressure generated can be used maximally to apply tip pressure against an object [ 73 ]. The turgor pressure of hyphae has been determined in very few fungal species.
In Achlya bisexualis and Armillaria gallica hyphae, it was found to be 0. In a rare study of force applied by a human pathogen, the oomycete Pythium insidiosum , it was calculated that the pressure generated by the hyphal tip was 0. Similar calculations have been undertaken for plant pathogens, and together the evidence indicates that host tissue penetration is likely to involve a combination of hyphal tip pressure and the hydrolytic activity of fungal exoenzymes [ , ].
Turgor pressure and the secretion of wall tensioning and degradative enzymes must be coordinated at the site of growth to promote tissue penetration. Analyses of targets of the induced microRNAs, from the different libraries, revealed that these molecules may potentially regulate in the cell wall, by repressing genes involved in the synthesis and degradation of glucans and chitin.
Therefore, this work describes a putative post transcriptional regulation, mediated by microRNAs in P. Small non-coding RNAs play an essential regulatory role in biological systems, without being translated into proteins.
Of these RNAs, microRNAs are small RNAs ranging in size from 21 to 24 nt and their action is to post-transcriptionally regulate the expression of target genes involved in different processes such as cell proliferation, tumorigenesis, and infection Bartel, The regulation of gene expression by microRNAs is an evolutionarily conserved mechanism, which may have evolved from parasitic infections, since parasites developed strategies to interfere with host microRNA Hakimi and Cannella, In fungi, expression of microRNAs is regulated under a wide range of different conditions, such as changes in temperature Bai et al.
Several studies have also demonstrated the importance of those molecules during infection of the human host by pathogenic fungi Croston et al.
Despite a current paucity of studies in microRNAs of fungi, data on the importance of those molecules are increasing. MicroRNAs produced by Aspergillus fumigatus have target proteins involved in the control of the metabolism, transport, and signal transduction. In addition a mRNA encoding a protein responsible for ustiloxin B biosynthesis is specifically targeted, which binds to tubulin and interferes with the cellular function of microtubules Bai et al.
MicroRNA produced by Metarhizium anisopliae , target mRNAs encoding proteins involved in sporulation and may influence the process of conidia formation Zhou et al. In addition, microRNAs-like have also been described in vesicles secreted by pathogenic fungi such as Paracoccidioides brasiliensis, Cryptococcus neoformans, Candida albicans , and in the non-pathogenic fungus Saccharomyces cerevisiae. The presence of these small RNAs in vesicles may be associated with cell signaling processes or even more so in the pathogenesis of these microorganisms Peres da Silva et al.
Species of the Paracoccidioides complex Matute et al. Countries such as Brazil, Ecuador, and Venezuela have a high incidence of the disease. The disease mainly occurs in rural areas, causing health problems with high mortality rates and often leaving patients with severe sequels Coutinho et al. Studies have demonstrated that species belonging to the Paracoccidioides complex, possess plasticity in adapting to different environmental conditions Nunes et al. Transcriptional studies on individual members of the complex revealed that remodeling of the gene expression during the dimorphic transition event, from mycelium to yeast cells, is a process that precedes the colonization in the host McEwen et al.
In addition, proteins involved in cellular signaling pathways such as MAPK and calmodulin are induced Nunes et al. Other genes have also been described as essential during the dimorphic transition, including histidine kinase drk1 Chaves et al. Blocking the function of the corresponding proteins by specific inhibitors, impairs the dimorphic transition process. In addition, cyclic AMP levels influence the morphogenesis of those fungi, and the PKA inhibitor H99 blocks the dimorphic transition process Chen et al.
In addition to the morphological changes triggered by temperature in species of the Paracoccidioides complex, transcriptional data also demonstrated differential gene expression in each specific stage.
Metabolism in the mycelium and yeast cells of P. In addition, genes of the glyoxylate cycle such as isocitrate lyase are preferentially expressed in the yeast cells Felipe et al.
In agreement, proteomic data from P. The mycelium proteins related to aerobic energy production and cellular defense against oxidizing agents, such as mitochondrial peroxiredoxin and superoxide, dismutase dependent on manganese were increased. At 22 h of the dimorphic transition of P. The latter is assumed to produce substrates for glycolysis in the yeast phase that follows subsequently Rezende et al. Although, transcriptional and proteomic data reveal a fine-tuned regulation of cellular metabolism and physiology in the specific morphological stages and the dimorphic transition phase, the mechanisms regulating the expression of those genes and proteins has not yet been fully elucidated.
In members of the Paracoccidioides species complex, proteins known to be involved in the biogenesis of microRNAs and the silencing of target genes in other fungi, seem to be conserved at least in P. Considering the relevance of microRNAs in regulating the essential mechanisms of adaptation and survival, a deeper understanding and knowledge about this species complex is important. Therefore, the objective of this study was to characterize the presence and differential expression of microRNAs in P.
By investigating the differential expression of microRNAs, from cDNA libraries, of the different morphological stages, we revealed the presumed role of these molecules, in the adaptation processes of members of the Paracoccidioides complex. Similarly, the mycelium was collected after 15 days, from a solid medium and transferred to the liquid Sabouraud medium. For the transition experiments, the mycelium of P. The transition time of 22 h for subsequent experiments was deduced from counting the number of yeast cells at 0, 22, 48, 72, and 96 h using a Neubauer chamber Bastos et al.
Sequences in FASTQ format were obtained for each library: the mycelium, the mycelium-to-yeast transition cells, and the yeast cells. Initially, the quality of the sequences was evaluated with the FastQC program. Poor quality sequences, as well as the adapters used for library preparation, were removed using the Trimmomatic program Bolger et al.
The processed sequences were then mapped to the reference genome of P. The output mapping file was analyzed by the miRDeep2. The numbers of reads mapped to each miRNA were evaluated with the quantifier.
The result was a counting matrix with each miRNA in the rows and libraries the mycelium, mycelium-to-yeast transition, and the yeast phases. This count matrix was used for statistical tests of differential expressions among libraries, using DESeq2 Love et al. A likelihood ratio test LRT was performed to compare the full model including all the phases against the reduced model only intercept. This LRT was used to find any difference among the phases.
In addition, Negative Binomial Wald Tests were performed to compare all three morphological phases in a pairwise manner. The functional classification of the targets was performed through the Blast2GO Conesa et al. Oligonucleotides for the genes involved in biogenesis of microRNAs, such as dicers and argonauts de Curcio et al. Standard curves were generated by a dilution of 1: 5 of the cDNA and the relative expression levels of the transcripts were calculated using the standard curve method for relative quantification Bookout et al.
The steps used in this study to predict the microRNAs in P. The procedure for extracting RNAs of the different phases of P. The time of extraction of the RNA in the transition phase from the mycelium-to-yeast cells was previously determined by Bastos et al.
At first, the level of expression of the transcripts encoding proteins involved in the processing of microRNAs, was evaluated in the mycelium, transition from the mycelium-to-yeast and the yeast cells. The transcript coding for Dcr-1 was up-regulated during the transition and in the yeast phase, while Dcr-2 was downregulated during the mycelium-to-yeast transition.
The transcript coding for the argonaut Ago-1 was down-regulated during the transition phase, but up-regulated in the yeast phase, whereas the transcript coding for Ago-2 was down-regulated both during the transition and in the yeast phase, although in the yeast phase, the mRNA levels were higher than during the transition phase Supplementary Figures 2A—D.
The differential expression of transcripts encoding proteins related to pre-miRNA processing, suggests that the production of those RNAs could be regulated in the phases of the mycelium, yeast and during the P. Similar differential regulation of dicers and argonauts was observed in the mycelium and yeast phase of Penicillium marneffei.
The gene coding for the dicer 2 protein was induced in the mycelium phase and silencing of this gene blocked the synthesis of specific microRNAs of this morphological stage. This demonstrated that induction of these genes correlates to the synthesis and production of mature microRNAs Lau et al. An analysis of microRNAs in different morphological stages of P. The number of raw small-RNA sequences obtained ranges from 18,, to 25,, among the different libraries Table 1.
An analysis of the quality of reads obtained during sequencing, through the FastQC database, demonstrated sequences of high quality with the vast majority of the bases having PHRED Scores above 30, which indicated an average of one sequencing error per 1, base pairs. Although the RNAseq data already presented a high quality, additional processing was performed with the Trimmomatic, which was mainly used to remove the attached adapter sequences present in the libraries.
The number of sequences for the different conditions, before and after processing, is shown in Table 1. After obtaining the RNAseq data and processing the sequences, they were mapped in the genome of P.
The mapped sequences were also analyzed by miRDeep2 to evaluate the presence of sequences with the potential to be precursors of microRNAs, which would be processed to generate mature microRNAs. Table 2 shows the precursor, mature, and star sequences of the microRNAs among the three analyzed conditions.
Among the cDNA libraries, 49 microRNAs were identified with a mirDeep2 score above 5, which was recommended by software to increase the likelihood of true positive predictions. Interestingly, two microRNAs were similar in sequence; those sequences may possibly derive from a tandem duplication in the genome of P. The sequences of all the precursors presented characteristics of hairpin structures with minimum free folding energy values similar to those described in other microorganisms Jiang et al.
These differences in the regulation of microRNA expression suggest that such molecules may influence each individual morphological stage. Figure 1. Heat map of identified microRNAs. Among the three libraries 49 microRNAs were identified and 44 were differentially expressed.
For each library, biological triplicates were generated. Comparing cDNA libraries from the mycelium and the mycelium-to-yeast transition, 16 microRNAs were differentially expressed, with seven induced in the mycelium and nine in the transition phase Supplementary Table 2.
By comparing the microRNAs present in the yeast cells and in the transition phase, 38 microRNAs were differentially expressed, whereby 18 were induced during the mycelium-to-yeast cell transition and 20 were more abundant in the yeast cells Supplementary Table 3. Comparisons between the mycelium and the yeast cells identified 39 microRNAs differentially regulated, 19 were induced in the yeast cells and 20 in the mycelium Supplementary Table 4.
To analyze the processes regulated by microRNAs in the different morphological phases, we investigated the targets of the microRNAs. Due to the large number of microRNAs differentially regulated in the cDNA libraries, we first focused on microRNAs with high expression values in the mycelium and the transition phase, but low expression in the yeast cells.
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