Finally, we show enhanced activities of multiple nucleic acid enzymes including two ribozymes and a deoxyribozyme, underscoring the generality of this approach, in which functional nucleic acids like aptamers and ribozymes, and in some cases key cosolutes localize within the coacervate microenvironments.Membraneless compartments, such as complex coacervates, have been hypothesized as plausible prebiotic micro-compartments due to their ability to sequester RNA; however, their compatibility with essential RNA World chemistries is unclear. We show that such compartments can enhance key prebiotically-relevant RNA chemistries. We demonstrate that template-directed RNA polymerization is sensitive to polycation identity, with polydiallyldimethylammonium chloride (PDAC) outperforming poly, poly(lysine), and poly(arginine) in polycation/RNA coacervates. Differences in RNA diffusion rates between PDAC/RNA and oligoarginine/RNA coacervates imply distinct biophysical environments. Template-directed RNA polymerization is relatively insensitive to Mg Membraneless compartments have been theorized to be prebiotic micro-compartments as they spontaneously encapsulate RNA and proteins. Here, the authors report membraneless compartments can enhance RNA chemistries, affecting template directed RNA polymerization and stimulating nucleic acid enzymes. Discovery of multiple natural and artificial ribozymes and aptamers has demonstrated RNA’s intrinsic ability to catalyze and regulate chemical reactions9. It is generally accepted that the transition from rich prebiotic chemistry to the earliest lifeforms must have included simpler compartments and protocells as intermediates to highly evolved compartmentalized systems consisting of genetic and catalytic polymers. Several lipid and fatty acid-containing membrane systems have been shown to facilitate compartmentalization11 and support template-directed polymerization of RNA12. However, permeability and transport in lipid-based vesicles is highly sensitive to the size of molecules, and especially unfavorable as the cargo becomes charged and larger13.The RNA world hypothesis posits roles for RNA as catalysts and carriers of genetic information on the primordial Earth16 because of their ability to assemble spontaneously from a variety of oligomeric or polymeric components and to encapsulate otherwise dilute solutes, enabling local concentrations of potentially functional molecules such as nucleic acids at many orders of magnitude higher than is present in the overall solution17. LLPS also allows diffusion both within and between the compartment and the surrounding environment, even for large and charged molecules such as RNAs19. Complex coacervates, which arise from phase separation of oppositely charged polyions, have been hypothesized as model protocells, or alternate biophysical compartments for prebiotic reactions21. Membraneless compartments such as the nucleolus and RNP granules exist in modern cells and have their own biological functions24. Formation of these RNA and protein-rich intracellular liquid phases relies on not only charge–charge interactions, but also π–π stacking, H-bonding, cation–π, and dipole–dipole contacts25. It is possible that biological membraneless organelles may have arisen as simple abiogenic phase-separated systems such as complex coacervates.Liquid–liquid phase-separated (LLPS) systems have been hypothesized as model prebiotic compartments27. High concentrations of nucleotides have been reported for coacervates containing polylysine and ATP16. Similarly, coacervates of poly (PAH) and ATP strongly concentrate RNA molecules, nucleotides, and Mg2+ ions17. Compartmentalization in aqueous two-phase systems consisting of the uncharged polymers polyethylene glycol (PEG) and dextran has been shown to enhance ribozyme catalysis due to increased local concentration28. However, RNA functions in complex coacervates, which have quite different composition and properties than aqueous two-phase systems, are largely unexplored. Importantly, RNA partitioning within complex coacervates generally involves ion-pairing interactions with polycationic components18, which may inhibit necessary RNA functions by disrupting native RNA structures. In fact, certain biological membraneless compartments have been shown to destabilize RNA duplexes29. The hammerhead ribozyme has recently been shown to retain some activity inside polylysine/carboxymethyldextran complex coacervates, with rates 60-fold slower than in the absence of coacervates30.The highly charged nature of RNA suggests that it could have participated in formation of complex coacervates with polycations on early-Earth, thus providing primitive compartments for functional RNAs such as ribozymes and RNA aptamers. Polycations such as polyamines have been shown to condense nucleic acids and form complex coacervates with nucleotides, RNA, and other anionic polymers2+ levels. The polyamines used in this study have varying charge-densities and propensity to interact with nucleic acids. Poly (PAH-260) has a high charge density with ~17 charges per kDa (see Reagents and synthesis in Methods), whereas oligoarginine (R10), polydiallyldimethylammonium chloride (PDAC-53), and oligolysine (K10) have much lower charge densities at ~6.2, 5.2, and 6.3 charges per kDa, respectively. We show that fluorescent RNA aptamers remain bright inside coacervates, thus maintaining their native fold. Finally, we demonstrate that RNA-cleavage reactions catalyzed by multiple ribozymes and a DNAzyme are enhanced in the presence of coacervates. Thus, depending on their chemical composition, complex coacervates could have not only supported RNA copying and RNA aptamer–ligand binding, but also enhanced ribozyme activity on early-Earth while efficiently compartmentalizing RNA and other biomolecules from bulk solutions.In this report, we study the effect of coacervate composition on template-directed polymerization of RNA, and explored RNA aptamer and ribozyme activities within membraneless compartments formed by complex coacervates. We show that the identity of cationic polymers in the coacervates significantly affects template-directed polymerization of RNA molecules. We first demonstrate that coacervates made with specific polyamines can inhibit template-directed RNA polymerization, while some enhance the reaction at sub-optimal Mg10) also showed inhibition at ≥500 µM total positive charge, with product yield reduced by more than half compared to reactions in the absence of any cationic polymers. Although R10 has lower charge density than PAH-260 (~6.2 vs 17 charge/kDa), the level of inhibition is similar for these polycations. Recently, 10-mer oligoarginine was reported to inhibit ribozyme-catalyzed RNA polymerization at 10 µM31; suggesting that inhibition of RNA chemistries by oligoarginine may be a prevalent phenomenon. Ribozyme-catalyzed RNA polymerization has been reported to be enhanced in the presence of 10-mer oligolysine and other heteropeptides derived from the ribosomal core proteins31; however, we did not observe any significant enhancement or inhibition of template-directed polymerization of RNA in the presence of a 10-mer oligolysine (K10), which has a charge density of 6.3 charge/kDa. Template-directed polymerization was however inhibited by larger 100-mer polylysine (K100), as well as protamine, which is a natural arginine-rich protein (~5 charge/kDa) , a quaternary amine that has been reported to form weaker/more labile ion-pairing interactions as compared to PAH-53 Fig. . Further-53 Fig. , right. 11) was used as the polyanion to drive phase separation of PDAC-53, K10, or R10 at 10 mM, 1:1 charge balance  with different polycations. Oligoaspartic acid was chosen because Asp is considered one of the early amino acids33. Product yields after 1.5 h were similar for reactions performed in buffer, and in PDAC/rA11, PDAC/D10, and K10/D10 coacervates . These data suggest that oligopeptides can also serve the role of polyanion to form coacervates while supporting template-directed polymerization.To demonstrate that the template-directed polymerization reaction occurs inside complex coacervates and not the dilute phase, we centrifuged the reactions and separated the bulk supernatant and the condensed phase. Reactions were allowed to proceed independently in the separated phases. Quantification of radiolabeled signal from the gel image for both coacervates and supernatant revealed that >90% of the primer partitioned to the condensed phase for PDAC-53 and Rion Fig. . These d34. FRAP of a Cy3-labeled RNA primer  were 27 ± 3 s for PDAC/rA11 coacervates and 101 ± 6 s for R10/rA11 coacervates. Structural analyses have shown that arginine dominates as the most frequent amino acid at the binding interface of RNA aptamers and their protein targets35, presumably due to its ability to interact with nucleic acids via multiple mechanisms. Strong arginine–RNA interactions could also explain slower RNA diffusion within this condensed phase and poor RNA polymerization (see above). FRAP experiments where coacervates contained Cy3-labeled RNA primer complexed with the template showed a similar trend, with the half-times for fluorescence recovery (t1/2) of 49 ± 5 s and 250 ± 64 s for PDAC-53/rA11 and R10/rA11 coacervates, respectively  experiments, which can report on physical properties of molecules inside biogenic and abiogenic membraneless compartmentsmer Fig.  revealed2+ concentration inside PAH/ATP coacervates17. To test whether PDAC/rA11 coacervates also concentrate Mg2+, we measured Mg2+ levels inside the condensed phase using Atomic Absorption Spectroscopy (AAS). We estimated the volumes of PDAC/rA11 coacervates by centrifuging the bulk coacervate solution and comparing to standards, where 20 µL of bulk solution gave about 1 µL coacervate phase , which was restored by PDAC-53 to the same extent as in no added Mg2+, as expected (4% vs 27%) . Addition of 1 mM EDTA to reactions containing 1 mM MgCl2 inhibited the polymerization, while it did not significantly affect reactions containing 5 mM MgCl2  Fig. . Moreove36, uses a precise 3D RNA structure to bind to the dye 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) and activate its fluorescence37. In vitro transcription reactions of Spinach aptamer have been performed within spermidine/polyU complex coacervates38. We sought to understand whether, and how well, PDAC-53/rA11 coacervates could support RNA aptamer activity. When DFHBI was added alone to PDAC-53/rA11 coacervates, no fluorescence was observed within the droplets, showing that coacervates alone do not induce fluorescence from DFHBI  aptamer and its inactive mutant to verify aptamer-induced fluorescence . Importantly, no fluorescence was observed in coacervates with the G63C/G87C binding site mutant   Fig. 37. Addit11 coacervates provide suitable reaction conditions for ribozyme catalysis. We evaluated full-length hammerhead ribozyme (HHRz) cleavage reactions  for PDAC-53/rA11 coacervates compared to R10. As expected, almost no signal was observed in the supernatant phase after partitioning of coacervate phase  between the ribozyme and the substrate to be 220 ± 10 nM at 1 mM MgCl2 and 28 ± 3 nM at 25 mM MgCl2 in the background of 2.5 mM KCl  coacervates by using hammerhead ribozyme with a fluorescent tag, and comparing the fluorescence inside coacervates to standards. PDAC-53/D10 coacervates indeed concentrated the ribozyme, where the concentration of enzyme strand inside the coacervate was measured to be 45 ± 3, 110 ± 5, and 140 ± 9 µM when only 10, 25, and 50 nM of fluorescently labeled ribozyme was added to the bulk coacervates, respectively  of PEG and dextran by our labs10 coacervates. When concentrations of the enzyme strand were reduced to 1 nM and 5 nM in 1 mM Mg2+, i.e., below bulk KD to remain under kcat/KM conditions, ribozyme cleavage was significantly reduced with only maximal cleavage of 4–6%  was selected to cleave RNA molecules by in vitro evolution5 h Fig. . However5 h Fig. . Similar 2+ Fig. . Identic 2+ Fig. . Taken t10, and K100 in the absence of counterions suggests that the highly charged polyamine alone is capable of interfering with template-directed RNA polymerization. It is likely that the extensive charge–charge interactions and H-bonding disrupt the primer–template complex and/or disorient the 3′ OH such that the 3′ O of primer and the P-5′ O bonds are not in line for the nucleophilic attack. Oligoarginine significantly inhibited the reaction whereas oligolysine with the same number of residues did not, although the longer oligolysine (K100) did. Arginine can interact extensively with nucleic acids, especially guanine nucleotides in bidentate interactions with O-6 and N-735, possibly interfering with the base-pairing within the primer–template complex and also potentially with the 2-Me-ImpG monomer. The quaternary amine PDAC lacks H-bonding capabilities and has a buried positive charge and therefore may not actively interact with nucleic acids in an inhibitory manner. Although the prebiotic origin of PDAC is not yet known, pyrrolidine, which resembles the unmethylated monomer of PDAC, has been detected in meteorite samples43. While PDAC/RNA coacervates outperformed others, it should be emphasized that templated-polymerization did occur in K10/RNA coacervates, albeit at slightly reduced levels. Furthermore, when coacervates coexisted with the dilute phase, templated-polymerization in K10/RNA and PDAC/RNA were identical to buffer conditions while encapsulating over 65% of the RNA in the coacervate phase. These observations coupled with the recent report of hammerhead catalysis in pLys/CM-Dex coacervates30 suggest that RNA chemistries can occur in a wide range of coacervate compositions and that the chemistry of a particular coacervate will impact its effectiveness as a reaction compartment.In this study, we have combined model prebiotic compartmentalization systems of coacervates and essential RNA functions of template-directed RNA polymerization, RNA aptamer–ligand interactions, and ribozyme catalysis. Inhibition of polymerization by PAH-260, R2+, PDAC-53/rA11 coacervates and PDAC-53 alone stimulated template-directed polymerization compared to buffer. Since PDAC does not have proton donating or accepting capabilities to act as general acid or general base, the enhancement in reaction could be from charge stabilization of the transition state provided by the positively charged nitrogens of PDAC. These data suggest that coacervate systems can actively participate in the chemistry rather than simply acting as a “compartment” for reactions. To the best of our knowledge, polyamine-dependent enhancement of template-directed polymerization has not been reported; however, enhancement of ribozyme cleavage from spermidine at limiting44 and in the absence41 of magnesium has been observed. Typically, molar amounts of monovalent are required to substitute for magnesium in ribozyme reactions46. Coacervates may provide the unique ionic conditions required for activity by concentrating ions. We estimated roughly a 5-fold increase in Mg2+ inside PDAC/rA11 coacervates, and Mg2+ bound weakly to the polyanions may still be available for template-directed polymerization and other related chemistries47.One of the intriguing observations was that at reduced or no Mg10 coacervates as observed in FRAP experiments indicate that specific coacervates may provide unique environments and have varying degree of RNA–coacervate interactions. Apparent diffusion coefficients reported here for PDAC-53/rA11 and R10/rA11 are about 400-fold lower compared to spermine/polyU RNA systems where different 15-mer RNAs were used as fluorescent probes18. This is likely due to differences in coacervate composition; for example, about 40-fold difference in fluorescence recovery time-constant from FRAP experiments for the same RNA probe in pLys/ATP and pLys/CM-Dextran coacervates has been reported30.Differential RNA diffusions in PDAC and R11 coacervates support folded RNA secondary and tertiary structures, which is required for many RNA functions. We have shown herein that PDAC/rA11 coacervates retain activity of hammerhead ribozyme. Reduced ribozyme activity has recently been reported for the hammerhead cleavage in coacervates made with polylysine and carboxylated dextran30 at saturating enzyme concentrations. Under those conditions, about 13-fold to 60-fold reductions in observed rate constants were reported depending on whether the reactions were carried out in microdroplets or in bulk coacervates. Interestingly, in our study, at low ribozyme concentrations, we observe enhancements in ribozyme cleavage yields in the presence of coacervates at all times points. Enhancement of the hammerhead ribozyme was also observed for K10/D10 coacervates. On a similar note, stimulation of the hairpin ribozyme by spermine-containing coacervates and enhancement of the DNAzyme cleavage by PDAC/D10 coacervates suggest that coacervate-dependent activation may be useful for reactions catalyzed by diverse biopolymers and that coacervate-dependent activation is not limited to PDAC-containing coacervates. Taken together, our study suggests that coacervates can potentiate functions of RNA and other biopolymers under sub-optimal enzyme and ion conditions by concentrating biomolecules and solutes, which would have been critical in the early RNA world where concentrations of functional RNAs may have been scarce.An important consideration for a model protocell during the RNA world is that it should be able to maintain activities of compartmentalized aptamers or ribozymes within the protocells. The activity of the fluorescent RNA aptamer inside the coacervate droplets unambiguously shows that PDAC/rA49. In an “RNA World”, a ribozyme with kinase50 or phosphatase activity could have performed similar roles by modulating charge densities on polymers through covalent modification4. Transient coacervation of metabolic enzymes and their substrates could have provided micro-reactor compartments for specific reactions that may otherwise be inaccessible in dilute solutions.It has been previously demonstrated that protein kinases can phosphorylate polyions such as cationic peptides or ADP to trigger dissolution of complex coacervates, which can be reversed by phosphatase enzymes10, and K10 are 8.851, 12.4852, and 10.5352, respectively. RNA primer and templates were purchased from IDT . Oligoadenosine (rA11) was purchased from Trilink Biotechnologies . Guanosine monophosphate was purchased from MP Biomedicals. 2-Me-ImpG was synthesized as previously described53, with some modifications. In brief, 100 mg of GMP-free acid and 240 mg of 2-methyl imidazole were added to 20 ml of DMSO and stirred at 50 °C. To the solution of GMP, 0.75 g of triphenylphosphine, 0.85 g of aldrithiol-2, and 1.08 mL of triethylamine were added. Reactions were allowed to proceed for 1 h at room temperature. Then, the mixture was added dropwise to 100 mL solution of 1:1 acetone, diethyl ether containing 1.1 g sodium perchlorate to precipitate 2-Me-ImpG. Solution was then centrifuged at 5000 × g for 5 min and the resulting pellet was washed 4× with ~20 mL 1:1 acetone:diethyl ether solution and then twice with ~20 mL diethyl ether. 2-Me-ImpG was obtained as dried powder by vacuum centrifugation.Polyamine hydrochloride (PAH) 15 kDa, protamine sulfate, and polyacrylic acid (1.8 kDa) were purchased from Sigma (St. Louis). Polydiallyldimethylammonium chloride (PDAC) 8.5 kDa was purchased from Polysciences . Oligolysine, oligoarginine, and oligoaspartic acid were purchased from Alamanda polymers  as chloride or sodium salts. Charge density calculated by assuming that all the protonable nitrogen atoms in the polymer are protonated at the reaction pH. pKas for monomer ionizable groups of PAH, R2). For reactions not containing any coacervates, cationic polymers were then added to indicated concentrations, 2-Me-ImpG was added to a final concentration of 10 mM, followed by addition of RNA primer–template complex (1 µM template and < 50 nM 5ʹ labeled primer). For reactions done under coacervation conditions, the anionic component of the coacervate system was added following the addition of water and buffer. The total negative charge from the rA11 polyanion was held at 7.5 mM. 2-Me-ImpG was then added to a final concentration of 10 mM. Coacervation was induced by addition of different cationic polymers with total positive charge at 10 mM. Reactions were initiated by addition of RNA primer–template complex and incubated at room temperature for indicated times. The total negative charge from the RNA primer–template complex was <20 µM.Template-directed reactions were assembled by first adding water and buffer , where A is the sum of intensities for polymerized bands, and B is the intensity of unreacted primer. For kinetic experiments, data were fit to first order exponentialf(t) is the fraction product formed at indicated time t, fmax is the plateau, and kobs is the observed rate constant. All fits were performed in GraphPad Prism 7. For reactions shown in Fig. g after addition of the RNA:primer–template complex. The resulting supernatant and the condensed phases were then separated and reactions were allowed to proceed in the respective phases for 5 h at room temperature.Reactions were stopped by adding equal volume of “stopping solution”, which contained 8.3 mM polyacrylic acid 1.8 kDa, 166 mM EDTA and 333 mM NaOH. Finally, one reaction volume of 90% formamide and 50 mM EDTA solution was added after which samples were heated at 85 °C for 1 min and separated by electrophoresis on a 20% denaturing polyacrylamide gel containing 7 M Urea. Gels were dried and exposed to phosphorscreens, which were then scanned by Typhoon FLA 9000 (GE). Fraction of polymerized RNA primer was calculated by using the equation 2), rA11 and then PDAC-53. Ribozyme (10 µM) and substrate (~100 nM) were added in folding buffer that did not contain any magnesium . The ribozyme–substrate complex was heated at 85 °C for 3 min and allowed to cool at room temperature for 3 min. Reactions were initiated by adding ribozyme:substrate complex to the Mg2+-containing coacervate solutions. Samples were immediately centrifuged at 14,000 × g for 1 min. Supernatant and the coacervate phases were separated and allowed to react for 1 h at room temperature. Reactions were stopped for supernatant solution by adding one reaction volume of “stopping solution”, which contained 8.3 mM polyacrylic acid 1.8 kDa, 166 mM EDTA and one reaction volume of formamide loading dye (90% formamide and 50 mM EDTA). For coacervates, water  was first added followed by addition of stopping solution. Samples were then heated at 90 °C for 3 min and coacervate phase was disrupted by pipette mixing immediately after heating. Reactions were then separated by electrophoresis on a 15% denaturing polyacrylamide gel containing 8 M Urea.Coacervates  were assembled by adding water, buffer , oligoaspartic acid D10 and PDAC-53. Renatured ribozyme was first added followed by the substrate to initiate the reactions. Reactions were then immediately aliquoted into tubes, and stopped at indicated times. Reactions were then separated by electrophoresis on a 15% denaturing polyacrylamide gel containing 8 M Urea. Plots of fraction cleaved were fit to Eq. . Ribozyme and substrate were allowed to equilibrate at room temperature for 3 min and kept on ice. To obtain the zero time-point, 10× substrate was added to water. Coacervates  were assembled by adding water, buffer . Solution was left at room temperature for 5 min to equilibrate. Indicated coacervates were prepared, and renatured primer–template complex was added such that the primer concentration was 0.5 µM in the coacervate solution. Overall, 8 µL of the solution was placed on a glass slide and mounted on a Leica TCS SP5 laser scanning confocal inverted microscope (LSCM) with a ×63 objective lens. A 543 nm laser was used for excitation and emission spectra were collected from 560 to 600 nm. Leica LAS AF software was used to acquire the images.Standards of indicated Cy-3-labeled RNA primer were made in solutions of 25 mM Tris–HCl pH 8.0, 1 mM MgCl2. Cy3-labeled-RNA primer was added to a final concentration of 5 µM. Sample was then centrifuged to pellet the condensed phase Samples were placed on UV transilluminator (FOTODYNE Inc.) and irradiated with 312 nm UV while the images were collected. Coacervate phase volume was compared against the known standards by visual inspection.Standards of 0.5, 1, 1.5, and 2 µL were prepared by adding indicated amount of Cy3-labeled-RNA primer. These were then centrifuged briefly to ensure that the samples were at the bottom of the tube. A total of 20 µL bulk coacervates of PDAC and rA11 were prepared at 10 mM charge balance in 25 mM Tris–HCl pH 8.0 and 1 mM MgCl2+ were made using serially diluted commercial 1 M Stock MgCl2 (Sigma) in water. Coacervates (20 µL) were prepared in solutions containing 25 mM Tris·HCl and 5 mM MgCl2 at 10 mM charge balance. To measure depleted Mg2+, samples were centrifuged for 14,000 × g for 2 min, and 10 µL of the supernatant was added to 9.99 mL of water. This was also done for samples that did not undergo centrifugation. Samples were then analyzed using a Shimadzu Flame Atomic Absorption Spectrometer 7000. The uncertainty in coacervate volume was not accounted for in estimation of magnesium concentration.Standards of 0, 1, 2.5, 5, and 10 µM Mg2 and 2.5 mM KCl. Hammerhead ribozyme was labeled on the 5′ end with fluorescein as described54. In short, in vitro transcribed RNA were treated with calf intestinal phosphatase (NEB) and phosphorylated using polynucleotide kinase (NEB) using ATP-γS. RNAs were then treated with 5-iodoacetamido-fluorescein (Sigma). Labeled RNA was purified using Sephadex G-25 column (GE). Efficiency of labeling and concentration of fluorescently tagged RNA was measured by comparing absorbance at 260 and 490 nm.  and calculated to be 35%. Coacervates of PDAC-53 and D10 were made at in 1 mM MgCl2 and 2.5 mM KCl and indicated concentrations of fluorescently labeled RNA was added to the droplets. Samples were then imaged as previously described. Overall, 488 nm laser was used for excitation, and the emission window was set from 510 to 550 nm.Standards of indicated fluorescein were made in solutions of 1 mM MgCl2 to a final concentration of 1.0 mM or 25 mM and subsequently placed on ice. The RNA was fractionated using native PAGE at 25 °C and imaged via phosphorimager. Fraction of substrate bound at indicated enzyme concentration was determined by quantification of the bands (ImageJ). Data were fit tofb is the fraction bound, Bmax is the maximum bound, Kd is the dissociation constant, X is the concentration of the enzyme and C is a constant.Samples containing 25 mM Tris–HCl pH 8.0, 2.5 mM KCl, 4 pM substrate, and various concentrations of enzyme ranging from 0 to 17.5 μM were incubated at 85 °C for 3 min and then placed at room temperature for a minimum of 3 min. Each sample was then mixed with MgCl19 by using the equation:F(t) is the normalized fluorescence intensity of the ROI at the given time. S(t) is the intensity within the chosen sample ROI for bleaching; R(t), the intensity within a different droplet that is not bleached; and B(t), is the intensity of the background ROI.After preparing coacervates as described, droplets for FRAP analysis were chosen in the FRAP Wizard interface in the Leica LAS AF software. A region of interest (ROI) of 1 µM diameter was defined for fluorescence measurements during pre-bleach, bleach, and post-bleach sequences. Overall, 5 frames at 0.50 s/frame pre-bleach, 5 frames at 0.19 s/frame during bleach, and 300 frames at 0.5 s/frame post-bleach were acquired. Laser power was set at 100% for 458, 476, 488, 514, 543, and 633 nm lasers for bleach. Fluorescence intensities during the pre-bleach, bleach, and post-bleach sequences were measured in FIJI (Fiji Is Just ImageJ) to generate recovery curves. Recovery data were normalized as described by Jia et al.f(t) is the normalized fluorescence at time t, A is the amplitude of recovery, and C is the Y-intercept. Half-lives (t(1/2)) of fluorescence recovery was calculated by using t(1/2) = ln(2)*τ. Apparent diffusion coefficient (Dapp) was calculated by the formula Dapp = (0.88ω2)/4·t1/2, where ω is the radius of the bleached ROI.Data were then fit to the first order exponentialGUGUGGGCUCAGAUGUCGAGUAGAGUGUGGGCUCCCUC) was transcribed in vitro, purified by PAGE and concentrated by ethanol precipitation. The bolded and underlined Gs were mutated to Cs for the inactive mutant. RNA was renatured by heating in water at 85 °C for 3 min, followed by immediately adding the 10× buffer . RNA was left at room temperature for 5 min after which, the DFHBI dye was added, and samples were left on ice until ready to be mixed with coacervate solutions. PDAC and rA11 coacervates were made as previously described. Overall, 1 µL of the folded RNA was added to 14 µL of the coacervate solution, and 10 µL of this sample was used to prepare slides. For samples in which sdB or DFHBI were added separately, coacervates were first formed after adding either DFHBI or folded sdB, then the other component was added. 15% of 453 and 476 nm lasers were used together for excitation, and the emission window was set at 500–550 nm. For bulk measurements, fluorescence of samples in the presence and absence of coacervates was measured at 15 °C in Applied Biosystem StepOne Plus qPCR machine using emission filter for Fluorescein amidite (FAM) (Em. Max = 520 nm).The stabilized dimeric broccoli aptamer  or 2.5 mM MgCl2 and 2.5 mM KCl (low Mg). 10× solution of ribozyme was added to indicated final concentrations followed by addition of the 5× substrate–substrate-binding strand complex to initiate the reaction. Reactions were stopped by adding two volumes of the stopping solution which contained 8.3 mM polyacrylic acid 1.8 kDa, 166 mM EDTA, and one volume of 90% formamide loading dye.10× solutions of ribozyme (loop B) was prepared in 25 mM Tris–HCl pH 7.5 1.25 mM MgCl10 were first assembled in 25 mM Tris pH 8.0 with either 2.5 mM MgCl2 and 2.5 mM KCl or 5 mM MgCl2 and 5 mM KCl. 10× solution of ribozyme was added to indicated final concentrations followed by addition of the 10× substrate strand to initiate the reaction. Reactions were stopped by adding two volumes of the stopping solution which contained 8.3 mM polyacrylic acid 1.8 kDa, 166 mM EDTA, and one volume of 90% formamide loading dye.10× solutions of DNAzyme was prepared in 25 mM Tris–HCl pH 8.0 and 2.5 mM KCl. DNAzyme solutions were first heated to 85 °C for 3 min and equilibrated at room temperature for 5–10 min. Similarly, 10× solutions of the substrate was also prepared in the same buffer and renatured separately. PDAC-53, DFurther information on experimental design is available in the Supplementary InformationReporting SummaryPeer Review File


Deterioration of RNA in dry-stored seeds occurs slowly and contemporaneously with loss of viability, making RNA quality an excellent marker for changes in seed quality during long-term storage. R2=0.91, P<0.0001). Despite this relationship, germinable and non-germinable seeds within cohorts could not be distinguished unless the RIN was <3.5, indicating substantial deterioration. Our work demonstrates that seed RNA incurs damage over time, observable in fragment size distributions. Under the experimental conditions used here, RIN appears to be a promising surrogate for germination tests used to monitor viability of stored seeds.This study investigates the relationship between germination ability and damage to RNA in soybean seeds (cv ‘Williams 82’) stored dry at 5 °C for 1–27 years. Total germination of 14 age cohorts harvested between 2015 and 1989 ranged from 100% to 3%. Germination decline followed classic seed viability kinetics, with symptomatic seed aging beginning after 17 years of storage. RNA integrity was assessed in dry seeds by electrophoresis of total RNA, followed by calculation of the RNA integrity number , which evaluates RNA fragment size distributions. Analysis of RNA extracted from cotyledons, embryonic axes, plumules, and seed coats across the range of age cohorts showed consistent RNA degradation: older seeds had over-representation of small RNAs compared with younger seeds, which had nearly a 2:1 ratio of 25S and 18S rRNAs. RIN values for cotyledons and embryonic axes from the same seed were correlated. Decline in RIN tracked reduced germination, with a pronounced decrease in RIN after 17 years of storage. This led to a high correlation between the mean RIN of cotyledon RNA and the total germination percentage ( Seed longevity is regulated by a combination of interacting genetic and environmental factors, making the phenotype difficult to measure or predict. In addition to multilocus genetic inheritance e.g. .de novo transcription is not  that fail to germinate or that germinate slowly as storage time progresses. Yet, something must have happened to make the previously germinable seed unable to germinate  as an accepted, standardized method to quantify RNA integrity  seeds grown in the US midwest between 1989 and 2015. Seeds were purchased or donated from various commercial or public sources: Illinois Foundation (1989–1996), United States Department of Agriculture soybean germplasm collection O8U-I-SI (1996–2008), and Missouri Foundation (2009–2015). Seeds were stored at 5 °C and ~35% relative humidity (RH) until use.Test material was soybean (Germination of 14 different age cohorts (1–27 years) was measured in 2016. Seeds were germinated in rolls of moist germination paper  incubated in the dark using a 16/8 h temperature cycle of 25 °C/15 °C. Each germination assay comprised three rolls of 20 seeds each for a total of 60 seeds. Six days after sowing, seeds were scored as apparently healthy (radicle >5 mm), stunted (radicle emerging from the seed coat but <5 mm), or not germinated (no radicle emergence). Total germination for a seed lot was calculated by pooling counts of apparently healthy and stunted seed. Radicle length was also measured to indicate growth rate. Germination data  were treated as proportion data , and theRNA was characterized from 10 of the 14 age cohorts, chosen to encompass the measured range of total germination (from 3% to 100%). RNA was extracted from dry embryonic axes, plumules, cotyledons, and seed coats from 2–10 individual seeds from each cohort ; each tissue and seed was analyzed separately. Plumule tissue included no more than 1 mm of the axis below the cotyledonary node. Approximately 30 mg of cotyledon was sampled either from the portion directly subtending the embryonic axis  or from the portion distal to the embryonic axis. When the distal portion of the cotyledon was sampled, the remaining axis and cotyledon were tested for germination. In this way, we identified cotyledons from seeds that were and were not germinable. Hundreds of cotyledons were sampled in age cohorts with high germination (i.e. the 2- and 16-year-old seed lots).–1, under liquid nitrogen and in the presence of PVP-40, added at a mass comparable with the seed sample (i.e. ~1 mg).Embryonic axis and cotyledon samples were ground in microcentrifuge tubes with a micropestle under liquid nitrogen in the presence of 1–2 mg of polyvinylpyrrolidone-40 (PVP-40). Plumule and seed coat samples were ground for 1 min using a steel shot #40 BB and a Retsch  Bead Mill set at 30 oscillations sRNA was extracted from ground embryonic axis and cotyledon tissues using a modification of the MMY method  SDS, 75 mM NaCl, 25 mM EDTA, 2% 2-mercaptoethanol] were added. The samples were again mixed gently, then centrifuged at 2000 g for 3 min. The supernatant was transferred to a new tube and 500 µl of TRIzol® Reagent  and 100 µl of chloroform were added. After vortexing, samples were incubated for 2 min. They were then centrifuged at 16 000 g for 10 min. The aqueous phase was transferred to a new tube, and an equal volume of chloroform was added. After mixing, samples were incubated on a nutator for 10 min and then centrifuged at 16 000 g for 10 min. The chloroform extraction of the aqueous phase was repeated once. After the second chloroform extraction, the aqueous phase was transferred to a new tube and an equal volume of isopropanol was added. After mixing, RNA was precipitated by placing the samples at −20 °C for 15 min, then centrifuging them at 20 000 g for 10 min at 4 °C. The supernatant was decanted and the pellet was washed twice with 70% ethanol. The pellet was dried and resuspended in 20 µl of water. Samples were processed in batches of 11 and included an embryonic axis and cotyledon (same seed) from five age cohorts, and a control sample comprised of pooled cotyledon tissue from seeds harvested in 2014. All assessments of RNA quality, including RIN calculations (described below), were consistent among control samples and confirmed there were no batch effects (data not shown).For the modified MMY extraction method  RNA 6000 Pico chips and the Plant RNA Pico assay (software version B.02.08.SI648 R3) according to the manufacturer’s protocols. Agilent 2100 software was used to draw electropherogram baselines and assess peak areas of different fragment sizes.The concentration of extracted RNA was measured by absorbance using a Nanodrop  1000 spectrophotometer. Samples were diluted to 1 ng µlRNA integrity is initially estimated by the prevalence and ratio of the 25S and 18S rRNA fractions, which are the most abundant RNAs in a total RNA preparation. The ratio of 25S rRNA to 18S rRNA is 2:1 in high-quality RNA, while other prevalent RNAs are detected as discrete bands including plastid rRNAs, 5S rRNA, and tRNAs. Peak areas were normalized for each electropherogram to reflect the percentage of total RNA area encompassed by each peak. The effect of storage time on distribution of RNA fragment size was tested with ANOVA using JMP 12.2 .Degradation of RNA is recognized as smears or additional peaks in electropherograms, indicating that molecules were fragmented. The RIN  considers the rRNA ratio, the height of the 25S rRNA peak, and the presence of peaks in interpeak regions (between the 25S and 18S rRNA peaks and between the 5S and 18S rRNA peaks)  and before 1999 . We used ANOVA to test the effect of tissue type, age cohort, and germination percentage on RIN values, and correlated RIN values obtained for embryonic axes and cotyledons of the same seed with the total and apparently healthy germination percentage of a population of 60 seeds. To test whether RNA quality was informative regarding the germination potential of the seed, RNA was extracted from a small portion of the cotyledon cut from the seed before it was sown. Sown seeds (with a portion of the cotyledon removed) were then scored as germinating or not germinating, and RIN values from each class and cohort were compared using Student’s t-test. These statistical comparisons were performed in JMP 12.2.Student’s P<0.05, tested with analysis of means for variances–Levene (absolute deviation from the median) to account for a non-normal distribution; Dry seeds from all age cohorts appeared the same, with no detectable difference in shape or size and no apparent browning in older seeds. Germination tests were required to detect differences among age cohorts. These assays revealed no differences among cohorts when tested within 6 months of harvest; initially, all seed lots gave total, apparently healthy, and radicle length measurements similar to values reported here for the 2015 cohort (1-year-old sample in –1 tissue among age cohorts) was much lower than RNA yield from plumules (average of 6377 ± 1184 ng mg–1 tissue among age cohorts). RNA yield from embryonic axes (including plumule) was greater than that from cotyledons, which was much greater than that from seed coats. Based on the RNA yield per tissue, tissue contribution to the RNA signal from a whole soybean seed would be 72–87, 6–19, 13–28, and 0.2–0.3% for the cotyledon, plumule, embryonic axis, and seed coat, respectively. Though RNA yield tends to be lower in older  compared with younger  cohorts, the differences in yield were not significant for any tissue .RNA yield varied considerably among tissues . RNA yieElectrophoretic patterns from 2-year-old seeds (2014 harvest) reflected general characteristics of high-quality RNA extracted from plants . Peaks #n=4, variation=SE) (n=5), and 4.5 ± 0.6 . RIN values of RNA extracted from embryonic axes were 8.4 ± 0.3 , 6.5 ± 0.3 , and 4.9 ± 0.5  (n=5 for each cohort) were 6.6 ± 0.4 (2014 cohort), 6.1 ± 0.1 , and 4.9 ± 0.1 . RIN values of RNA extracted from seed coats (n=2 for each cohort) were 6.9 (2014 cohort), 4.7 , and 2.85 . A summary of the mean RIN values for each tissue type pooled for seeds stored for ≤17 years (post-1999 harvest) and >17 years (pre-1999 harvest) demonstrates extensive change for seed coats , comparable change in RIN for cotyledons, embryonic axes, and plumules of seeds harvested post-1999 , and a significant effect of tissue type on RIN values for seeds harvested pre-1999   with RNA extracted from 27- (1989 cohort) and 20- (1996 cohort) year-old seeds, the latter two cohorts having low total germination rates of 30% and 3%, respectively . In gene cohort wtion=SE) , 5.5 ± 0ds, n=4) . Electrods, n=4) , 4. Noti, ANOVA) .R2=0.28, n=48, P=0.002, RINcot=2.798 + 0.592×RINaxis), but there was considerable unexplained variation, reflected by the low R2 of the regression, non-zero intercept, and slope <1 .To obtain a more detailed view of RNA quality, we selected 10 of the 14 age cohorts (four or five seeds from each cohort) to represent seed lots with total germination ranging from 100% to 3% . RIN valF=20.2, P<0.0001, ANOVA). The changes in the fast RNA and 18S rRNA classes are less pronounced than the changes in the small RNA and 25S rRNA classes. No change is detected in the relative abundance of plastid rRNAs.The trend of decreasing large RNAs and increasing small RNAs with storage time was further characterized by calculating average peak areas for each RNA class  from the axis and cotyledon electropherograms for eight age cohorts . As seedP>0.05, analysis of means for variances–Levene (absolute deviation from the median)]. Changes in RNA integrity, as measured by RIN, only appear after year 16 . In fact, mean RIN values closely associate with total or apparently healthy germination percentage, though correlations were strongest for the linear regression between total germination and RIN from cotyledon extracts  and weakest for the linear regression between apparently healthy germination and RIN from axis extracts . Log-transforming germination data produced better correlations with apparently healthy germination (not shown).Data presented herein show a clear decline in germination potential in soybean seeds over a 27 year period that became noticeable only after ~17 years of storage. The data also show the tendency for RNA fragments to decrease in size over the 27 year period, but the time course for RNA changes remains unclear: are changes noticeable before 17 years? There is no significant difference in mean RIN for age cohorts between 1 and 16 years [P=0.008, Student’s t-test).The physical appearance of the dry seed does not help distinguish between seeds which are dead or alive, and so the transition between germinable and non-germinable seeds  goes mostly unnoticed unless seeds are repeatedly viability tested or, as in this study, a unique collection of age cohorts is available for contemporaneous sampling. Our work shows that RIN in cotyledon tissues correlates closely with total germination , C. TherThe results of this study illustrate why predictions of seed longevity and early detection of seed aging are elusive. Under fairly routine storage conditions, the transition from asymptomatic to rapid aging took ~5 (stunting) to 17 (lost viability) years in a seed known to be short lived . HoweverRNA integrity was measured by the proportion of sizes of RNA fragments within an extract. In principle, this is similar to well-studied effects of time on DNA quality, in which progressive fragmentation is anticipated over a much longer time period . AnalogoA striking feature of the germination time course is the variation in germination behavior among seed lots (age cohorts) once the viability threshold (near 17 years) is surpassed. For example, the germination percentage of the 20 year cohort is near 0 and germination percentages of the 23 and 24 year cohorts are similar to those of recently harvested seeds . RNA intOur conclusion about the close correlation of RIN with germination potential among age cohorts is based on RNA analyses of embryonic axes and cotyledons . Higher The question of a ‘death signature’ is prominent for cryptobiotic organisms such as dry seeds . It remaDetection of dying seeds is another profound issue. There is very little distinction in RIN among seeds which are alive or dead within healthy age cohorts , leadingOur results also point out that germination assays only provide a snapshot of seed health and do not inform about the viability threshold that both defines seed longevity and clearly varies among age cohorts. Similarly, RNA integrity does not portend the upcoming aging threshold. The ultimate goal of finding a biological marker for longevity is more attainable now with a good marker of seed deterioration.The relative sizes of RNA molecules decrease with storage time in soybean seeds stored under dry, refrigerated conditions for up to 27 years. This is an indication that RNA is degrading with time, presumably by oxidative reactions, and this degradation occurs contemporaneously with progressive loss of germination potential within the seed lot. The work provides some exciting possibilities to explore cryptobiotic organisms, in which changed physiological status is not observable until samples are thawed or water is added. The work also has some extremely practical implications in the context of phenotyping the seed longevity trait as well as ensuring that banked seeds can be monitored for health but that the collection is not depleted by that effort.


RNA localization involves cis-motifs that are recognized by RNA-binding proteins (RBP), which then mediate localization to specific sub-cellular compartments. RNA localization is critical for many different cell functions, e.g., in neuronal dendrites, localization is a critical step for long-lasting synaptic potentiation. However, there is little consensus regarding which RNAs are localized and the role of alternative isoforms in localization. A comprehensive catalog of localized RNA can help dissect RBP/RNA interactions and localization motifs. Here, we utilize a single cell sub-cellular RNA sequencing approach to profile differentially localized RNAs from individual cells across multiple single cells to help identify a consistent set of localized RNA in mouse neurons.Using independent RNA sequencing from soma and dendrites of the same neuron, we deeply profiled the sub-cellular transcriptomes to assess the extent and variability of dendritic RNA localization in individual hippocampal neurons, including an assessment of differential localization of alternative 3′UTR isoforms. We identified 2225 dendritic RNAs, including 298 cases of 3′UTR isoform-specific localization. We extensively analyzed the localized RNAs for potential localization motifs, finding that B1 and B2 SINE elements are up to 5.7 times more abundant in localized RNA 3′UTRs than non-localized, and also functionally characterized the localized RNAs using protein structure analysis.We integrate our list of localized RNAs with the literature to provide a comprehensive list of known dendritically localized RNAs as a resource. This catalog of transcripts, including differentially localized isoforms and computationally hypothesized localization motifs, will help investigators further dissect the genome-scale mechanism of RNA localization.The online version of this article (10.1186/s12915-019-0630-z) contains supplementary material, which is available to authorized users. RNA localization is critical to many inter-cellular processes. Neurons are an excellent system to study RNA localization because their extreme polar morphology (neurites of the neurons) creates clear spatial differentiation of the localized RNA and makes it relatively easy to isolate the localized RNA. In addition, RNA localization is critical to neuronal function in which neurons require local protein synthesis within the dendrites to produce long-lasting synaptic potentiation –3. In orThere are several major challenges in profiling the dendritic transcriptome: (1) cleanly separating the somatic and dendritic compartments so that they can be profiled separately, (2) differentiating transcript variation  in addition to localization, (3) accounting for single cell variation in both somatic expression and dendritic localization, and 4) distinguishing actively translocated RNA from randomly diffused RNA. Here, we approach these challenges by performing simultaneous RNA sequencing of the somatic and dendritic compartments of single neurons from primary cultures to allow for a direct contrast of the dendritic transcriptome with its parent soma and to enable the assessment of heterogeneity of localization across neurons. Approaching the problem by single-neuron-matched sub-cellular sequencing has two advantages. First, by matching the dendrite and soma samples, we are able to more clearly identify differentially expressed, and therefore likely to be actively translocated, RNA. Second, since randomly diffused RNA is likely to be different in different individual cells, by examining the variability and consistency across the individual cells, we are again likely to identify actively translocated RNA. Our approach also allows us to examine individual cell variability in localization as well as effects of isoform usage. Given that substantial gene expression heterogeneity has already been observed on the whole-neuron level , it woul distinguGrin1, Mtap2, and Neurod6, with little expression of other brain cell type markers   Fig. . NotablyActb, Bdnf, Calm1, Dlg4, Grin1, and Map2. To differentiate from the 387 differentially expressed genes described above, we refer to this set as the constitutive dendritic (consDend) RNAs, and the previous set as the differentially expressed dendritic (deDend) RNAs. The consDend RNAs covered many of the same ontology functions as the deDend RNAs, such as mitochondria and translation, but additionally were strongly enriched for a large number of synaptic and localization-related GO terms . Overall, the differences between the deDend and consDend lists suggest that at the single-cell level, RNAs with important dendritic and synaptic functions are often not localized to the point of having higher expression concentration in the dendrites relative to the soma, but are nonetheless consistently present in the dendrites at a lower level.Differential expression analysis may not identify all localized RNAs because not all localized RNAs are expected to have higher expression concentration in the dendrites than the soma. This may be particularly relevant when expression is profiled at the single-cell level, since factors such as bursting transcription and variable rates of localization can lead to high variability in the relative amounts of RNA in each compartment at the time of collection. Therefore, we additionally identified RNAs that were consistently present in the dendrites across the profiled cells, since these RNAs are likely to have important dendrite function even if they are not differentially at higher concentration in the dendrites compared to the soma. We found 1863 RNAs in at least 90% of the dendrite samples, which included well-characterized localized RNAs such as Serhl, Ptpn14, Liph, Mre11, Aox3, Casp4, Ddx58), most of which do not currently have a defined dendritic function, although mutations in Mre11 have been previously associated with Ataxia-telangiectasia-like disorder 1 {3,},’ which requires three consecutive matches to the pattern “three or more G’s followed by 1–7 of any nucleotide” and then ending with a fourth set of three or more G’s. The background set was the same as described in the previous section.De novo identification of enriched RNA secondary structures was performed using NoFold . SlidingGAGGCAGGCGGATTTCTGAGTTCGAGGCCAGCCTGGTCTACAGAGTGAGTTCCAGGACAGCCAGGGCTACACAGAGAAACCCTGTCTCB1 sequence:((((((((....(((((((((((..(((...(((((.((........))..)))))...))).)))))...))))))...))))))))B1 structure:GCTGGTGAGATGGCTCAGTGGGTAAGAGCACCCGACTGCTCTTCCGAAGGTCAGGAGTTCAAATCCCAGCB2 sequence:(((((.((..((((((....((.(((((((......))))))))).........))).)))..)))))))B2 structure:Bitscore cutoffs for high-quality matches were set to 50 for B1 and 35 for B2 based on the length of the model. Enrichment was computed using Fisher’s exact test based on the number of high-quality matches in the localized set compared to the non-localized background (same background as above). Only one match was counted per gene for the purposes of enrichment testing.For each predicted dendritic RNA, we obtained the canonical protein sequence, if any, from UniProt . The canEach protein was split into domains based on DomainFinder Gene3D predictions , 52. If Additional file 1:GO annotations. Full list of enriched GO terms for differentially expressed (deDend and deSoma) and consistent dendrite (consDend) lists for biological processes (BP), molecular functions (MF), and cellular components (CC). Related to Fig. Additional file 2:Heatmap of subsampled variability of localization, related to Fig. Additional file 3:Re-analysis of differential localization of 3′UTR isoforms using 250 nt merge distance. (PDF 78 kb)Additional file 4:Table S1. Predicted transmembrane structures. Table S2. Predicted RNA-binding structures. Table S3. Predicted structures commonly found in synaptic proteins. (PDF 189 kb)Expanded structure analysis of potential locally translated proteins. Additional file 5:Full list of predicted protein structural folds for dendritic genes found in this study. (XLSX 506 kb)Additional file 6:Table S4. Full list of dendritic genes from current study and seven previous publications. Table S5. Full deDend gene list. Table S6. Full consDend gene list. Table S7. Full isoDend gene list. (XLSX 346 kb)Catalog of dendritic genes. Additional file 7:Overview of 3′UTR definition, quantification, selection of top two isoforms, and calculation of distal fraction. (PDF 275 kb)


Candida, Aspergillus, and Cryptococcus species are an increasing problem worldwide, associated with very high mortality rates. The successful prevalence of these human pathogens is due to their ability to thrive in stressful host niche colonization sites, to tolerate host immune system-induced stress, and to resist antifungal drugs. This review focuses on the key role played by multidrug resistance (MDR) transporters, belonging to the ATP-binding cassette (ABC), and the major facilitator superfamilies (MFS), in mediating fungal resistance to pathogenesis-related stresses. These clearly include the extrusion of antifungal drugs, with C. albicans CDR1 and MDR1 genes, and corresponding homologs in other fungal pathogens, playing a key role in this phenomenon. More recently, however, clues on the transcriptional regulation and physiological roles of MDR transporters, including the transport of lipids, ions, and small metabolites, have emerged, linking these transporters to important pathogenesis features, such as resistance to host niche environments, biofilm formation, immune system evasion, and virulence. The wider view of the activity of MDR transporters provided in this review highlights their relevance beyond drug resistance and the need to develop therapeutic strategies that successfully face the challenges posed by the pleiotropic nature of these transporters.Fungal infections caused by  Aspergillus, Cryptococcus, and Candida genera constitute the most relevant human fungal pathogens. Infections caused by these pathogens are especially severe in immunocompromised patients, particularly HIV-infected patients, cancer patients, and transplant recipients -β-estradiol was not affected by the truncation of domain 12 of CaCdr1, a mutation that affects drug transport, suggesting that Cdr1 displays different binding sites for these different substrates [Candida albicans strain SC5314 and the vaginal isolate GC15 in Roswell Park Memorial Institute (RPMI)-free supplemented with supraphysiological (10−5 M) or physiological (10−10 M) concentrations of 17-β-estradiol and estradiol, respectively, during different time points, exhibit the upregulation of CaCDR1 and CaCDR2. When both genes were disrupted, the C. albicans cells exposed to 17-β-estradiol had a decreased germ tube formation [For example, the bstrates . Howeverbstrates . Interesbstrates ,108,109.bstrates ,111. CanS. cerevisiae, sterol uptake requires the involvement of the ABC transporters Pdr11 and Aus1. These are necessary for the nonvesicular movement, from the plasma membrane to the endoplasmatic reticulum, a movement needed for the sterol ester synthesis required for sterol uptake [S. cerevisiae Upc2 transcription factor is known to induce the AUS1 and PDR11 expression under anaerobiosis conditions, when the sterol uptake is essential. The absence of these two genes leads to defects in sterol uptake [S. cerevisiae, no connection to MDR has been observed for these transporters; in Cryptococcus gattii, Pdr11 was found to be necessary for fluconazole resistance in the VGII clinical strain [C. glabrata Aus1 has been linked to sterol transport associated with the presence of azole drugs, being upregulated in anaerobic conditions [C. glabrata strains defective in ergosterol [C. glabrata clinical isolates [C. glabrata cells to survive ergosterol defects with host sterols, like cholesterol [C. glabrata virulence, as Aus1 is necessary in a mice model of disseminated infection, being significantly upregulated in such conditions [C. glabrata to survive in the human host, ensuring its virulence and antifungal resistance in mucosal niches.In l uptake . The S. l uptake . Althougl strain ; and C. nditions . In factgosterol  and in Cisolates . In factlesterol . Sterol nditions . TherefoC. albicans and C. glabrata. The MFS-MDR transporters Qdr1, Qdr2, and Qdr3 of C. albicans were found to be localized in the plasma membrane, with preferential localization of Qdr1 and Qdr2 in lipid rafts. These transporters do not participate in the resistance to any type of known antifungals [C. glabrata and in S. cerevisiae are recognized drug transporters [C. albicans. When absent, CaQDR1, CaQDR2, or CaQDR3 lead to the accumulation of phosphatidylinositol and phosphatidylserine, as well as of sphingolipids, thus indicating a role of these transporters in lipid homeostasis [C. glabrata Tpo1_2 MFS-MDR transporter has also been linked to fatty acid and sterol homeostasis, especially during biofilm formation. The absence of CgTPO1_2 upon biofilm formation leads to a decrease of very long fatty acids. Moreover, ergosterol content in the ∆cgtpo1_2 deletion mutant was found to be 30% higher than in its parental strain [C. glabrata seems to be important for to the adaptation to low pH environments with organic acids, as alterations in the membrane lipid composition have been described as a mechanism to surpass the stress on such environment [Major facilitator superfamilies multidrug transporters have also been found to contribute to lipid homeostasis in ifungals , althougsporters ,121,122.eostasis . On the l strain . This phironment .C. glabrata in the oral cavity. One of the mechanisms used by the host to resist candidiasis is the release of the antimicrobial peptide histatin-5 in the saliva [C. glabrata to this antimicrobial peptide [C. albicans Flu1 MFS transporter involved in the resistance towards fluconazole, ketoconazole, and itraconazole [CaFLU1 significantly defects cellular growth and biofilm formation upon the exposure to this antimicrobial peptide [The CgTpo1_2 homolog, CgTpo1_1, is also an MFS drug transporter with an interesting proposed role in the survival of e saliva , as desc peptide . Anotherconazole . In fact peptide . Both stS. cerevisiae are also different and interesting. This transporter was found to be responsible for the secretion of a lipopeptide mating pheromone, designated a-factor, but also for the resistance of this species to valinomycin, an antibiotic peptide [C. albicans homolog of Ste6, designated Hst6, was found to be able to complement the role of S. cerevisiae Ste6 in a ∆scste6 deletion mutant [C. albicans remains to be discovered, as this species is not known to secrete mating pheromones. On the other hand, the Ste6 homolog in C. neoformans has been shown to be involved in the mating process of this pathogen [A. fumigatus Afu3g03430, which is also similar to S. cerevisiae Ste6, has been indicated as being involved in iron uptake [The roles of the Ste6 ABC transporter of  peptide . Interesn mutant . Neverthpathogen . In turnn uptake ,130,131.n uptake .S. cerevisiae [C. glabrata, MFS-MDR transporters have also been found to contribute to the resistance to polyamines, which may become toxic above certain concentrations, such as those found in the human urogenital tract [Candida species in this niche is likely facilitated by two homologs of ScTpo1 in C. glabrata, CgTpo1_1 and CgTpo1_2, which have been identified as determinants in spermine resistance [C. glabrata azole resistance, presents the same physiological role as its S. cerevisiae homolog, Tpo3, in polyamine resistance [Another example of the importance of MDR transporters in the adaptation to human niches is their role in polyamine resistance. Polyamines like spermine, spermidine, and putrescine are essential organic polycations, which are usually involved in the regulation of nucleic acid and protein synthesis, as well as cell growth . The plarevisiae ,134,135.al tract . The presistance . In turnsistance .Candida colonization and successful infection. Interestingly, S. cerevisiae MFS-MDR transporters, like Aqr1 and Qdr1, are important determinants in short-chain monocarboxylic acid resistance [S. cerevisiae Aqr1 has also been implicated in the excretion of homoserine, threonine, alanine, glutamate, and aspartate, probably through vesicles, releasing the amino acids to the extracellular environment through exocytosis [S. cerevisiae Aqr1 in C. glabrata, CgAqr1, has been linked to flucytosine and clotrimazole resistance, as well as resistance against acetic acid [C. glabrata to acetic acid [Since the vaginal tract is characterised by an acidic pH of about 4, as well as the presence of weak acids, like acetic and lactic acids ,100, thesistance ,137. S. ocytosis . The homtic acid . Such a tic acid ,140, beitic acid  point ouTo persist in the human host, fungal pathogens need to survive in the first site of infection, and disseminate to other niches of the host. To achieve this goal, several barriers imposed by the niches need to be surpassed, as well as the ones imposed by the immune response . To faceS. cerevisiae Pdr5 and Yor1 ABC transporters [C. albicans CaCdr1, CaCdr2, and CaCdr3 have been shown to play a role in phospholipid translocation when expressed in S. cerevisiae. CaCdr1 and CaCdr2 conduct an in-to-out movement of phospholipids through the plasma membrane bilayer in an energy-dependent manner, while CaCdr3, which is not involved in antifungal drug resistance, produces the opposite movement, from the outer to the inner leaflet of the plasma membrane [C. albicans evasion to the host immune system, as some phospholipids have been identified as lipid antigens and precursors of lysophospholipids, also known to be lipid antigens recognized by human natural killer T lymphocytes [C. albicans relies on CaQdr2 and CaQdr3 transporters, given that they are necessary for the maintenance of phosphatidylinositol, which is a lipid antigen recognized by the CD1 antigen-presenting cells from the host [CaQDR2 and CaQDR3 genes have shown to attenuated C. albicans virulence in a murine model of hematogenously disseminated candidiasis [For example, similarly to the sporters , C. albimembrane . The traphocytes ,143. Forthe host . Interesdidiasis .+ fluxes [Candida species display an activation of oxidative stress response in this environment, as well as other survival mechanisms [C. glabrata cells are known to be able to resist and proliferate inside mammalian macrophages for a very long time [C. glabrata is known to count with two transporters from the MFS-MDR family, CgAqr1 and CgDtr1, which are responsible for the resistance to weak acids and oxidative stress [S. cerevisiae [C. glabrata in the macrophages and, indirectly, to increase virulence. Indeed, the CgDTR1 expression is upregulated in cells phagocytozed by Galleria mellonella, hemocytes and the overexpression of this gene leads to an increase in the survival of C. glabrata cells in G. mellonella hemocytes. Furthermore, CgDtr1 was found to be necessary for the full virulence of C. glabrata in the infection model G. mellonella [When fungal evasion is not effective, pathogens are phagocyted and a new environment is set upon them, to which they have to adapt to survive. Different host cells are able to perform phagocytosis, including monocytes/macrophages, neutrophils, and dendritic cells ,146. Allchanisms . In partong time . Togethee stress ,139. CgAe stress , like itrevisiae , and CgDCryptococcus neoformans survival in phagocytic cells also depends on its reaction to the new environment with low pH, ROS, and nitric oxide [C. neoformans phagocytosis by macrophages in vitro, the overexpression of the CnAFR1 gene takes place [C. neoformans, in intravenous and in inhalation mouse models [CnAFR1 gene [C. neoformans cells phagocyted by peritoneal macrophages [Cryptococcus species survival in the host.ic oxide . Upon C.es place . The Afres place ,39,58, bFR1 gene . Moreoverophages , highligCryptococcus gattii seems to count with drug transporters for its full virulence in the brain and the pulmonary tissue, like the MFS Mdr1 and the ABC Pdr11 transporters, determinants of C. gattii MDR [MDR1 gene was found to be upregulated in cells recovered from the brain and lungs of infected mice, while PDR11 was overexpressed in cells recovered from the lungs of infected mice [C. gattii virulence and adaptation to the host environment, although the exact mechanism underlying this observation is unclear.ttii MDR ,114. Theted mice . Both MdA. fumigatus infects primarily the lungs disseminating to any possible organ [A. fumigatus. From all these, some have been identified as antifungal resistant players [A. fumigatus transporters, with a high similarity to the S. cerevisiae Pdr5 drug efflux pump. abcB is necessary for full virulence of A. fumigatus in the G. mellonella model of infection and the overexpression of the abcA gene leads to an augmentation of virulence in the presence of voriconazole, in the same infection model [Likewise, le organ . Such inle organ , which,  players ,29,55,56 players . abcA anon model .C. albicans Mdr1 MFS transporter is well known for its role in MDR [C. albicans in immunocompetent and immunocompromised mice models [C. albicans in a mouse model [The e in MDR ,153,154.e models , althougse model .C. albicans biofilm, which, although not contributing for the total biomass produced, help to shape the structure of the biofilm [C. glabrata, the CgTpo1_2 has been highlighted as an important player in biofilm formation, being necessary for the normal expression of CgALS1, CgEAP1, and CgEPA1 genes encoding adhesins. Its role in biofilm formation seems to be linked to its importance in fatty acids homeostasis, as it affects the incorporation of very long chain fatty acids, which are usually found in C. glabrata biofilms [A. fumigatus Mdr4 transporter has been identified as upregulated upon voriconazole exposure and biofilm formation conditions [EPA1 [Several MDR transporters have also been shown to have roles in biofilm formation, an important feature that allows the persistence of fungal pathogens in the host . One spe biofilm . In C. gbiofilms . In turnnditions ,158, indns [EPA1 ,160.C. albicans and C. glabrata is reviewed. Although a similar analysis would be very interesting for A. fumigatus and C. neoformans, the available information is very limited and scattered. On the contrary, information on C. albicans and C. glabrata has been recently gathered in the PathoYeastract database (www.pathoyeastract.org) [Recently, clues on the physiological roles of MDR transporters, including the transport of lipids, ions, and small metabolites, have emerged from transcriptional regulation data, linking these transporters to important pathogenesis features such as resistance to host niche environments, biofilm formation, immune system evasion, and virulence. In this section, all available information on the transcriptional regulation of ABC/MFS-MDR transporter genes in the human pathogens act.org) , enablinC. albicans. In fact, the number of regulatory associations with MDR transporter genes involving transcription factors virtually not related to MDR is much higher than that involving recognized MDR transcription factors. Surprisingly, with regards to multidrug resistance, only 36% (12 out of 33) of the MDR-related transporters in C. albicans are reported to be regulated by at least one MDR-related transcription factor, more specifically, 57% (8 out of 14) of the ABC transporters and nearly 21% (4 out of 19) of the MFS-MDR transporters  of the C. albicans MDR transporters have been found to be regulated by at least one stress-related transcription factor, with the transcription factors CaCap1 and CaSko1 regulating the highest number of transporters   B. These albicans ,163, whialbicans , and thealbicans . Among oalbicans ; CaRpn4 albicans ; and Casalbicans , but alsalbicans .C. albicans   C. Nobiled CaNAG4 ,172,173.d CaNAG4 .C. albicans, the ability to undergo morphological switching from yeast cells to hyphae, in response to various environmental signals, is an important virulence factor that contributes to biofilm formation, invasion and dissemination of Candida in host tissues, and resistance to macrophage and neutrophil engulfment [CDR genes and MDR1 , including the CDR gene family and CaMDR1  genes [CaMDR1 shows no detectable phospholipid exchange activity [CaQDR1, CaQDR2, and CaQDR3 [CgTPO1_2 [Additionally, CaUpc2, the master regulator of the ergosterol biosynthesis (G) genes , and CaHG) genes , regulatactivity . Howeverd CaQDR3 , or CgTPCgTPO1_2 , does afC. glabrata, only a few regulators of MDR transporter genes have yet been unveiled, reflecting the fact that the study of transcriptional regulation in this yeast is still in its infancy, especially in biological processes beyond drug resistance. The transcription factor CgPdr1 is described as the master regulator of multidrug resistance in this organism, regulating the expression of several MDR transporter genes [S. cerevisiae. The overexpression of CgSTB5 in C. glabrata represses azole resistance, while its deletion produces a shy intensification in resistance. Expression analysis assays established that CgStb5p shares many transcriptional targets with CgPdr1, but, unlike the second, it is a negative regulator of pleiotropic drug resistance [In the case of er genes . CgPdr1 sistance ,182. TheC. glabrata. Five stress-responsive transcription factors are reported to regulate the expression of at least one MDR transporter gene  and MFS (39.3%)-MDR transporters were found to be required for full virulence in infection models, several of which, however, for unknown reasons. It will be interesting to ascertain the precise function of these transporters in the context of virulence.This review, however, highlights the observation that both ABC and MFS-MDR transporters contribute to the ability of fungal pathogens to colonize the host and evade host induced defenses, by executing other functions not directly connected with MDR. The study of the transcriptional networks controlling the expression of drug transporters in A. fumigatus, for which only 2.1% of the MFS-MDR transporters have been characterised, or C. tropicalis, for which only 4.5% of the ABC drug efflux pumps have been studied.Despite their crucial role in several aspects of pathogenesis, there is still a striking lack of knowledge about the function of the majority of the MDR transporters. Indeed, among the 241 predicted ABC drug transporters in the fungal pathogens approached in this review, only 10.8%, that is 26, have been characterised. In the case of the predicted MFS-MDR transporters, only 5.8% (28 out of 479), have been functionally analysed. This observation highlights the pressing need to invest in the study of these two families of drug transporters, with special emphasis on less studied organisms, which in this context includes Overall, this review gathers evidence of the multitasking capacity of MDR transporters in fungal pathogens, while highlighting their key role in the successful colonization, persistence, and virulence in the human host. The truly pleiotropic activity of the ABC and MFS-MDR transporters underlines their importance in fungal pathogenesis, and highlights them as very promising drug targets for the development of new antifungals.


Stylophora pistillata branches). The substrates were deployed in a marginal coral reef located in the Gulf of Papagayo  for four months during the northern winter upwelling period (December 2013 to March 2014), and consecutively sampled after each month. Due to the upwelling environmental conditions within the Eastern Tropical Pacific, this region serves as a natural laboratory to study ecological processes such as bioerosion, which may reflect climate change scenarios. Time-series analyses showed a rapid settlement of bioeroders, particularly of lithophagine bivalves of the genus Lithophaga/Leiosolenus , within the first two months of exposure. The observed enhanced calcium carbonate loss of coral substrate (>30%) may influence seawater carbon chemistry. This is evident by measurements of an elevated seawater pH (>8.2) and aragonite saturation state (Ωarag >3) at Matapalo Reef during the upwelling period, when compared to a previous upwelling event observed at a nearby site in distance to a coral reef (Marina Papagayo). Due to the resulting local carbonate buffer effect of the seawater, an influx of atmospheric CO2 into reef waters was observed. Substrates showed no secondary cements in thin-section analyses, despite constant seawater carbonate oversaturation (Ωarag >2.8) during the field experiment. Micro Computerized Tomography (μCT) scans and microcast-embeddings of the substrates revealed that the carbonate loss was primarily due to internal macrobioerosion and an increase in microbioerosion. This study emphasizes the interconnected effects of upwelling and carbonate bioerosion on the reef carbonate budget and the ecological turnovers of carbonate producers in tropical coral reefs under environmental change.Coral reefs persist in an accretion-erosion balance, which is critical for understanding the natural variability of sediment production, reef accretion, and their effects on the carbonate budget. Bioerosion (i.e. biodegradation of substrate) and encrustation  influence the carbonate budget and the ecological functions of coral reefs, by substrate formation/consolidation/erosion, food availability and nutrient cycling. This study investigates settlement succession and carbonate budget change by bioeroding and encrusting calcifying organisms on experimentally deployed coral substrates (skeletal fragments of  At the same time the biogenic skeletal CaCO3 is degraded by means of bioerosion .–27.25–27ate sand . Such fiPocillopora branches; Stylophora pistillata grown in the marine experimental facility at the Leibniz Centre for Tropical Marine Research (ZMT) in Germany was used in the field experiment (CITES permit number 10314/IV/SATS-LN/2009). Despite being non-native in the ETP, S. pistillata is a branching species with calices of comparable size . This was done to avoid abnormal causes for an attraction of bioerosive/encrusting settlers . Subsequently, the cleaned substrates were weighed  before being deployed in the reef.Similar to the sedimentary substrate at the study site . This cS. pistillata substrates were fixed within custom made plastic frames with angler line, whereby a hole was drilled pre-experimentally in the middle part of each substrate  resulting in a reduced number of replicates for some of the months. Originally, substrates were deployed in a higher temporal replication at the described study site and also at Bahía Santa Elena , located north of Matapalo Reef. Due to major loss of substrates, this study has to focus on the results from Matapalo Reef during the upwelling period. th 2013. The sample was recovered on February 13th 2014 after two months of exposure. Other substrates deployed at Bahía Santa Elena were lost after the second month.Exposure experiments were conducted during the northern winter upwelling period from December 2013 to April 2014. For this purpose, a total of 16 ubstrate . The fraManta) was measured between December 2013 and April 2014 by deploying a Manta 2 Water Quality Multiprobe  0.5 m above the reef substrate. Stuhldreier et al. [Manta measurements did not meet the accuracy requested in Dickson et al. [T) and total dissolved inorganic carbon (DIC) were determined with a titration unit VINDTA 3C , which includes a UIC CO2 coulometer detector . The VINDTA 3C was calibrated using the Dickson Certified Reference Material (Batch 127) [2SYS was used to calculate the pHVINDTA , the pCO2 and aragonite saturation state (Ωarag). For the calculations, the daily mean seawater temperature and salinity obtained from the Manta multiprobe were used, except on February 3rd and March 31st 2014. At these two days the Manta multiprobe was not deployed and a WTW sensor was used to measure seawater temperature and salinity [Seawater nutrient concentration and physico-chemical parameters, such as seawater temperature and salinity, were measured by Stuhldreier et al.  directlyr et al.  providedn et al. , discrettch 127) . Sáncheztch 127)  describetch 127)  and the salinity .2O2 (30%) for 72 h, which removed organic material (3 removed per substrate and day). Additionally, percentages of CaCO3 loss rate per substrate, and monthly means were calculated. A one-way ANOVA test using JMP (version 9.0.2) was conducted to statistically assess the change in CaCO3 during the four months exposure period. Homogeneity of variance of the means is assumed  based on the Levene’s test (Prob > F = 0.05) followed by a Tukey HSD means comparison for each month, which distinguished if means were significantly different from each other. However, it is noted that there is a small sample size and therefore a likelihood of a type II error.All coral substrates retrieved were air dried and shipped back to ZMT for further analyses. At ZMT, the coral substrates were digitally photographed and weighed after bleaching with Hmaterial . Net ero3 body was mounted with modeling clay to facilitate beam hardening correction during the reconstruction process. Substrates were scanned using a Skyscan® 1772 μCT scanner  with a voxel size of 7–8 μm in 0.9 mm rotational steps and 360° rotation. The raw scan data was reconstructed at ZMT Bremen using the software nRecon with 43% beam hardening correction, no data smoothing and maximum ring artifact reduction accuracy. Voxel-based 3D volume models were visualized with the software CTVox and a color map was applied to discriminate morphological changes due to encrustation and bioerosion (Micro Computerized Tomography (μCT) scans were conducted from one control substrate (pre-experiment) and from one substrate of each exposure period (i.e. from each of the monthly recoveries) throughout the field experiment. On top of the substrates a small CaCOoerosion .Microbioerosion was investigated using Scanning Electron Microscopy (SEM) of cast-embeddings and petrographic thin-sections of the coral substrates. Partially etched (5% HCl solution for approx. 30s) epoxy-resin casts were prepared in a vacuum chamber following the protocol in Wisshak , except For the investigation of microbioerosion from thin-sections, longitudinal and latitudinal petrographic thin-sections of the previously μCT scanned coral substrates were prepared. For this, substrates were embedded in epoxy and subsequently sections were polished to a thickness of 45 μm. Thin-sections for SEM analyses were gold-sputtered for 30 s and analyzed using the Back-Scattered Electron detector (BSE) at 10 keV.For analyses of surface microbioerosion, coral substrates were mounted on SEM stubs with conductible modeling clay (Leit-C plast). The surface of the substrates was then examined using low-vacuum mode and the BSE detector at 20 keV.pCO2 increased during this upwelling event in 2009. In contrast, during the upwelling event observed in February 2014 at Matapalo Reef, the pHManta increased from 8.11 to 8.30  was 27.2°C . In Febr to 8.30 . Unfortu in 2014 . Prior tand 2013 ,39. Evenurements  at Marinurements  and the arag derived from AT and DIC measurement ranged between 2.8 and 3.7 . This indicates an influx of atmospheric CO2 into the seawater surrounding Matapalo Reef. In contrast, during the upwelling event in 2009 at Marina Papagayo [pCO2 exceeded atmospheric CO2 and thus CO2 was emitted. In addition to upwelling, the intrusion of subsurface water via enhanced wind mixing increased seawater pCO2 from ~320 μatm to ~600 μatm during the non-upwelling period in 2009 [At Matapalo Reef, Ω0.4 μatm . During  in 2009 .Mean concentrations of nitrate were 0.09 ± 0.10 μmol/L in the first month, 0.97 ± 0.87 μmol/L in the second month, 2.72 ± 1.47 μmol/L in the third month (upwelling pulse), and 0.63 ± 0.24 μmol/L in the fourth month . With thHomotrema rubrum), lithophagine bivalves (Lithophaga (Leiosolenus) cf. aristata ; [H. rubrum was present from the second month onward ; , Leon Hoh onward , encrusth onward .Lithophaga/Leiosolenus  after the four months exposure period . No gros3 substrate budget (accretion minus bioerosion) shows an overall negative trend with a mean loss of 0.5 ± 0.2 mg CaCO3 d-1 over the four months period of the experiment, which over the exposure period equates to a mean ~9% CaCO3 substrate loss per month  also correlates with a shift in settlement community. The community shift is represented by a change from phototrophic (e.g. CCA) to larger organotrophic calcifying genera  that settled especially during the last two months. Primarily, bioerosion from bivalves (genus Lithophaga/Leiosolenus) and microbioerosion caused net CaCO3 loss of original coral substrate . Thus, these calcifying settlers biased the total CaCO3 loss of the coral substrates, which in this study was not investigated separately.The time-series analysis of the net bioerosion CaCOer month ; Table 2p <0.01; . Howeverate Figs . HoweverpCO2, lowered pH and decreased Ωarag [arag generally decreased concordantly with lower seawater temperatures, reflecting a strong influence of the corrosive subsurface (i.e. upwelled) waters on the seawater carbon chemistry at Marina Papagayo  could accordingly be explained by a stronger response of photoautotrophic organisms and bioeroders to the intrusion of corrosive and nutrient-enriched seawater. Such an amplified response to the intrusion of cold subsurface water could also explain why the pH did not drop during the main upwelling event in February 2014. The reason why CaCO3 dissolution can occur despite CaCO3 over-saturation is that the conditions measured in the water column likely differ from conditions at the substrate-seawater interface , which is reflected in the measured seawater parameters (i.e. elevated seawater pH and Ωarag).Low seawater temperature, low dissolved oxygen concentration and enhanced nutrient concentrations provide evidence that several cold water intrusions (i.e. upwelling event) influenced the study site at Matapalo Reef during the period from December 2013 to April 2014 ,24. Dataed Ωarag ,39. As iPapagayo . Comparend Ωarag . Photosyct; e.g. ). Seawat3 blocks, and thus may represent a realistic scenario of sedimentary infestation. The skeletal morphology of the coral substrate used is comparable with the Pocillopora coral rubble at the study site . Bivalvte size; . No borete size; ). Infestte size; ,53. In rte size; .3 cements was not observed during the four months exposure period. Although Matapalo Reef is a relatively sheltered near-shore environment, it does experience a relatively low seawater CaCO3 saturation state  fills the role of stabilizing the reef framework [3 recrystallization  and micritization of the original coral skeleton was present in close vicinity to microborings . Another important cause for the rapid CaCO3 substrate loss through time is endolithic microbioerosion. Number and penetration depth of microbioerosion traces also increased considerably with exposure time  at the substrate-seawater interface. This assumption is supported by the fact that the onset of intense CaCO3 substrate loss correlates with enhanced settlement of organotrophic species such as serpulids, bryozoans, barnacles, lithophagine bivalves (i.e. metabolic respiration) from the second month onward, favored by elevated nutrient conditions with the onset of upwelling. To a minor part bioerosive grazing and predation  may have contributed to the observed erosion pattern. The complete loss of some substrates, especially for the substrates exposed for four months, may well be complete crumbling due to external and internal bioerosion, the lack of intragranular cementation and sufficient external encrustation.The observed CaCO3 loss of ~36% total dry weight after four months of exposure. This resembles a mean loss of >1 mg CaCO3 d-1 in coral substrates that were exposed in the reef for the whole experimental period (3 loss per day was significantly higher in the substrates exposed for four months compared to the substrates exposed only up to three months (<0.5 mg CaCO3 d-1), which indicates an enhancement of the bioerosive activity during the fourth month and after the onset of upwelling . This indicates an effect of bioerosion on the carbonate buffer capacity of the seawater .The organisms of the encruster and macrobioeroder community build CaCOseawater , S12 FigCaulerpa;Bioerosion rates in ETP reefs are among the highest recorded in the world ,55,56. TEspecially in marginal reefs of the ETP, such transitions may occur frequently due to disruptive environmental events, resulting in periods of stagnation and (re-)commencement of coral growth ,24. The 3 erosion of the substrate by bioeroders increased markedly with the onset of upwelling. Derived from our time-series experiment, bioerosion caused a negative CaCO3 substrate budget. Dissolution of CaCO3 agrees with the elevated Ωarag and pH observed at Matapalo Reef, when compared to the site at Marina Papagayo, which is located in ~200 m distance to a reef. The resulting local carbonate buffer effect favored an influx of atmospheric CO2 into reef waters. This may suggest that even in upwelling influenced reef zones, ocean waters are still capable to take up atmospheric CO2, and presently mitigate and conceal the global concentration rise caused by anthropogenic sources.In this study, we present the rapid development and alternate succession of an encruster and bioeroder community on coral substrate in an ETP coral reef. Foremost the rapid settlement of lithophagine bivalves as the main macrobioeroders of the substrates is particular to coral reefs in the ETP. CaCO3 substrate budget due to enhanced bioerosion, paired with the absence of secondary cementation, may have negative consequences for net reef accretion. However, if marginal reef ecosystems are protected from further and upcoming anthropogenic impacts and are granted sufficient time to recover, natural regeneration processes stimulated by settlement communities of encrusting and bioeroding organisms may still assist in the remediation of such temporarily stressed coral reefs.For the ecosystem scope, the settlement community provides important functions, such as habitat formation, and substrate consolidation. The community may even have an effect on the reef´s seawater carbon chemistry, enhancing the carbonate buffer capacity. With these functions settlement communities give plasticity to marginal coral reefs where dynamic environmental conditions, such as upwelling, can temporarily impair coral growth. The rapid bioerosion observed in ETP reefs thus provides a possible future scenario for tropical coral reefs affected by ocean acidification and eutrophication. Up to now, encrusting and bioeroding organisms complement the resilience potential of marginal reefs as an important part of their “historically-developed” community structures. However, these communities will likely become altered due to climate change, and marginal reef ecosystems may become locked in eutrophic, bioerosive conditions. The resulting negative CaCOS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Fig(JPG)Click here for additional data file.S2 FigCaulerpa. Water depth ~5 m bsl.Crustose coralline red algae (CCA) encrusting the coral rubble substrate forming rhodoliths, and growth of the green macro-alga genus (JPG)Click here for additional data file.S3 Figa) control, and after b) one month, c) two months, d) three months, and e) four month of exposure. Note the increase in borings and the loss of skeletal structure  over time. Scale bar 50 μm.(TIFF)Click here for additional data file.S4 Fig(MKV)Click here for additional data file.S5 Fig(MKV)Click here for additional data file.S6 Fig(MKV)Click here for additional data file.S7 Fig(MKV)Click here for additional data file.S8 Fig(MKV)Click here for additional data file.S9 FigAfter a, b) one month; c, d) two months; e, f) three months; g, h) four months of exposure.(JPG)Click here for additional data file.S10 FigNote the enhanced settlement of balanids (acorn barnacles), i.e. competition for space, and the presence of lithophagine bivalves.(MKV)Click here for additional data file.S11 FigScale bar 500 μm.(TIFF)Click here for additional data file.S12 Figarag and temperature, and c) Ωarag and AT/DIC at Marina Papagayo  pH and temperature, b) Ωata from ,39) compata from , this st(EPS)Click here for additional data file.