Bacterial degradability of an intrafeather unmelanized ornament: a role for feather‐degrading bacteria in sexual selection?

The impact of feather‐degrading bacilli on feathers depends on the presence or absence of melanin. In vitro studies have demonstrated that unmelanized (white) feathers are more degradable by bacteria than melanized (dark) ones. However, no previous study has looked at the possible effect of feather‐degrading bacilli on the occurrence of patterns of unmelanized patches on otherwise melanized feathers. The pied flycatcher Ficedula hypoleuca Pallas, 1764 is a sexually dimorphic passerine with white wing bands consisting of unmelanized patches on dark flight feathers. These patches are considered to be a sexually selected trait in Ficedula flycatchers, especially in males, where the patches are more conspicuous (larger and possibly whiter) than in females. Using in vitro tests of feather bacterial degradation, we compared the degradability of unmelanized and melanized areas of the same feather for 127 primaries collected from the same number of individuals in a population breeding in central Spain (58 males and 69 females). In addition, we also looked for sex differences in feather degradability. Based on honest signalling theory and on the fact that there is stronger sexual selection for males to signal feather quality than in females, we predicted that unmelanized areas should be more degradable by bacteria than melanized ones within the same feather, and that these unmelanized areas should also be more degradable in males than in females. We confirmed both predictions. Microstructural differences between cross‐section dimensions of unmelanized and melanized barbs, but not differences in the density of barbs within unmelanized and melanized areas of feathers in males and females, could partly explain differences in degradability. This is the first study to show differences in bacterial degradability among markings on the same feather and among unmelanized feather patches between males and females as predicted by sexual selection theory. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 409–419.     

Melanization of appressoria is critical for the pathogenicity of <Emphasis Type="Italic">Diplocarpon rosae</Emphasis>

Previous studies have shown the role of melanized appressoria in the pathogenicity of various fungi. Diplocarpon rosae is a worldwide outdoor fungal pathogen of rose plants causing black spot disease of rose leaves. To fully understand how this fungus colonizes its host, which is critical for the development of an efficient and sustainable disease management program, we studied the fungal (especially the appressoria) structures of D. rosae in detail at an early stage of infection. Using both microscopic and biochemical analyses, we observed strong melanized appressoria formation localized at the point of D. rosae penetration, which forms the pathogen–plant interface. Treatment of infected plants with melanin biosynthesis inhibitors (MBIs) prevented melanization of D. rosae appressoria and positively correlated with significant reductions in black spot disease symptoms, suggesting that melanization of appressoria might be a critical factor for the pathogenicity of D. rosae. Our findings were confirmed and validated by the lack of melanized appressorial ring formation on an artificial surface and on a D. rosae-non host plant system, Arabidopsis thaliana. Our findings suggest that localized melanization of appressoria is a crucial factor for the pathogenicity of D. rosae and treatment of the fungus with MBIs seems to be a promising disease management alternative for black spot disease of roses.     

THE INDUCTION OF MELANIZATION IN GOLDFISH SCALES WITH ACTH, IN VITRO : Cellular and Subcellular Changes

The induction of melanization in xanthic goldfish scales with ACTH in vitro has been studied by light and electron microscopy utilizing ammoniated silver nitrate staining of premelanin and melanin. The melanized cells (melanophores and melanocytes) and the yellow pigmented cells (lipophores and the newly described lipocytes) were found to possess many similarities at the levels of cellular and subcellular structure. The latter cells contain characteristic cytoplasmic bodies which react positively to the premelanin stain. Changes accompanying ACTH stimulation of goldfish scales in tissue culture suggest that these bodies in the lipocytes and lipophores can become melanized. Electron micrographs illustrate the intermediate staining of newly formed melanin granules in an induced melanocyte and the appearance of a transitional melanolipophore. It is postulated that ACTH can promote the association of the enzyme tyrosinase with the preformed structure of unmelanized granules.     

Primary and secondary metabolism regulates lipolysis in appressoria of Colletotrichum orbiculare

The conidia of Colletotrichum orbiculare, the causal agent of cucumber anthracnose, develop appressoria that are pigmented with melanin for host plant infection. Premature appressoria contain abundant lipid droplets (LDs), but these disappear during appressorial maturation, indicating lipolysis inside the appressorial cells. The lipolysis and melanization in appressoria require the peroxin PEX6, suggesting the importance of peroxisomal metabolism in these processes. To investigate the relationships between appressorial lipolysis and fungal metabolic pathways, C. orbiculare knockout mutants of MFE1, which encodes a peroxisomal multifunctional enzyme, were generated in this study, and the phenotype of the mfe1 mutants was investigated. In contrast to the wild-type strain, which forms melanized appressoria, the mfe1 mutants formed colorless nonmelanized appressoria with abundant LDs, similar to those of pex6 mutants. This indicates that fatty acid β-oxidation in peroxisomes is critical for the appressorial melanization and lipolysis of C. orbiculare. Soraphen A, a specific inhibitor of acetyl-CoA carboxylase, inhibited appressorial lipolysis and melanization, producing phenocopies of the mfe1 mutants. This suggests that the conversion of acetyl-CoA, derived from fatty acid β-oxidation, to malonyl-CoA is required for the activation of lipolysis in appressoria. Surprisingly, we found that genetically blocking PKS1-dependent polyketide synthesis, an initial step in melanin biosynthesis, also impaired appressorial lipolysis. In contrast, genetically or pharmacologically blocking the steps in melanin synthesis downstream from PKS1 did not abolish appressorial lipolysis. These findings indicate that melanin biosynthesis, as well as fatty acid β-oxidation, is involved in the regulation of lipolysis inside fungal infection structures.     

MoGrr1, a novel F-box protein,is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

The production of asexual spores plays a critical role in rice blast disease. However, the mechanisms of the genes involved in the conidiogenesis pathway are not well understood. F-box proteins are specific adaptors to E3 ubiquitin ligases that determine the fate of different substrates in ubiquitin-mediated protein degradation and play diverse roles in fungal growth regulation. Here, we identify a Saccharomyces cerevisiae Grr1 homolog, MoGrr1, in Magnaporthe oryzae. Targeted disruption of Mogrr1 resulted in defects in vegetative growth, melanin pigmentation, conidial production, and resistance to oxidative stress, and these mutants consequently exhibited attenuated virulence to host plants. Microscopy studies revealed that the inability to form conidiophores is responsible for the defect in conidiation. Although the Mogrr1 mutants could develop melanized appressoria from hyphal tips, the appressoria were unable to penetrate into plant tissues due to insufficient turgor pressure within the appressorium, thereby attenuating the virulence of the mutants. Quantitative RT-PCR results revealed significantly decreased expression of chitin synthase-encoding genes, which are involved in fungal cell wall integrity, in the Mogrr1 mutants. The Mogrr1 mutants also displayed reduced expression of central components of the MAP kinase and cAMP signaling pathways, which are required for appressorium differentiation. Furthermore, domain complementation analysis indicated that two putative protein-interacting domains in MoGrr1 play essential roles during fungal development and pathogenicity. Taken together, our results suggest that MoGrr1 plays essential roles in fungal development and is required for the full virulence of M. oryzae.     

Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Neurospora crassa contains all four enzymes for the synthesis of DHN (dihydroxynaphthalene), the substrate for melanin formation. We show that the DHN melanin pathway functions during N. crassa female development to generate melanized peridium and ascospore cell walls. N. crassa contains one polyketide synthase (PER-1), two polyketide hydrolases (PKH-1 and PKH-2), two THN (tetrahydroxynaphthalene) reductases (PKR-1 and PKR-2), and one scytalone dehydratase (SCY-1). We show that the PER-1, PKH-1, PKR-1 and SCY-1 are required for ascospoer melanization. We also identified the laccase that functions in the conversion of DHN into melanin via a free radical oxidative polymerization reaction, and have named the gene lacm-1 (laccase for melanin formation-1). In maturing perithecia, we show that LACM-1 is localized to the peridium cell wall space while the DHN pathway enzymes are localized to intracellular vesicles. We present a model for melanin formation in which melanin is formed within the cell wall space and the cell wall structure is similar to “reinforced concrete” with the cell wall glucan, chitin, and glycoproteins encased within the melanin polymer. This arrangement provides for a very strong and resilient cell wall and protects the glucan/chitin/glycoprotein matrix from digestion from enzymes and damage from free radicals.     

Effects of melanin upon susceptibility of Cryptococcus to antifungals

Melanin is a recognized virulence factor in Cryptococcus neoformans; several pathogenetic mechanisms have been suggested. We studied melanin as an antifungal resistance factor. The growth of laccase-active strains of C. neoformans and C. albidus in L-DOPA resulted in the production of black pigment. The formal minimal inhibitory concentrations (MICs) of amphotericin B and fluconazole were not changed by melanization. However, when we examined those wells which contained inhibited cells, we found live cells only in wells containing melanized C. neoformans. In contrast, melanization did not protect C. albidus from killing by amphotericin B. In an amphotericin B time-kill study of C. neoformans, significantly more melanized cells than non-melanized survived for the first few hours. Fluorescence microscopy and flow cytometry analyses showed that fewer melanized cells were stained with the fluorescent dye MitoRed. Incubation of MitoRed (the model) or amphotericin B with melanin extracted from C. neoformans decreased the free concentrations of these substances. Fluconazole, in contrast, was not removed from solution by melanin. This suggests that neoformans cryptococcal melanin deposited amphotericin B in the cell wall binds, reducing its effective concentrations.     

Resistance of melanized feathers to bacterial degradation: is it really so black and white?

Melanins are common feather pigments that contribute to signaling and crypsis. Melanins may also help feathers resist feather‐degrading bacteria (FDB). Two recent studies (Goldstein et al. 2004, Grande et al. 2004) tested the resistance of melanized versus unmelanized feathers to FDB using in vitro experiments, but draw opposite conclusions. Goldstein et al. (2004) concluded that melanized feathers resist FDB more than unmelanized feathers, while Grande et al. (2004) concluded that unmelanized feathers resist FDB more than melanized feathers. To resolve this conflict in the literature, we replicated previous studies but included additional tests not previously used. We inoculated melanized and unmelanized feathers of domestic geese Anser anser domesticus, with the FDB Bacillus licheniformis and measured bacterial activity every two days over two weeks. Three metrics of bacterial activity on feathers were measured: soluble protein content around feathers in solution, bacterial growth on feathers, and loss of feather mass. The latter two metrics were not considered in the aforementioned studies, which indirectly measured bacterial activity. We conducted two trials, one in which feathers were sterilized by autoclaving before inoculation (Goldstein et al. 2004, Grande et al. 2004), and a second in which feathers were sterilized by ethylene oxide gas. This allowed us to test whether autoclaving, done in previous studies, influences bacterial activity on feathers and could confound results. In both trials, unmelanized feathers degraded earlier, supported greater bacterial growth, and lost more mass than melanized feathers. These results support the findings of Goldstein et al. (2004); melanized feathers are more resistant to FDB than unmelanized feathers. Thus, using direct metrics of bacterial activity, we resolve a current conflict in the literature. We also found that autoclaving feathers influences FDB activity on them, and thus autoclaving should be avoided in future studies.     

Inhibition of melanin biosynthesis by cerulenin in appressoria ofColletotrichum lagenarium

Cerulenin [(2S) (3R)2,3-epoxy-4-oxo-7,10-dodecadienoylamide], a polyketide synthesis inhibitor, inhibited appressorial pigmentation ofColletotrichum lagenarium at concentrations higher than 10 μg/ml. The inhibitory concentrations of cerulenin inhibited neither spore germination nor appressorium formation but did inhibit penetration of nitrocellulose membranes by penetrating hyphae from appressoria. The colorless appressoria germinated laterally on nitrocellulose membranes and rarely penetrated them. In the presence of cerulenin, treatment with scytalone, a melanin precursor for this fungus, restored appressorial pigmentation and also penetration by appressoria of nitrocellulose membranes. In Czapek liquid medium, mutant 8015 which is defective in the enzyme involved in the conversion of scytalone to 1,3,8-trihydroxynaphthalene in the melanin biosynthetic pathway produced scytalone after 9 days of incubation when hyphal growth reached the maximum level. Application of 10 μg/ml of cerulenin at 9 days of incubation inhibited further production of scytalone by mutant 8015. From these results, it is concluded that appressorial melanin ofC. lagenarium was synthesizedvia the polyketide pathway.     

The crucial role of the Pls1 tetraspanin during ascospore germination in Podospora anserina provides an example of the convergent evolution of morphogenetic processes in fungal plant pathogens and saprobes

Pls1 tetraspanins were shown for some pathogenic fungi to be essential for appressorium-mediated penetration into their host plants. We show here that Podospora anserina, a saprobic fungus lacking appressorium, contains PaPls1, a gene orthologous to known PLS1 genes. Inactivation of PaPls1 demonstrates that this gene is specifically required for the germination of ascospores in P. anserina. These ascospores are heavily melanized cells that germinate under inducing conditions through a specific pore. On the contrary, MgPLS1, which fully complements a ΔPaPls1 ascospore germination defect, has no role in the germination of Magnaporthe grisea nonmelanized ascospores but is required for the formation of the penetration peg at the pore of its melanized appressorium. P. anserina mutants with mutation of PaNox2, which encodes the NADPH oxidase of the NOX2 family, display the same ascospore-specific germination defect as the ΔPaPls1 mutant. Both mutant phenotypes are suppressed by the inhibition of melanin biosynthesis, suggesting that they are involved in the same cellular process required for the germination of P. anserina melanized ascospores. The analysis of the distribution of PLS1 and NOX2 genes in fungal genomes shows that they are either both present or both absent. These results indicate that the germination of P. anserina ascospores and the formation of the M. grisea appressorium penetration peg use the same molecular machinery that includes Pls1 and Nox2. This machinery is specifically required for the emergence of polarized hyphae from reinforced structures such as appressoria and ascospores. Its recurrent recruitment during fungal evolution may account for some of the morphogenetic convergence observed in fungi.     

Immunomagnetic Purification of Colletotrichum lindemuthianum Appressoria

We developed a method to purify appressoria of the bean anthracnose fungus Colletotrichum lindemuthianum for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.     

Electron microscopic observation of cell wall structure during appressorium formation in Colletotrichum lagenarium

Summary The formation of cell walls during the appressorium formation inColletotrichum lagenarium was observed by electron microscope on the materials prepared by replicas and sectioning. The outer layer of conidia cell walls ruptured at the time of germination and the inner layer bulged out to form a germ tube. The germ tubes and primordia of appressoria had smooth surface and were consisted of one-layered cell wall. However, as the appressorium matured, the electron dense materials appeared on the outer surface of the cell wall which grew into granules. These granules are believed to form the outer layer of appressoria. The under side of the appressorium in contact with the glass surface showed a round pore.Contribution No. 191.     

Nonpathogenic Strains of Colletotrichum lindemuthianum Trigger Progressive Bean Defense Responses during Appressorium-Mediated Penetration

The fungal bean pathogen Colletotrichum lindemuthianum differentiates appressoria in order to penetrate bean tissues. We showed that appressorium development in C. lindemuthianum can be divided into three stages, and we obtained three nonpathogenic strains, including one strain blocked at each developmental stage. H18 was blocked at the appressorium differentiation stage; i.e., no genuine appressoria were formed. H191 was blocked at the appressorium maturation stage; i.e., appressoria exhibited a pigmentation defect and developed only partial internal turgor pressure. H290 was impaired in appressorium function; i.e., appressoria failed to penetrate into bean tissues. Furthermore, these strains could be further discriminated according to the bean defense responses that they induced. Surprisingly, appressorium maturation, but not appressorium function, was sufficient to induce most plant defense responses tested (superoxide ion production and strong induction of pathogenesis-related proteins). However, appressorium function (i.e., entry into the first host cell) was necessary for avirulence-mediated recognition of the fungus.     

Infection Structure–Specific Expression of β-1,3-Glucan Synthase Is Essential for Pathogenicity of Colletotrichum graminicola and Evasion of β-Glucan–Triggered Immunity in Maize

β-1,3-Glucan and chitin are the most prominent polysaccharides of the fungal cell wall. Covalently linked, these polymers form a scaffold that determines the form and properties of vegetative and pathogenic hyphae. While the role of chitin in plant infection is well understood, the role of β-1,3-glucan is unknown. We functionally characterized the β-1,3-glucan synthase gene GLS1 of the maize (Zea mays) pathogen Colletotrichum graminicola, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging, and aniline blue fluorochrome staining. This hemibiotroph sequentially differentiates a melanized appressorium on the cuticle and biotrophic and necrotrophic hyphae in its host. Massive β-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and β-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, downregulation of β-1,3-glucan synthesis increased cell wall elasticity, and the appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Constitutive expression of GLS1 led to exposure of β-1,3-glucan on biotrophic hyphae, massive induction of broad-spectrum defense responses, and significantly reduced disease symptom severity. Thus, while β-1,3-glucan synthesis is required for cell wall rigidity in appressoria and fast-growing necrotrophic hyphae, its rigorous downregulation during biotrophic development represents a strategy for evading β-glucan–triggered immunity.     

Isolation of melanized cell lines with stable phenotypes from a goldfish erythrophoroma cell line and cryopreservation of these cells by the use of autologous serum

Summary Cell lines with stable melanized phenotypes were isolated from a goldfish erythrophoroma cell line. These cell lines include several interesting phenotypes: a) reversible dedifferentiation and redifferentiation in response to withdrawal and addition of fish serum; b) irreversible dedifferentiation in response to withdrawal of fish serum; c) independence on fish serum for melanization; d) dependence on fish serum for growth; and e) pigment aggregation in response to epinephrine. We also report that cryopreservation of all the melanized cell lines, but not any of the unmelanized cell lines, requires the presence of fish serum. This raises the possibility that there may be advantages to use autologous serum for the cryopreservation of sensitive cell lines. This research was supported by grant DK 13724 from the National Institutes of Health, Bethesda, MD     

Melanin is crucial for growth of the black yeast Hortaea werneckii in its natural hypersaline environment

Melanin has an important role in the ability of fungi to survive extreme conditions, like the high NaCl concentrations that are typical of hypersaline environments. The black fungus Hortaea werneckii that has been isolated from such environments has 1,8-dihydroxynaphthalene-melanin incorporated into the cell wall, which minimises the loss of glycerol at low NaCl concentrations. To further explore the role of melanin in the extremely halotolerant character of H. werneckii, we studied the effects of several melanin biosynthesis inhibitors on its growth, pigmentation and cell morphology. The most potent inhibitors were a 2,3-dihydrobenzofuran derivative and tricyclazole, which restricted the growth of H. werneckii on high-salinity media, as shown by growth curves and plate-drop assays. These inhibitors promoted release of the pigments from the H. werneckii cell surface and changed the medium colour. Inhibitor-treated H. werneckii cells exposed to high salinity showed both decreased and increased cell lengths. We speculate that this absence of melanin perturbs the integrity of the cell wall in H. werneckii, which affects its cell division and exposes it to the harmful effects of high NaCl concentrations. Surprisingly, melanin had no effect on H. werneckii survival under H₂O₂ oxidative stress.     

The osmotic flow of an electrolyte through a charged porous membrane

A pore model in which the pore wall has a continuous distribution of electrical charge is used to investigate the osmotic flow through a charged permeable membrane separating electrolyte solutions of unequal concentrations. The pore is treated as a long, circular, cylindrical duct. The analysis is based on a continuum formulation in which a dilute electrolyte solution is described by the coupled Nernst-Planck/Poisson creeping flow equations. Account is taken of the significant size of the electrolyte ions (assumed to be rigid spheres) when compared with the diameter of the membrane pores. Analytical solutions for the ion concentrations, hydrostatic pressure and electrostatic potential in the electrolyte solutions are given and an intra-pore flow solution is derived. A mathematical expression for the osmotic reflection coefficient as a function of the solute ion: pore diameter ratio λ and the solute fluxes is obtained. Approximate solutions are quoted which relate the solute fluxes and the solution electrostatic potentials at the membrane surfaces to the bulk solution concentrations, the membrane pore charge and pore geometry. The osmotic reflection coefficient is thus determined as a function of these parameters.