Chlamydospore Formation Research Articles

Oropharyngeal candidiasis prevalence among HIV-infected patients at the teaching hospital of Treichville (Abidjan, Côte d’Ivoire)

The aim of this study was to determine the mycological profile of oropharyngeal candidiasis among HIV-infected patients from Internal Medicine, Infectious and Tropical Diseases, and Pneumo-Phthisiology Diseases departments of the Teaching Hospital of Treichville in Abidjan, Côte d'Ivoire. This was a cross-sectional study carried out on patients with lesions suggestive of oropharyngeal candidiasis from October 2010 to April 2011. Oral swabs were cultured, and Candida species were identified using a germ tube test, a chlamydospore formation assay, and the API 20C system. A total of 286 patients were included, among whom 99.1% were infected with HIV. The prevalence of oropharyngeal candidiasis was 79.4% (CI95%=74.4-83.8). Five different species of Candida were identified, with the predominant species being Candida albicans (95.2%). Most affected patients were female (54.6%, P<0.0001) between the ages of 30 and 45 (78.4%, P<0.0001). The most lesion types observed were thrush (87.8%). Patients infected with HIV1 (95.6%), with a primary school level of education (52.8%), under antiretroviral therapy (88.5%) and with tuberculosis as an associated pathology (62.5%) were the most commonly affected. Patients were mostly under ART first line treatment (86.4%) and at the beginning of the treatment (86.4%). Oropharyngeal candidiasis is frequent among HIV-infected patients. Better and early management of this vulnerable population should allow for a reduction in the high prevalence observed.

Structure of Ralsolamycin, the Interkingdom Morphogen from the Crop Plant Pathogen Ralstonia solanacearum.

Ralsolamycin, an inducer of chlamydospore formation in fungi, was recently reported from the plant pathogenic bacterium Ralstonia solanacearum. Although interpretation of tandem mass data and bioinformatics enabled a preliminary chemical characterization, the full structure of ralsolamycin was not resolved. We now report the recovery of this secondary metabolite from an engineered R. solanacearum strain. The structure of ralsolamycin was elucidated by extensive spectroscopic analyses. Chemical derivatization as well as bioinformatics were used to assign the absolute stereochemistry. Our results identified an erroneous genome sequence, thereby emphasizing the value of chemical methods to complement bioinformatics-based procedures in natural product research.

Chlamydospore Specific Proteins of Candida albicans

Polymorphic yeast, Candida albicans, forms thick-walled structures called chlamydospores in order to survive under adverse conditions. We present proteomic profile changes occurring during chlamydospore formation. Chlamydospores were induced by inoculating C. albicans cells (grown for 48 h) on rice extract and semisolid agar containing tween 80 (1%), and were overlaid by a polyethene sheet to induce microaerophilic conditions at 30 °C. Proteins extracted from chlamydospores and hyphae (producing chlamydospores) were identified by LC-MS/MS analysis. Present datasets include proteomic data (Swath spectral libraries) of chlamydospores and yeast phase cells, as well as methodologies and tools used for the data generation. Further analysis is expected to provide an opportunity to understand modulations in metabolic processes, molecular architecture (i.e., cell wall, membrane, and cytoskeleton) and stress response pathways leading to chlamydospore formation and thus facilitating survival of C. albicans under adverse conditions.

In vitro susceptibility of Candida albicans clinical isolates to eight antifungal agents in Ouagadougou (Burkina Faso)

In recent years, the infection Candidaalbicans infection worldwide has risen, and the incidence of resistance to traditional antifungal therapies is also increasing. The aim of this study was to evaluate in vitro susceptibility of C.albicans clinical isolates to eight antifungal agents in Ouagadougou. A cross-sectional study was conducted from January 2013 to December 2015 at Yalgado Ouédraogo University Teaching Hospital. Two hundred seven strains have been isolated from 347 symptomatic patients received in different clinical services. Samples were cultured on Sabouraud Dextrose Agar supplemented with Cloramphenicol. Isolates were diagnosed as C.albicans using germ tube test, chlamydospore formation on Corn Meal Agar, and Api-Candida test (Biomérieux). Antifungal susceptibility testing was performed by disk diffusion method and isolates classified as susceptible, susceptible dose-dependent and resistant. Three hundred forty-seven (347) patients are included in this study. Two hundred and six (206) out of 347 collected samples (59.36%) were found positive for C.albicans. The strains were mostly isolated from vulvovaginal (49%) and oral infections (40.3%). The highest resistance rates of azoles were obtained with fluconazole (66.5%), itraconazole (52.3%) and ketoconazole (22.9%) when all clinical isolates were included. The resistance rates of fluconazole, itraconazole and ketoconazole remain highest for vulvovaginal and oral isolates. The rate of resistance to the polyene amphotericin B was 32.0% for all clinical isolates and was 56.4% for vulvovaginal strains. Resistance rate to nystatin was 6.3% for all clinical isolates. Cross-resistance analysis with data of all clinical strains revealed that the incidence of resistance to ketoconazole and itraconazole in fluconazole-resistant isolates was significantly higher than recorded for fluconazole-susceptible isolates. In vitro C.albicans antifungal susceptibility test in this study showed relatively high resistance to commonly and widely used azoles (fluconazole, ketoconazole). Most C.albicans clinical isolates were susceptible to nystatin.

Identification of suitable reference genes during the formation of chlamydospores in Clonostachys rosea 67-1.

Clonostachys rosea is a potential biocontrol fungus that can produce highly resistant chlamydospores under specific conditions. To investigate the genes related to chlamydospore formation, we identified reliable reference genes for quantification of gene expression in C. rosea 67‐1 during sporulation. In this study, nine reference genes, actin (ACT), elongation factor 1 (EF1), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), histone (HIS), RNA polymerase II CTD phosphatase Fcp1 (RPP), succinate‐semialdehyde dehydrogenase (SSD), TATA‐binding protein (TBP), ubiquitin (UBQ), and ubiquitin‐conjugating enzyme (UCE), were selected and cloned from 67‐1, and their expression stability during chlamydospore formation was determined using reverse transcription quantitative PCR and assessed using the software geNorm, NormFinder and BestKeeper. The Ct values of the candidates ranged from 19.9 to 29.7, among which HIS,ACT and SSD exhibited high expression levels. The statistical analysis showed that ACT and SSD were most stably expressed, while UBQ and GAPDH showed relatively large variations under different culture conditions. Calculation of pairwise variation value indicated that two reference genes were required for precise quantification. Finally, ACT and SSD were selected to normalize gene expression during chlamydospore production in C. rosea 67‐1. To the best of our knowledge, this is the first report of SSD as a reference gene. This study will facilitate the accurate quantification of differentially expressed genes during the generation of chlamydospores and contribute to the investigation of the molecular mechanism underlying chlamydospore formation in C. rosea.

Comparison of duplex PCR and phenotypic analysis in differentiating Candida dubliniensis from Candida albicans from oral samples

Candida dubliniensis shares a wide range of phenotypic characteristics with Candida albicans including a common trait called germ tube positivity. Hence, laboratory differentiation of these two species is cumbersome. Duplex PCR analyses for C. albicans and C. dubliniensis was performed directly on DNA extracted from a total of 122 germ tube positive isolates derived from 100 concentrated oral rinse samples from a random cohort of diabetics attending a clinic in Sri Lanka. These results were confirmed by DNA sequencing of internal transcribed spacer (ITS) region of rDNA of the yeasts. Performance efficacy of duplex PCR was then compared with phenotypic identification using a standard battery of phenotypic tests. Of the 122 germ tube positive isolates three were identified by duplex PCR as C. dubliniensis and the remainder as C. albicans. On the contrary, when the standard phenotypic tests, sugar assimilation and chlamydospore formation, were used to differentiate the two species 13 germ tube positive isolates were erroneously identified as C. dubliniensis. Duplex PCR was found to be rapid, sensitive and more specific than phenotypic identification methods in discriminating C. dubliniensis from C. albicans. This is also the first report on the oral carriage of C. dubliniensis in a Sri Lankan population.

Modulation of Inter-kingdom Communication by PhcBSR Quorum Sensing System in Ralstonia solanacearum Phylotype I Strain GMI1000.

Ralstonia solanacearum is a ubiquitous soil-borne plant pathogenic bacterium, which frequently encounters and interacts with other soil cohabitants in competition for environmental niches. Ralsolamycin, which is encoded by the rmy genes, has been characterized as a novel inter-kingdom interaction signal that induces chlamydospore development in fungi. In this study, we provide the first genetic evidence that the rmy gene expression is controlled by the PhcBSR quorum sensing (QS) system in strain GMI1000. Mutation of phcB could lead to significant reduction of the expression levels of the genes involved in ralsolamycin biosynthesis. In addition, both the phcB and rmy mutants were attenuated in induction of chlamydospore formation in Fusarium oxysporum f. cubense and diminished in the ability to compete with the sugarcane pathogen Sporisorium scitamineum. Agreeable with the pattern of QS regulation, transcriptional expression analysis showed that the transcripts of the rmy genes were increased along with the increment of the bacterial population density. Taken together, the above findings provide new insights into the regulatory mechanisms that the QS system involves in governing the ralsolamycin inter-kingdom signaling system.

Congolese Rhizospheric Soils as a Rich Source of New Plant Growth-Promoting Endophytic Piriformospora Isolates.

In the last decade, there has been an increasing focus on the implementation of plant growth-promoting (PGP) organisms as a sustainable option to compensate for poor soil fertility conditions in developing countries. Trap systems were used in an effort to isolate PGP fungi from rhizospheric soil samples collected in the region around Kisangani in the Democratic Republic of Congo. With sudangrass as a host, a highly conducive environment was created for sebacinalean chlamydospore formation inside the plant roots resulting in a collection of 51 axenically cultured isolates of the elusive genus Piriformospora (recently transferred to the genus Serendipita). Based on morphological data, ISSR fingerprinting profiles and marker gene sequences, we propose that these isolates together with Piriformospora williamsii constitute a species complex designated Piriformospora (= Serendipita) ‘williamsii.’ A selection of isolates strongly promoted plant growth of in vitro inoculated Arabidopsis seedlings, which was evidenced by an increase in shoot fresh weight and a strong stimulation of lateral root formation. This isolate collection provides unprecedented opportunities for fundamental as well as translational research on the Serendipitaceae, a family of fungal endophytes in full expansion.

Study of the Differentiation of Fusarium oxysporum f.sp. albedinis Chlamydospores on Different Culture Media

Chlamydospores of Fusarium oxysporum f.sp. albedinis are well known as fungus survival spores in soil, also act as a causal agent of Bayoud disease of the date palm. Till now, there are a little study in the literature about the favorable media for the formation of chlamydospores and the stages of chlamydogenesis. This study shows that rice flour based medium (45 chlamydospores per mm 2), oatmeal (31 chlamydospores per mm 2) and bean flour (27 chlamydospores per mm 2) tested for the first time, are among the media inducing the formation of chlamydospores in large numbers. The optimum formation of chlamydospores is observed on rice flour-based medium after 7 days of Original Research Article Smail et al.; ARRB, 18(4): 1-9, 2017; Article no.ARRB.35653 2 incubation at 18°C and pH 7. The effect of light does not seem very important on chlamydogenesis. However, relative humidity has a significant effect on the growth and chlamydogenesis of F. oxysporum f.sp. albedinis. The tested G 1 isolate grows and forms chlamydospores at relative humidities of 70 and 100%. The number of chlamydospores varies between 40.01 and 44.05 chlamydospores/mm 2. To form chlamydospores, cultures of F. oxysporum f.sp. albedinis need to be in contact with the favorable culture medium. Thus, the cultures develop and form more chlamydospores on a single layer of cellophane deposited on a culture medium, but show slower growth and do not form chlamydospores on two layers of cellophane. Chlamydospores of Fusarium oxysporum f.sp. albedinis are always at the extremity of the mycelial filaments. They differ in one to four cells in superimposed position. A terminal hyphal bulge leads to the formation of the terminal cell. The second cell is formed at the base of the first, and the process continues so that one can sometimes observed chlamydospores in bead-like chains.

Candida species Rewired Hyphae Developmental Programs for Chlamydospore Formation.

Chlamydospore formation is a characteristic of many fungal species, among them the closely related human-pathogenic dimorphic yeasts Candida albicans and C. dubliniensis. Whereas function and regulation of filamentation are well-studied in these species, the basis of chlamydospore formation is mostly unknown. Here, we investigate the contribution of environmental and genetic factors and identified central proteins involved in species-specific regulation of chlamydosporulation. We show that specific nutrient levels strongly impact chlamydospore initiation, with starvation favoring sporulation and elevated levels of saccharides or peptone inhibiting it. Thresholds for these nutritional effects differ between C. albicans and C. dubliniensis, which explain species-specific chlamydospore formation on certain diagnostic media. A C. albicans nrg1Δ mutant phenocopied C. dubliniensis, putting Nrg1 regulation at the basis of species-specific chlamydospore formation under various conditions. By screening a series of potential chlamydospore regulators, we identified the TOR and cAMP pathways as crucial for sporulation. As rapamycin treatment blocked chlamydosporulation, a low basal Tor1 activity seems to be essential. In addition, TOR effector pathways play an important role, and loss of the NCR (nitrogen catabolite repression) gene regulators Gat1 and Gln3 reduced chlamydospore formation. A severe reduction was seen for a C. albicans gcn4Δ deletion strain, implicating a link between regulation of amino acid biosynthesis and chlamydospore development. On the other hand, deletion of the GTPase gene RAS1 and the adenylyl cyclase gene CYR1 caused a defect in chlamydospore formation that was mostly rescued by cAMP supplementation. Thus, cAMP-signaling is a second major pathway to control chlamydospore production. Finally, we confirmed light exposure to have a repressive effect on chlamydosporulation. However, permanent illumination only reduced, but not abolished chlamydospore production of C. albicans whereas C. dubliniensis sporulation was unaffected. In summary, we describe novel environmental factors which determine chlamydosporulation and propose a first model for the regulatory network of chlamydospore formation by different Candida species.

Candida albicans ISW2 Regulates Chlamydospore Suspensor Cell Formation and Virulence In Vivo in a Mouse Model of Disseminated Candidiasis.

Formation of chlamydospores by Candida albicans was an established medical diagnostic test to confirm candidiasis before the molecular era. However, the functional role and pathological relevance of this in vitro morphological transition to pathogenesis in vivo remain unclear. We compared the physical properties of in vitro-induced chlamydospores with those of large C. albicans cells purified by density gradient centrifugation from Candida-infected mouse kidneys. The morphological and physical properties of these cells in kidneys of mice infected intravenously with wild type C. albicans confirmed that chlamydospores can form in infected kidneys. A previously reported chlamydospore-null Δisw2/Δisw2 mutant was used to investigate its role in virulence and chlamydospore induction. Virulence of the Δisw2/Δisw2 mutant strain was reduced 3.4-fold compared to wild type C. albicans or the ISW2 reconstituted strain. Altered host inflammatory reactions to the null mutant further indicate that ISW2 is a virulence factor in C. albicans. ISW2 deletion abolished chlamydospore formation within infected mouse kidneys, whereas the reconstituted strain restored chlamydospore formation in kidneys. Under chlamydospore inducing conditions in vitro, deletion of ISW2 significantly delayed chlamydospore formation, and those late induced chlamydospores lacked associated suspensor cells while attaching laterally to hyphae via novel spore-hypha septa. Our findings establish the induction of chlamydospores by C. albicans during mouse kidney colonization. Our results indicate that ISW2 is not strictly required for chlamydospores formation but is necessary for suspensor cell formation. The importance of ISW2 in chlamydospore morphogenesis and virulence may lead to additional insights into morphological differentiation and pathogenesis of C. albicans in the host microenvironment.

First report of Fusarium oxysporum species complex infection in zebrafish culturing system

Fusarium oxysporum species complex (FOSC) is a highly diverse fungus. Recently, F.oxysporum infection was identified from zebrafish (Danio rerio) culturing system in Korea. Initially, a rapid whitish smudge was appeared in the water with the fungal blooming on walls of fish tanks. Microscopic studies were conducted on fungal hyphae, colony pigmentation and chlamydospore formation and the presence of macro- and microspores confirmed that the isolated fungus as F.oxysporum. Furthermore, isolated F.oxysporum was confirmed by internal transcribed spacer sequencing which matched (100%) to nine F.oxysporum sequences available in GenBank. Experimental hypodermic injection of F.oxysporum into adult zebrafish showed the development of fungal mycelium and pathogenicity similar to signs observed. Histopathologic results revealed a presence of F.oxysporum hyphae in zebrafish muscle. Fusarium oxysporum growth was increased with sea salt in a concentration-dependent manner. Antifungal susceptibility results revealed that F.oxysporum is resistant to copper sulphate (up to 200μgmL-1 ) and sensitive to nystatin (up to 40μgmL-1 ). This is the first report of FOSC from zebrafish culture system, suggesting it appears as an emerging pathogen, thus posing a significant risk on zebrafish facilities in the world.

Ralstonia solanacearum lipopeptide induces chlamydospore development in fungi and facilitates bacterial entry into fungal tissues.

Ralstonia solanacearum is a globally distributed soil-borne plant pathogenic bacterium, which shares a broad ecological range with many plant- and soil-associated fungi. We sought to determine if R. solanacearum chemical communication directs symbiotic development of polymicrobial consortia. R. solanacearum produced a diffusible metabolite that induced conserved morphological differentiation in 34 species of fungi across three diverse taxa (Ascomycetes, Basidiomycetes and Zygomycetes). Fungi exposed to this metabolite formed chlamydospores, survival structures with thickened cell walls. Some chlamydospores internally harbored R. solanacearum, indicating a newly described endofungal lifestyle for this important plant pathogen. Using imaging mass spectrometry and peptidogenomics, we identified an undescribed lipopeptide, ralsolamycin, produced by an R. solanacearum non-ribosomal peptide synthetase-polyketide synthase hybrid. Inactivation of the hybrid non-ribosomal peptide synthetase-polyketide synthase gene, rmyA, abolished ralsolamycin synthesis. R. solanacearum mutants lacking ralsolamycin no longer induced chlamydospore development in fungal coculture and invaded fungal hyphae less well than wild-type. We propose that ralsolamycin contributes to the invasion of fungal hyphae and that the formation of chlamydospores may provide not only a specific niche for bacterial colonization but also enhanced survival for the partnering fungus.

The Oral Carriage of Candida in Oral Cancer Patients of Indian Origin Undergoing Radiotherapy and/or Chemotherapy.

Oral cancer is a challenging disease in Indian subcontinent because of increased use of tobacco and associated products. Although surgery is the main treatment modality, radiotherapy (RT) and chemotherapy (CT) are employed in inaccessible cases. Both RT & CT will result in painful and debilitating adverse effects in oral cavity e.g., mucositis, ulceration, dysgeusia, xerostomia and opportunistic infections. One of the most common opportunistic infection is caused by fungus Candida. Our aim was to investigate the incidence of oral colonization of Candida species with differentiation between carrier and infective state of the organism. We also investigate the effect of treatment modality (RT and CT) on the incidence of Candida, in oral cancer patients, undergoing RT and/or CT, in order to prevent and treat the Candida infection in a better way. It was a cross-sectional case-control study; done in Gujarat, India. Fifty patients of oral cancer undergoing RT, CT alone or combined were investigated and compared with the healthy controls. The samples were collected from mid-dorsum of tongue by using imprint culture technique. The samples were inoculated on Sabouraud's dextrose agar medium and the organisms were identified by wet mount, germ tube test, chlamydospore formation and sugar fermentation tests. There was significant increase in oral Candida colonization from 20% in healthy controls to 70% in oral cancer patients undergoing RT and/or CT (p = 0.001, < 0.05). A significant increase in infective state of Candida (71.4%) was noted (p = 0.001, < 0.05) with predominance of non-albicans species of Candida, chiefly C. tropicalis (42.8%). RT and CT leads to increased oral colonization and infection by Candida with a shift towards growth of non-albicans species. As the pattern of candidal species infection is changing, such studies are important for better diagnosis and treatment planning to gain good control over the disease.

The GRF10 homeobox gene regulates filamentous growth in the human fungal pathogen Candida albicans.

Candida albicans is the most common human fungal pathogen and can cause life-threatening infections. Filamentous growth is critical in the pathogenicity of C. albicans, as the transition from yeast to hyphal forms is linked to virulence and is also a pivotal process in fungal biofilm development. Homeodomain-containing transcription factors have been linked to developmental processes in fungi and other eukaryotes. We report here on GRF10, a homeobox transcription factor-encoding gene that plays a role in C. albicans filamentation. Deletion of the GRF10 gene, in both C. albicans SN152 and BWP17 strain backgrounds, results in mutants with strongly decreased hyphal growth. The mutants are defective in chlamydospore and biofilm formation, as well as showing dramatically attenuated virulence in a mouse infection model. Expression of the GRF10 gene is highly induced during stationary phase and filamentation. In summary, our study emphasizes a new role for the homeodomain-containing transcription factor in morphogenesis and pathogenicity of C. albicans.

Resistance sources, resistance screening techniques and disease management for Fusarium wilt in cotton

Fusarium wilt (FW), caused by the fungal pathogen Fusarium oxysporum f. sp. vasinfectum (FOV), is a significant economic constraint to cotton production worldwide. The pathogen has a wide geographical distribution, and its biological characteristics have enabled its establishment of eight races in several agro-ecosystems spanning from regions with high precipitation to semi-arid and arid irrigated zones. Management of Fusarium wilt is challenging because of the ability of FOV to persist in production systems through formation of long-lived chlamydospores and through its parasitic and pathogenic associations with cotton and other rotational cops and weeds. The interaction of FOV with nematodes constitutes additional constraints to the management of the disease. Breeding and utilizing FW-resistant cultivars has proven to be the most cost-effective control method. Numerous approaches and methods have been used in screening cotton for resistance to FW. This review provides a background on accumulated knowledge over the past two decades on sources of FW resistance and methods of screening for resistance including inoculation and evaluation protocols in cotton. The review also provides an overview of the biology and management of FOV.

Dolichol phosphate mannose synthase from the pathogenic yeast Candida albicans is a multimeric enzyme

BackgroundDolichol phosphate mannose synthase (DPMS) is a key enzyme in N- and O-linked glycosylations and glycosylphosphatidylinositol (GPI)-anchor synthesis. DPMS generates DPM, the substrate for mentioned processes, by the transfer of mannosyl residue from GDP-Man to dolichol phosphate. Here we describe the role of DPMS for Candida albicans physiology with emphasis on the cell wall composition and morphogenesis. MethodsC. albicans genes for DPMS subunits were cloned, tagged and expressed in Saccharomyces cerevisiae. The C. albicans strains with controlled expression of DPM genes were constructed and analyzed. Gene expression and enzyme activities were measured using RT-PCR and radioactive substrate. Sensitivities against chemical agents were tested with microdilution method. The composition of the cell wall was estimated by HPLC. Glycosylation status of the marker protein was analyzed by Western blot. Morphological differentiation of the strains was checked on the media promoting hyphae and chlamydospore formation. ResultsWe demonstrate that C. albicans DPMS consists of three interacting subunits, among which Dpm1 and Dpm3 are indispensable, whereas Dpm2 increases enzymatic activity. Lowered expression of DPMS genes results in decreased DPMS activity, increased susceptibility to cell wall perturbing agents and in altered cell wall composition. Mutants Tetp-DPM1 and Tetp-DPM3 show defective protein glycosylation and are impaired in hyphae and chlamydospore formation. Major conclusionDPMS from C. albicans, opposite to S. cerevisiae, belongs to the family of DPMS with multimeric protein structure. General significanceThis work provides important data about factors required for a proper protein glycosylation and for morphogenesis of pathogenic yeast C. albicans.

Biocontrol of tomato plant diseases caused by Fusarium solani using a new isolated Aspergillus tubingensis CTM 507 glucose oxidase

The present study focuses on the potential of glucose oxidase (GOD) as a promising biocontrol agent for fungal plant pathogens. In fact, a new GOD producing fungus was isolated and identified as an Aspergillus tubingensis. GOD (125 AU) has been found to inhibit Fusarium solani growth and spore production. Indeed, GOD caused the reduction of spores, the formation of chlamydospores, the induction of mycelial cords and the vacuolization of mycelium. In vivo assays, GOD acted as a curative treatment capable of protecting the tomato plants against F. solani diseases. In fact, the incidence was null in the curative treatment with GOD and it is around 45% for the preventive treatment. The optimization of media composition and culture conditions led to a 2.6-fold enhancement in enzyme activity, reaching 81.48U/mL. This study has demonstrated that GOD is a potent antifungal agent that could be used as a new biofungicide to protect plants from diseases.

Mapping of functional domains and characterization of the transcription factor Cph1 that mediate morphogenesis in Candida albicans

Cph1, a transcription factor of the Mitogen Activated Protein (MAP) kinase pathway, regulates morphogenesis in human fungal pathogen Candida albicans. Here, by following a systemic deletion approach, we have identified functional domains and motifs of Cph1 that are involved in transcription factor activity and cellular morphogenesis. We found that the N-terminal homeodomain is essential for the DNA binding activity; however, C-terminal domain and polyglutamine motif (PQ) are indispensable for the transcriptional activation function. Complementation analysis of the cph1Δ null mutant using various deletion derivatives revealed functional significance of the N- and C-terminal domains and PQ motif in filamentation process, chlamydospore formation and sensitivity to the cell wall interfering compounds. Genome-wide identification of the Cph1 binding site and quantitative RT-PCR transcript analysis in cph1Δ null mutant revealed that a number of genes which are associated with the filamentous growth, maintaining cell wall organization and mitochondrial function, and the genes of the pH response pathway are the transcriptional targets of Cph1. The data also suggest that Cph1 may function as a positive or negative regulator depending on the morphological state and physiological conditions. Moreover, differential expression of the upstream MAP kinase pathway genes in wild type and cph1Δ null mutant indicated the existence of a feedback regulation.

Colonização do xilema de eucalipto por Ceratocystis spp. isolado de diferentes hospedeiros

Ceratocystis sp. é um fungo que coloniza o xilema, agente causal de murcha e seca em diversas plantas lenhosas. O presente trabalho teve como objetivo acompanhar a colonização deste fungo na superfície de plantas de eucalipto (Eucalyptus sp.). Foram utilizados 5 isolados de Ceratocystis de diferentes hospedeiros (eucalipto, cacau, manga, teca e atemóia). Para isso, plantas de eucaliptos (clone 219) tiveram parte do seu sistema vascular exposto. Nesta região foi depositada uma suspensão contendo 106 esporos. Como testemunha, uma planta foi inoculada somente com água destilada autoclavada. Após a inoculação, estas plantas foram mantidas em câmara úmida a 25 oC no escuro. Parte da área inoculada foi coletada em intervalos de tempo pré-determinados (6, 12 e 24 horas), e fixadas em solução de "Karnovsky". As amostras foram preparadas e analisadas ao microscópio eletrônico de varredura. Todos os isolados foram capazes de germinar, penetrar e se desenvolver nos elementos de vasos de plantas de eucalipto no período de 6 horas. O isolado de cacau foi o que aparentemente teve o desenvolvimento mais lento dentro dos períodos estudados. Foi observada a germinação de ascósporos, clamidósporos e de conídios do tipo cilíndrico neste período. Doze horas após a inoculação ocorreu aumento da quantidade de micélio de todos os isolados testados. Nos casos dos isolados de manga e eucalipto foi possível observar a formação de novos clamidósporos. Vinte e quatro horas após a inoculação, com exceção do isolado de cacau, todos os outros isolados já apresentavam a formação de conidióforos cilíndricos. Este estudo comprova que isolados deste fungo, mesmo que provindos de outros hospedeiros, são capazes de se desenvolver no xilema do eucalipto.