RESUMO
The Phytophthora genus is composed, mainly, of plant pathogens. This genus belongs to the Oomycete class, also known as "pseudo-fungi", within the Chromista Kingdom. Phytophthora spp. is highlighted due to the significant plant diseases that they cause, which represents some of the most economically and cultural losses, such as European chestnut ink disease, which is caused by P. cinnamomi. Currently, there have been four genome assemblies placed at the National Center for Biotechnology Information (NCBI), although the progress to understand and elucidate the pathogenic process of P. cinnamomi by its genome is progressing slowly. In this review paper, we aim to report and discuss the recent findings related to P. cinnamomi and its genomic information. Our research is based on paper databases that reported probable functions to P. cinnamomi proteins using sequence alignments, bioinformatics, and biotechnology approaches. Some of these proteins studied have functions that are proposed to be involved in the asexual sporulation and zoosporogenesis leading to the host colonization and consequently associated with pathogenicity. Some remarkable genes and proteins discussed here are related to oospore development, inhibition of sporangium formation and cleavage, inhibition of flagellar assembly, blockage of cyst germination and hyphal extension, and biofilm proteins. Lastly, we report some biotechnological approaches using biological control, studies with genome sequencing of P. cinnamomi resistant plants, and gene silencing through RNA interference (iRNA).
Assuntos
Biotecnologia/métodos , Biologia Computacional/métodos , Genômica/métodos , Oomicetos/genética , Phytophthora/genética , Parede Celular/microbiologia , Interações Hospedeiro-Patógeno , Oomicetos/fisiologia , Phytophthora/classificação , Phytophthora/fisiologia , Doenças das Plantas/microbiologia , Esporos/genéticaRESUMO
In nature, there are >200 species of fungi with hallucinogenic properties. These fungi are classified as Psilocybe, Gymnopilus, and Panaeolus which contain active principles with hallucinogenic properties such as ibotenic acid, psilocybin, psilocin, or baeocystin. In Chile, fungi seizures are mainly of mature specimens or spores. However, clandestine laboratories have been found that process fungus samples at the mycelium stage. In this transient stage of growth (mycelium), traditional taxonomic identification is not feasible, making it necessary to develop a new method of study. Currently, DNA analysis is the only reliable method that can be used as an identification tool for the purposes of supporting evidence, due to the high variability of DNA between species. One way to identify the species of a distinctive DNA fragment is to study PCR products analyzed by real time PCR and sequencing. One of the most popular sequencing methods of forensic interest at the generic and intra-generic levels in plants is internal transcribed spacer (ITS). With real time PCR it is possible to distinguish PCR products by differential analysis of their melting temperature (Tm) curves. This paper describes morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory. The fungus species were identified using scanning electron microscopy (SEM), mass spectrometry, HRM analysis, and ITS sequencing. The sporological studies showed a generally smooth surface and oval shape, with maximum length 10.1⯵m and width 6.4⯵m. The alkaloid Psilocyn was identified by mass spectrometry, while HRM analysis and ITS sequencing identified the species as Psilocybe cubensis. A genetic match was confirmed between the HRM curves obtained from the mycelia (evidence) and biological tissue extracted from the fruiting bodies. Mycelia recovered from the evidence and fruiting bodies (control) were genetically indistinguishable.
Assuntos
Alucinógenos/análise , Micélio/genética , Psilocybe/classificação , Psilocibina/análogos & derivados , Chile , DNA Fúngico/análise , Tráfico de Drogas , Genética Forense , Cromatografia Gasosa-Espectrometria de Massas , Microscopia Eletrônica de Varredura , Psilocibina/análise , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Análise de Sequência de RNA , Esporos/genéticaRESUMO
Myxosporean are endoparasitic cnidarians of wide distribution and responsible for important economic losses in fisheries and aquaculture. A new myxosporean species, Henneguya peruviensis n. sp., is herein described as obtained from the gill filaments of Hyphessobrycon loretoensis caught in the Nanay River, Department of Loreto, Peru. The parasite was found in 37 of 45 (82.2%) examined H. loretoensis. The new species was characterized based on morphological features and 18S rDNA gene sequence data. The sequencing of the 18S rDNA gene from the spores of H. peruviensis n. sp. resulted in 1632 nucleotides and this sequence did not match any of the myxozoan available in the GenBank. Phylogenetic analysis showed that H. peruviensis n. sp. closed together with H. leporinicola. Nonetheless, the 18S rDNA sequences of H. peruviensis n. sp. and H. leporinicola have only 82% similarity. This is the first description and molecular study of a Myxozoa parasitizing fish of the genus Hyphessobrycon in the Amazon basin. Given the importance of the ornamental fish industry in translocation of aquatic organisms worldwide, the international movement of myxosporeans in infected fish is discussed in terms of disease outbreaks and the need for preventative action.
Assuntos
Characidae/parasitologia , DNA Ribossômico/genética , Doenças dos Peixes/parasitologia , Brânquias/parasitologia , Myxozoa/genética , Animais , Aquicultura , Peixes/parasitologia , Myxozoa/anatomia & histologia , Peru , Filogenia , Rios , Esporos/genéticaRESUMO
The absence of base excision repair (BER) proteins involved in processing ROS-promoted genetic insults activates a DNA damage scanning (DisA)-dependent checkpoint event in outgrowing Bacillus subtilis spores. Here, we report that genetic disabling of transcription-coupled repair (TCR) or nucleotide excision repair (NER) pathways severely affected outgrowth of ΔdisA spores, and much more so than the effects of these mutations on log phase growth. This defect delayed the first division of spore's nucleoid suggesting that unrepaired lesions affected transcription and/or replication during outgrowth. Accordingly, return to life of spores deficient in DisA/Mfd or DisA/UvrA was severely affected by a ROS-inducer or a replication blocking agent, hydrogen peroxide and 4-nitroquinoline-oxide, respectively. Mutation frequencies to rifampin resistance (Rifr ) revealed that DisA allowed faithful NER-dependent DNA repair but activated error-prone repair in TCR-deficient outgrowing spores. Sequencing analysis of rpoB from spontaneous Rifr colonies revealed that mutations resulting from base deamination predominated in outgrowing wild-type spores. Interestingly, a wide range of base substitutions promoted by oxidized DNA bases were detected in ΔdisA and Δmfd outgrown spores. Overall, our results suggest that Mfd and DisA coordinate excision repair events in spore outgrowth to eliminate DNA lesions that interfere with replication and transcription during this developmental period.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/genética , Dano ao DNA , Reparo do DNA , Esporos/crescimento & desenvolvimento , Esporos/genética , Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Replicação do DNA , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana , Mutação , Espécies Reativas de Oxigênio/toxicidade , Rifampina/farmacologia , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In a survey of myxozoan parasites of ornamental freshwater fish from the Rio Negro river, it was found that seven of 30 (23.3 %) Corydoras melini specimens examined had plasmodia of a new Myxidium species (Myxidium amazonense n. sp.) in the gallbladder. The fish were caught in the Rio Negro river, in the municipality of Santa Isabel do Rio Negro, in the state of Amazonas, Brazil. The plasmodia had a tubular shape, which was organized as a spiral spring with several turns in the gallbladder. The development of the myxospores was asynchronic, with disporic pansporoblasts. Mature myxospores were elongated, with 17.0 ± 0.9 (16.1-17.9) µm in length and 3.7 ± 0.7 (3.0-4.4) µm in width, and lightly arcuate from the valval view, with their bodies tapering slowly until ending in rounded extremities. The valval surface had nine to ten grooves in each valve. The polar capsules, one at either end of the spore, had a length of 5.4 ± 0.5 (4.9-5.9) µm and a width of 3.4 ± 0.6 (2.8-4.0) µm. Ultrastructural analysis showed that the wall of the plasmodia had numerous microvilli-like structures, pinocytotic canals, and cytoplasmic bridges connecting the pansporoblasts to each other and to the ectoplasm zone. Phylogenetic analysis, based on a small subunit ribosomal RNA (ssrRNA), identified the new species as a sister species of Myxidiumceccarelli, the unique South American Myxidium species whose ssrRNA sequence is available in the NCBI database. This study is the first description of Myxidium species in ornamental freshwater fish from Amazon.
Assuntos
Doenças dos Peixes/parasitologia , Myxozoa/isolamento & purificação , Doenças Parasitárias em Animais/parasitologia , Subunidades Ribossômicas Menores/genética , Animais , Sequência de Bases , Brasil , Peixes-Gato/parasitologia , Vesícula Biliar/parasitologia , Dados de Sequência Molecular , Myxozoa/classificação , Myxozoa/genética , Myxozoa/ultraestrutura , Filogenia , Rios/parasitologia , Esporos/classificação , Esporos/genética , Esporos/isolamento & purificação , Esporos/ultraestruturaRESUMO
In this study, we investigated the reverse transcriptase subunit of telomerase in the dimorphic fungus Ustilago maydis. This protein (Trt1) contains 1371 amino acids and all of the characteristic TERT motifs. Mutants created by disrupting trt1 had senescent traits, such as delayed growth, low replicative potential, and reduced survival, that were reminiscent of the traits observed in est2 budding yeast mutants. Telomerase activity was observed in wild-type fungus sporidia but not those of the disruption mutant. The introduction of a self-replicating plasmid expressing Trt1 into the mutant strain restored growth proficiency and replicative potential. Analyses of trt1 crosses in planta suggested that Trt1 is necessary for teliospore formation in homozygous disrupted diploids and that telomerase is haploinsufficient in heterozygous diploids. Additionally, terminal restriction fragment analysis in the progeny hinted at alternative survival mechanisms similar to those of budding yeast.
Assuntos
Telomerase/biossíntese , Telomerase/genética , Ustilago/enzimologia , Sequência de Aminoácidos , Replicação do DNA/genética , Diploide , Regulação Fúngica da Expressão Gênica , Esporos/genética , Telomerase/isolamento & purificação , Ustilago/genética , Ustilago/crescimento & desenvolvimentoRESUMO
During a survey of myxozoan parasites of freshwater fish from the São Francisco River in Minas Gerais State, Brazil, plasmodia of Myxidium ceccarellii n. sp. were found in gallbladders of two out of six specimens (22%) of Leporinus elongatus (Anastomidae). Parasite plasmodia were translucent and greenish, with disporic sporoblasts that develop asynchronously. Mature myxospores were ellipsoidal in frontal and lateral views, with slightly pointed ends. The surfaces of each valve had four to six longitudinal grooves. Spores dimensions were as follows: length 17.7 ± 0.5 µm (17.1-18.1), width 10.4 ± 0.47 µm (9.8-11.3), and thickness 10.1 ± 0.27 µm (9.6-10.4). Two polar capsules, one at either end of the spore, had the length of 6.3 ± 0.5 µm (5.7-7.0) and width of 6.4 ± 0.44 µm (5.7-6.9), with four to five polar filament turns. Some aberrant spores had one or three polar capsules. Partial sequencing of M. ceccarellii n. sp. small subunit ribosomal RNA (ssrRNA) gene resulted in 1,845 bp. This is the first molecular study of a Myxidium species that parasitizes a South American freshwater fish. Phylogenetic reconstruction using ssrRNA gene sequences showed that M. ceccarellii n. sp. was positioned basally in a recognized clade of myxozoans that infect the biliary systems of nonfish vertebrates.
Assuntos
Caraciformes/parasitologia , Doenças dos Peixes/parasitologia , Vesícula Biliar/parasitologia , Doenças Parasitárias em Animais/parasitologia , Filogenia , RNA Ribossômico/classificação , Animais , Brasil , Myxozoa/ultraestrutura , RNA Ribossômico/genética , Subunidades Ribossômicas Menores/genética , Rios , Análise de Sequência de DNA , Esporos/genética , Esporos/ultraestruturaRESUMO
Phytophthora capsici from seven provinces of China were investigated for their mating type, hyphal growth, zoospore production, and virulence. All of the morphological characteristics and the results of polymerase chain reaction confirmed that these isolates were indeed Phytophthora capsici. The test of mating type showed that the mating types of 19 representative isolates from China varied. The hyphal growth and the amount of zoospores produced from these isolates differed and there was no evident relationship between them, which indicated the existence of genetic diversity among the isolates in China. Also, the isolates that were more virulent on the pepper cultivars that we checked produced more zoospores than other isolates.
Assuntos
Phytophthora/genética , Capsicum/microbiologia , Phytophthora/isolamento & purificação , Phytophthora/patogenicidade , Phytophthora/fisiologia , Reprodução/genética , Esporos/genética , Esporos/fisiologia , Virulência/genéticaRESUMO
Spores of Clostridium difficile are essential for infection, persistence and transmission of C. difficile infections (CDI). Proteins of the surface of C. difficile spores are thought to be essential for initiation and persistence of CDI. In this work, we demonstrate that three C. difficile collagen-like exosporium proteins (BclA) encoded in the C. difficile 630 genome are expressed during sporulation and localize to the spore via their N-terminal domains. Using polyclonal antibodies against the N- and C-terminal domains and full length BclA1 we demonstrate that BclA1 is likely to be localized to the exosporium layer, presumably undergoes post-translational cleavages and might be cross-linked with other exosporium proteins. The collagen-like region of recombinant BclA1 and BclA2 was susceptible to collagenase degradation. Collagenase digestion assay of C. difficile spores suggests that, similarly as in Bacillus anthracis BclA, the N-terminal domain and the C-terminal domain of BclA1 might be buried in the basal layer and oriented to the exosporium surface, respectively. We also demonstrate that the collagen-like BclAs proteins do not contribute to the spore hydrophobicity and its absence slightly increased the adherence of spores to Caco-2 cells. BclA1 was also shown to have poor immunogenic properties. These results provide the first study on the BclA1 collagen-like proteins of C. difficile spores.
Assuntos
Proteínas de Bactérias/análise , Clostridioides difficile/química , Proteínas de Membrana/análise , Esporos/química , Aderência Bacteriana , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Células CACO-2 , Clostridioides difficile/genética , Perfilação da Expressão Gênica , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Peso Molecular , Processamento de Proteína Pós-Traducional , Esporos/genéticaRESUMO
Pythium insidiosum causes pythiosis, a life-threatening disease that occurs in tropical regions and affects man and animals. Although pythiosis in Brazil had been described in various animal species, the first human case was only recently reported. The present study aimed to characterize the morphologic and molecular characteristics of a new equine isolate of P. insidiosum and compare them with those of the first Brazilian human isolate. Both isolates were recovered from the same region of the country. Macroscopic and microscopic features were evaluated in two culture media. Sporangia formation and zoospore release were obtained after culturing the isolates with fragments of grasses and crops in an appropriate liquid induction medium. The molecular analysis of the isolates consisted of the complete sequencing of the ITS-5.8S rDNA region and sequences of both showed identical composition of 836 bp and 99% similarity with the isolates M16, 65, M12, 339 and 394 deposited at GenBank. Simple mycological procedures such as the production of sporangia and zoospores may distinguish P. insidiosum from zygomycetes. The rDNA sequencing indicates that, in Brazil, both humans and animals might be infected by a common genotype of the pathogen.
Assuntos
Doenças dos Cavalos/microbiologia , Infecções/microbiologia , Infecções/veterinária , Pythium/citologia , Pythium/isolamento & purificação , Animais , Brasil , DNA de Algas/genética , DNA Espaçador Ribossômico/genética , Cavalos , Humanos , Masculino , Dados de Sequência Molecular , Filogenia , Pythium/classificação , Pythium/genética , RNA Ribossômico 5,8S/genética , Esporos/citologia , Esporos/genética , Esporos/isolamento & purificaçãoRESUMO
Chromatin-remodeling factors regulate the establishment of transcriptional programs during plant development. Although 42 genes encoding members of the SWI2/SNF2 family have been identified in Arabidopsis thaliana, <10 have been assigned a precise function on the basis of a mutant phenotype, and none have been shown to play a specific role during the gametophytic phase of the plant life cycle. A. thaliana chromatin-remodeling protein 11 (CHR11) encodes an imitation of switch (ISWI)-like chromatin-remodeling protein abundantly expressed during female gametogenesis and embryogenesis in Arabidopsis. To determine the function of CHR11 in wild-type plants, we introduced a hairpin construct leading to the production of double-stranded RNA, which specifically degraded the endogenous CHR11 mRNA by RNA interference (RNAi). Transcription of the RNAi-inducing hairpin RNA was driven by either a constitutive cauliflower mosaic virus 35S promoter (CaMV35S) acting at most stages of the sporophytic phase or a newly identified specific promoter acting at the onset of the female gametophytic phase (pFM1). All adult transformants that constitutively lacked sporophytic CHR11 activity showed reduced plant height and small cotyledonary embryos with limited cell expansion. In contrast, RNAi lines in which CHR11 was specifically silenced at the onset of female gametogenesis (megagametogenesis) had normal height and embryo size but had defective female gametophytes arrested before the completion of the mitotic haploid nuclear divisions. These results show that CHR11 is essential for haploid nuclear proliferation during megagametogenesis and cell expansion during the sporophytic phase, demonstrating the functional versatility of SWI2/SNF2 chromatin-remodeling factors during both generations of the plant life cycle.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Divisão do Núcleo Celular/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas Cromossômicas não Histona/fisiologia , Gametogênese/fisiologia , Adenosina Trifosfatases , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/biossíntese , Proteínas de Arabidopsis/genética , Divisão do Núcleo Celular/genética , Tamanho Celular , Montagem e Desmontagem da Cromatina/genética , Proteínas Cromossômicas não Histona/biossíntese , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/fisiologia , Gametogênese/genética , Haploidia , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Esporos/citologia , Esporos/genética , Esporos/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
Cytogenetic studies carried out in the tetraploid accession BRA001068 of Brachiaria decumbens, also known as cv. Basilisk, revealed an unusual pattern of microsporogenesis. The spindle in metaphase I and anaphase I became heavily stained with propionic carmine. In telophase I, the interzonal microtubules continued to be intensely stained, and during the phragmoplast formation the fibers were pushed to the cell wall, persisting until prophase II, even after cytokinesis. Due to its tetraploid condition, the accession presented many cells with precocious chromosome migration to the poles in metaphase I and laggards in anaphase I that gave rise to micronuclei in telophase I. While in other polyploid accessions of Brachiaria micronuclei remained in this condition until the second cytokinesis, the micronuclei in this accession organized their own spindle in the second division. In several microsporocytes, the micronuclei with their minispindle were divided further into microcytes by additional cytokinesis. Some curious planes of cytokinesis were found in some cells, with partitioning of cytoplasm into cells of irregular shape. The result consisted of a high frequency of abnormal products of meiosis. Quadrivalents were observed in diakinesis at low frequency, which suggests a segmental allotetraploid and the inability of both genomes to co-ordinate their activities, leading to multiple spindle and precocious cellularization. In spite of abnormal meiotic products reducing pollen fertility, seed production was normal. Enough normal pollen was available to fertilize the central-cell nucleus of the embryo sac and produce normal endosperm in this pseudogamous aposporous apomictic accession.