Proteína PR-4 de pimenteira-do-reino (piper nigrum l.): expressão heteróloga em sistema bacteriano e avaliação funcional

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into 17 independent families, PR-1 to PR-17. Antifungal and enzymatic activities have been described for some of these proteins. Among them, the PR-4 family comprises class I and II of plant chitinases. cDNA clone that encode a PR-4 of the class II, designated as PnPR-4, was previously isolated, in others studies, from Piper nigrum L. (black pepper). This is an important crop for Brazil, mainly in Pará state, however its production has decreased because root rot (Fusariose) disease caused by fungus Fusarium solani f. sp. Piperis. In this study, PnPR-4 cDNA clone was used for production of the recombinant protein, named PnPR-4, by bacterial expression system. Open Read Frame (ORF) of the PnPR-4 protein, was amplified from the cDNA clone by PCR assays, then ORF was linked in the expression vector pET-29(a) and introduced, by eletroporation, into E. coli Rosetta (DE3). The production of target protein, in transformed cells, was induced by 1 mM IPTG, at 37°C for 5h. After production, enzymatic activity of recombinant PnPR-4 was evaluated by Detection of Chitinase Activity after Gel Electrophoresis Polyacrylamide. Activity was evaluated in pH: 5.0 and pH: 7.8 for showing the stability of the recombinant protein. Molecular mass of Recombinant protein, PnPR-4, was 13.5kDa, according with others PR-4 produced by heterologous expression in bacterial system. In assay of enzymatic activity, PnPR-4 presented chitinolytic activity, both in pH: 5.0 or 7.8. This is a characteristic of plant quitinases, activity in a broad range of pHs. Where, optimal activity occurs between pH: 3.0 and 5.0. For PnPR-4, the optimal pH was 5.0, however in pH: 7.8 the recombinant protein had activity, demonstrating the stability of enzyme. These results showing that bacterial system of heterologous expression is effective for production of the recombinant protein PnPR-4, that it is an enzyme with chitinolytic activity and highly stable. Thus, PnPR-4, is now, a new enzyme which can be used in plant biotechnology in the combat against plant's pathogenic fungi.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorCNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESPA - Fundação Amazônia de Amparo a Estudos e PesquisasProteínas Relacionadas à Patogênese (PR) são induzidas em resposta ao ataque de patôgenos. Proteínas PR são agrupadas em 17 famílias, PR-1 a PR-17. Atividades antifúngicas e enzimáticas foram descritas para algumas dessas proteínas. Entre elas, a família PR-4 compõe as classes I e II de quitinases de plantas. Um clone de cDNA que codifica uma PR-4 da classe II, nomeada PnPR-4, foi isolado, em estudos anteriores, de Piper nigrum L. (pimenteira do reino). Esta é uma importante cultura para Brasil, principalmente no estado do Pará, no entanto sua produção tem diminuído devido à doença conhecida como podridão da raiz (Fusariose) causada pelo fungo Fusarium solani f. sp. Piperis. Neste trabalho, o cDNA da PnPR-4 foi usado para a produção da proteína recombinante, chamada de PnPR-4. O quadro de leitura aberta (ORF) da PnPR-4, foi amplificado por meio de ensaio de PCR e, em seguida a ORF foi ligada ao vetor de expressão pET-29(a) e introduzido, por eletroporação, em E. coli Rosetta (DE3). Nas células transformadas, a produção da proteína de interesse foi induzida por IPTG 1mM à 37°C por 5h. Após a produção, a atividade enzimática da proteina recombinante PnPR-4 foi avaliada pela detecção da atividade enzimática de quitinase em gel de poliacrilamida após eletroforese. A atividade foi avaliada em pH: 5.0 e pH:7.8 para demonstra a estabilidade da proteína recombinante. A massa molecular da PnPR-4 foi de 13.5 kDa, estando de acordo com outras PR-4 produzidas pelo sistema de expressão heterologa em sistema bacteriano. No ensaio de atividade enzimática, a PnPR-4 apresentou atividade quitinolitica, tanto em pH:5.0 ou pH:7.8. Esta é uma característica de chitinases de plantas, atividade em uma ampla faixa de pHs. Onde, a atividade ótima ocorre entre pH: 3.0 e 5.0. Na proteína recombianante, o pH ótimo foi 5.0, no entato ela teve atividade em pH 7.8, demonstrando a estabilidade da enzima. Estes resultados mostram que o sistema bacteriano de expressão heteróloga é eficaz para a produção da proteína recombinante PnPR-4, que é uma enzima com atividade quitinolitica e altamente estável. Assim, a PnPR-4 é uma nova enzima que pode ser usado em biotecnologia vegetal no combate contra fungos patogênicos da planta

Figure 3 in Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region

Figure 3. Schematic drawing of a spore of Kudoa viseuensis n. sp. in apical view (right) and side view (left). Scale bar = 5 μmPublished as part of Monteiro, Elideth Pacheco, Da Silva, Diehgo Tuloza, Sanches, Osimar, Hamoy, Igor & Matos, Edilson Rodrigues, 2019, Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region, pp. 7-16 in Acta Protozoologica 58 (1) on page 12, DOI: 10.4467/16890027AP.19.002.10833, http://zenodo.org/record/835694

Kudoa viseuensis Monteiro & Da Silva & Sanches & Hamoy & Matos 2019, n. sp.

Kudoa viseuensis n. sp. ZooBank: lsid:zoobank.org:act: AF886D76-EC95- 4EA7-8F0D-DA2D4B956A60 Host: Batrachoides surinamensis (Bloch and Schnei- der, 1801). Infection site: Pseudocysts in the somatic musculature. Type locality: Brazil, state of Pará, municipality of Viseu (1°08’ S, 46°05’ W). Prevalence: 86% (52/60) of the examined hosts were infected. Etymology: the specific name, viseuensis, refers to the municipality of Viseu, where the specimens were captured. Type specimen: A glass slide with a 5 μm-thick his- tological section stained in Haematoxylin and Eosin, containing the spores of the new species was deposited in the Zoology Museum of the National Institute of Amazonian Research (INPA) in Manaus, Amazonas, Brazil, under catalog number CNIDARIA – INPA 038. Description of the spores: The spores of Kudoa viseuensis n. sp. were 7.2±0.2 μm in length and 5.2±0.2 μm in width (Figure 3). In the apical view, the polar capsules were 1.8±0.2 μm in length and 1.3±0.1 μm in width. When observed laterally, the mean length was 2.7±0.2 μm and the width was 1.3±0.1 μm (Table 1). Table 1 compares the dimensions of the spores and polar capsules (and the shape of the spores) of other Kudoa species with Kudoa viseuensis n. sp. (Cnidaria: Myxozoa: Myxosporea: Multivalvulida: Kudoidae). Kudoa viseuensis n. sp. was restricted to the skeletal musculature of the host, and was not found in any other organ of the host fish. The morphological comparisons indicated that Kudoa viseuensis n. sp. is most similar to K. orbicularis (Azevedo et al. 2016) in terms of the width of the spore and the polar capsule, although other dimensions are clearly distinct from those of this species, and other Kudos species, studies previously. These findings, to- gether with the molecular data (see below) lend support to the present description of the new species. Phylogenetic analyses A partial sequence of 1400 base pairs (bps) of the SSU rDNA gene was obtained from the spores of Kudoa viseuensis n. sp., found in the musculature of B. surinamensis. This sequence was deposited in GenBank under accession number MK256272. The phylogenetic tree generated by Bayesian Inference defined a major clade, denominated clade A, composed of species of the genus Kudoa (Figure 4), which is subdivided into two clades, denominated A1 and A2, with high support (posterior probabilities). Each of these clades was influenced strongly by the tissue tropism of the parasites, with Kudoa viseuensis n. sp. being included in subclade A2. Subclade A1 is composed of Kudoa species that parasitise the musculature, brain, and intestine of fish- es. Kudoa viseuensis n. sp. is included in subclade A2, which is basal to A1. Kudoa viseuensis n. sp. parasitizes the musculature of the marine fish B. surinamensis, and clusters with Kudoa orbicularis (Azevedo et al. 2016), a parasite of the musculature of the freshwater fish, Chaetobranchopsis orbicularis. In this arrangement, K. orbicularis is the sister species of Kudoa viseuensis n. sp., and the two species not only share the infection site, but are also found in the same geographic region. ND – Not available; L* – Lateral; A* – Apic The outgroup is composed of species of the genus Unicapsula Davis, 1924, a member of the order Multivalvulida Schulman, 1959, which infects the musculature of its hosts. A new alignment was run for the pairwise comparison of key Kudoa species with Kudoa viseuensis n. sp. (Table 2). The smallest pairwise genetic distance (p) found in this analysis between Kudoa viseuensis n. sp. and the other Kudoa species was 3.9% in the case of K. orbicularis (KM192365), while the greatest distance was 4.9% for K. rosenbuschi (AY623795).Published as part of Monteiro, Elideth Pacheco, Da Silva, Diehgo Tuloza, Sanches, Osimar, Hamoy, Igor & Matos, Edilson Rodrigues, 2019, Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region, pp. 7-16 in Acta Protozoologica 58 (1) on pages 9-13, DOI: 10.4467/16890027AP.19.002.10833, http://zenodo.org/record/835694

Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region

Monteiro, Elideth Pacheco, Da Silva, Diehgo Tuloza, Sanches, Osimar, Hamoy, Igor, Matos, Edilson Rodrigues (2019): Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region. Acta Protozoologica 58 (1): 7-16, DOI: 10.4467/16890027AP.19.002.10833, URL: http://dx.doi.org/10.4467/16890027ap.19.002.1083

Figure 4 in Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region

Figure 4. Phylogenetic tree derived from Bayesian Inference (BI), based on the partial sequences of the SSU rDNA gene of Kudoa vi- seuensis n. sp. and closely-related myxosporans. The GenBank access numbers are shown next to the species names, and the numbers at each node are the BI posterior probabilities. The new species is highlighted in bold type. Abbreviations: Msl – muscle; Dtr – digestive tract; Nsy – nervous system.Published as part of Monteiro, Elideth Pacheco, Da Silva, Diehgo Tuloza, Sanches, Osimar, Hamoy, Igor & Matos, Edilson Rodrigues, 2019, Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region, pp. 7-16 in Acta Protozoologica 58 (1) on page 12, DOI: 10.4467/16890027AP.19.002.10833, http://zenodo.org/record/835694

Figure 2 in Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region

Figure 2. Light photomicrograph: (A) longitudinal histology section of the skeletal musculature of B. surinamensis containing a pseudocyst (*), along the axis of the muscle, showing the substitution of the fiber by the parasite; (B) transversal section showing the pseudocyst of the mixosporean occupying the central portion of the muscle fiber (arrowhead), typical of an individual infection; (C) Multiple infection of pseudocysts within a single muscle fiber, separated from one another and the muscle tissue by a fine conjunctive membrane (arrows). Scale bars: 40 µm.Published as part of Monteiro, Elideth Pacheco, Da Silva, Diehgo Tuloza, Sanches, Osimar, Hamoy, Igor & Matos, Edilson Rodrigues, 2019, Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region, pp. 7-16 in Acta Protozoologica 58 (1) on page 10, DOI: 10.4467/16890027AP.19.002.10833, http://zenodo.org/record/835694

Figure 1 in Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region

Figure 1. Light photomicrograph: (A) Whitish pseudocyst (arrowhead) found in the musculature of B. surinamensis. Scale bar: 1000 µm; (B) pseudocyst (c) and numerous mature spores (e) observed following the rupture of the pseudocyst. Scale bar: 100 µm; (C) Fresh, pseu- do-square spores (e) of Kudoa viseuensis n. sp. Scale bar: 20 µm; Inset: polar capsules (PC) in lateral (L) and apical (A) views (DIC). Scale bar: 10 µm.Published as part of Monteiro, Elideth Pacheco, Da Silva, Diehgo Tuloza, Sanches, Osimar, Hamoy, Igor & Matos, Edilson Rodrigues, 2019, Morphological and Molecular Characteristics of Kudoa viseuensis n. sp. (Myxosporea: Multivalvulida), Found in the Muscle of Batrachoides surinamensis (Teleostei: Batrachoididae) in the Brazilian Amazon Region, pp. 7-16 in Acta Protozoologica 58 (1) on page 10, DOI: 10.4467/16890027AP.19.002.10833, http://zenodo.org/record/835694

Identificação de duplicatas em acessos de mandioca coletados na Região Norte do Brasil por meio de marcadores microssatélites

Duplicates are common in germplasm banks and their identification is needed to facilitate germplasm bank management and to reduce maintenance costs. The aim of this work was to identify duplicates of cassava from a germplasm bank in Eastern Amazon, which had been previously characterized both morphological and agronomically. In order to be genotyped with 15 microsatellite loci, 36 accessions were selected. These accessions were classified into 13 groups of similar morpho-agronomical characteristics. All loci were polymorphic, and 75 alleles were identified, with an average of five alleles per loci and HE = 0.66. There were determined 34 pairs of genotypes with identical multiloci profiles and the probability of genetic identity was 1.1x10-12 with probability of exclusion of 99.9999%. Among these duplicates, there are accessions sampled on different years and places, but with different names and accessions with the same name sampled in different places and years. The study identified genotypes that are grown in different places and that have been maintained over the years by local farmers.FAPESPA - Fundação Amazônia de Amparo a Estudos e PesquisasCNPq - Conselho Nacional de Desenvolvimento Científico e TecnológicoEMBRAPA - Empresa Brasileira de Pesquisa AgropecuáriaDuplicatas costumam ocorrer em bancos de germoplasma e a sua identificação é necessária para facilitar o manejo dos bancos ativos de germoplasma (BAGs) e diminuir custos de manutenção. O objetivo deste trabalho foi identificar duplicatas de mandioca determinadas previamente pela caracterização morfo-agronômica, em um BAG da Amazônia Oriental. Foram selecionados 36 acessos que se agrupavam em 13 grupos de similaridade morfo-agronômica para serem genotipados com 15 locos microssatélites. Todos os locos foram polimórficos, sendo obtidos 75 alelos, com média de cinco alelos por loco e HE = 0,66. Foram encontrados 34 pares de genótipos que apresentaram perfis multilocos idênticos e a probabilidade de identidade genética foi de 1,1x10-12 com probabilidade de exclusão de 99,9999%. Entre essas duplicatas, estão materiais coletados em épocas e locais diferentes, e com diferentes denominações e acessos com o mesmo nome coletados em diferentes locais e anos. O estudo identificou genótipos que vem sendo cultivados em diferentes locais e que vêm sendo mantidos pelos agricultores ao longo dos anos.SILVA, D. T. Universidade Federal do Par