Cloning and expression analysis of an endo‑1,3‑β‑d‑glucosidase from Phytophthora cinnamomi

Phytophthora is considered one of the most destructive genus for many agricultural plant species worldwide, with a strong environmental and economic impact. Phytophthora cinnamomi is a highly aggressive Phytophthora species associated with the forest decline and responsible for the ink disease in chestnut trees (Castanea sativa Miller), a culture which is extremely important in Europe. This pathogenicity occurs due to the action of several enzymes like the hydrolysis of 1,3-β-glucans at specific sites by the enzyme endo-1,3-β-d-glucosidase. The aim of this work to analyze the heterologous expression in two microorganisms, Escherichia coli and Pichia pastoris, of an endo-1,3-β-d-glucosidase encoded by the gene ENDO1 (AM259651) from P. cinnamomi. Different plasmids were used to clone the gene on each organism and the real-time quantitative polymerase chain reaction was used to determine its level of expression. Homologous expression was also analyzed during growth in different carbon sources (glucose, cellulose, and sawdust) and time-course experiments were used for endo-1,3-β-d-glucosidase production. The highest expression of the endo-1,3-β-d-glucosidase gene occurred in glucose after 8 h of induction. In vivo infection of C. sativa by P. cinnamomi revealed an increase in endo-1,3-β-d-glucosidase expression after 12 h. At 24 h its expression decreased and at 48 h there was again a slight increase in expression, and more experiments in order to further explain this fact are underway.info:eu-repo/semantics/publishedVersio

Isolation and Sequencing of Actin1, Actin2 and Tubulin1 Genes Involved in Cytoskeleton Formation in Phytophthora cinnamomi

Oomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. On the Nordeste Transmontano region (northeast Portugal), the Castanea sativa chestnut culture is extremely important. The biggest productivity and yield break occurs due to the ink disease, caused by Phytophthora cinnamomi which is one of the most widely distributed Phytophthora species, with nearly 1000 host species. The knowledge about molecular mechanisms responsible for pathogenicity is an important tool in order to combat associate diseases of this pathogen. Complete open reading frames (ORFs) of act1, act2 and tub1 genes who participate in cytoskeleton formation in P. cinnamomi were achieved by high-efficiency thermal asymmetric interlaced (HE-TAIL) polymerase chain reaction (PCR). act1 gene comprises a 1128 bp ORF, encoding a deduced protein of 375 amino acids (aa) and 41,972 kDa. act2 ORF comprises 1083 bp and encodes a deduced protein of 360 aa and 40,237 kDa. tub1 has a total length of 2263 bp and encodes a 453 aa protein with a molecular weight of 49.911 kDa. Bioinformatics analyses shows that actin1 is ortholog to the act1 genes of Phytophthora infestans, Phytophthora megasperma and Phytophthora melonis; actin2 is ortholog to the act2 genes of P. infestans, Phytophthora brassicae, P. melonis and Pythium splendens and tubulin1 shows the highest orthology to P. infestans and P. capsici α-tubulin genes. Analysed 3D structure of the three putative proteins revealed a spatial conformation highly similar to those described for orthologous proteins obtained by X-ray diffraction.COMBATINTA/SP2.P11/02 Interreg IIIA–Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Fund

Genes found in partial sequencing of Phytophthora cinnamomi

Members of the oomycete cause extensive losses in agriculture and widespread degradation in natural plant communities, being responsible for the death of thousands of trees every year. Two of the representative species are Phytophthora infestans, which causes late blight of potato, and Phytophthora cinnamomi, which causes chestnut ink disease, responsible for losses on sweet chestnut production in Europe. Genome sequencing efforts have been focused on the study of three species: P. infestans, P. sojae and P. ramorum. Phytophthora infestans has been developed as the model specie for the genus, possessing excellent genetic and genomics resources including genetic maps, BAC libraries, and EST sequences. Our research team is trying to sequence the genome of P. cinnamomi in order to gain a better understanding of this oomycete, to study changes in plant-pathogen relationships including those resulting from climate change and trying to decrease the pathogen’s impact on crops and plants in natural ecosystems worldwide. We present here a preliminary report of partially sequenced genomic DNA from P. cinnamomi encoding putative protein-coding sequences and tRNAs. Database analysis reveals the presence of genes conserved in oomycetes.Os oomicetas causam enormes perdas na agricultura e degradação generalizada em áreas de crescimento natural, sendo responsáveis pela morte de milhares de árvores por ano. Duas das espécies representativas de oomicetas são Phytophthora infestans, que provoca o míldio da batateira, e Phytophthora cinnamomi, que causa a doença da tinta do castanheiro, responsável por perdas de produção de castanha na Europa. Os esforços de sequenciação de genomas de oomicetas têm estado focados no estudo de três espécies: P. infestans, P. sojae e P. ramorum. Phytophthora infestans foi considerada como espécie modelo para o género, pois possui excelentes recursos genéticos e de genómica, incluindo mapas genéticos, bibliotecas BAC, e sequências de EST. Esta equipa de investigadores procura sequenciar o genoma de P. cinnamomi, a fim de obter uma melhor compreensão deste oomiceta, para estudar mudanças nas relações plantapatogénio, incluindo as resultantes das alterações climáticas, e tentando diminuir o impacto do patogénio nas culturas e plantas em ecossistemas naturais em todo o mundo. É apresentada uma abordagem preliminar do DNA genómico parcialmente sequenciado a partir de P. cinnamomi, o qual codifica possíveis sequências de proteínas e tRNAs. As análises em bases de dados revelam a presença de genes conservados em oomicetas.The Project COMBATINTA/SP2.P11/02 Interreg IIIA – Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Found, supported this work

Characterization of molecular factors from plants pathogen Phytophthora cinnamomi

The culture of the chestnut tree is extremely important in the northern region of Portugal, occupying a significant proportion of useful agricultural area. The annual average chestnut production in Portugal can reach 20 000 tons. New plantation areas have increased in the last few decades. However the ink disease caused by the oomycete Phytophthora cinnamomi has damage and killed many trees and up to now no concrete solutions have been offered to control the illness. As a consequence, the disease propagation in the orchards of chestnut trees has been causing severe productivity and yield breaks. In addition to the economical losses, the importance of sociological and landscape aspects for the region cannot be neglected. Oomycetes species can manipulate biochemical and physiological processes in their host plants through a diverse array of virulence or avirulence molecules, known as effectors. In susceptible plants, these effectors promote infection by suppressing defense responses, enhancing susceptibility, or inducing disease symptoms. Alternatively, in resistant plants, effectors are recognized by the products of plant resistance genes, resulting in host cell death and effective defence responses known as the hypersensitive response (HR). We've identified and characterized some proteins involved in mechanisms of infection by Phytophthora cinnamomi: endo-1,3-beta-glucanase (complete cds), exo-glucanase (partial cds); glucanase inhibitor protein (GIP) (complete cds); necrosis-inducing Phytophthora protein 1 (NPP1) (complete cds) and transglutaminase. Several technologies, such reverse transcriptase PCR, in vivo expression technology, and Bioinformatics tools have been used to study the expression of selected genes from fungi during infection. In this work we intend to integrate the necessary bioinformatics tools that were used in this investigation. These tools include the use of Databases and associated homology programs as Fasta and Clustal, and several programs for sequence analysis and design of experiments such PCR. RT-PCR studies demonstrate that P. cinnamomi elicitins have higher expression in substrates such cellulose and sawdust. The studies of expression of these genes in vivo infection, with cell lines of Castanea sativa, reveal the intimate relationship between plants and phytopathogens has led to the coevolution of a number of complex strategies for attack and defense. For a pathogen to colonize a host successfully, it must develop mechanisms either to evade detection or, failing that, to subvert the defense responses.Sociedade Portuguesa de Microbiologia, Sociedade Portuguesa de BiotecnologiaProjects COMBATINTA/SP2.P11/02 Interreg IIIA – Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Fund, and “Identification, characterization and role of molecular factors associated with the mechanisms of infection of Fagaceae species by Phytophthora cinnamomi” (PTDC/AGR-AAM/67628/2006) financed by FCT, supported this work

Identification of a Phytophthora cinnamomi glucanase inhibitor protein: a molecular factor associated to infection mechanism.

The oomycete P. cinnamomi, the causal agent of Castanea sativa ink disease, is one of the most destructive species of Phytophthora genus, and has been associated with the decline of several forest, ornamental and fruit trees and shrubs, causing enormous economic losses worldwide. Phytophthora cell walls are composed of glucans and have no chitin. Production of glucanase inhibitor proteins (GIPs) by Phytophthora species are thought to provide them a counter-defense against plant hosts β-1,3-glucanases (Rose et al., 2002), that otherwise would degrade these pathogen cell walls. GIPs belongs to the chymotrypsin family of serine proteases but are catalytically nonfunctional because one or more residues of the essential catalytic triad are absent. We report the identification of the gene encoding the first known P. cinnamomi GIP, presumably involved in the pathogen infection mechanism. Total genomic DNA was obtained from strain P. cinnamomi Pr120 and polymerase chain reaction was used to amplify a 308bp fragment of the GIP gene, using degenerate oligonucleotide primers, which were designed based on homology of previous published sequences of Phytophthora sp. GIP’s from EMBL databases. Full gene sequence length (1171bp) was obtained by flanking the known sequence with asymmetric PCR. P. cinnamomi GIP gene encodes a 269 amino acids protein with 28,818.2Da and a calculated global iso-electric point value of 8.54. It shares great identity and similarity with already described GIPs of P. sojae and P. infestans (E-values from 3.4e-49 to 2.6e-38), showing the importance of these proteins as effectors in plant-pathogen infection process. The UniProtKB/TrEMBL accession number for the sequence reported in this paper is B0B0H5_PHYCI

Photoacclimation of picophytoplankton in the central Cantabrian Sea

Photoacclimation of picophytoplankton was studied in the mixed layer of 3 stations in the central Cantabrian Sea (southern Bay of Biscay). Picophytoplankton chl a:carbon ratios (θ) presented minimum values during summer, when irradiance, temperature, and biomass of prokaryotes reached maximum values and inorganic nutrient concentrations were low. Conversely, the maximum θ were reached during winter, coincident with lowest annual irradiance but maximum concentration of inorganic nutrients and higher relative biomass of eukaryotes. Changes in θ were modeled using irradiance as an independent variable. Exponentially decreasing functions of θ with irradiance were significant only when the mean temperatures in the mixed layer were above 14°C. These functions presented light-saturated minimum ratios (θmin) that decreased linearly with temperature and low-light maximum ratios (θmax) that increased exponentially with temperature. Such relationships were used to establish an empirical model that reproduced the seasonality of picophytoplankton θ in the mixed layer, with minima in summer and maxima in winter. A maximum potential θ, θN,T-max, was determined to estimate picophytoplankton growth rates in the central Cantabrian Sea. Combinations of picophytoplankton growth rates and biomass in the mixed layer were used to estimate areal picophytoplankton primary production rates in the euphotic zone that presented a bimodal seasonal cycle, with maxima in late winter (ca. 100 mg C m-2 d-1) and in late autumn (>200 mg C m-2 d-1) and mean annual values around 120 mg C m-2 d-1.En prens

Synthetic studies on alotamide A: construction of N‐demethylalotamide A

Financiado para publicación en acceso aberto: Universidade de Vigo/CISUGSeveral approaches to the synthesis of cyclodepsipeptide natural product alotamide A are described, eventually affording a very advanced N-demethylated analogue of the targeted natural product. The difficulties found in our endeavors on the synthesis of alotamide A have allowed us to gather some valuable information regarding the most convenient synthetic step for each key transformation. The intramolecular Csp2−Csp2 Stille cross-coupling and the macrolactam formation were found to be reliable protocols for the final construction of the alotamide A skeleton.Agencia Estatal de Investigación | Ref. PID2019-107855RB−I00Xunta de Galicia | Ref. ED431 C 2021/45Xunta de Galicia | Ref. ED431G/0

Bispyrrolidinoindoline Epi(poly)thiodioxopiperazines (BPI-ETPs) and simplified mimetics: structural characterization, bioactivities, and total synthesis

Within the 2,5-dioxopiperazine-containing natural products generated by “head-to-tail” cyclization of peptides, those derived from tryptophan allow further structural diversification due to the rich chemical reactivity of the indole heterocycle, which can generate tetracyclic fragments of hexahydropyrrolo[2,3-b]indole or pyrrolidinoindoline skeleton fused to the 2,5-dioxopiperazine. Even more complex are the dimeric bispyrrolidinoindoline epi(poly)thiodioxopiperazines (BPI-ETPs), since they feature transannular (poly)sulfide bridges connecting C3 and C6 of their 2,5-dioxopiperazine rings. Homo- and heterodimers composed of diastereomeric epi(poly)thiodioxopiperazines increase the complexity of the family. Furthermore, putative biogenetically generated downstream metabolites with C11 and C11’-hydroxylated cores, as well as deoxygenated and/or oxidized side chain counterparts, have also been described. The isolation of these complex polycyclic tryptophan-derived alkaloids from the classical sources, their structural characterization, the description of the relevant biological activities and putative biogenetic routes, and the synthetic efforts to generate and confirm their structures and also to prepare and further evaluate structurally simple analogs will be reported.Xunta de Galicia | Ref. GRC ED431C 2021/045Ministerio de Economía | Ref. PID2019-107855RB-I00Xunta de Galicia | Ref. ED-431G/02-FEDE

Intervención Fisioterápica en un caso de Esclerosis Múltiple

Tratamiento fisioterápico en una paciente diagnosticada de Esclerosis Múltiple del tipo Recurrente Remitente. Valoración y revisión en nueve meses, demostrando la efectividad de la fisioterapia en este tipo de pacientes, a través de este caso clínico

Sequence analysis and expression of genes involved cytoskeleton and infection mechanisms in the plant pathogen Phytophthora cinnamomi

Oomycetes from the genus Phytophthora are plant pathogens that are devastating for agriculture and natural ecosystems. The biggest productivity and yield break occurs due to the ink disease; caused by Phytophthora cinnamomi, which is one of the most widely distributed Phytophthora species, with nearly 1 000 host species. The knowledge about the molecular mechanisms responsible for its pathogenicity is an important tool in order to fight diseases associated with this pathogen