Itabuna, Brazil
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Da Hora Junior B.T.,State University of Santa Cruz | Poloni J.D.F.,University of Caxias do Sul | Poloni J.D.F.,Federal University of Rio Grande do Sul | Lopes M.A.,State University of Santa Cruz | And 11 more authors.
Molecular BioSystems | Year: 2012

This study reports on expression analysis associated with molecular systems biology of cacao-Moniliophthora perniciosa interaction. Gene expression data were obtained for two cacao genotypes (TSH1188, resistant; Catongo, susceptible) challenged or not with the fungus M. perniciosa and collected at three time points through disease. Using expression analysis, we identified 154 and 227 genes that are differentially expressed in TSH1188 and Catongo, respectively. The expression of some of these genes was confirmed by RT-qPCR. Physical protein-protein interaction (PPPI) networks of Arabidopsis thaliana orthologous proteins corresponding to resistant and susceptible interactions were obtained followed by cluster and gene ontology analyses. The integrated analysis of gene expression and systems biology allowed designing a general scheme of major mechanisms associated with witches' broom disease resistance/susceptibility. In this sense, the TSH1188 cultivar shows strong production of ROS and elicitors at the beginning of the interaction with M. perniciosa followed by resistance signal propagation and ROS detoxification. On the other hand, the Catongo genotype displays defense mechanisms that include the synthesis of some defense molecules but without success in regards to elimination of the fungus. This phase is followed by the activation of protein metabolism which is achieved with the production of proteasome associated with autophagy as a precursor mechanism of PCD. This work also identifies candidate genes for further functional studies and for genetic mapping and marker assisted selection. © 2012 The Royal Society of Chemistry.


Pereira Menezes S.,State University of Santa Cruz | de Andrade Silva E.M.,State University of Santa Cruz | Matos Lima E.,State University of Santa Cruz | Oliveira de Sousa A.,State University of Santa Cruz | And 7 more authors.
BMC Plant Biology | Year: 2014

Background: The production and accumulation of pathogenesis-related proteins (PR proteins) in plants in response to biotic or abiotic stresses is well known and is considered as a crucial mechanism for plant defense. A pathogenesis-related protein 4 cDNA was identified from a cacao-Moniliophthora perniciosa interaction cDNA library and named TcPR-4b.Results: TcPR-4b presents a Barwin domain with six conserved cysteine residues, but lacks the chitin-binding site. Molecular modeling of TcPR-4b confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. In the cacao genome, TcPR-4b belonged to a small multigene family organized mainly on chromosome 5. TcPR-4b RT-qPCR analysis in resistant and susceptible cacao plants infected by M. perniciosa showed an increase of expression at 48 hours after infection (hai) in both cacao genotypes. After the initial stage (24-72 hai), the TcPR-4b expression was observed at all times in the resistant genotypes, while in the susceptible one the expression was concentrated at the final stages of infection (45-90 days after infection). The recombinant TcPR-4b protein showed RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Moreover, TcPR-4b presented antifungal action against M. perniciosa, and the reduction of M. perniciosa survival was related to ROS production in fungal hyphae.Conclusion: To our knowledge, this is the first report of a PR-4 showing simultaneously RNase, DNase and antifungal properties, but no chitinase activity. Moreover, we showed that the antifungal activity of TcPR-4b is directly related to RNase function. In cacao, TcPR-4b nuclease activities may be related to the establishment and maintenance of resistance, and to the PCD mechanism, in resistant and susceptible cacao genotypes, respectively. © 2014 Pereira Menezes et al.; licensee BioMed Central Ltd.


Dias C.V.,State University of Santa Cruz | Dias C.V.,Mars Center for Cocoa Science | Mendes J.S.,State University of Santa Cruz | dos Santos A.C.,State University of Santa Cruz | And 9 more authors.
Plant Physiology and Biochemistry | Year: 2011

In plant-pathogen interaction, the hydrogen peroxide (H 2O 2) may play a dual role: its accumulation inhibits the growth of biotrophic pathogens, while it could help the infection/colonization process of plant by necrotrophic pathogens. One of the possible pathways of H 2O 2 production involves oxalic acid (Oxa) degradation by apoplastic oxalate oxidase. Here, we analyzed the production of H 2O 2, the presence of calcium oxalate (CaOx) crystals and the content of Oxa and ascorbic acid (Asa) - the main precursor of Oxa in plants - in susceptible and resistant cacao (Theobroma cacao L.) infected by the hemibiotrophic fungus Moniliophthora perniciosa. We also quantified the transcript level of ascorbate peroxidase (Apx), germin-like oxalate oxidase (Glp) and dehydroascorbate reductase (Dhar) by RT-qPCR. We report that the CaOx crystal amount and the H 2O 2 levels in the two varieties present distinct temporal and genotype-dependent patterns. Susceptible variety accumulated more CaOx crystals than the resistant one, and the dissolution of these crystals occurred in the early infection steps and in the final stage of the disease in the resistant and the susceptible variety, respectively. High expression of the Glp and accumulation of Oxa were observed in the resistant variety. The content of Asa increased in the inoculated susceptible variety, but remained constant in the resistant one. The susceptible variety presented reduced Dhar expression. The role of H 2O 2 and its formation from Oxa via Apx and Glp in resistant and susceptible variety infected by M. perniciosa were discussed. © 2011 Elsevier Masson SAS.


Gramacho K.P.,Cocoa Research Center | Schmidt R.A.,University of Florida | Miller T.,University of Florida
Forests | Year: 2013

Single-gall and mixed-gall inocula from fusiform rust (Cronartium quercuum fusiforme) galls in field studies were used in greenhouse tests to investigate their pathogenic variability and the temporal and spatial stability of fusiform rust incidence of resistant slash pine (Pinus elliottii var. elliottii) seedlings. Analyses of variance showed significant main effects for families and inocula and for the interactions of families and inocula indicating abundant pathogenic variation and differential family resistance. Averaged over all families and inocula, there were no significant differences between sequential inocula, i.e., inocula from successive generations of the pathogen. However, when analyzed separately Family R1 with segregated major gene resistance showed increased rust incidence with successive generations of the pathogen. Also Family R1 accounted for a major portion of the temporal (51.4%) and spatial (49.6%) interaction sum of squares. In contrast the other resistant families each accounted for a minor portion of this statistic. These results indicate pathogen virulence toward major gene resistance, possible selection for virulence and the relative instability of Family R1 compared with other resistance families. © 2013 by the authors.


PubMed | Cocoa Research Center and University of Regensburg
Type: Journal Article | Journal: Integrative zoology | Year: 2016

Size and shape of sperm cells vary tremendously throughout the animal kingdom. The adaptive significance of this variation is not fully understood. In addition to sperm-female interactions and the environmental conditions, the risk of sperm competition might affect number, morphology and other quality traits of sperm. In the male-diphenic ant Cardiocondyla obscurior, winged sneaker males have limited sperm number, because their testes degenerate shortly after adult emergence, as is typical for males of social Hymenoptera. In contrast, wingless fighter males continuously replenish their sperm supply due to their exceptional lifelong spermatogenesis. While winged males usually have to compete with several other winged males for virgin queens, wingless males are able to monopolize queens by killing all other rivals. Hence, this presents a unique system to investigate how alternative reproductive tactics and associated physiology affect sperm morphology and viability. We found that sperm-limited males invest into sperm number instead of sperm size. Variance in sperm length is smaller in winged males, probably reflecting that they have to compete with several other males. Finally, sperm viability is equally high in both male phenotypes.


Camillo L.R.,State University of Santa Cruz | Filadelfo C.R.,Laboratorio Of Biomorfologia Parasitaria | Monzani P.S.,University of Northern Paraná | Correa R.X.,State University of Santa Cruz | And 4 more authors.
Plant Physiology and Biochemistry | Year: 2013

The level of hydrogen peroxide (H2O2) in plants signalizes the induction of several genes, including that of ascorbate peroxidase (APX-EC 1.11.1.11). APX isoenzymes play a central role in the elimination of intracellular H2O2 and contribute to plant responses to diverse stresses. During the infection process in Theobroma cacao by Moniliophthora perniciosa oxidative stress is generated and the APX action recruited from the plant. The present work aimed to characterize the T.cacao APX involved in the molecular interaction of T.cacao-M.perniciosa. The peroxidase activity was analyzed in protein extracts from cocoa plants infected by M.perniciosa and showed the induction of peroxidases like APX in resistant cocoa plants. The cytosolic protein of T.cacao (GenBank: ABR68691.2) was phylogenetically analyzed in relation to other peroxidases from the cocoa genome and eight genes encoding APX proteins with conserved domains were also analyzed. The cDNA from cytosolic APX was cloned in pET28a and the recombinant protein expressed and purified (rTc-cAPX). The secondary structure of the protein was analyzed by Circular Dichroism (CD) displaying high proportion of α-helices when folded. The enzymatic assay shows stable activity using ascorbate and guaiacol as an electron donor for H2O2 reduction. The pH 7.5 is the optimum for enzyme activity. Chromatographic analysis suggests that rTc-cAPX is a homodimer in solution. Results indicate that the rTc-cAPX is correctly folded, stable and biochemically active. The purified rTc-cAPX presented biotechnological potential and is adequate for future structural and functional studies. © 2013 The Authors.


ArgOlo Santos Carvalho H.,State University of Santa Cruz | Figueredo Ribeiro L.,State University of Santa Cruz | Pirovani C.P.,State University of Santa Cruz | Peres Gramacho K.,Cocoa Research Center | And 2 more authors.
Physiological and Molecular Plant Pathology | Year: 2013

We report the first analysis of polygalacturonase regulation in the basidiomycete Moniliophthora perniciosa. Non-secreted and secreted polygalacturonase activity was obtained from M.perniciosa cultivated on bran-based solid medium or liquid media containing additional carbon sources or cacao extracts (infected or not by the fungus). Polygalacturonase activity assays were carried out under different temperatures and incubation periods. The best secreted polygalacturonase activity was obtained when the enzymatic assay was made at 50°C for 10min. Moreover, the polygalacturonase activity was enhanced when the fungus was cultivated on potato dextrose medium, in the presence of additional fermentable carbon sources, in the presence of cacao pulp or non infected cacao extracts. © 2013 Elsevier Ltd.


Aleixo S.,State University of North Fluminense Darcy Ribeiro | Gama-Rodrigues A.C.,State University of North Fluminense Darcy Ribeiro | Costa M.G.,State University of North Fluminense Darcy Ribeiro | Sales M.V.S.,State University of North Fluminense Darcy Ribeiro | And 2 more authors.
Agroforestry Systems | Year: 2016

Little is known about the phosphorus (P) fractions and P lability of agroforest soils in tropical regions, particularly those of cacao (Theobroma cacao L.) agroforests. We hypothesized that the effect of P fertilization on the distribution of P fractions in the soil based on the source-sink relationship differs for different cacao agroforestry systems. The cacao agroforestry systems studied were the following: open cacao-cabruca, closed cacao-cabruca, cacao + erythrina, mixed cacao + rubber tree, and cacao + rubber tree intercropping. A natural forest and an unfertilized pasture were used as reference systems. The P fractions were determined using the Hedley sequential extraction method, and the P transformation processes were evaluated via structural equation modeling. The impact of low P fertilizer input on the P fractions varied according to the specific environmental conditions of each cacao production system. Consequently, there was high dissimilarity among all of the cacao sites. In all of the cacao agroforestry systems, there was an increase in inorganic P (Pi), especially the labile fraction (resin-Pi and NaHCO3-Pi), but organic P (Po) increased only in the cacao + erythrina system and in the rubber tree planting row of the cacao + rubber tree intercropping system. As a result, the fitted structural models indicated that the inter-relationships of the geochemical processes were more important for determining the P availability than the biological processes. However, the Po concentrations and relative proportion were high in all of the cacao agroforestry systems, thereby revealing the high potential of supplying P to plants via the mineralization process in the eventual removal of mineral fertilization. © 2016 Springer Science+Business Media Dordrecht


Santana J.O.,State University of Santa Cruz | Freire L.,State University of Santa Cruz | de Sousa A.O.,State University of Santa Cruz | Fontes Soares V.L.,State University of Santa Cruz | And 2 more authors.
Plant Physiology and Biochemistry | Year: 2016

Legumains are cysteine proteases related to plant development, protein degradation, programmed cell death, and defense against pathogens. In this study, we have identified and characterized three legumains encoded by Theobroma cacao genome through in silico analyses, three-dimensional modeling, genetic expression pattern in different tissues and as a response to the inoculation of Moniliophthora perniciosa fungus. The three proteins were named TcLEG3, TcLEG6, and TcLEG9. Histidine and cysteine residue which are part of the catalytic site were conserved among the proteins, and they remained parallel in the loop region in the 3D modeling. Three-dimensional modeling showed that the propeptide, which is located in the terminal C region of legumains blocks the catalytic cleft. Comparing dendrogram data with the relative expression analysis, indicated that TcLEG3 is related to the seed legumain group, TcLEG6 is related with the group of embryogenesis activities, and protein TcLEG9, with processes regarding the vegetative group. Furthermore, the expression analyses proposes a significant role for the three legumains during the development of Theobroma cacao and in its interaction with M. perniciosa. © 2015 Universidade Estadual de Santa Cruz, CNPJ: 40738999/0001-95.


PubMed | State University of Santa Cruz and Cocoa Research Center
Type: | Journal: Plant physiology and biochemistry : PPB | Year: 2016

Legumains are cysteine proteases related to plant development, protein degradation, programmed cell death, and defense against pathogens. In this study, we have identified and characterized three legumains encoded by Theobroma cacao genome through in silico analyses, three-dimensional modeling, genetic expression pattern in different tissues and as a response to the inoculation of Moniliophthora perniciosa fungus. The three proteins were named TcLEG3, TcLEG6, and TcLEG9. Histidine and cysteine residue which are part of the catalytic site were conserved among the proteins, and they remained parallel in the loop region in the 3D modeling. Three-dimensional modeling showed that the propeptide, which is located in the terminal C region of legumains blocks the catalytic cleft. Comparing dendrogram data with the relative expression analysis, indicated that TcLEG3 is related to the seed legumain group, TcLEG6 is related with the group of embryogenesis activities, and protein TcLEG9, with processes regarding the vegetative group. Furthermore, the expression analyses proposes a significant role for the three legumains during the development of Theobroma cacao and in its interaction with M.perniciosa.

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