Papers by Aurizângela Sousa
Investigation of the presence and enzymatic activity of fungi in stored corn (Zea mays L.) and soybean [Glycine max (L.) Merrill] grains
Caderno Pedagógico, Apr 11, 2024
Ethanol test to evaluate the physiological quality of forest seeds
Figshare, 2022
PLOS ONE, 2015
The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors... more The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors, such as cystatins, can be perceived as part of the molecular battlefield in plantpathogen interaction. In cacao, four cystatins were identified and characterized by our group. We identified 448 proteases in cacao genome, whereof 134 were cysteine-proteases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immunoblottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected by Moniliophthora perniciosa during the transition from the biotrophic to the saprophytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant to M. perniciosa, respectively, but greater intensity and additional isoforms were observed in TSH1188. The fungal protein MpNEP induced PLCP isoform expression in tobacco leaves, according to the cross reaction with anti-TcCYSPR04. Several protein isoforms were detected at 72 hours after treatment with MpNEP. We captured an active PLCP from cacao tissues, using a recombinant cacao cystatin immobilized in CNBr-Sepharose. Mass spectrometry showed that this protein corresponds to TcCYSPR04. A homology modeling was obtained for both proteins. In order to become active, TcCYSPR04 needs to lose its inhibitory domain. Molecular docking showed the physical-chemical complementarities of the interaction between the cacao enzyme and its inhibitor. We propose that TcCYSPR04 and its interactions with cacao cystatins are involved in the senescence and necrosis events related to witches' broom symptoms. This molecular interaction may be the target for future interventions to control witches' broom disease.
Physiology and molecular biology of plants : an international journal of functional plant biology, 2018
L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and indu... more L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in , coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that is the most stable gene in different tissues and stages of seed development and i...
Analysis of the accumulation of transcripts of the genes encoding enzymes involved in oxidative stress in sweet orange variety “Westin” plants infected and non-infected with CTV
<p>The columns marked with ** indicate that the values among treatments presented significa... more <p>The columns marked with ** indicate that the values among treatments presented significant differences through Tukey test (p ≤ 0.001. The vertical columns indicate the average values of Cts which were calculated through method 2<sup>-ΔΔCt</sup> (LIVAK E SCHMITTGEN, 2001), the bars above the columns indicate the standard error of averages.</p
Enzyme activity in “Westin” sweet oranges infected and non-infected with CTV
<p><b>A,</b> Catalase <b>B,</b> Superoxide Dismutase <b>C,<... more <p><b>A,</b> Catalase <b>B,</b> Superoxide Dismutase <b>C,</b> Guaiacol Peroxidase. The vertical columns indicate the average absorbance values (n = 4). The bars above the columns represent the standard error of averages. The asterisk (*) indicates that the values presented a significant difference through Tukey test, taking into account p ≤ 0.05.</p
Peroxidase activity in “Westin” sweet orange infected and non-infected with CTV, with the use of ascorbic acid and guaiacol as electron donors
<p><b>A, B, and C</b> AsA—exhaustion times in the reaction at 10 mmol.L<sup&... more <p><b>A, B, and C</b> AsA—exhaustion times in the reaction at 10 mmol.L<sup>-1</sup>, 20 mmol.L<sup>-1</sup> and 30 mmol.L<sup>-1</sup>, respectively. The arrows indicate the times at which GPX activity was started.</p
Amplification of CTV viral particles through RT-PCR in order to confirm the presence of the virus in infected samples, and to confirm its absence in non-infected samples
<p><b>M,</b> molecular weight marker; <b>NIC-</b> and <b>IC-,... more <p><b>M,</b> molecular weight marker; <b>NIC-</b> and <b>IC-,</b> negative controls of the reaction for each of the primers (1 and 2). <b>NI1</b> and <b>I1</b>, non-infected and infected samples (primer 1—CN487/489), respectively. <b>NI2</b> e <b>I2</b>, non-infected and infected samples (primer 2—CN488/491), respectively. The arrows show the amplified bands as per their expected sizes. The reaction was confirmed in a 1% agarose gel.</p
Distribution of differentially-expressed proteins of sweet orange variety “Westin” in response to <i>Citrus tristeza virus</i> (CTV), according to their expression levels
<p>The gray segment of the bar corresponds to up-accumulated proteins and in light gray, th... more <p>The gray segment of the bar corresponds to up-accumulated proteins and in light gray, the down-accumulated ones.</p
Functional categorization and cell location of identified differentially expressed proteins in infected and non-infected sweet orange variety “Westin” subjects
<p><b>A,</b> Functional categorization of significantly different proteins. <... more <p><b>A,</b> Functional categorization of significantly different proteins. <b>B,</b> Cell location of differentially expressed proteins.</p
Differentially expressed proteins among infected and non-infected samples of “Westin” sweet orange, identified through Mass Spectrometry (ms/ms)
<p>* Exclusives Spots from Infected Samples</p><p>The peptides were sequenced t... more <p>* Exclusives Spots from Infected Samples</p><p>The peptides were sequenced through ms/ms</p><p>Score corresponding to the coverage value, as calculated by Mascot.</p><p>Differentially expressed proteins among infected and non-infected samples of “Westin” sweet orange, identified through Mass Spectrometry (ms/ms).</p
Additional file 6: Figure S4. of Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao
Background detection by western blotting by overincubating the membranes. The anbidodies against ... more Background detection by western blotting by overincubating the membranes. The anbidodies against Catalase and HSP70 were very specific. However, the antibody against Catalase has revealed background bands that were homogeneously transferred to the membrane at 0, 2 and 4 hag. (TIFF 498 kb)
Additional file 5: Table S2. of Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao
Total spots identified in the MS/MS. In sequence, mw corresponds to a molecular mass values, the ... more Total spots identified in the MS/MS. In sequence, mw corresponds to a molecular mass values, the isoelectric point pI estimated; number of peptides by MS / MS; Score of sequence, sequence coverage and peptide sequence. All values were calculated by Mascot at http:. // www.matrixscience.com . (XLS 118 kb)
Additional file 4: Figure S3. of Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao
Distribution of the spots and their respective isoelectric point in the 0, 2 and 4 hag. (TIFF 741... more Distribution of the spots and their respective isoelectric point in the 0, 2 and 4 hag. (TIFF 741 kb)
Additional file 3: Figure S2. of Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao
Distribution of the spots and their respective molecular weight in the 0, 2 and 4 hag. (TIFF 2477... more Distribution of the spots and their respective molecular weight in the 0, 2 and 4 hag. (TIFF 2477 kb)
Additional file 2: Figure S1. of Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao
Sequence alignment of the target proteins identified in the western blots and their homologous in... more Sequence alignment of the target proteins identified in the western blots and their homologous in plant recognized by the antibodies. A – Sequence alignment between BiP from A. thaliana and the HSP70 from Moniliophthora perniciosa. B – Sequence alignment of the ATP synthase from A. thaliana and its homologous in Moniliophthora perniciosa. C – Sequence alignment of the catalase A from A. thaliana and its homologous in Moniliophthora perniciosa. (JPEG 3987 kb)
Additional file 1: Table S1. of Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao
Ratio values for each protein spot used to perform the hierarchical clustering analysis. (XLS 61 kb)
PLOS ONE, 2015
Citrus Tristeza disease, caused by CTV (Citrus tristeza virus), committs citrus plantations aroun... more Citrus Tristeza disease, caused by CTV (Citrus tristeza virus), committs citrus plantations around the world and specifically attacks phloem tissues of the plant. The virus exists as a mixture of more or less severe variants, which may or may not cause symptoms of Tristeza. The objective of this study was to analyze the changes caused by CTV in the proteome of stems of sweet orange, as well as in the activity and gene expression of antioxidant enzymes. The CTV-infected sweet orange displayed mild symptoms, which were characterized by the presence of sparse stem pitting throughout their stems. The presence of virus was confirmed by RT-PCR. Proteomic analysis by 2DE-PAGE-MS / MS revealed the identity of 40 proteins differentially expressed between CTV-infected and-non-infected samples. Of these, 33 were up-regulated and 7 were down-regulated in CTV-infected samples. Among the proteins identified stands out a specific from the virus, the coat protein. Other proteins identified are involved with oxidative stress and for this their enzymatic activity was measured. The activity of superoxide dismutase (SOD) was higher in CTV-infected samples, as catalase (CAT) showed higher activity in uninfected samples. The activity of guaiacol peroxidase (GPX) did not vary significantly between samples. However, ascorbate peroxidase (APX) was more active in the infected samples. The relative expression of the genes encoding CAT, SOD, APX and GPX was analyzed by quantitative real time PCR (RT-qPCR). The CTV-infected samples showed greater accumulation of transcripts, except for the CAT gene. This gene showed higher expression in the uninfected samples. Taken together, it can be concluded that the CTV affects the protein profile and activity and gene expression of antioxidant enzymes in plants infected by this virus.
BMC Plant Biology, 2014
Background: The production and accumulation of pathogenesis-related proteins (PR proteins) in pla... more 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.
Protein Profile
<p>Differential profile (according to the statistical analysis performed by the software) o... more <p>Differential profile (according to the statistical analysis performed by the software) of the expression of identified proteins between non infected (A) and infected (B) treatments of “Westin” sweet orange.</p
Uploads
Papers by Aurizângela Sousa