Unlocking Cowpea's Defense Responses: Conserved Transcriptional Signatures in the Battle against CABMV and CPSMV Viruses.

Cowpea aphid-borne mosaic virus (CABMV) and Cowpea severe mosaic virus (CPSMV) threaten cowpea commercial production. This study aimed to analyze Conserved Transcriptional Signatures (CTS) in cowpea's genotypes that are resistant to these viruses. CTS covered up- (UR) or down-regulated (DR) cowpea transcripts in response to CABMV and CPSMV mechanical inoculations. The conservation of cowpea's UR defense response was primarily observed with the one hpi treatments, with decreased CTS representatives as time elapsed. This suggests that cowpea utilizes generic mechanisms during its early interaction with the studied viruses, and subsequently employs more specialized strategies for each viral agent. The potential action of the CTS-UR emphasizes the importance of redox balance, ethylene and jasmonic acid pathways. Additionally, the CTS-UR provides evidence for the involvement of R genes, PR proteins, and PRRs receptors-extensively investigated in combating bacterial and fungal pathogens-in the defense against viral inoculation. AP2-ERF, WRKY, and MYB transcription factors, as well as PIP aquaporins and MAPK cascades, also emerged as significant molecular players. The presented work represents the first study investigating conserved mechanisms in the cowpea defense response to viral inoculations, highlighting relevant processes for initial defense responses.

Blood parasite load by qPCR as therapeutic monitoring in visceral leishmaniasis patients in Brazil: a case series study.

BACKGROUND: This study aimed to describe the kinetics of Leishmania parasite load determined using kinetoplast DNA (kDNA)-based quantitative polymerase chain reaction (qPCR) in visceral leishmaniasis (VL) patients. METHODS: Parasite load in blood was assessed by qPCR at five time points, up to 12 months post-diagnosis. Sixteen patients were followed up. RESULTS: A significant reduction in the parasite load was observed after treatment (P < 0.0001). One patient had an increased parasite load 3 months post-treatment and relapsed clinically at month six. CONCLUSIONS: We have described the use of kDNA-based qPCR in the post-treatment follow-up of VL cases.

Blood parasite load by qPCR as therapeutic monitoring in visceral leishmaniasis patients in Brazil: a case series study

ABSTRACT Background: This study aimed to describe the kinetics of Leishmania parasite load determined using kinetoplast DNA (kDNA)-based quantitative polymerase chain reaction (qPCR) in visceral leishmaniasis (VL) patients. Methods: Parasite load in blood was assessed by qPCR at five time points, up to 12 months post-diagnosis. Sixteen patients were followed up. Results: A significant reduction in the parasite load was observed after treatment (P < 0.0001). One patient had an increased parasite load 3 months post-treatment and relapsed clinically at month six. Conclusions: We have described the use of kDNA-based qPCR in the post-treatment follow-up of VL cases.

Recombinant osmotin inclusion bodies from Calotropis procera produced in E. coli BL21(DE3) prevent acute inflammation in a mouse model of listeriosis.

BACKGROUND: The osmotin from the medicinal plant Calotropis procera (CpOsm) has characteristics similar to adiponectin, a human protein with immunoregulatory actions. PURPOSE: This study aimed to investigate whether recombinant osmotin inclusion bodies from C. procera (IB/rCpOsm) produced in E. coli BL21(DE3) can prevent infection-induced inflammation. A virulent strain of Listeria monocytogenes was used as an infection model. METHODS: Cells of E. coli BL21(DE3) carrying the plasmid pET303-CpOsm were used to express the recombinant osmotin, which accumulated at reasonable levels as inclusion bodies (IB/rCpOsm). IB/rCpOsm were purified from induced cells and SDS-polyacrylamide gel electrophoresis followed by mass spectrometry analyses confirmed the identity of the major protein band (23 kDa apparent molecular mass) as CpOsm. Peritoneal macrophages (pMØ) from Swiss mice were cultured with IB/rCpOsm (1 or 10 µg/ml) in 96-well plates and then infected with L. monocytogenes. IB/rCpOsm (0.1, 1 or 10 mg/kg) was also administered intravenously to Swiss mice, which were then infected intraperitoneally with L. monocytogenes. RESULTS: Pretreatment of the pMØ with IB/rCpOsm significantly increased cell viability after infection and reduced the intracellular bacterial load. The infiltration of neutrophils into the peritoneal cavity of mice pretreated with IB/rCpOsm at 10 mg/kg (but not 0.1 and 1 mg/kg) was reduced after infection. In these mice, the bacterial load was high in the peritoneal fluid and the liver, but histological damage was discrete. The treatments with IB/rCpOsm at 10 mg/kg significantly increased the expression of the anti-inflammatory cytokine IL-10. CONCLUSION: This study shows that recombinant osmotin inclusion bodies from C. procera were bioactive and prompted anti-inflammatory actions at therapeutic dosages in the L. monocytogenes infection model.

Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane.

One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes ( αTUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24 h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, αTUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFP α1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100 mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches.

Early transcriptional response of soybean contrasting accessions to root dehydration.

Drought is a significant constraint to yield increase in soybean. The early perception of water deprivation is critical for recruitment of genes that promote plant tolerance. DeepSuperSAGE libraries, including one control and a bulk of six stress times imposed (from 25 to 150 min of root dehydration) for drought-tolerant and sensitive soybean accessions, allowed to identify new molecular targets for drought tolerance. The survey uncovered 120,770 unique transcripts expressed by the contrasting accessions. Of these, 57,610 aligned with known cDNA sequences, allowing the annotation of 32,373 unitags. A total of 1,127 unitags were up-regulated only in the tolerant accession, whereas 1,557 were up-regulated in both as compared to their controls. An expression profile concerning the most representative Gene Ontology (GO) categories for the tolerant accession revealed the expression "protein binding" as the most represented for "Molecular Function", whereas CDPK and CBL were the most up-regulated protein families in this category. Furthermore, particular genes expressed different isoforms according to the accession, showing the potential to operate in the distinction of physiological behaviors. Besides, heat maps comprising GO categories related to abiotic stress response and the unitags regulation observed in the expression contrasts covering tolerant and sensitive accessions, revealed the unitags potential for plant breeding. Candidate genes related to "hormone response" (LOX, ERF1b, XET), "water response" (PUB, BMY), "salt stress response" (WRKY, MYB) and "oxidative stress response" (PER) figured among the most promising molecular targets. Additionally, nine transcripts (HMGR, XET, WRKY20, RAP2-4, EREBP, NAC3, PER, GPX5 and BMY) validated by RT-qPCR (four different time points) confirmed their differential expression and pointed that already after 25 minutes a transcriptional reorganization started in response to the new condition, with important differences between both accessions.

SymGRASS: a database of sugarcane orthologous genes involved in arbuscular mycorrhiza and root nodule symbiosis.

BACKGROUND: The rationale for gathering information from plants procuring nitrogen through symbiotic interactions controlled by a common genetic program for a sustainable biofuel production is the high energy demanding application of synthetic nitrogen fertilizers. We curated sequence information publicly available for the biofuel plant sugarcane, performed an analysis of the common SYM pathway known to control symbiosis in other plants, and provide results, sequences and literature links as an online database. METHODS: Sugarcane sequences and informations were downloaded from the nucEST database, cleaned and trimmed with seqclean, assembled with TGICL plus translating mapping method, and annotated. The annotation is based on BLAST searches against a local formatted plant Uniprot90 generated with CD-HIT for functional assignment, rpsBLAST to CDD database for conserved domain analysis, and BLAST search to sorghum's for Gene Ontology (GO) assignment. Gene expression was normalized according the Unigene standard, presented as ESTs/100 kb. Protein sequences known in the SYM pathway were used as queries to search the SymGRASS sequence database. Additionally, antimicrobial peptides described in the PhytAMP database served as queries to retrieve and generate expression profiles of these defense genes in the libraries compared to the libraries obtained under symbiotic interactions. RESULTS: We describe the SymGRASS, a database of sugarcane orthologous genes involved in arbuscular mycorrhiza (AM) and root nodule (RN) symbiosis. The database aggregates knowledge about sequences, tissues, organ, developmental stages and experimental conditions, and provides annotation and level of gene expression for sugarcane transcripts and SYM orthologous genes in sugarcane through a web interface. Several candidate genes were found for all nodes in the pathway, and interestingly a set of symbiosis specific genes was found. CONCLUSIONS: The knowledge integrated in SymGRASS may guide studies on molecular, cellular and physiological mechanisms by which sugarcane controls the establishment and efficiency of endophytic associations. We believe that the candidate sequences for the SYM pathway together with the pool of exclusively expressed tentative consensus (TC) sequences are crucial for the design of molecular studies to unravel the mechanisms controlling the establishment of symbioses in sugarcane, ultimately serving as a basis for the improvement of grass crops.

Expression Analysis of Sugarcane Aquaporin Genes under Water Deficit.

The present work is a pioneer study specifically addressing the aquaporin transcripts in sugarcane transcriptomes. Representatives of the four aquaporin subfamilies (PIP, TIP, SIP, and NIP), already described for higher plants, were identified. Forty-two distinct aquaporin isoforms were expressed in four HT-SuperSAGE libraries from sugarcane roots of drought-tolerant and -sensitive genotypes, respectively. At least 10 different potential aquaporin isoform targets and their respective unitags were considered to be promising for future studies and especially for the development of molecular markers for plant breeding. From those 10 isoforms, four (SoPIP2-4, SoPIP2-6, OsPIP2-4, and SsPIP1-1) showed distinct responses towards drought, with divergent expressions between the bulks from tolerant and sensitive genotypes, when they were compared under normal and stress conditions. Two targets (SsPIP1-1 and SoPIP1-3/PIP1-4) were selected for validation via RT-qPCR and their expression patterns as detected by HT-SuperSAGE were confirmed. The employed validation strategy revealed that different genotypes share the same tolerant or sensitive phenotype, respectively, but may use different routes for stress acclimation, indicating the aquaporin transcription in sugarcane to be potentially genotype-specific.

New insights in the sugarcane transcriptome responding to drought stress as revealed by superSAGE.

In the scope of the present work, four SuperSAGE libraries have been generated, using bulked root tissues from four drought-tolerant accessions as compared with four bulked sensitive genotypes, aiming to generate a panel of differentially expressed stress-responsive genes. Both groups were submitted to 24 hours of water deficit stress. The SuperSAGE libraries produced 8,787,315 tags (26 bp) that, after exclusion of singlets, allowed the identification of 205,975 unitags. Most relevant BlastN matches comprised 567,420 tags, regarding 75,404 unitags with 164,860 different ESTs. To optimize the annotation efficiency, the Gene Ontology (GO) categorization was carried out for 186,191 ESTs (BlastN against Uniprot-SwissProt), permitting the categorization of 118,208 ESTs (63.5%). In an attempt to elect a group of the best tags to be validated by RTqPCR, the GO categorization of the tag-related ESTs allowed the in silico identification of 213 upregulated unitags responding basically to abiotic stresses, from which 145 presented no hits after BlastN analysis, probably concerning new genes still uncovered in previous studies. The present report analyzes the sugarcane transcriptome under drought stress, using a combination of high-throughput transcriptome profiling by SuperSAGE with the Solexa sequencing technology, allowing the identification of potential target genes during the stress response.