High-throughput analysis reveals disturbances throughout the cell caused by Arabidopsis UCP1 and UCP3 double knockdown.

Three genes encoding mitochondrial uncoupling proteins (UCPs) have been described in Arabidopsis thaliana (UCP1 to UCP3). In plants, UCPs may act as an uncoupler or as an aspartate/glutamate exchanger. For instance, much of the data regarding UCP functionality were obtained for the UCP1 and UCP2 isoforms compared with UCP3. Here, to get a better understanding about the concerted action of UCP1 and UCP3 in planta, we investigated the transcriptome and metabolome profiles of ucp1 ucp3 double mutant plants during the vegetative phase. For that, 21-day-old mutant plants, which displayed the most evident phenotypic alterations compared to wild type (WT) plants, were employed. The double knockdown of UCP1 and UCP3, isoforms unequivocally present inside the mitochondria, promoted important transcriptional reprogramming with alterations in the expression of genes related to mitochondrial and chloroplast function as well as those responsive to abiotic stress, suggesting disturbances throughout the cell. The observed transcriptional changes were well integrated with the metabolomic data of ucp1 ucp3 plants. Alterations in metabolites related to primary and secondary metabolism, particularly enriched in the Alanine, Aspartate and Glutamate metabolism, were detected. These findings extend our knowledge of the underlying roles played by UCP3 in concert with UCP1 at the whole plant level.

Antimicrobial resistance and genetic background of non-typhoidal Salmonella enterica strains isolated from human infections in São Paulo, Brazil (2000-2019).

Salmonella enterica causes Salmonellosis, an important infection in humans and other animals. The number of multidrug-resistant (MDR) phenotypes associated with Salmonella spp. isolates is increasing worldwide, causing public health concern. Here, we aim to characterize the antimicrobial-resistant phenotype of 789 non-typhoidal S. enterica strains isolated from human infections in the state of São Paulo, Brazil, along 20 years (2000-2019). Among the non-susceptible isolates, 31.55, 14.06, and 13.18% were resistant to aminoglycosides, tetracycline, and ß-lactams, respectively. Moreover, 68 and 11 isolates were considered MDR and Extended Spectrum ß-Lactamase (ESBL) producers, respectively, whereas one isolate was colistin-resistant. We selected four strains to obtain a draft of the Genome Sequence; one S. Infantis (ST32), one S. Enteritidis (ST11), one S. I 4,[5],12:i:- (ST19), and one S. Typhimurium (ST313). Among them, three presented at least one of the following antimicrobial resistance genes (AMR) linked to mobile DNA: blaTEM-1B, dfrA1, tetA, sul1, floR, aac(6')-laa, and qnrE1. This is the first description of the plasmid-mediated quinolone resistance (PMQR) gene qnrE1 in a clinical isolate of S. I 4,[5],12:i:-. The S. Typhimurium is a colistin-resistant isolate, but did not harbor mcr genes, but it presented mutations within the mgrB, pmrB, and pmrC regions that might be linked to the colistin-resistant phenotype. The virulence pattern of the four isolates resembled the virulence pattern of the highly pathogenic S. Typhimurium UK-1 reference strain in assays involving the in vivo Galleria mellonella model. In conclusion, most isolates studied here are susceptible, but a small percentage present an MDR or ESBL-producer and pathogenic phenotype. Sequence analyses revealed plasmid-encoded AMR genes, such as ß-lactam and fluoroquinolone resistance genes, indicating that these characteristics can be potentially disseminated among other bacterial strains.

Molecular characterization of antimicrobial resistance in Klebsiella pneumoniae isolated from Brazilian dairy herds.

In this observational study, phenotypic and genotypic patterns of antimicrobial resistance (AMR) in Klebsiella pneumoniae isolated from intramammary infections, clinical mastitis, fresh feces, rectal swabs, animal hindlimbs, and bulk tank milk samples from Brazilian dairy herds were investigated. In addition, we identified specific genetic variants present among extended-spectrum ß-lactamase (ESBL) producers. We obtained 169 isolates of K. pneumoniae from 2009 to 2011 on 24 Brazilian dairy farms located in 4 Brazilian states. The AMR profile of all isolates was determined using disk-diffusion assays. The antimicrobial panel included drugs commonly used as mastitis treatment in Brazilian dairy herds (gentamicin, cephalosporins, sulfamethoxazole-trimethoprim, tetracycline) as well as antimicrobials of critical importance for human health (meropenem, ceftazidime, fluoroquinolones). The K. pneumoniae isolates resistant to tetracycline, fluoroquinolones, sulfamethoxazole-trimethoprim, or chloramphenicol were screened for presence of drug-specific AMR genes [tet, qnr, aac(6')-Ib, floR, catA2, cm1A, dfr, sul] using PCR. In addition, we identified ESBL genes present among ESBL-producers by using whole genome sequencing. Genomes were assembled and annotated, and patterns of AMR genes were investigated. Resistance was commonly detected against tetracycline (22.5% of all isolates), streptomycin (20.7%), and sulfamethoxazole-trimethoprim (9.5%). Antimicrobial resistance rates were higher in K. pneumoniae isolated from intramammary infections in comparison with isolates from feces (19.2 and 0% of multidrug resistance in intramammary and fecal isolates, respectively). In contrast, no difference in AMR rates was observed when contrasting hind limbs and isolates from intramammary infections. The genes tetA, sul2, and floR were the most frequently observed AMR genes in K. pneumoniae resistant to tetracycline, sulfamethoxazole-trimethoprim, and chloramphenicol, respectively. The tetA gene was present exclusively in isolates from milk. The genes blaCTX-M8 and blaSHV-108 were present in 3 ESBL-producing K. pneumoniae, including an isolate from bulk tank milk. The 3 isolates were of sequence type 281 and had similar mobile genetic elements and virulence genes. Our study reinforced the epidemiological importance and dissemination of blaCTX-M-8 pST114 plasmid in food-producing animals in Brazil.

Unraveling the complex genome of Saccharum spontaneum using Polyploid Gene Assembler.

The Polyploid Gene Assembler (PGA), developed and tested in this study, represents a new strategy to perform gene-space assembly from complex genomes using low coverage DNA sequencing. The pipeline integrates reference-assisted loci and de novo assembly strategies to construct high-quality sequences focused on gene content. Pipeline validation was conducted with wheat (Triticum aestivum), a hexaploid species, using barley (Hordeum vulgare) as reference, that resulted in the identification of more than 90% of genes and several new genes. Moreover, PGA was used to assemble gene content in Saccharum spontaneum species, a parental lineage for hybrid sugarcane cultivars. Saccharum spontaneum gene sequence obtained was used to reference-guided transcriptome analysis of six different tissues. A total of 39,234 genes were identified, 60.4% clustered into known grass gene families. Thirty-seven gene families were expanded when compared with other grasses, three of them highlighted by the number of gene copies potentially involved in initial development and stress response. In addition, 3,108 promoters (many showing tissue specificity) were identified in this work. In summary, PGA can reconstruct high-quality gene sequences from polyploid genomes, as shown for wheat and S. spontaneum species, and it is more efficient than conventional genome assemblers using low coverage DNA sequencing.

New insights into Phakopsora pachyrhizi infection based on transcriptome analysis in planta.

Asian soybean rust (ASR) is one of the most destructive diseases affecting soybeans. The causative agent of ASR, the fungus Phakopsora pachyrhizi, presents characteristics that make it difficult to study in vitro, limiting our knowledge of plant-pathogen dynamics. Therefore, this work used leaf lesion laser microdissection associated with deep sequencing to determine the pathogen transcriptome during compatible and incompatible interactions with soybean. The 36,350 generated unisequences provided an overview of the main genes and biological pathways that were active in the fungus during the infection cycle. We also identified the most expressed transcripts, including sequences similar to other fungal virulence and signaling proteins. Enriched P. pachyrhizi transcripts in the resistant (PI561356) soybean genotype were related to extracellular matrix organization and metabolic signaling pathways and, among infection structures, in amino acid metabolism and intracellular transport. Unisequences were further grouped into gene families along predicted sequences from 15 other fungi and oomycetes, including rust fungi, allowing the identification of conserved multigenic families, as well as being specific to P. pachyrhizi. The results revealed important biological processes observed in P. pachyrhizi, contributing with information related to fungal biology and, consequently, a better understanding of ASR.

Suppression of Plant Immunity by Fungal Chitinase-like Effectors.

Crop diseases caused by fungi constitute one of the most important problems in agriculture, posing a serious threat to food security [1]. To establish infection, phytopathogens interfere with plant immune responses [2, 3]. However, strategies to promote virulence employed by fungal pathogens, especially non-model organisms, remain elusive [4], mainly because fungi are more complex and difficult to study when compared to the better-characterized bacterial pathogens. Equally incomplete is our understanding of the birth of microbial virulence effectors. Here, we show that the cacao pathogen Moniliophthora perniciosa evolved an enzymatically inactive chitinase (MpChi) that functions as a putative pathogenicity factor. MpChi is among the most highly expressed fungal genes during the biotrophic interaction with cacao and encodes a chitinase with mutations that abolish its enzymatic activity. Despite the lack of chitinolytic activity, MpChi retains substrate binding specificity and prevents chitin-triggered immunity by sequestering immunogenic chitin fragments. Remarkably, its sister species M. roreri encodes a second non-orthologous catalytically impaired chitinase with equivalent function. Thus, a class of conserved enzymes independently evolved as putative virulence factors in these fungi. In addition to unveiling a strategy of host immune suppression by fungal pathogens, our results demonstrate that the neofunctionalization of enzymes may be an evolutionary pathway for the rise of new virulence factors in fungi. We anticipate that analogous strategies are likely employed by other pathogens.

Ceratocystis cacaofunesta genome analysis reveals a large expansion of extracellular phosphatidylinositol-specific phospholipase-C genes (PI-PLC).

BACKGROUND: The Ceratocystis genus harbors a large number of phytopathogenic fungi that cause xylem parenchyma degradation and vascular destruction on a broad range of economically important plants. Ceratocystis cacaofunesta is a necrotrophic fungus responsible for lethal wilt disease in cacao. The aim of this work is to analyze the genome of C. cacaofunesta through a comparative approach with genomes of other Sordariomycetes in order to better understand the molecular basis of pathogenicity in the Ceratocystis genus. RESULTS: We present an analysis of the C. cacaofunesta genome focusing on secreted proteins that might constitute pathogenicity factors. Comparative genome analyses among five Ceratocystidaceae species and 23 other Sordariomycetes fungi showed a strong reduction in gene content of the Ceratocystis genus. However, some gene families displayed a remarkable expansion, in particular, the Phosphatidylinositol specific phospholipases-C (PI-PLC) family. Also, evolutionary rate calculations suggest that the evolution process of this family was guided by positive selection. Interestingly, among the 82 PI-PLCs genes identified in the C. cacaofunesta genome, 70 genes encoding extracellular PI-PLCs are grouped in eight small scaffolds surrounded by transposon fragments and scars that could be involved in the rapid evolution of the PI-PLC family. Experimental secretome using LC-MS/MS validated 24% (86 proteins) of the total predicted secretome (342 proteins), including four PI-PLCs and other important pathogenicity factors. CONCLUSION: Analysis of the Ceratocystis cacaofunesta genome provides evidence that PI-PLCs may play a role in pathogenicity. Subsequent functional studies will be aimed at evaluating this hypothesis. The observed genetic arsenals, together with the analysis of the PI-PLC family shown in this work, reveal significant differences in the Ceratocystis genome compared to the classical vascular fungi, Verticillium and Fusarium. Altogether, our analyses provide new insights into the evolution and the molecular basis of plant pathogenicity.

EUCANEXT: an integrated database for the exploration of genomic and transcriptomic data from Eucalyptus species.

Database URL: http://www.lge.ibi.unicamp.br/eucalyptusdb.

Impacts of the overexpression of a tomato translationally controlled tumor protein (TCTP) in tobacco revealed by phenotypic and transcriptomic analysis.

KEY MESSAGE: Overexpression of a tomato TCTP impacts plant biomass production and performance under stress. These phenotypic alterations were associated with the up-regulation of genes mainly related to photosynthesis, fatty acid metabolism and water transport. The translationally controlled tumor protein (TCTP) is a multifaceted and highly conserved eukaryotic protein. In plants, despite the existence of functional data implicating this protein in cell proliferation and growth, the detailed physiological roles of many plant TCTPs remain poorly understood. Here we focused on a yet uncharacterized TCTP from tomato (SlTCTP). We show that, when overexpressed in tobacco, SlTCTP may promote plant biomass production and affect performance under salt and osmotic stress. Transcriptomic analysis of the transgenic plants revealed the up-regulation of genes mainly related to photosynthesis, fatty acid metabolism and water transport. This induced photosynthetic gene expression was paralleled by an increase in the photosynthetic rate and stomatal conductance of the transgenic plants. Moreover, the transcriptional modulation of genes involved in ABA-mediated regulation of stomatal movement was detected. On the other hand, genes playing a pivotal role in ethylene biosynthesis were found to be down-regulated in the transgenic lines, thus suggesting deregulated ethylene accumulation in these plants. Overall, these results point to a role of TCTP in photosynthesis and hormone signaling.

Whole-Genome Sequence of Salmonella enterica Serovar Enteritidis Phage Type 4, Isolated from a Brazilian Poultry Farm.

The draft genome of Salmonella enterica serovar Enteritidis phage type 4 (PT4) strain IOC4647/2004, isolated from a poultry farm in São Paulo state, was obtained with high-throughput Illumina sequencing platform, generating 4,173,826 paired-end reads with 251 bp. The assembly of 4,804,382 bp in 27 scaffolds shows strong similarity to other S Enteritidis strains.

Contrasting nitrogen fertilization treatments impact xylem gene expression and secondary cell wall lignification in Eucalyptus.

BACKGROUND: Nitrogen (N) is a main nutrient required for tree growth and biomass accumulation. In this study, we analyzed the effects of contrasting nitrogen fertilization treatments on the phenotypes of fast growing Eucalyptus hybrids (E. urophylla x E. grandis) with a special focus on xylem secondary cell walls and global gene expression patterns. RESULTS: Histological observations of the xylem secondary cell walls further confirmed by chemical analyses showed that lignin was reduced by luxuriant fertilization, whereas a consistent lignin deposition was observed in trees grown in N-limiting conditions. Also, the syringyl/guaiacyl (S/G) ratio was significantly lower in luxuriant nitrogen samples. Deep sequencing RNAseq analyses allowed us to identify a high number of differentially expressed genes (1,469) between contrasting N treatments. This number is dramatically higher than those obtained in similar studies performed in poplar but using microarrays. Remarkably, all the genes involved the general phenylpropanoid metabolism and lignin pathway were found to be down-regulated in response to high N availability. These findings further confirmed by RT-qPCR are in agreement with the reduced amount of lignin in xylem secondary cell walls of these plants. CONCLUSIONS: This work enabled us to identify, at the whole genome level, xylem genes differentially regulated by N availability, some of which are involved in the environmental control of xylogenesis. It further illustrates that N fertilization can be used to alter the quantity and quality of lignocellulosic biomass in Eucalyptus, offering exciting prospects for the pulp and paper industry and for the use of short coppices plantations to produce second generation biofuels.

Transcriptional analysis of genes involved in nodulation in soybean roots inoculated with Bradyrhizobium japonicum strain CPAC 15.

BACKGROUND: Biological nitrogen fixation in root nodules is a process of great importance to crops of soybean [Glycine max (L.) Merr.], as it may provide the bulk of the plant's needs for nitrogen. Legume nodulation involves several complex steps and, although studied for many decades, much remains to be understood. RESULTS: This research aimed at analyzing the global expression of genes in soybean roots of a Brazilian cultivar (Conquista) inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. To achieve this, we used the suppressive subtractive hybridization (SSH) technique combined with Illumina sequencing. The subtractive library (non-inoculated x inoculated) of soybean roots resulted in 3,210 differentially expressed transcripts at 10 days after inoculation were studied. The data were grouped according to the ontologies of the molecular functions and biological processes. Several classes of genes were confirmed as related to N2 fixation and others were reported for the first time. CONCLUSIONS: During nodule formation, a higher percentage of genes were related to primary metabolism, cell-wall modifications and the antioxidant defense system. Putative symbiotic functions were attributed to some of these genes for the first time.

Xylem transcription profiles indicate potential metabolic responses for economically relevant characteristics of Eucalyptus species.

BACKGROUND: Eucalyptus is one of the most important sources of industrial cellulose. Three species of this botanical group are intensively used in breeding programs: E. globulus, E. grandis and E. urophylla. E. globulus is adapted to subtropical/temperate areas and is considered a source of high-quality cellulose; E. grandis grows rapidly and is adapted to tropical/subtropical climates; and E. urophylla, though less productive, is considered a source of genes related to robustness. Wood, or secondary xylem, results from cambium vascular differentiation and is mostly composed of cellulose, lignin and hemicelluloses. In this study, the xylem transcriptomes of the three Eucalyptus species were investigated in order to provide insights on the particularities presented by each of these species. RESULTS: Data analysis showed that (1) most Eucalyptus genes are expressed in xylem; (2) most genes expressed in species-specific way constitutes genes with unknown functions and are interesting targets for future studies; (3) relevant differences were observed in the phenylpropanoid pathway: E. grandis xylem presents higher expression of genes involved in lignin formation whereas E. urophylla seems to deviates the pathway towards flavonoid formation; (4) stress-related genes are considerably more expressed in E. urophylla, suggesting that these genes may contribute to its robustness. CONCLUSIONS: The comparison of these three transcriptomes indicates the molecular signatures underlying some of their distinct wood characteristics. This information may contribute to the understanding of xylogenesis, thus increasing the potential of genetic engineering approaches aiming at the improvement of Eucalyptus forest plantations productivity.

Global analyses of Ceratocystis cacaofunesta mitochondria: from genome to proteome.

BACKGROUND: The ascomycete fungus Ceratocystis cacaofunesta is the causal agent of wilt disease in cacao, which results in significant economic losses in the affected producing areas. Despite the economic importance of the Ceratocystis complex of species, no genomic data are available for any of its members. Given that mitochondria play important roles in fungal virulence and the susceptibility/resistance of fungi to fungicides, we performed the first functional analysis of this organelle in Ceratocystis using integrated "omics" approaches. RESULTS: The C. cacaofunesta mitochondrial genome (mtDNA) consists of a single, 103,147-bp circular molecule, making this the second largest mtDNA among the Sordariomycetes. Bioinformatics analysis revealed the presence of 15 conserved genes and 37 intronic open reading frames in C. cacaofunesta mtDNA. Here, we predicted the mitochondrial proteome (mtProt) of C. cacaofunesta, which is comprised of 1,124 polypeptides - 52 proteins that are mitochondrially encoded and 1,072 that are nuclearly encoded. Transcriptome analysis revealed 33 probable novel genes. Comparisons among the Gene Ontology results of the predicted mtProt of C. cacaofunesta, Neurospora crassa and Saccharomyces cerevisiae revealed no significant differences. Moreover, C. cacaofunesta mitochondria were isolated, and the mtProt was subjected to mass spectrometric analysis. The experimental proteome validated 27% of the predicted mtProt. Our results confirmed the existence of 110 hypothetical proteins and 7 novel proteins of which 83 and 1, respectively, had putative mitochondrial localization. CONCLUSIONS: The present study provides the first partial genomic analysis of a species of the Ceratocystis genus and the first predicted mitochondrial protein inventory of a phytopathogenic fungus. In addition to the known mitochondrial role in pathogenicity, our results demonstrated that the global function analysis of this organelle is similar in pathogenic and non-pathogenic fungi, suggesting that its relevance in the lifestyle of these organisms should be based on a small number of specific proteins and/or with respect to differential gene regulation. In this regard, particular interest should be directed towards mitochondrial proteins with unknown function and the novel protein that might be specific to this species. Further functional characterization of these proteins could enhance our understanding of the role of mitochondria in phytopathogenicity.

A web-based bioinformatics interface applied to the GENOSOJA Project: Databases and pipelines.

The Genosoja consortium is an initiative to integrate different omics research approaches carried out in Brazil. Basically, the aim of the project is to improve the plant by identifying genes involved in responses against stresses that affect domestic production, like drought stress and Asian Rust fungal disease. To do so, the project generated several types of sequence data using different methodologies, most of them sequenced by next generation sequencers. The initial stage of the project is highly dependent on bioinformatics analysis, providing suitable tools and integrated databases. In this work, we describe the main features of the Genosoja web database, including the pipelines to analyze some kinds of data (ESTs, SuperSAGE, microRNAs, subtractive cDNA libraries), as well as web interfaces to access information about soybean gene annotation and expression.

Subtractive libraries for prospecting differentially expressed genes in the soybean under water deficit.

Soybean has a wide range of applications in the industry and, due to its crop potential, its improvement is widely desirable. During drought conditions, soybean crops suffer significant losses in productivity. Therefore, understanding the responses of the soybean under this stress is an effective way of targeting crop improvement techniques. In this study, we employed the Suppressive Subtractive Hybridization (SSH) technique to investigate differentially expressed genes under water deficit conditions. Embrapa 48 and BR 16 soybean lines, known as drought-tolerant and -sensitive, respectively, were grown hydroponically and subjected to different short-term periods of stress by withholding the nutrient solution. Using this approach, we have identified genes expressed during the early response to water deficit in roots and leaves. These genes were compared among the lines to assess probable differences in the plant transcriptomes. In general, similar biochemical processes were predominant in both cultivars; however, there were more considerable differences between roots and leaves of Embrapa 48. Moreover, we present here a fast, clean and straightforward method to obtain drought-stressed root tissues and a large enriched collection of transcripts expressed by soybean plants under water deficit that can be useful for further studies towards the understanding of plant responses to stress.

Identification of SNPs in RNA-seq data of two cultivars of Glycine max (soybean) differing in drought resistance.

The legume Glycine max (soybean) plays an important economic role in the international commodities market, with a world production of almost 260 million tons for the 2009/2010 harvest. The increase in drought events in the last decade has caused production losses in recent harvests. This fact compels us to understand the drought tolerance mechanisms in soybean, taking into account its variability among commercial and developing cultivars. In order to identify single nucleotide polymorphisms (SNPs) in genes up-regulated during drought stress, we evaluated suppression subtractive libraries (SSH) from two contrasting cultivars upon water deprivation: sensitive (BR 16) and tolerant (Embrapa 48). A total of 2,222 soybean genes were up-regulated in both cultivars. Our method identified more than 6,000 SNPs in tolerant and sensitive Brazilian cultivars in those drought stress related genes. Among these SNPs, 165 (in 127 genes) are positioned at soybean chromosome ends, including transcription factors (MYB, WRKY) related to tolerance to abiotic stress.