Effects on gene expression during maize-Azospirillum interaction in the presence of a plant-specific inhibitor of indole-3-acetic acid production.

Amongst the sustainable alternatives to increase maize production is the use of plant growth-promoting bacteria (PGPB). Azospirillum brasilense is one of the most well-known PGPB being able to fix nitrogen and produce phytohormones, especially indole-3-acetic acid - IAA. This work investigated if there is any contribution of the bacterium to the plant's IAA levels, and how it affects the plant. To inhibit plant IAA production, yucasin, an inhibitor of the TAM/YUC pathway, was applied. Plantlets' IAA concentration was evaluated through HPLC and dual RNA-Seq was used to analyze gene expression. Statistical differences between the group treated with yucasin and the other groups showed that A. brasilense inoculation was able to prevent the phenotype caused by yucasin concerning the number of lateral roots. Genes involved in the auxin and ABA response pathways, auxin efflux transport, and the cell cycle were regulated by the presence of the bacterium, yucasin, or both. Genes involved in the response to biotic/abiotic stress, plant disease resistance, and a D-type cellulose synthase changed their expression pattern among two sets of comparisons in which A. brasilense acted as treatment. The results suggest that A. brasilense interferes with the expression of many maize genes through an IAA-independent pathway.

Transcriptomic Response of the Diazotrophic Bacteria Gluconacetobacter diazotrophicus Strain PAL5 to Iron Limitation and Characterization of the fur Regulatory Network.

Gluconacetobacter diazotrophicus has been the focus of several studies aiming to understand the mechanisms behind this endophytic diazotrophic bacterium. The present study is the first global analysis of the early transcriptional response of exponentially growing G. diazotrophicus to iron, an essential cofactor for many enzymes involved in various metabolic pathways. RNA-seq, targeted gene mutagenesis and computational motif discovery tools were used to define the G. diazotrophicusfur regulon. The data analysis showed that genes encoding functions related to iron homeostasis were significantly upregulated in response to iron limitations. Certain genes involved in secondary metabolism were overexpressed under iron-limited conditions. In contrast, it was observed that the expression of genes involved in Fe-S cluster biosynthesis, flagellar biosynthesis and type IV secretion systems were downregulated in an iron-depleted culture medium. Our results support a model that controls transcription in G. diazotrophicus by fur function. The G. diazotrophicusfur protein was able to complement an E. colifur mutant. These results provide new insights into the effects of iron on the metabolism of G. diazotrophicus, as well as demonstrate the essentiality of this micronutrient for the main characteristics of plant growth promotion by G. diazotrophicus.

High-Resolution Translatome Analysis Reveals Cortical Cell Programs During Early Soybean Nodulation.

Nodule organogenesis in legumes is regulated temporally and spatially through gene networks. Genome-wide transcriptome, proteomic, and metabolomic analyses have been used previously to define the functional role of various plant genes in the nodulation process. However, while significant progress has been made, most of these studies have suffered from tissue dilution since only a few cells/root regions respond to rhizobial infection, with much of the root non-responsive. To partially overcome this issue, we adopted translating ribosome affinity purification (TRAP) to specifically monitor the response of the root cortex to rhizobial inoculation using a cortex-specific promoter. While previous studies have largely focused on the plant response within the root epidermis (e.g., root hairs) or within developing nodules, much less is known about the early responses within the root cortex, such as in relation to the development of the nodule primordium or growth of the infection thread. We focused on identifying genes specifically regulated during early nodule organogenesis using roots inoculated with Bradyrhizobium japonicum. A number of novel nodulation gene candidates were discovered, as well as soybean orthologs of nodulation genes previously reported in other legumes. The differential cortex expression of several genes was confirmed using a promoter-GUS analysis, and RNAi was used to investigate gene function. Notably, a number of differentially regulated genes involved in phytohormone signaling, including auxin, cytokinin, and gibberellic acid (GA), were also discovered, providing deep insight into phytohormone signaling during early nodule development.

Herbaspirillum seropedicae strain HRC54 expression profile in response to sugarcane apoplastic fluid.

Bacterial transcriptome profiling in the presence of plant fluids or extracts during microbial growth may provide relevant information on plant-bacteria interactions. Here, RNA sequencing (RNA-Seq) was used to determine the transcriptomic profile of Herbaspirillum seropedicae strain HRC54 at the early stages of response to sugarcane apoplastic fluid. Differentially expressed gene (DEG) analysis was performed using the DESeq2 and edgeR packages, followed by functional annotation using Blast2GO and gene ontology enrichment analysis using the COG and KEGG databases. After 2 h of sugarcane apoplastic fluid addition to the H. seropedicae HRC54 culture, respectively, 44 and 45 genes were upregulated and downregulated. These genes were enriched in bacterial metabolism (e.g., oxidoreductase and transferase), ABC transporters, motility, secretion systems, and signal transduction. RNA-Seq expression profiles of 12 genes identified in data analyses were verified by RT-qPCR. The results suggested that H. seropedicae HRC54 recognized sugarcane apoplastic fluid as the host signal, and some DEGs were closely involved at the early stages of the establishment of plant-bacteria interactions. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02848-y.

Preventing Chagas disease: A new RT-qPCR method for rapid and specific quantification of viable Trypanosoma cruzi for food safety.

Without standardized methods for rapidly detecting in food matrices viable T. cruzi, foodborne outbreaks remain neglected. In this work, a reverse-transcriptase real-time PCR (RT-qPCR) mRNA-based technique was developed for the rapid and specific detection and quantification of viable Trypanosoma cruzi in açai fruits and juice. The method uses specific primer targeting region on the cyt b gene. The maximum recovery rate of T. cruzi from inoculated açai juice was 82.50%. The limit of detection and quantification in açai juice was 10 parasites/mL for RT-qPCR (mRNA-based) and qPCR (DNA-based). The RT-qPCR efficiency was estimated at 97.27% with an R2 of 0.994. The RT-qPCR was shown to be able to discriminate between viable and nonviable cells. This method provides a useful tool for rapid assessment of low concentrations of viable T. cruzi in naturally contaminated food samples, and can be applied industrially as a quality and security method.

Diverse Bacterial Genes Modulate Plant Root Association by Beneficial Bacteria.

The plant rhizosphere harbors a diverse population of microorganisms, including beneficial plant growth-promoting bacteria (PGPB), that colonize plant roots and enhance growth and productivity. In order to specifically define bacterial traits that contribute to this beneficial interaction, we used high-throughput transposon mutagenesis sequencing (TnSeq) in two model root-bacterium systems associated with Setaria viridis: Azoarcus olearius DQS4T and Herbaspirillum seropedicae SmR1. This approach identified ∼100 significant genes for each bacterium that appeared to confer a competitive advantage for root colonization. Most of the genes identified specifically in A. olearius encoded metabolism functions, whereas genes identified in H. seropedicae were motility related, suggesting that each strain requires unique functions for competitive root colonization. Genes were experimentally validated by site-directed mutagenesis, followed by inoculation of the mutated bacteria onto S. viridis roots individually, as well as in competition with the wild-type strain. The results identify key bacterial functions involved in iron uptake, polyhydroxybutyrate metabolism, and regulation of aromatic metabolism as important for root colonization. The hope is that by improving our understanding of the molecular mechanisms used by PGPB to colonize plants, we can increase the adoption of these bacteria in agriculture to improve the sustainability of modern cropping systems.IMPORTANCE There is growing interest in the use of associative, plant growth-promoting bacteria (PGPB) as biofertilizers to serve as a sustainable alternative for agriculture application. While a variety of mechanisms have been proposed to explain bacterial plant growth promotion, the molecular details of this process remain unclear. The current research supports the idea that PGPB use in agriculture will be promoted by gaining more knowledge as to how these bacteria colonize plants, promote growth, and do so consistently. Specifically, the research seeks to identify those bacterial genes involved in the ability of two, PGPB strains, Azoarcus olearius and Herbaspirillum seropedicae, to colonize the roots of the C4 model grass Setaria viridis. Applying a transposon mutagenesis (TnSeq) approach, we assigned phenotypes and function to genes that affect bacterial competitiveness during root colonization. The results suggest that each bacterial strain requires unique functions for root colonization but also suggests that a few, critical functions are needed by both bacteria, pointing to some common mechanisms. The hope is that such information can be exploited to improve the use and performance of PGPB in agriculture.

Transcriptome profiling of non-climacteric 'yellow' melon during ripening: insights on sugar metabolism.

BACKGROUND: The non-climacteric 'Yellow' melon (Cucumis melo, inodorus group) is an economically important crop and its quality is mainly determined by the sugar content. Thus, knowledge of sugar metabolism and its related pathways can contribute to the development of new field management and post-harvest practices, making it possible to deliver better quality fruits to consumers. RESULTS: The RNA-seq associated with RT-qPCR analyses of four maturation stages were performed to identify important enzymes and pathways that are involved in the ripening profile of non-climacteric 'Yellow' melon fruit focusing on sugar metabolism. We identified 895 genes 10 days after pollination (DAP)-biased and 909 genes 40 DAP-biased. The KEGG pathway enrichment analysis of these differentially expressed (DE) genes revealed that 'hormone signal transduction', 'carbon metabolism', 'sucrose metabolism', 'protein processing in endoplasmic reticulum' and 'spliceosome' were the most differentially regulated processes occurring during melon development. In the sucrose metabolism, five DE genes are up-regulated and 12 are down-regulated during fruit ripening. CONCLUSIONS: The results demonstrated important enzymes in the sugar pathway that are responsible for the sucrose content and maturation profile in non-climacteric 'Yellow' melon. New DE genes were first detected for melon in this study such as invertase inhibitor LIKE 3 (CmINH3), trehalose phosphate phosphatase (CmTPP1) and trehalose phosphate synthases (CmTPS5, CmTPS7, CmTPS9). Furthermore, the results of the protein-protein network interaction demonstrated general characteristics of the transcriptome of young and full-ripe melon and provide new perspectives for the understanding of ripening.

Herbaspirillum rubrisubalbicans as a Phytopathogenic Model to Study the Immune System of Sorghum bicolor.

Herbaspirillum rubrisubalbicans is the causal agent of red stripe disease (RSD) and mottle stripe disease of sorghum and sugarcane, respectively. In all, 63 genotypes of Sorghum bicolor were inoculated with H. rubrisubalbicans, with 59 showing RSD symptoms. Quantitative trait loci (QTL) analysis in a recombinant inbred line (RIL) population identified several QTL associated with variation in resistance to RSD. RNA sequencing analysis identified a number of genes whose transcript levels were differentially regulated during H. rubrisubalbicans infection. Among those genes that responded to H. rubrisubalbicans inoculation were many involved in plant-pathogen interactions such as leucine-rich repeat receptors, mitogen-activated protein kinase 1, calcium-binding proteins, transcriptional factors (ethylene-responsive element binding factor), and callose synthase. Pretreatment of sorghum leaves with the pathogen-associated molecular pattern (PAMP) molecules flg22 and chitooctaose provided protection against subsequent challenge with the pathogen, suggesting that PAMP-triggered immunity plays an important role in the sorghum immunity response. These data present baseline information for the use of the genetically tractable H. rubrisubalbicans-sorghum pathosystem for the study of innate immunity and disease resistance in this important grain and bioenergy crop. Information gained from the use of this system is likely to be informative for other monocots, including those more intractable for experimental study (e.g., sugarcane).

Transcriptome profile of Corynebacterium pseudotuberculosis in response to iron limitation.

BACKGROUND: Iron is an essential micronutrient for the growth and development of virtually all living organisms, playing a pivotal role in the proliferative capability of many bacterial pathogens. The impact that the bioavailability of iron has on the transcriptional response of bacterial species in the CMNR group has been widely reported for some members of the group, but it hasn't yet been as deeply explored in Corynebacterium pseudotuberculosis. Here we describe for the first time a comprehensive RNA-seq whole transcriptome analysis of the T1 wild-type and the Cp13 mutant strains of C. pseudotuberculosis under iron restriction. The Cp13 mutant strain was generated by transposition mutagenesis of the ciuA gene, which encodes a surface siderophore-binding protein involved in the acquisition of iron. Iron-regulated acquisition systems are crucial for the pathogenesis of bacteria and are relevant targets to the design of new effective therapeutic approaches. RESULTS: Transcriptome analyses showed differential expression in 77 genes within the wild-type parental T1 strain and 59 genes in Cp13 mutant under iron restriction. Twenty-five of these genes had similar expression patterns in both strains, including up-regulated genes homologous to the hemin uptake hmu locus and two distinct operons encoding proteins structurally like hemin and Hb-binding surface proteins of C. diphtheriae, which were remarkably expressed at higher levels in the Cp13 mutant than in the T1 wild-type strain. These hemin transport protein genes were found to be located within genomic islands associated with known virulent factors. Down-regulated genes encoding iron and heme-containing components of the respiratory chain (including ctaCEF and qcrCAB genes) and up-regulated known iron/DtxR-regulated transcription factors, namely ripA and hrrA, were also identified differentially expressed in both strains under iron restriction. CONCLUSION: Based on our results, it can be deduced that the transcriptional response of C. pseudotuberculosis under iron restriction involves the control of intracellular utilization of iron and the up-regulation of hemin acquisition systems. These findings provide a comprehensive analysis of the transcriptional response of C. pseudotuberculosis, adding important understanding of the gene regulatory adaptation of this pathogen and revealing target genes that can aid the development of effective therapeutic strategies against this important pathogen.

Transcriptional effects in the estuarine guppy Poecilia vivipara exposed to sanitary sewage in laboratory and in situ.

The urban growth has increased sanitary sewage discharges in coastal ecosystems, negatively affecting the aquatic biota. Mangroves, one of the most human-affected coastal biomes, are areas for reproduction and nursing of several species. In order to evaluate the effects of sanitary sewage effluents in mangrove species, this study assessed the hepatic transcriptional responses of guppy fish Poecilia vivipara exposed to sanitary sewage 33% (v:v), using suppressive subtraction hybridization (SSH), high throughput sequencing of RNA (Ion-proton) and quantification of transcript levels by qPCR of some identified genes in fish kept in a sewage-contaminated environment. Genes identified are related predominantly to xenobiotic biotransformation, immune system and sexual differentiation. The qPCR results confirmed the induction of cytochrome P450 1A (CYP1A), glutathione S transferase A-like (GST A-like) methyltransferase (MET) and UDP glycosyltransferase 1A (UDPGT1A), and repression of complement component C3 (C3), doublesex and mab-3 related transcription factor 1 (DMRT1), and transferrin (TF) in the laboratory experiment. In the field exposure, the transcript levels of CYP1A, DMRT1, MET, GST A-like and UDPGT1A were higher in fishes exposed at the contaminated sites compared to the reference site. Chemical analysis in fish from the laboratory and in situ experiments, and surface sediment from the sewage-contaminated sites revealed relevant levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and linear alkylbenzenes (LABs). These data reinforce the use of P. vivipara as a sentinel for monitoring environmental contamination in coastal regions.

Genome sequencing of Burkholderia contaminans LTEB11 reveals a lipolytic arsenal of biotechnological interest.

Burkholderia contaminans LTEB11 is a Gram-negative betaproteobacterium isolated as a contaminant of a culture in mineral medium supplemented with vegetable oil. Here, we report the genome sequence of B. contaminans LTEB11, identifying and analyzing the genes involved in its lipolytic machinery and in the production of other biotechnological products.

Sugarcane apoplast fluid modulates the global transcriptional profile of the diazotrophic bacteria Paraburkholderia tropica strain Ppe8.

The stalk apoplast fluid of sugarcane contains different sugars, organic acids and amino acids that may supply the demand for carbohydrates by endophytic bacteria including diazotrophs P. tropica (syn. B. tropica) strain Ppe8, isolated from sugarcane, is part of the bacterial consortium recommended as inoculant to sugarcane. However, little information has been accumulated regarding this plant-bacterium interaction considering that it colonizes internal sugarcane tissues. Here, we made use of the RNA-Seq transcriptomic analysis to study the influence of sugarcane stalk apoplast fluid on Ppe8 gene expression. The bacterium was grown in JMV liquid medium (100 ml), divided equally and then supplemented with 50 ml of fresh JMV medium or 50 ml of apoplast fluid extracted from sugarcane variety RB867515. Total RNA was extracted 2 hours later, the rRNAs were depleted and mRNAs used to construct libraries to sequence the fragments using Ion Torrent technology. The mapping and statistical analysis were carried out with CLC Genomics Workbench software. The RNA-seq data was validated by RT-qPCR using the reference genes fliP1, paaF, and groL. The data analysis showed that 544 genes were repressed and 153 genes were induced in the presence of apoplast fluid. Genes that induce plant defense responses, genes related to chemotaxis and movements were repressed in the presence of apoplast fluid, indicating that strain Ppe8 recognizes the apoplast fluid as a plant component. The expression of genes involved in bacterial metabolism was regulated (up and down), suggesting that the metabolism of strain Ppe8 is modulated by the apoplast fluid. These results suggest that Ppe8 alters its gene expression pattern in the presence of apoplast fluid mainly in order to use compounds present in the fluid as well as to avoid the induction of plant defense mechanisms. This is a pioneer study showing the role played by the sugarcane apoplast fluid on the global modulation of genes in P. tropica strain Ppe8.

qPCR for the detection of foodborne Trypanosoma cruzi.

Here we presented a potential real-time PCR (qPCR) method with public health importance and relevance for detection of Trypanosoma cruzi in açai pulp. There is not a current process to identify T. cruzi in açai, that ensures innocuity of this food concerning oral transmission. First, six new primers were designed using the DNA sequences of T. cruzi y152 and Emerald strains obtained from GenBank. For primers evaluation and titration they were validated regarding the amplification and not with the fluorophore chosen 1ngµL-1 of the T. cruzi DNA as target. For determination of the ideal concentration the titration of the primers drawn in this study showed T. cruzi DNA amplification in five primer pairs at concentrations 100, 200 and 300nM and DNA fixed concentrations at 1ngµL-1. For standardization all reactions were performed in triplicate with 5.0µL and positives and negatives controls were included in every run. As positive control DNA from two genotypes TcI and TcII were used. As negative control the reaction product without DNA of the parasite was used. The best primer concentration, for the expected fragments, was 300nM. From six primers improved the Ep1F/Ep1R primer detected 1×10-4ngµL-1 for both genotype of the parasite. The Bp1F/Bp1R showed amplification for 1.70.10-7ngµL-1 for TcI and 4.31.10-8ngµL-1 for TcII, based on the standard curve. The last step we tested the selected primers in qPCR for monitoring T. cruzi in açai pulp experimentally contaminated. The recovery rate for the TcII was 71%, whereas in açai samples contaminated with TcI it was 76%.

Genome Sequence of Type Strain Fonsecaea multimorphosa CBS 980.96T, a Causal Agent of Feline Cerebral Phaeohyphomycosis.

A draft genome sequence of type strain Fonsecaea multimorphosa CBS 980.96T was obtained. This species was first isolated from a cat with cerebral phaeohyphomycosis in Queensland, Australia.

Iron deficiency resistance mechanisms enlightened by gene expression analysis in Paenibacillus riograndensis SBR5.

Despite its importance in growth and cell division, iron metabolism is still poorly understood in microorganisms, especially in Gram-positive bacteria. In this work, we used RNA sequencing technology to elucidate global mechanisms involved in iron starvation resistance in Paenibacillus riograndensis SBR5, a potential plant growth-promoting bacterium. Iron deficiency caused several changes in gene expression, and 150 differentially expressed genes were found: 71 genes were overexpressed and 79 genes were underexpressed. Eight genes for which expression was at least twice as high or twice as low in iron-limited condition compared with iron-sufficient condition were chosen for RT-qPCR analysis to validate the RNA seq data. In general, most genes exhibited the same pattern of expression after 24 h of P. riograndensis growth under iron-limiting condition. Our results suggest that, during iron deficiency, bacteria express several genes related to nutrient uptake when they start to grow to obtain all of the molecules necessary for maintaining major cellular processes. However, once iron becomes highly limiting and is no longer able to sustain exponential growth, bacteria begin to express genes related to several processes, like sporulation and DNA protection, as a way of resisting this stress.

Proteomic analysis of Herbaspirillum seropedicae cultivated in the presence of sugar cane extract.

Bacterial endophytes of the genus Herbaspirillum colonize sugar cane and can promote plant growth. The molecular mechanisms that mediate plant- H. seropedicae interaction are poorly understood. In this work, we used 2D-PAGE electrophoresis to identify H. seropedicae proteins differentially expressed at the log growth phase in the presence of sugar cane extract. The differentially expressed proteins were validated by RT qPCR. A total of 16 differential spots (1 exclusively expressed, 7 absent, 5 up- and 3 down-regulated) in the presence of 5% sugar cane extract were identified; thus the host extract is able to induce and repress specific genes of H. seropedicae. The differentially expressed proteins suggest that exposure to sugar cane extract induced metabolic changes and adaptations in H. seropedicae presumably in preparation to establish interaction with the plant.