Comparative genomics reveals probable adaptations for xylose use in Thermoanaerobacterium saccharolyticum.

Second-generation ethanol, a promising biofuel for reducing greenhouse gas emissions, faces challenges due to the inefficient metabolism of xylose, a pentose sugar. Overcoming this hurdle requires exploration of genes, pathways, and organisms capable of fermenting xylose. Thermoanaerobacterium saccharolyticum is an organism capable of naturally fermenting compounds of industrial interest, such as xylose, and understanding evolutionary adaptations may help to bring novel genes and information that can be used for industrial yeast, increasing production of current bio-platforms. This study presents a deep evolutionary study of members of the firmicutes clade, focusing on adaptations in Thermoanaerobacterium saccharolyticum that may be related to overall fermentation metabolism, especially for xylose fermentation. One highlight is the finding of positive selection on a xylose-binding protein of the xylFGH operon, close to the annotated sugar binding site, with this protein already being found to be expressed in xylose fermenting conditions in a previous study. Results from this study can serve as basis for searching for candidate genes to use in industrial strains or to improve Thermoanaerobacterium saccharolyticum as a new microbial cell factory, which may help to solve current problems found in the biofuels' industry.

Wide distribution of D-xylose dehydrogenase in yeasts reveals a new element in the D-xylose metabolism for bioethanol production.

D-xylose utilization by yeasts is an essential feature for improving second-generation ethanol production. However, industrial yeast strains are incapable of consuming D-xylose. Previous analyzes of D-xylose-consuming or fermenting yeast species reveal that the genomic features associated with this phenotype are complex and still not fully understood. Here we present a previously neglected yeast enzyme related to D-xylose metabolism, D-xylose dehydrogenase (XylDH), which is found in at least 105 yeast genomes. By analyzing the XylDH gene family, we brought evidence of gene evolution marked by purifying selection on codons and positive selection evidence in D-xylose-consuming and fermenting species, suggesting the importance of XylDH for D-xylose-related phenotypes in yeasts. Furthermore, although we found no putative metabolic pathway for XylDH in yeast genomes, namely the absence of three bacterial known pathways for this enzyme, we also provide its expression profile on D-xylose media following D-xylose reductase for two yeasts with publicly available transcriptomes. Based on these results, we suggest that XylDH plays an important role in D-xylose usage by yeasts, likely being involved in a cofactor regeneration system by reducing cofactor imbalance in the D-xylose reductase pathway.

Plant microbiomes harbor potential to promote nutrient turnover in impoverished substrates of a Brazilian biodiversity hotspot.

The substrates of the Brazilian campos rupestres, a grassland ecosystem, have extremely low concentrations of phosphorus and nitrogen, imposing restrictions to plant growth. Despite that, this ecosystem harbors almost 15% of the Brazilian plant diversity, raising the question of how plants acquire nutrients in such a harsh environment. Here, we set out to uncover the taxonomic profile, the compositional and functional differences and similarities, and the nutrient turnover potential of microbial communities associated with two plant species of the campos rupestres-dominant family Velloziaceae that grow over distinct substrates (soil and rock). Using amplicon sequencing data, we show that, despite the pronounced composition differentiation, the plant-associated soil and rock communities share a core of highly efficient colonizers that tend to be highly abundant and is enriched in 21 bacterial families. Functional investigation of metagenomes and 522 metagenome-assembled genomes revealed that the microorganisms found associated to plant roots are enriched in genes involved in organic compound intake, and phosphorus and nitrogen turnover. We show that potential for phosphorus transport, mineralization, and solubilization are mostly found within bacterial families of the shared microbiome, such as Xanthobacteraceae and Bryobacteraceae. We also detected the full repertoire of nitrogen cycle-related genes and discovered a lineage of Isosphaeraceae that acquired nitrogen-fixing potential via horizontal gene transfer and might be also involved in nitrification via a metabolic handoff association with Binataceae. We highlight that plant-associated microbial populations in the campos rupestres harbor a genetic repertoire with potential to increase nutrient availability and that the microbiomes of biodiversity hotspots can reveal novel mechanisms of nutrient turnover.

Phylogenomics and gene selection in Aspergillus welwitschiae: Possible implications in the pathogenicity in Agave sisalana.

Aspergillus welwitschiae causes bole rot disease in sisal (Agave sisalana and related species) which affects the production of natural fibers in Brazil, the main worldwide producer of sisal fibers. This fungus is a saprotroph with a broad host range. Previous research established A. welwitschiae as the only causative agent of bole rot in the field, but little is known about the evolution of this species and its strains. In this work, we performed a comparative genomics analysis of 40 Aspergillus strains. We show the conflicting molecular identity of this species, with one sisal-infecting strain sharing its last common ancestor with Aspergillus niger, having diverged only 833 thousand years ago. Furthermore, our analysis of positive selection reveals sites under selection in genes coding for siderophore transporters, Sodium­calcium exchangers, and Phosphatidylethanolamine-binding proteins (PEBPs). Herein, we discuss the possible impacts of these gene functions on the pathogenicity in sisal.

Machine learning and comparative genomics approaches for the discovery of xylose transporters in yeast.

BACKGROUND: The need to mitigate and substitute the use of fossil fuels as the main energy matrix has led to the study and development of biofuels as an alternative. Second-generation (2G) ethanol arises as one biofuel with great potential, due to not only maintaining food security, but also as a product from economically interesting crops such as energy-cane. One of the main challenges of 2G ethanol is the inefficient uptake of pentose sugars by industrial yeast Saccharomyces cerevisiae, the main organism used for ethanol production. Understanding the main drivers for xylose assimilation and identify novel and efficient transporters is a key step to make the 2G process economically viable. RESULTS: By implementing a strategy of searching for present motifs that may be responsible for xylose transport and past adaptations of sugar transporters in xylose fermenting species, we obtained a classifying model which was successfully used to select four different candidate transporters for evaluation in the S. cerevisiae hxt-null strain, EBY.VW4000, harbouring the xylose consumption pathway. Yeast cells expressing the transporters SpX, SpH and SpG showed a superior uptake performance in xylose compared to traditional literature control Gxf1. CONCLUSIONS: Modelling xylose transport with the small data available for yeast and bacteria proved a challenge that was overcome through different statistical strategies. Through this strategy, we present four novel xylose transporters which expands the repertoire of candidates targeting yeast genetic engineering for industrial fermentation. The repeated use of the model for characterizing new transporters will be useful both into finding the best candidates for industrial utilization and to increase the model's predictive capabilities.

Adaptive evolution of Moniliophthora PR-1 proteins towards its pathogenic lifestyle.

BACKGROUND: Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches' broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. RESULTS: Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. CONCLUSIONS: Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.

Ethanol production process driving changes on industrial strains.

Ethanol production has key differences between the two largest producing countries of this biofuel, Brazil and the USA, such as feedstock source, sugar concentration and ethanol titers in industrial fermentation. Therefore, it is highly probable that these specificities have led to genome adaptation of the Saccharomyces cerevisiae strains employed in each process to tolerate different environments. In order to identify particular adaptations, in this work, we have compared the genomes of industrial yeast strains widely used to produce ethanol from sugarcane, corn and sweet sorghum, and also two laboratory strains as reference. The genes were predicted and then 4524 single-copy orthologous were selected to build the phylogenetic tree. We found that the geographic location and industrial process were shown as the main evolutionary drivers: for sugarcane fermentation, positive selection was identified for metal homeostasis and stress response genes, whereas genes involved in membrane modeling have been connected with corn fermentation. In addition, the corn specialized strain Ethanol Red showed an increased number of copies of MAL31, a gene encoding a maltose transporter. In summary, our work can help to guide new strain chassis selection for engineering strategies, to produce more robust strains for biofuel production and other industrial applications.

Peripheral oxytocin injection modulates vomeronasal sensory activity and reduces pup-directed aggression in male mice.

Behaviors are shaped by hormones, which may act either by changing brain circuits or by modifying sensory detection of relevant cues. Pup-directed behaviors have been previously shown to change via action of hormones at the brain level. Here, we investigated hormonal control of pup-induced activity in the vomeronasal organ, an olfactory sensory structure involved in the detection of non-volatile chemosignals. Vomeronasal activity decreases as males switch from a pup-aggressive state to a non-aggressive parenting state, after they socially contact a female. RNA sequencing, qPCR, and in situ hybridization were used to identify expression, in the vomeronasal sensory epithelium, of candidate GPCR hormone receptors chosen by in silico analyses and educated guesses. After identifying that oxytocin and vasopressin receptors are expressed in the vomeronasal organ, we injected the corresponding hormones in mice and showed that oxytocin administration reduced both pup-induced vomeronasal activity and aggressive behavior. Conversely, injection of an oxytocin receptor antagonist in female-primed male animals, which normally exhibit reduced vomeronasal activity, significantly increased the number of active vomeronasal neurons. These data link oxytocin to the modulation of olfactory sensory activity, providing a possible mechanism for changes in male behavior after social experience with females.

Novel xylose transporter Cs4130 expands the sugar uptake repertoire in recombinant Saccharomyces cerevisiae strains at high xylose concentrations.

BACKGROUND: The need to restructure the world's energy matrix based on fossil fuels and mitigate greenhouse gas emissions stimulated the development of new biobased technologies for renewable energy. One promising and cleaner alternative is the use of second-generation (2G) fuels, produced from lignocellulosic biomass sugars. A major challenge on 2G technologies establishment is the inefficient assimilation of the five-carbon sugar xylose by engineered Saccharomyces cerevisiae strains, increasing fermentation time. The uptake of xylose across the plasma membrane is a critical limiting step and the budding yeast S. cerevisiae is not designed with a broad transport system and regulatory mechanisms to assimilate xylose in a wide range of concentrations present in 2G processes. RESULTS: Assessing diverse microbiomes such as the digestive tract of plague insects and several decayed lignocellulosic biomasses, we isolated several yeast species capable of using xylose. Comparative fermentations selected the yeast Candida sojae as a potential source of high-affinity transporters. Comparative genomic analysis elects four potential xylose transporters whose properties were evaluated in the transporter null EBY.VW4000 strain carrying the xylose-utilizing pathway integrated into the genome. While the traditional xylose transporter Gxf1 allows an improved growth at lower concentrations (10 g/L), strains containing Cs3894 and Cs4130 show opposite responses with superior xylose uptake at higher concentrations (up to 50 g/L). Docking and normal mode analysis of Cs4130 and Gxf1 variants pointed out important residues related to xylose transport, identifying key differences regarding substrate translocation comparing both transporters. CONCLUSIONS: Considering that xylose concentrations in second-generation hydrolysates can reach high values in several designed processes, Cs4130 is a promising novel candidate for xylose uptake. Here, we demonstrate a novel eukaryotic molecular transporter protein that improves growth at high xylose concentrations and can be used as a promising target towards engineering efficient pentose utilization in yeast.

Toxoplasmagondii oocysts, Giardia cysts and Cryptosporidium oocysts in outdoor swimming pools in Brazil.

The accidental ingestion of treated recreational water is an important transmission route of waterborne protozoa worldwide. The present study aimed to provide the first evaluation of swimming pools in Brazil, analysing the presence of pathogenic protozoa (Toxoplasma gondii, Cryptosporidium spp. and Giardia spp.) by parasitological and molecular methods. A total of 57 samples were collected from 21 public swimming pools, either directly from the pool or filter backwash water and concentrated using the membrane filtration technique. Giardia cysts and Cryptosporidium oocysts were visualized by direct immunofluorescence assay after purification by immunomagnetic separation. Toxoplasma gondii oocysts were detected by autofluorescence visualization using the supernatant discarded during the purification step as a sample. Positive samples were submitted to molecular analysis. The molecular markers were used: SSU-rRNA, tpi, gdh and bg, for Giardia DNA amplification, and 18S rRNA gene fragment amplification was used for the Cryptosporidium oocysts. The 529-bp repeat element (REP529) fragment and the 35-fold repetitive B1 gene were employed as a target for T. gondii. Amplified products were submitted to sequencing and phylogenetic analysis. Giardia cysts were detected in 19.0% and Cryptosporidium oocysts in 9.5% of swimming pools. In one swimming pool (4.7%), both protozoa were detected on at least one occasion. Structures similar to T. gondii oocysts were detected in 33.3% of the samples, ranging from one to 23 per slide. Giardia was confirmed by DNA amplification in three swimming pools; Giardia duodenalis Assemblage A was identified by the phylogenetic positioning of the ß-giardin gene. Toxoplasma gondii DNA was detected in 14.2% of swimming pools. The present study represents the first report of the occurrence of T. gondii oocysts in swimming pools. Recreational activity in swimming pools contaminated by chlorine-resistant protozoa can represent a high risk of infection for bathers and swimmers.

Giardia spp. and Cryptosporidium spp. removal efficiency of a combined fixed-film system treating domestic wastewater receiving hospital effluent.

Giardia and Cryptosporidium have caused numerous outbreaks of diarrhea as a result of the ingestion of water contaminated with sewage. In Brazil, the efficiency of Giardia and Cryptosporidium removal by combined fixed-film systems has rarely been studied. The aims of the present study were therefore to verify the removal efficiency of Giardia and Cryptosporidium by a combined system (anaerobic/anoxic filter and aerated submerged biofilter) and to perform the genetic characterization of these parasites. The (oo)cysts were detected by centrifuge concentration and membrane filtration from raw sewage, effluents, adhered biomass, and sludge samples. Immunofluorescence assay and differential interference contrast microscopy were used for the visualization of the (oo)cysts. Nested PCR was applied to confirm Giardia and Cryptosporidium. Giardia and Cryptosporidium were detected in 27% and 5.5% of the 144 analyzed samples of raw sewage and effluents, respectively. A total of 33,000 cysts/L were recovered in the adhered biomass samples (n = 25) from different points of the aerated submerged biofilter, while 6000 oocysts/L were registered in a single point. An average of 11,800 cysts/L were found in the sludge samples (n = 5). The combined system exhibited a removal efficiency of Giardia cysts of 1.8 ± 1.0 log removal. The C and BIV assemblages of Giardia were identified in the raw sewage while AII was found in the treated effluent sample. It was not possible to calculate the removal efficiency of Cryptosporidium oocysts by the combined system. The combined system exhibited some potential as a suitable treatment for the removal of parasites from sewage.

Positive Selection Evidence in Xylose-Related Genes Suggests Methylglyoxal Reductase as a Target for the Improvement of Yeasts' Fermentation in Industry.

Xylose assimilation and fermentation are important traits for second generation ethanol production. However, some genomic features associated with this pentose sugar's metabolism remain unknown in yeasts. Comparative genomics studies have led to important insights in this field, but we are still far from completely understanding endogenous yeasts' xylose metabolism. In this work, we carried out a deep evolutionary analysis suited for comparative genomics of xylose-consuming yeasts, searching for of positive selection on genes associated with glucose and xylose metabolism in the xylose-fermenters' clade. Our investigation detected positive selection fingerprints at this clade not only among sequences of important genes for xylose metabolism, such as xylose reductase and xylitol dehydrogenase, but also in genes expected to undergo neutral evolution, such as the glycolytic gene phosphoglycerate mutase. In addition, we present expansion, positive selection marks, and convergence as evidence supporting the hypothesis that natural selection is shaping the evolution of the little studied methylglyoxal reductases. We propose a metabolic model suggesting that selected codons among these proteins caused a putative change in cofactor preference from NADPH to NADH that alleviates cellular redox imbalance. These findings provide a wider look into pentose metabolism of yeasts and add this previously overlooked piece into the intricate puzzle of oxidative imbalance. Although being extensively discussed in evolutionary works the awareness of selection patterns is recent in biotechnology researches, rendering insights to surpass the reached status quo in many of its subareas.

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.

Occurrence and molecular characterization of Giardia duodenalis cysts and Cryptosporidium oocysts in raw water samples from the Rímac River, Peru.

Giardia and Cryptosporidium are potentially pathogenic protozoa which are ubiquitous in ambient surface water. The present study included 60 samples of surface water from three sampling sites from the Rímac River, Lima and Callao, Peru, to detect the occurrence of Giardia spp. and Cryptosporidium spp. and to perform molecular characterization of specimens found. Water samples were concentrated using the membrane filtration technique, and following elution, cysts and oocysts were visualized by direct immunofluorescence assay (IFA). For molecular characterization, tpi and bg gene fragments and 18S rRNA were amplified by nested PCR for Giardia and Cryptosporidium, respectively, followed by sequencing and phylogenetic analysis. Giardia cysts were found in 93.3% of the analyzed samples, whereas Cryptosporidium oocysts were detected in 15%. The positivity of the Giardia cysts was 86.6% (n = 26) in 2014, while Cryptosporidium oocysts were not detected. In 2015, both protozoa were found in raw water samples, with all 30 samples collected positive for Giardia cysts (100.0%) and 9 positive for Cryptosporidium oocysts (30.0%). Oocysts were detected in 20.0% of water samples from sites 1 (mean 5.25 oocysts/L) and 2 (mean 52.3 oocysts/L), while at site 3, oocysts were detected in 50.0% of raw water samples (mean 193.6 oocysts/L). The presence of Giardia duodenalis assemblage A was confirmed in several samples by the phylogenetic positioning of the bg and tpi genes, and the sub-assemblage AII was predominant (8/9). Sequencing for Cryptosporidium resulted in profiles compatible with Cryptosporidium hominis, Cryptosporidium meleagridis, and Cryptosporidium baileyi. This is the first time that the presence of G. duodenalis assemblage A/sub-assemblage AII and Cryptosporidium species has been reported in surface water samples in Peru. These Cryptosporidium species and the Giardia duodenalis assemblage are associated with human disease which highlights the potential risk to public health and the need to increase environmental monitoring measures to protect this water body.

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.

Conversion of an inactive xylose isomerase into a functional enzyme by co-expression of GroEL-GroES chaperonins in Saccharomyces cerevisiae.

BACKGROUND: Second-generation ethanol production is a clean bioenergy source with potential to mitigate fossil fuel emissions. The engineering of Saccharomyces cerevisiae for xylose utilization is an essential step towards the production of this biofuel. Though xylose isomerase (XI) is the key enzyme for xylose conversion, almost half of the XI genes are not functional when expressed in S. cerevisiae. To date, protein misfolding is the most plausible hypothesis to explain this phenomenon. RESULTS: This study demonstrated that XI from the bacterium Propionibacterium acidipropionici becomes functional in S. cerevisiae when co-expressed with GroEL-GroES chaperonin complex from Escherichia coli. The developed strain BTY34, harboring the chaperonin complex, is able to efficiently convert xylose to ethanol with a yield of 0.44 g ethanol/g xylose. Furthermore, the BTY34 strain presents a xylose consumption rate similar to those observed for strains carrying the widely used XI from the fungus Orpinomyces sp. In addition, the tetrameric XI structure from P. acidipropionici showed an elevated number of hydrophobic amino acid residues on the surface of protein when compared to XI commonly expressed in S. cerevisiae. CONCLUSIONS: Based on our results, we elaborate an extensive discussion concerning the uncertainties that surround heterologous expression of xylose isomerases in S. cerevisiae. Probably, a correct folding promoted by GroEL-GroES could solve some issues regarding a limited or absent XI activity in S. cerevisiae. The strains developed in this work have promising industrial characteristics, and the designed strategy could be an interesting approach to overcome the non-functionality of bacterial protein expression in yeasts.

De Novo Assembly of Candida sojae and Candida boidinii Genomes, Unexplored Xylose-Consuming Yeasts with Potential for Renewable Biochemical Production.

Candida boidinii and Candida sojae yeasts were isolated from energy cane bagasse and plague-insects. Both have fast xylose uptake rate and produce great amounts of xylitol, which are interesting features for food and 2G ethanol industries. Because they lack published genomes, we have sequenced and assembled them, offering new possibilities for gene prospection.

High-resolution transcript profiling of the atypical biotrophic interaction between Theobroma cacao and the fungal pathogen Moniliophthora perniciosa.

Witches' broom disease (WBD), caused by the hemibiotrophic fungus Moniliophthora perniciosa, is one of the most devastating diseases of Theobroma cacao, the chocolate tree. In contrast to other hemibiotrophic interactions, the WBD biotrophic stage lasts for months and is responsible for the most distinctive symptoms of the disease, which comprise drastic morphological changes in the infected shoots. Here, we used the dual RNA-seq approach to simultaneously assess the transcriptomes of cacao and M. perniciosa during their peculiar biotrophic interaction. Infection with M. perniciosa triggers massive metabolic reprogramming in the diseased tissues. Although apparently vigorous, the infected shoots are energetically expensive structures characterized by the induction of ineffective defense responses and by a clear carbon deprivation signature. Remarkably, the infection culminates in the establishment of a senescence process in the host, which signals the end of the WBD biotrophic stage. We analyzed the pathogen's transcriptome in unprecedented detail and thereby characterized the fungal nutritional and infection strategies during WBD and identified putative virulence effectors. Interestingly, M. perniciosa biotrophic mycelia develop as long-term parasites that orchestrate changes in plant metabolism to increase the availability of soluble nutrients before plant death. Collectively, our results provide unique insight into an intriguing tropical disease and advance our understanding of the development of (hemi)biotrophic plant-pathogen interactions.

Quality of seeds produced by Psychotria tenuinervis (Rubiaceae): distance from anthropogenic and natural edges of Atlantic forest fragment.

The aim of this study was to investigate whether there were differences in the genetic variability and rate and velocity of the seed germination produced by Psychotria tenuinervis located at anthropogenic edges, natural edges, and in the forest interior. The populations of P. tenuinervis showed no differences in genetic variability or structure among the three habitats. There was, however, an indication of inbreeding, which was significantly higher in natural edges than in anthropogenic edges and the forest interior. Within-habitat variation was considerable, but there were no differences in seed mass or rate and velocity of germination among the three habitats. These results suggest that seed characteristics were not influenced by the genetic pattern of P. tenuinervis and that other characteristics of the forest fragment, such as gaps, edge age, and type of matrix exert more influence on seed mass and germination than the distance from the edges.

Population genetics of Collisella subrugosa (Patellogastropoda: Acmaeidae): evidence of two scales of population structure.

Marine invertebrate populations usually show high levels of genetic variability that has frequently been associated with spatial and temporal environmental heterogeneity. One of the most heterogeneous marine environments is the intertidal zone, the habitat of Collisella subrugosa, the most widespread and abundant Brazilian limpet. C. subrugosa has planktonic larvae that can disperse over long distances, what can promote gene flow among shores, working against interpopulational differentiation. In this study we investigated the genetic variability and populational substructure of C. subrugosa through analysis of 24 allozyme loci in 14 samples (590 individuals) collected along 2,700 km of the Brazilian coast. The genetic variability was high ([Formula: see text] and [Formula: see text]), as expected for intertidal species. Genetic differentiation among samples was low (F (ST) = 0.03) what may reflect intensive gene flow associated with larval dispersal. However, we detected an isolation-by-distance pattern of population substructure in one sampled region. High levels of heterozygote deficiency were also observed for many loci in each sample. Alternative hypothesis are discussed, and the "breeding groups" is suggested to explain these pattern, indicating the main cause as environmental heterogeneity.