Metabolomics of the interaction between a consortium of entomopathogenic fungi and their target insect: Mechanisms of attack and survival.

One of the most concerning pests that attack strawberries in Brazil is Duponchelia fovealis (Zeller), a non-native moth with no registered control methods to date. Our group recently observed that a fungal consortium formed by two strains of Beauveria bassiana (Balsamo) increased the mortality of D. fovealis more than inoculation with each strain on its own. However, the molecular interaction between the fungal consortium and the caterpillars is unknown. Thus, in this work, we sought to pioneer the evaluation of the molecular interaction between a fungal consortium of B. bassiana and D. fovealis caterpillars. We aimed to understand the biocontrol process involved in this interaction and the defense system of the caterpillar. Seven days after D. fovealis were inoculated with the consortium, the dead and surviving caterpillars were analyzed using GC-MS and LC-MS. Some of the metabolites identified in dead caterpillars have primarily antioxidant action. Other metabolites may have insecticidal potential, such as diltiazem-like and tamsulosin-like compounds, as well as 2,5-dimethoxymandelic acid. In surviving caterpillars, the main mechanisms are pro-inflammatory from 2-Palmitoylglycerol metabolite and the antifungal action of the metabolite Aegle marmelos Alkaloid-C. The metabolites identified in dead caterpillars may explain the increased mortality caused by the consortium due to its antioxidant mechanism, which can suppress the caterpillars' immune system, and insecticide action. In surviving caterpillars, the main resistance mechanisms may involve the stimulus to the immunity and antifungal action.

Integrative Analysis of the Ethanol Tolerance of Saccharomyces cerevisiae.

Ethanol (EtOH) alters many cellular processes in yeast. An integrated view of different EtOH-tolerant phenotypes and their long noncoding RNAs (lncRNAs) is not yet available. Here, large-scale data integration showed the core EtOH-responsive pathways, lncRNAs, and triggers of higher (HT) and lower (LT) EtOH-tolerant phenotypes. LncRNAs act in a strain-specific manner in the EtOH stress response. Network and omics analyses revealed that cells prepare for stress relief by favoring activation of life-essential systems. Therefore, longevity, peroxisomal, energy, lipid, and RNA/protein metabolisms are the core processes that drive EtOH tolerance. By integrating omics, network analysis, and several other experiments, we showed how the HT and LT phenotypes may arise: (1) the divergence occurs after cell signaling reaches the longevity and peroxisomal pathways, with CTA1 and ROS playing key roles; (2) signals reaching essential ribosomal and RNA pathways via SUI2 enhance the divergence; (3) specific lipid metabolism pathways also act on phenotype-specific profiles; (4) HTs take greater advantage of degradation and membraneless structures to cope with EtOH stress; and (5) our EtOH stress-buffering model suggests that diauxic shift drives EtOH buffering through an energy burst, mainly in HTs. Finally, critical genes, pathways, and the first models including lncRNAs to describe nuances of EtOH tolerance are reported here.

Small Extracellular Vesicles from Hypoxic Triple-Negative Breast Cancer Cells Induce Oxygen-Dependent Cell Invasion.

Hypoxia, a condition of low oxygenation frequently found in triple-negative breast tumors (TNBC), promotes extracellular vesicle (EV) secretion and favors cell invasion, a complex process in which cell morphology is altered, dynamic focal adhesion spots are created, and ECM is remodeled. Here, we investigated the invasive properties triggered by TNBC-derived hypoxic small EV (SEVh) in vitro in cells cultured under hypoxic (1% O2) and normoxic (20% O2) conditions, using phenotypical and proteomic approaches. SEVh characterization demonstrated increased protein abundance and diversity over normoxic SEV (SEVn), with enrichment in pro-invasive pathways. In normoxic cells, SEVh promotes invasive behavior through pro-migratory morphology, invadopodia development, ECM degradation, and matrix metalloprotease (MMP) secretion. The proteome profiling of 20% O2-cultured cells exposed to SEVh determined enrichment in metabolic processes and cell cycles, modulating cell health to escape apoptotic pathways. In hypoxia, SEVh was responsible for proteolytic and catabolic pathway inducement, interfering with integrin availability and gelatinase expression. Overall, our results demonstrate the importance of hypoxic signaling via SEV in tumors for the early establishment of metastasis.

A systems biology view of wood formation in Eucalyptus grandis trees submitted to different potassium and water regimes.

Wood production in fast-growing Eucalyptus grandis trees is highly dependent on both potassium (K) fertilization and water availability but the molecular processes underlying wood formation in response to the combined effects of these two limiting factors remain unknown. E. grandis trees were submitted to four combinations of K-fertilization and water supply. Weighted gene co-expression network analysis and MixOmics-based co-regulation networks were used to integrate xylem transcriptome, metabolome and complex wood traits. Functional characterization of a candidate gene was performed in transgenic E. grandis hairy roots. This integrated network-based approach enabled us to identify meaningful biological processes and regulators impacted by K-fertilization and/or water limitation. It revealed that modules of co-regulated genes and metabolites strongly correlated to wood complex traits are in the heart of a complex trade-off between biomass production and stress responses. Nested in these modules, potential new cell-wall regulators were identified, as further confirmed by the functional characterization of EgMYB137. These findings provide new insights into the regulatory mechanisms of wood formation under stressful conditions, pointing out both known and new regulators co-opted by K-fertilization and/or water limitation that may potentially promote adaptive wood traits.

Sugarcane must fed-batch fermentation by Saccharomyces cerevisiae: impact of sterilized and non-sterilized sugarcane must.

The presence of microbial contaminants is common in the sugarcane ethanol industry and can decrease process yield, reduce yeast cell viability and induce yeast cell flocculation. To evaluate the effect of microbial contamination on the fermentation process, we compared the use of sterilized and non-sterilized sugarcane must in the performance of Saccharomyces cerevisiae with similar fermentation conditions to those used in Brazilian mills. Non-sterilized sugarcane must had values of 103 and 108 CFU mL-1 of wild yeast and bacterial contamination, respectively; decreased total reducing sugar (TRS); and increased lactic and acetic acids, glycerol and ethanol concentrations during storage. During fermentation cycles with sterilized and non-sterilized sugarcane must, S. cerevisiae viability did not change, whereas ethanol yield varied from 74.1 to 80.2%, but it did not seem to be related to must microbial contamination. Ethanol productivity decreased throughout the fermentation cycles and was more pronounced in the last two fermentation cycles with non-sterilized must, but that may be related to the decrease in must TRS. High values of the ratio of total acid production per ethanol were reported at the end of the last two fermentation cycles conducted with non-sterilized must. Additionally, the values of wild yeast contamination increased from 102 to 103 CFU mL-1 and bacterial contamination increased from 104 to 106 CFU mL-1 when comparing the first and last fermentation cycles with non-sterilized must. In addition to the increase in microbial contamination and acid concentration, ethanol yield and yeast viability rates were not directly affected by the microbial contamination present in the non-sterilized sugarcane must.

Plant Cell Wall Proteomics: A Focus on Monocot Species, Brachypodium distachyon, Saccharum spp. and Oryza sativa.

Plant cell walls mostly comprise polysaccharides and proteins. The composition of monocots' primary cell walls differs from that of dicots walls with respect to the type of hemicelluloses, the reduction of pectin abundance and the presence of aromatic molecules. Cell wall proteins (CWPs) differ among plant species, and their distribution within functional classes varies according to cell types, organs, developmental stages and/or environmental conditions. In this review, we go deeper into the findings of cell wall proteomics in monocot species and make a comparative analysis of the CWPs identified, considering their predicted functions, the organs analyzed, the plant developmental stage and their possible use as targets for biofuel production. Arabidopsis thaliana CWPs were considered as a reference to allow comparisons among different monocots, i.e., Brachypodium distachyon, Saccharum spp. and Oryza sativa. Altogether, 1159 CWPs have been acknowledged, and specificities and similarities are discussed. In particular, a search for A. thaliana homologs of CWPs identified so far in monocots allows the definition of monocot CWPs characteristics. Finally, the analysis of monocot CWPs appears to be a powerful tool for identifying candidate proteins of interest for tailoring cell walls to increase biomass yield of transformation for second-generation biofuels production.

Hyper response to ovarian stimulation affects the follicular fluid metabolomic profile of women undergoing IVF similarly to polycystic ovary syndrome.

INTRODUCTION: During in vitro fertilization (IVF), the hyper response to controlled ovarian stimulation (COS) is a common characteristic among patients diagnosed with polycystic ovary syndrome (PCOS), although non-diagnosed patients may also demonstrate this response. OBJECTIVES: In an effort to investigate follicular metabolic characteristics associated with hyper response to COS, the present study analyzed follicular fluid (FF) samples from patients undergoing IVF. METHODS: FF samples were obtained from patients with PCOS and hyper response during IVF (PCOS group, N = 15), patients without PCOS but with hyper response during IVF (HR group, N = 44), and normo-responder patients receiving IVF (control group, N = 22). FF samples underwent Bligh and Dyer extraction, followed by metabolomic analysis by ultra-performance liquid chromatography mass spectrometry, considering two technical replicates. Clinical data was analyzed by ANOVA and chi-square tests. The metabolomic dataset was analyzed by multivariate statistics, and the significance of biomarkers was confirmed by ANOVA. RESULTS: Clinical data showed differences regarding follicles production, oocyte and embryo quality. From the 15 proposed biomarkers, 14 were of increased abundance in the control group and attributed as fatty acids, diacylglycerol, triacylglycerol, ceramide, ceramide-phosphate, phosphatidylcholine, and sphingomyelin. The PCOS patients showed increased abundance of a metabolite of m/z 144.0023 that was not attributed to a class. CONCLUSION: The clinical and metabolic similarities observed in the FF of hyper responders with and without PCOS diagnosis indicate common biomarkers that could assist on the development of accessory tools for assessment of IVF parameters.

Integrated analysis of gene expression from carbon metabolism, proteome and metabolome, reveals altered primary metabolism in Eucalyptus grandis bark, in response to seasonal variation.

BACKGROUND: Seasonal variation is presumed to play an important role in the regulation of tree growth, especially for Eucalyptus grandis, a fast-growing tree. This variation may induce changes in the whole tree at transcriptional, protein and metabolite levels. Bark represents an important group of tissues that protect trees from desiccation and pathogen attack, and it has been identified as potential feedstock for lignocellulosic derived biofuels. Despite the growing interest, little is known about the molecular mechanisms that regulates bark metabolism, particularly in tropical countries. RESULTS: In this study we report the changes observed in the primary metabolism of E. grandis bark during two contrasting seasons in Brazil, summer (wet) and winter (dry), through the combination of transcripts (RT-qPCR), proteome (2-DE gels) and metabolome (GC-MS) analysis, in an integrated manner. Twenty-four genes, involved in carbon metabolism, were analyzed in the two seasons. Eleven were up-regulated in summer, three were up-regulated in winter and ten did not show statistical differences in the expression pattern. The proteomic analysis using 2-DE gels showed 77 proteins expressing differences in abundance, with 38 spots up-regulated in summer and 37 in winter. Different metabolites significantly accumulated during winter. CONCLUSIONS: This study revealed a metabolic reconfiguration in the primary metabolism of E. grandis bark, triggered by seasonal variation. Transcripts and protein data suggests that during winter carbohydrate formation seems to be favored by tree metabolism. Glucose, fructose and sucrose accumulated at significant levels during the winter.

Cell wall proteome of sugarcane stems: comparison of a destructive and a non-destructive extraction method showed differences in glycoside hydrolases and peroxidases.

BACKGROUND: Sugarcane has been used as the main crop for ethanol production for more than 40 years in Brazil. Recently, the production of bioethanol from bagasse and straw, also called second generation (2G) ethanol, became a reality with the first commercial plants started in the USA and Brazil. However, the industrial processes still need to be improved to generate a low cost fuel. One possibility is the remodeling of cell walls, by means of genetic improvement or transgenesis, in order to make the bagasse more accessible to hydrolytic enzymes. We aimed at characterizing the cell wall proteome of young sugarcane culms, to identify proteins involved in cell wall biogenesis. Proteins were extracted from the cell walls of 2-month-old culms using two protocols, non-destructive by vacuum infiltration vs destructive. The proteins were identified by mass spectrometry and bioinformatics. RESULTS: A predicted signal peptide was found in 84 different proteins, called cell wall proteins (CWPs). As expected, the non-destructive method showed a lower percentage of proteins predicted to be intracellular than the destructive one (33% vs 44%). About 19% of CWPs were identified with both methods, whilst the infiltration protocol could lead to the identification of 75% more CWPs. In both cases, the most populated protein functional classes were those of proteins related to lipid metabolism and oxido-reductases. Curiously, a single glycoside hydrolase (GH) was identified using the non-destructive method whereas 10 GHs were found with the destructive one. Quantitative data analysis allowed the identification of the most abundant proteins. CONCLUSIONS: The results highlighted the importance of using different protocols to extract proteins from cell walls to expand the coverage of the cell wall proteome. Ten GHs were indicated as possible targets for further studies in order to obtain cell walls less recalcitrant to deconstruction. Therefore, this work contributed to two goals: enlarge the coverage of the sugarcane cell wall proteome, and provide target proteins that could be used in future research to facilitate 2G ethanol production.

Physiological and transcriptional analyses of developmental stages along sugarcane leaf.

BACKGROUND: Sugarcane is one of the major crops worldwide. It is cultivated in over 100 countries on 22 million ha. The complex genetic architecture and the lack of a complete genomic sequence in sugarcane hamper the adoption of molecular approaches to study its physiology and to develop new varieties. Investments on the development of new sugarcane varieties have been made to maximize sucrose yield, a trait dependent on photosynthetic capacity. However, detailed studies on sugarcane leaves are scarce. In this work, we report the first molecular and physiological characterization of events taking place along a leaf developmental gradient in sugarcane. RESULTS: Photosynthetic response to CO2 indicated divergence in photosynthetic capacity based on PEPcase activity, corroborated by activity quantification (both in vivo and in vitro) and distinct levels of carbon discrimination on different segments along leaf length. Additionally, leaf segments had contrasting amount of chlorophyll, nitrogen and sugars. RNA-Seq data indicated a plethora of biochemical pathways differentially expressed along the leaf. Some transcription factors families were enriched on each segment and their putative functions corroborate with the distinct developmental stages. Several genes with higher expression in the middle segment, the one with the highest photosynthetic rates, were identified and their role in sugarcane productivity is discussed. Interestingly, sugarcane leaf segments had a different transcriptional behavior compared to previously published data from maize. CONCLUSION: This is the first report of leaf developmental analysis in sugarcane. Our data on sugarcane is another source of information for further studies aiming to understand and/or improve C4 photosynthesis. The segments used in this work were distinct in their physiological status allowing deeper molecular analysis. Although limited in some aspects, the comparison to maize indicates that all data acquired on one C4 species cannot always be easily extrapolated to other species. However, our data indicates that some transcriptional factors were segment-specific and the sugarcane leaf undergoes through the process of suberizarion, photosynthesis establishment and senescence.

Cell wall proteomics of sugarcane cell suspension cultures.

The use of cell walls to produce cellulosic ethanol from sugarcane bagasse is a new challenge. A better knowledge of proteins involved in cell wall remodelling is essential to improve the saccharification processes. Cell suspension cultures were used for this first cell wall proteomics study of sugarcane. Proteins extracted from cell walls were identified using an adapted protocol. They were extracted using 0.2 M CaCl2 and 2 M LiCl after purification of cell walls. The proteins were then identified by the innovative nanoACQUITY UPLC MS/MS technology and bioinformatics using the translated SUCEST EST cluster database of sugarcane. The experiments were reproduced three times. Since Sorghum bicolor is the closest plant with a fully sequenced genome, homologous proteins were searched for to complete the annotation of proteins, that is, prediction of subcellular localization and functional domains. Altogether, 69 different proteins predicted to be secreted were identified among 377 proteins. The reproducibility of the experiments is discussed. These proteins were distributed into eight functional classes. Oxidoreductases such as peroxidases were well represented, whereas glycoside hydrolases were scarce. This work provides information about the proteins that could be manipulated through genetic transformation, to increase second-generation ethanol production.

Alterations of protein expression in conditions of copper-deprivation for Paracoccidioides lutzii in the presence of extracellular matrix components.

BACKGROUND: Paracoccidioides spp is a fungi genus and the agent of paracoccidioidomycosis. The strategies of infection used by these pathogens involve the expression of proteins related to adaptation to the host, particularly regarding the uptake of micronutrients. This study analyzed the adhesion of Paracoccidioides lutzii during conditions of copper (Cu) and iron (Fe) deprivation, while also evaluating the proteins expressed in conditions of Cu depletion in the presence of four extracellular matrix (ECM) components (laminin, fibronectin and types I and IV collagen). RESULTS: We cultured the P. lutzii in a chemically defined media without Cu and Fe. The fungus was then placed in contact with different ECM components and adhesion was evaluated. A significant increase in binding to all ECM components was observed when the fungus was cultured without Cu; which might be related to some adhesins expression. A proteomic assay was developed and revealed 39 proteins expressed that are involved in processes such as virulence, protein synthesis, metabolism, energy, transcription, transport, stress response and the cell cycle when the fungus was interacting with the ECM components. The up-regulated expression of two important adhesins, enolase and 14-3-3, was observed at the fungal cell wall during the interaction with the ECM components, indicating the role of these proteins in the Paracoccidioides-host interaction. CONCLUSIONS: This study is important for determining prospective proteins that may be involved in the interaction of Paracoccidioides with a host. Understanding the adaptive response to different growth conditions, elucidating the processes of adhesion and cell invasion, and identifying the proteins that are differentially expressed during the fungus-host interaction may help elucidate mechanisms used for survival and growth of Paracoccidioides in various human tissues.

Genetic Variability in Puccinia psidii Populations as Revealed by PCR-DGGE and T-RFLP Markers.

Eucalyptus rust caused by Puccinia psidii is responsible for losses of approximately 20% of young Eucalyptus plants, depending on the environmental conditions and the geographic location. Despite its economic importance, there are few studies describing the genetic variability in P. psidii populations that infect different host plants. In the present study, we evaluated the ribosomal DNA internal transcribed spacer region (rDNA-ITS) using polymerase chain reaction denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism to assess the genetic variability in P. psidii populations infecting different Eucalyptus spp. and hybrids, as well as guava, jabuticaba, and syzygium. These culture-independent methods were efficient in differentiating populations based on the host species from which they were collected. In general, the results from both techniques showed that the populations collected from guava, jabuticaba, and syzygium were different from and had a greater level of diversity than the Eucalyptus rust populations. The sequencing of cloned rDNA-ITS fragments confirmed that the vast majority of the profiles generated were from P. psidii. This analysis also revealed interesting single-nucleotide polymorphisms. Therefore, these culture-independent methods are suitable for the rapid assessment of genetic variability within and between populations of this biotrophic fungus on a variety of host species and could be a tool to study the evolution of this pathogen and its interactions with host plants.

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.

Uterine secretome: What do the proteins say about maternal-fetal communication in buffaloes?

The aim was to compare the UF proteomics of pregnant and non-pregnant buffalo during early pregnancy. Forty-four females were submitted to hormonal estrus synchronization and randomly divided into two groups: pregnant (n = 30) and non-pregnant (n = 14). The pregnant group was artificially inseminated and divided into a further two groups: P12 (n = 15) and P18 (n = 15). Conceptus and uterine fluid samples were collected during slaughter at, respectively, 12 and 18 days after insemination. Of all the inseminated females, only eight animals in each group were pregnant, which reduced the sample of the groups to P12 (n = 8) and P18 (n = 8). The non-pregnant group was also re-divided into two groups at the end of synchronization: NP12 (n = 7) and NP18 (n = 7). The UF samples were processed for proteomic analysis. The results were submitted to multivariate and univariate analysis. A total of 1068 proteins were found in the uterine fluid in both groups. Our results describe proteins involved in the conceptus elongation and maternal recognition of pregnancy, and their action was associated with cell growth, endometrial remodeling, and modulation of immune and antioxidant protection, mechanisms necessary for embryonic maintenance in the uterine environment. SIGNIFICANCE: Uterine fluid is a substance synthesized and secreted by the endometrium that plays essential roles during pregnancy in ruminants, contributing significantly to embryonic development. Understanding the functions that the proteins present in the UF perform during early pregnancy, a period marked by embryonic implantation, and maternal recognition of pregnancy is of fundamental importance to understanding the mechanisms necessary for the maintenance of pregnancy. The present study characterized and compared the UF proteome at the beginning of pregnancy in pregnant and non-pregnant buffaloes to correlate the functions of the proteins and the stage of development of the conceptus and unravel their processes in maternal recognition of pregnancy. The proteins found were involved in cell growth and endometrial remodeling, in addition to acting in the immunological protection of the conceptus and performing antioxidant actions necessary for establishing a pregnancy.

Using design of experiments (DoE) to optimize performance and stability of biomimetic cell membrane-coated nanostructures for cancer therapy.

Introduction: Cell membrane-covered biomimetic nanosystems have allowed the development of homologous nanostructures to bestow nanoparticles with enhanced biointerfacing capabilities. The stability of these structures, however, still represents a challenge for the scientific community. This study is aimed at developing and optimizing cell derived membrane-coated nanostructures upon applying design of experiments (DoE) to improve the therapeutic index by homotypic targeting in cancer cells. Methods: Important physicochemical features of the extracted cell membrane from tumoral cells were assessed by mass spectrometry-based proteomics. PLGA-based nanoparticles encapsulating temozolomide (TMZ NPs) were successfully developed. The coating technology applying the isolated U251 cell membrane (MB) was optimized using a fractional two-level three-factor factorial design. All the formulation runs were systematically characterized regarding their diameter, polydispersity index (PDI), and zeta potential (ZP). Experimental conditions generated by DoE were also subjected to morphological studies using negative-staining transmission electron microscopy (TEM). Its short-time stability was also assessed. MicroRaman and Fourier-Transform Infrared (FTIR) spectroscopies and Confocal microscopy were used as characterization techniques for evaluating the NP-MB nanostructures. Internalization studies were carried out to evaluate the homotypic targeting ability. Results and Discussion: The results have shown that nearly 80% of plasma membrane proteins were retained in the cell membrane vesicles after the isolation process, including key proteins to the homotypic binding. DoE analysis considering acquired TEM images reveals that condition run five should be the best-optimized procedure to produce the biomimetic cell-derived membrane-coated nanostructure (NP-MB). Storage stability for at least two weeks of the biomimetic system is expected once the original characteristics of diameter, PDI, and ZP, were maintained. Raman, FTIR, and confocal characterization results have shown the successful encapsulation of TMZ drug and provided evidence of the effective coating applying the MB. Cell internalization studies corroborate the proteomic data indicating that the optimized NP-MB achieved specific targeting of homotypic tumor cells. The structure should retain the complex biological functions of U251 natural cell membranes while exhibiting physicochemical properties suitable for effective homotypic recognition. Conclusion: Together, these findings provide coverage and a deeper understanding regarding the dynamics around extracted cell membrane and polymeric nanostructures interactions and an in-depth insight into the cell membrane coating technology and the development of optimized biomimetic and bioinspired nanostructured systems.

Fungal consortium of two Beauveria bassiana strains increases their virulence, growth, and resistance to stress: A metabolomic approach.

The use of two or more microorganisms in a microbial consortium has been increasingly applied in the biological control of diseases and pests. Beauveria bassiana is one of the most widely studied fungal species in biological control, yet little is known about its role in fungal consortiums. In a previous study, our group found that a consortium formed by two strains of B. bassiana had significantly greater biocontrol potential against the polyphagous caterpillars Duponchelia fovealis (Lepidoptera: Crambidae) than either strain on its own. In this study, we use GC-MS and LC-MS/MS to evaluate and discuss the metabolomics of the consortium. A total of 21 consortium biomarkers were identified, corresponding to 14 detected by LC-MS/MS and seven by GC-MS. Antioxidant and anti-inflammatory mechanisms are the main properties of the metabolites produced by the consortium. These metabolites can depress the insect's immune system, increasing its vulnerability and, hence, the fungal virulence of the consortium. In light of these results, we propose an action model of insect mortality due to the metabolites secreted by the consortium. The model includes the inhibition of defense mechanisms such as pro-inflammatory interleukin secretion, cell migration, cell aggregation, Dif, Dorsal and Relish gene transcription, and JAK/STAT and JNK signaling pathways. It also promotes the cleaning of oxidative molecules, like ROS, NOS, and H2O2, and the induction of virulence factors.

Proteomics Reveals an Increase in the Abundance of Glycolytic and Ethanolic Fermentation Enzymes in Developing Sugarcane Culms During Sucrose Accumulation.

Sugarcane is an economically important crop contributing to the sugar and ethanol production of the world with 80 and 40%, respectively. Despite its importance as the main crop for sugar production, the mechanisms involved in the regulation of sucrose accumulation in sugarcane culms are still poorly understood. The aim of this work was to compare the quantitative changes of proteins in juvenile and maturing internodes at three stages of plant development. Label-free shotgun proteomics was used for protein profiling and quantification in internodes 5 (I5) and 9 (I9) of 4-, 7-, and 10-month-old-plants (4M, 7M, and 10M, respectively). The I9/I5 ratio was used to assess the differences in the abundance of common proteins at each stage of internode development. I9 of 4M plants showed statistically significant increases in the abundance of several enzymes of the glycolytic pathway and proteoforms of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC). The changes in content of the enzymes were followed by major increases of proteins related to O2 transport like hemoglobin 2, ROS scavenging enzymes, and enzymes involved in the ascorbate/glutatione system. Besides, intermediates from tricarboxylic acid cycle (TCA) were reduced in I9-4M, indicating that the increase in abundance of several enzymes involved in glycolysis, pentose phosphate cycle, and TCA, might be responsible for higher metabolic flux, reducing its metabolites content. The results observed in I9-4M indicate that hypoxia might be the main cause of the increased flux of glycolysis and ethanolic fermentation to supply ATP and reducing power for plant growth, mitigating the reduction in mitochondrial respiration due to the low oxygen availability inside the culm. As the plant matured and sucrose accumulated to high levels in the culms, the proteins involved in glycolysis, ethanolic fermentation, and primary carbon metabolism were significantly reduced.

Revealing the high variability on nonconserved core and mobile elements of Austropuccinia psidii and other rust mitochondrial genomes.

Mitochondrial genomes are highly conserved in many fungal groups, and they can help characterize the phylogenetic relationships and evolutionary biology of plant pathogenic fungi. Rust fungi are among the most devastating diseases for economically important crops around the world. Here, we report the complete sequence and annotation of the mitochondrial genome of Austropuccinia psidii (syn. Puccinia psidii), the causal agent of myrtle rust. We performed a phylogenomic analysis including the complete mitochondrial sequences from other rust fungi. The genome composed of 93.299 bp has 73 predicted genes, 33 of which encoded nonconserved proteins (ncORFs), representing almost 45% of all predicted genes. A. psidii mtDNA is one of the largest rust mtDNA sequenced to date, most likely due to the abundance of ncORFs. Among them, 33% were within intronic regions of diverse intron groups. Mobile genetic elements invading intron sequences may have played significant roles in size but not shaping of the rust mitochondrial genome structure. The mtDNAs from rust fungi are highly syntenic. Phylogenetic inferences with 14 concatenated mitochondrial proteins encoded by the core genes placed A. psidii according to phylogenetic analysis based on 18S rDNA. Interestingly, cox1, the gene with the greatest number of introns, provided phylogenies not congruent with the core set. For the first time, we identified the proteins encoded by three A. psidii ncORFs using proteomics analyses. Also, the orf208 encoded a transmembrane protein repressed during in vitro morphogenesis. To the best of our knowledge, we presented the first report of a complete mtDNA sequence of a member of the family Sphaerophragmiacea.

Network Analysis Combining Proteomics and Metabolomics Reveals New Insights Into Early Responses of Eucalyptus grandis During Rust Infection.

Eucalyptus rust is caused by the biotrophic fungus, Austropuccinia psidii, which affects commercial plantations of Eucalyptus, a major raw material for the pulp and paper industry in Brazil. In this manuscript we aimed to uncover the molecular mechanisms involved in rust resistance and susceptibility in Eucalyptus grandis. Epifluorescence microscopy was used to follow the fungus development inside the leaves of two contrasting half-sibling genotypes (rust-resistance and rust-susceptible), and also determine the comparative time-course of changes in metabolites and proteins in plants inoculated with rust. Within 24 h of complete fungal invasion, the analysis of 709 metabolomic features showed the suppression of many metabolites 6 h after inoculation (hai) in the rust-resistant genotype, with responses being induced after 12 hai. In contrast, the rust-susceptible genotype displayed more induced metabolites from 0 to 18 hai time-points, but a strong suppression occurred at 24 hai. Multivariate analyses of genotypes and time points were used to select 16 differential metabolites mostly classified as phenylpropanoid-related compounds. Applying the Weighted Gene Co-Expression Network Analysis (WGCNA), rust-resistant and rust-susceptible genotypes had, respectively, 871 and 852 proteins grouped into 5 and 6 modules, of which 5 and 4 of them were significantly correlated to the selected metabolites. Functional analyses revealed roles for photosynthesis and oxidative-dependent responses leading to temporal activity of metabolites and related enzymes after 12 hai in rust-resistance; while the initial over-accumulation of those molecules and suppression of supporting mechanisms at 12 hai caused a lack of progressive metabolite-enzyme responses after 12 hai in rust-susceptible genotype. This study provides some insights on how E. grandis plants are functionally modulated to integrate secondary metabolites and related enzymes from phenylpropanoid pathway and lead to temporal divergences of resistance and susceptibility responses to rust.