Expression profiling of Clostridium thermocellum B8 during the deconstruction of sugarcane bagasse and straw.

The gram-positive bacterium Clostridium thermocellum contains a set of carbohydrate-active enzymes that can potentially be employed to generate high-value-added products from lignocellulose. In this study, the gene expression profiling of C. thermocellum B8 was provided during growth in the presence of sugarcane bagasse and straw as a carbon source in comparison to growth using microcrystalline cellulose. A total of 625 and 509 genes were up-regulated for growth in the presence of bagasse and straw, respectively. These genes were mainly grouped into carbohydrate-active enzymes (CAZymes), cell motility, chemotaxis, quorum sensing pathway and expression control of glycoside hydrolases. These results show that type of carbon source modulates the gene expression profiling of carbohydrate-active enzymes. In addition, highlight the importance of cell motility, attachment to the substrate and communication in deconstructing complex substrates. This present work may contribute to the development of enzymatic cocktails and industrial strains for biorefineries based on sugarcane residues as feedstock.

Aedes aegypti Aag-2 Cell Proteome Modulation in Response to Chikungunya Virus Infection.

Chikungunya virus (CHIKV) is a single-stranded positive RNA virus that belongs to the genus Alphavirus and is transmitted to humans by infected Aedes aegypti and Aedes albopictus bites. In humans, CHIKV usually causes painful symptoms during acute and chronic stages of infection. Conversely, virus-vector interaction does not disturb the mosquito's fitness, allowing a persistent infection. Herein, we studied CHIKV infection of Ae. aegypti Aag-2 cells (multiplicity of infection (MOI) of 0.1) for 48 h through label-free quantitative proteomic analysis and transmission electron microscopy (TEM). TEM images showed a high load of intracellular viral cargo at 48 h postinfection (hpi), as well as an unusual elongated mitochondria morphology that might indicate a mitochondrial imbalance. Proteome analysis revealed 196 regulated protein groups upon infection, which are related to protein synthesis, energy metabolism, signaling pathways, and apoptosis. These Aag-2 proteins regulated during CHIKV infection might have roles in antiviral and/or proviral mechanisms and the balance between viral propagation and the survival of host cells, possibly leading to the persistent infection.

Two viruses from Stylosanthes guianensis may represent a new genus within Potyviridae.

Forage crops occupy large areas of tropical pastures for cattle feeding in Brazil. The use of stylos (Stylosanthes spp.) in these pastures, which are leguminous shrubs, has increased in the country due to their outstanding nutritional value and for being an efficient and alternative source for nitrogen fixation in the soil. In recent years, virus-like mosaic symptoms on S.guianensis leaves have often been observed in the field, indicating possible virus-like pathogen infections. In an effort to identify the causal agent, virus semi-purification protocol was performed using symptomatic S. guianensis leaves collected at EMBRAPA Beef Cattle Research Center. Total RNA extracted from this semi-purified preparation was submitted to high-throughput sequencing, which revealed complete genome sequences of novel viruses of the family Potyviridae. These viruses, tentatively named stylo mosaic-associated virus 1 (StyMaV-1) and stylo mosaic-associated virus 2 (StyMaV-2), shared 73 % CP aa identity and 77 % polyprotein aa identity with each other and, after that, being closest related to blackberry virus Y, genus Brambyvirus (only 41 % CP aa identity). Based on ICTV genus demarcation criteria, StyMaV-1 and StyMaV-2 represent new species of a new genus within the family Potyviridae. StyMaV-1 and StyMaV-2 are also not efficiently transmitted to other plant species by mechanical inoculation.

Dynamic proteomic analysis of Aedes aegypti Aag-2 cells infected with Mayaro virus.

BACKGROUND: Mayaro virus (MAYV) is responsible for a mosquito-borne tropical disease with clinical symptoms similar to dengue or chikungunya virus fevers. In addition to the recent territorial expansion of MAYV, this virus may be responsible for an increasing number of outbreaks. Currently, no vaccine is available. Aedes aegypti is promiscuous in its viral transmission and thus an interesting model to understand MAYV-vector interactions. While the life-cycle of MAYV is known, the mechanisms by which this arbovirus affects mosquito host cells are not clearly understood. METHODS: After defining the best conditions for cell culture harvesting using the highest virus titer, Ae. aegypti Aag-2 cells were infected with a Brazilian MAYV isolate at a MOI of 1 in order to perform a comparative proteomic analysis of MAYV-infected Aag-2 cells by using a label-free semi-quantitative bottom-up proteomic analysis. Time-course analyses were performed at 12 and 48 h post-infection (hpi). After spectrum alignment between the triplicates of each time point and changes of the relative abundance level calculation, the identified proteins were annotated and using Gene Ontology database and protein pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes. RESULTS: After three reproducible biological replicates, the total proteome analysis allowed for the identification of 5330 peptides and the mapping of 459, 376 and 251 protein groups, at time 0, 12 hpi and 48 hpi, respectively. A total of 161 mosquito proteins were found to be differentially abundant during the time-course, mostly related to host cell processes, including redox metabolism, translation, energy metabolism, and host cell defense. MAYV infection also increased host protein expression implicated in viral replication. CONCLUSIONS: To our knowledge, this first proteomic time-course analysis of MAYV-infected mosquito cells sheds light on the molecular basis of the viral infection process and host cell response during the first 48 hpi. Our data highlight several mosquito proteins modulated by the virus, revealing that MAYV manipulates mosquito cell metabolism for its propagation.

Tomato Chlorotic Spot Virus (TCSV) Putatively Incorporated a Genomic Segment of Groundnut Ringspot Virus (GRSV) Upon a Reassortment Event.

Tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV) share several genetic and biological traits. Both of them belong to the genus Tospovirus (family Peribunyaviridae), which is composed by viruses with tripartite RNA genome that infect plants and are transmitted by thrips (order Thysanoptera). Previous studies have suggested several reassortment events between these two viruses, and some speculated that they may share one of their genomic segments. To better understand the intimate evolutionary history of these two viruses, we sequenced the genomes of the first TCSV and GRSV isolates ever reported. Our analyses show that TCSV and GRSV isolates indeed share one of their genomic segments, suggesting that one of those viruses may have emerged upon a reassortment event. Based on a series of phylogenetic and nucleotide diversity analyses, we conclude that the parental genotype of the M segment of TCSV was either eliminated due to a reassortment with GRSV or it still remains to be identified.

Identification and genome analysis of tomato chlorotic spot virus and dsRNA viruses from coinfected vegetables in the Dominican Republic by high-throughput sequencing.

The Tomato chlorotic spot virus (TCSV) was first reported in the 1980s, having its occurrence limited to Brazil and Argentina. Due to an apparent mild severity in the past, molecular studies concerning TCSV were neglected. However, TCSV has disseminated over the USA and Caribbean countries. In Dominican Republic TCSV has been recently reported on important cultivated crops such as pepper and beans. In this work, we provide the first complete genome of a TCSV isolate from symptomatic plants in Dominican Republic, which was obtained by high-throughput sequencing. In addition, three dsRNA viruses from different virus families were identified coinfecting these plants Bell pepper endornavirus (BPEV), Southern tomato virus (STV) and Pepper cryptic virus 2 (PCV-2). Phylogenetic analysis showed that the Dominican Republic TCSV isolate has a close relationship with other TCSV isolates and a reassortant isolate between TCSV and Groundnut ringspot virus (GRSV), all found in USA. BPEV, STV and PCV-2 isolates from Dominican Republic were close related to corresponding American isolates. The possible biological implications of these virus-mixed infections are discussed.

The NSm proteins of phylogenetically related tospoviruses trigger Sw-5b-mediated resistance dissociated of their cell-to-cell movement function.

The cell-to-cell movement protein (NSM) of tomato spotted wilt virus (TSWV) has been recently identified as the effector of the single dominant Sw-5b resistance gene from tomato (Solanum lycopersicum L.). Although most TSWV isolates shows a resistance-inducing (RI) phenotype, regular reports have appeared on the emergence of resistance-breaking (RB) isolates in tomato fields, and suggested a strong association with two point mutations (C118Y and T120N) in the NSM protein. In this study the Sw-5b gene has been demonstrated to confer not only resistance against TSWV but to members of five additional, phylogenetically-related classified within the so-called "American" evolutionary clade, i.e., Alstroemeria necrotic streak virus (ANSV), chrysanthemum stem necrosis virus (CSNV), groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV) and tomato chlorotic spot virus (TCSV). Remarkably, bean necrotic mosaic virus (BeNMV), a recently discovered tospovirus classified in a distinct American subclade and circulating on the American continent, did not trigger a Sw-5b-mediated hypersensitive (HR) response. Introduction of point mutations C118Y and T120N into the NSM protein of TSWV, TCSV and CSNV abrogated the ability to trigger Sw-5b-mediated HR in both transgenic-N. benthamiana and tomato isolines harboring the Sw-5b gene whereas it had no effect on BeNMV NSM. Truncated versions of TSWV NSM lacking motifs associated with tubule formation, cell-to-cell or systemic viral movement were made and tested for triggering of resistance. HR was still observed with truncated NSM proteins lacking 50 amino acids (out of 301) from either the amino- or carboxy-terminal end. These data altogether indicate the importance of amino acid residues C118 and T120 in Sw-5b-mediated HR only for the NSM proteins from one cluster of tospoviruses within the American clade, and that the ability to support viral cell-to-cell movement is not required for effector functionality.

Cell death triggering and effector recognition by Sw-5 SD-CNL proteins from resistant and susceptible tomato isolines to Tomato spotted wilt virus.

Only a limited number of dominant resistance genes acting against plant viruses have been cloned, and further functional studies of these have been almost entirely limited to the resistance genes Rx against Potato virus X (PVX) and N against Tobacco mosaic virus (TMV). Recently, the cell-to-cell movement protein (NSM ) of Tomato spotted wilt virus (TSWV) has been identified as the avirulence determinant (Avr) of Sw-5b-mediated resistance, a dominant resistance gene which belongs to the class of SD-CC-NB-LRR (Solanaceae domain-coiled coil-nucleotide-binding-leucine-rich repeat, SD-CNL) resistance genes. On transient expression of the NSM protein in tomato and transgenic Nicotiana benthamiana harbouring the Sw-5b gene, a hypersensitive cell death response (HR) is triggered. Here, it is shown that high accumulation of the Sw-5b protein in N. benthamiana leaves, achieved by co-expression of the Sw-5b protein with RNA silencing suppressors (RSSs), leads to auto-activity in the absence of NSM . In a similar approach, Sw-5a, the highest conserved paralogue of Sw-5b from Solanum peruvianum, also triggered HR by auto-activation, whereas the highest conserved orthologue from susceptible S. lycopersicum, named Sw-5aS , did not. However, neither of the last two homologues was able to trigger an NSM -dependent HR. Truncated and mutated versions of these Sw-5 proteins revealed that the NB-ARC [nucleotide-binding adaptor shared by Apaf-1 (from humans), R proteins and CED-4 (from nematodes)] domain is sufficient for the triggering of HR and seems to be suppressed by the SD-CC domain. Furthermore, a single mutation was sufficient to restore auto-activity within the NB-ARC domain of Sw-5aS . When the latter domain was fused to the Sw-5b LRR domain, NSM -dependent HR triggering was regained, but not in the presence of its own Sw-5aS LRR domain. Expression analysis in planta revealed a nucleocytoplasmic localization pattern of Sw-5b, in which the SD-CC domain seems to be required for nuclear translocation. Although the Sw-5 N-terminal CC domain, in contrast with Rx, contains an additional SD, most findings from this study support a conserved role of domains within NB-LRR (NLR) proteins against plant viruses.

The Tomato spotted wilt virus cell-to-cell movement protein (NSM ) triggers a hypersensitive response in Sw-5-containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw-5b resistance gene copy.

Although the Sw-5 gene cluster has been cloned, and Sw-5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum 'SR1' plants transformed with a copy of the Sw-5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw-5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non-structural TSWV proteins tested, the TSWV cell-to-cell movement protein (NSM ) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non-replicative pEAQ-HT expression vector system. HR was induced in Sw-5b-transgenic N. benthamiana as well as in resistant near-isogenic tomato lines after agroinfiltration with a functional cell-to-cell movement protein (NSM ) from a resistance-inducing (RI) TSWV strain (BR-01), but not with NSM from a Sw-5 resistance-breaking (RB) strain (GRAU). This is the first biological demonstration that Sw-5-mediated resistance is triggered by the TSWV NSM cell-to-cell movement protein.

Characterization of bean necrotic mosaic virus: a member of a novel evolutionary lineage within the Genus Tospovirus.

BACKGROUND: Tospoviruses (Genus Tospovirus, Family Bunyaviridae) are phytopathogens responsible for significant worldwide crop losses. They have a tripartite negative and ambisense RNA genome segments, termed S (Small), M (Medium) and L (Large) RNA. The vector-transmission is mediated by thrips in a circulative-propagative manner. For new tospovirus species acceptance, several analyses are needed, e.g., the determination of the viral protein sequences for enlightenment of their evolutionary history. METHODOLOGY/PRINCIPAL FINDINGS: Biological (host range and symptomatology), serological, and molecular (S and M RNA sequencing and evolutionary studies) experiments were performed to characterize and differentiate a new tospovirus species, Bean necrotic mosaic virus (BeNMV), which naturally infects common beans in Brazil. Based upon the results, BeNMV can be classified as a novel species and, together with Soybean vein necrosis-associated virus (SVNaV), they represent members of a new evolutionary lineage within the genus Tospovirus. CONCLUSION/SIGNIFICANCES: Taken together, these evidences suggest that two divergent lineages of tospoviruses are circulating in the American continent and, based on the main clades diversity (American and Eurasian lineages), new tospovirus species related to the BeNMV-SVNaV clade remain to be discovered. This possible greater diversity of tospoviruses may be reflected in a higher number of crops as natural hosts, increasing the economic impact on agriculture. This idea also is supported since BeNMV and SVNaV were discovered naturally infecting atypical hosts (common bean and soybean, respectively), indicating, in this case, a preference for leguminous species. Further studies, for instance a survey focusing on crops, specifically of leguminous plants, may reveal a greater tospovirus diversity not only in the Americas (where both viruses were reported), but throughout the world.

An RNA-dependent RNA polymerase gene of a distinct Brazilian tospovirus.

The tospoviral RNA-dependent RNA polymerases (RdRp), or L proteins, perform several conserved functions during virus replication in host cells. In this study, an L segment sequence of 9,040 bp from a new tospovirus (family Bunyaviridae) naturally infecting bean (Phaseolus vulgaris L.) plants was characterized. It encodes the largest RdRp gene known yet for this genus, with deduced 2932aa and a molecular mass of approximately 336 kDa. A Lysine-rich C-terminal extension was found, which apart from our isolate, was only recognized in another recently discovered tospovirus infecting Fabaceae, Soybean vein necrosis associated virus (SVNaV). Due to its distinct biological features and L protein-based phylogenetic analysis showing an almost equidistant position in comparison to Eurasian and American Tospovirus groups, as well as the clustering with SVNaV, we suggest the tentative name Bean necrotic mosaic virus for this unique isolate.