Sequencing of small RNAs of the fern Pleopeltis minima (Polypodiaceae) offers insight into the evolution of the microrna repertoire in land plants.

MicroRNAs (miRNAs) are short, single stranded RNA molecules that regulate the stability and translation of messenger RNAs in diverse eukaryotic groups. Several miRNA genes are of ancient origin and have been maintained in the genomes of animal and plant taxa for hundreds of millions of years, playing key roles in development and physiology. In the last decade, genome and small RNA (sRNA) sequencing of several plant species have helped unveil the evolutionary history of land plants. Among these, the fern group (monilophytes) occupies a key phylogenetic position, as it represents the closest extant cousin taxon of seed plants, i.e. gymno- and angiosperms. However, in spite of their evolutionary, economic and ecological importance, no fern genome has been sequenced yet and few genomic resources are available for this group. Here, we sequenced the small RNA fraction of an epiphytic South American fern, Pleopeltis minima (Polypodiaceae), and compared it to plant miRNA databases, allowing for the identification of miRNA families that are shared by all land plants, shared by all vascular plants (tracheophytes) or shared by euphyllophytes (ferns and seed plants) only. Using the recently described transcriptome of another fern, Lygodium japonicum, we also estimated the degree of conservation of fern miRNA targets in relation to other plant groups. Our results pinpoint the origin of several miRNA families in the land plant evolutionary tree with more precision and are a resource for future genomic and functional studies of fern miRNAs.

Genome and transcriptome analysis of the Mesoamerican common bean and the role of gene duplications in establishing tissue and temporal specialization of genes.

BACKGROUND: Legumes are the third largest family of angiosperms and the second most important crop class. Legume genomes have been shaped by extensive large-scale gene duplications, including an approximately 58 million year old whole genome duplication shared by most crop legumes. RESULTS: We report the genome and the transcription atlas of coding and non-coding genes of a Mesoamerican genotype of common bean (Phaseolus vulgaris L., BAT93). Using a comprehensive phylogenomics analysis, we assessed the past and recent evolution of common bean, and traced the diversification of patterns of gene expression following duplication. We find that successive rounds of gene duplications in legumes have shaped tissue and developmental expression, leading to increased levels of specialization in larger gene families. We also find that many long non-coding RNAs are preferentially expressed in germ-line-related tissues (pods and seeds), suggesting that they play a significant role in fruit development. Our results also suggest that most bean-specific gene family expansions, including resistance gene clusters, predate the split of the Mesoamerican and Andean gene pools. CONCLUSIONS: The genome and transcriptome data herein generated for a Mesoamerican genotype represent a counterpart to the genomic resources already available for the Andean gene pool. Altogether, this information will allow the genetic dissection of the characters involved in the domestication and adaptation of the crop, and their further implementation in breeding strategies for this important crop.

Structure, composition and metagenomic profile of soil microbiomes associated to agricultural land use and tillage systems in Argentine Pampas.

Agriculture is facing a major challenge nowadays: to increase crop production for food and energy while preserving ecosystem functioning and soil quality. Argentine Pampas is one of the main world producers of crops and one of the main adopters of conservation agriculture. Changes in soil chemical and physical properties of Pampas soils due to different tillage systems have been deeply studied. Still, not much evidence has been reported on the effects of agricultural practices on Pampas soil microbiomes. The aim of our study was to investigate the effects of agricultural land use on community structure, composition and metabolic profiles on soil microbiomes of Argentine Pampas. We also compared the effects associated to conventional practices with the effects of no-tillage systems. Our results confirmed the impact on microbiome structure and composition due to agricultural practices. The phyla Verrucomicrobia, Plactomycetes, Actinobacteria, and Chloroflexi were more abundant in non cultivated soils while Gemmatimonadetes, Nitrospirae and WS3 were more abundant in cultivated soils. Effects on metabolic metagenomic profiles were also observed. The relative abundance of genes assigned to transcription, protein modification, nucleotide transport and metabolism, wall and membrane biogenesis and intracellular trafficking and secretion were higher in cultivated fertilized soils than in non cultivated soils. We also observed significant differences in microbiome structure and taxonomic composition between soils under conventional and no-tillage systems. Overall, our results suggest that agronomical land use and the type of tillage system have induced microbiomes to shift their life-history strategies. Microbiomes of cultivated fertilized soils (i.e. higher nutrient amendment) presented tendencies to copiotrophy while microbiomes of non cultivated homogenous soils appeared to have a more oligotrophic life-style. Additionally, we propose that conventional tillage systems may promote copiotrophy more than no-tillage systems by decreasing soil organic matter stability and therefore increasing nutrient availability.

A first insight into Pycnoporus sanguineus BAFC 2126 transcriptome.

Fungi of the genus Pycnoporus are white-rot basidiomycetes widely studied because of their ability to synthesize high added-value compounds and enzymes of industrial interest. Here we report the sequencing, assembly and analysis of the transcriptome of Pycnoporus sanguineus BAFC 2126 grown at stationary phase, in media supplemented with copper sulfate. Using the 454 pyrosequencing platform we obtained a total of 226,336 reads (88,779,843 bases) that were filtered and de novo assembled to generate a reference transcriptome of 7,303 transcripts. Putative functions were assigned for 4,732 transcripts by searching similarities of six-frame translated sequences against a customized protein database and by the presence of conserved protein domains. Through the analysis of translated sequences we identified transcripts encoding 178 putative carbohydrate active enzymes, including representatives of 15 families with roles in lignocellulose degradation. Furthermore, we found many transcripts encoding enzymes related to lignin hydrolysis and modification, including laccases and peroxidases, as well as GMC oxidoreductases, copper radical oxidases and other enzymes involved in the generation of extracellular hydrogen peroxide and iron homeostasis. Finally, we identified the transcripts encoding all of the enzymes involved in terpenoid backbone biosynthesis pathway, various terpene synthases related to the biosynthesis of sesquiterpenoids and triterpenoids precursors, and also cytochrome P450 monooxygenases, glutathione S-transferases and epoxide hydrolases with potential functions in the biodegradation of xenobiotics and the enantioselective biosynthesis of biologically active drugs. To our knowledge this is the first report of a transcriptome of genus Pycnoporus and a resource for future molecular studies in P. sanguineus.

Transgenic sweet orange plants expressing a dermaseptin coding sequence show reduced symptoms of citrus canker disease.

Citrus canker provoked by Xanthomonas axonopodis pv. citri is a bacterial disease causing severe losses in all citrus-producing areas around the world. Xanthomonas infection is considered as an endemic disease in Northeast and Northwest Argentina, affecting as much as 10% of commercial citrus plantations. There is not known natural resistance neither in orange varieties nor in rootstocks used for grafting of commercial cultivars. To introduce resistance to this disease, plants of Pineapple sweet orange were transformed with a genetic construct allowing constitutive accumulation of dermaseptin. In comparison with non-transformed plants, transgenic plants showed symptom reduction levels of up to 50% in in planta assays performed under controlled conditions.

The PAMPA datasets: a metagenomic survey of microbial communities in Argentinean pampean soils.

BACKGROUND: Soil is among the most diverse and complex environments in the world. Soil microorganisms play an essential role in biogeochemical cycles and affect plant growth and crop production. However, our knowledge of the relationship between species-assemblies and soil ecosystem processes is still very limited. The aim of this study was to generate a comprehensive metagenomic survey to evaluate the effect of high-input agricultural practices on soil microbial communities. RESULTS: We collected soil samples from three different areas in the Argentinean Pampean region under three different types of land uses and two soil sources (bulk and rhizospheric). We extracted total DNA from all samples and also synthetized cDNA from rhizospheric samples. Using 454-FLX technology, we generated 112 16S ribosomal DNA and 14 16S ribosomal RNA amplicon libraries totaling 1.3 M reads and 36 shotgun metagenome libraries totaling 17.8 million reads (7.7 GB). Our preliminary results suggested that water availability could be the primary driver that defined microbial assemblages over land use and soil source. However, when water was not a limiting resource (annual precipitation >800 mm) land use was a primary driver. CONCLUSION: This was the first metagenomic study of soil conducted in Argentina and our datasets are among the few large soil datasets publicly available. The detailed analysis of these data will provide a step forward in our understanding of how soil microbiomes respond to high-input agricultural systems, and they will serve as a useful comparison with other soil metagenomic studies worldwide.

Stacking of antimicrobial genes in potato transgenic plants confers increased resistance to bacterial and fungal pathogens.

Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens.

Field testing, gene flow assessment and pre-commercial studies on transgenic Solanum tuberosum spp. tuberosum (cv. Spunta) selected for PVY resistance in Argentina.

Solanum tuberosum ssp. tuberosum (cv. Spunta) was transformed with a chimeric transgene containing the Potato virus Y (PVY) coat protein (CP) sequence. Screening for PVY resistance under greenhouse conditions yielded over 100 independent candidate lines. Successive field testing of selected lines allowed the identification of two genetically stable PVY-resistant lines, SY230 and SY233, which were further evaluated in field trials at different potato-producing regions in Argentina. In total, more than 2,000 individuals from each line were tested along a 6-year period. While no or negligible PVY infection was observed in the transgenic lines, infection rates of control plants were consistently high and reached levels of up to 70-80%. Parallel field studies were performed in virus-free environments to assess the agronomical performance of the selected lines. Tubers collected from these assays exhibited agronomical traits and biochemical compositions indistinguishable from those of the non-transformed Spunta cultivar. In addition, an interspecific out-crossing trial to determine the magnitude of possible natural gene flow between transgenic line SY233 and its wild relative Solanum chacoense was performed. This trial yielded negative results, suggesting an extremely low probability for such an event to occur.

Potato virus X coat protein fusion to human papillomavirus 16 E7 oncoprotein enhance antigen stability and accumulation in tobacco chloroplast.

Cervical cancer linked to infection with human papillomavirus (HPV) is the third cause of cancer-related death in women. As the virus cannot be propagated in culture, vaccines have been based on recombinant antigens with inherited high-cost production. In a search of alternative cheap production system, E7 HPV type 16 protein, an attractive candidate for anticancer vaccine development, was engineered to be expressed in tobacco chloroplast. In addition, E7 coding sequence was fused to potato virus X coat protein (CP) to compare expression level. Results show that E7CP transcript accumulation reached lower levels than non-fused E7. However, antigen expression levels were higher for fusion protein indicating that CP stabilizes E7 peptide in the chloroplast stroma. These results support viability of transplastomic plants for antigen production and the relevance of improving recombinant peptide stability for certain transgenes to enhance protein accumulation in this organelle.

Phosphorylation of the TGBp1 movement protein of Potato virus X by a Nicotiana tabacum CK2-like activity.

The movement protein (MP) TGBp1 of the potexvirus Potato virus X (PVX) is a multifunctional protein required for cell-to-cell movement within the host plant. Recent work on other plant viruses has indicated that MP phosphorylation by host kinases can regulate MP function. In this study, we demonstrate that recombinant and native TGBp1 are phosphorylated by Nicotiana tabacum extracts from both PVX-infected and non-infected leaves. The phosphorylation activity present in plant extracts has distinctive characteristics of casein kinase 2 (CK2): it is inhibited by heparin, stimulated by polylysine, and uses either ATP or GTP as phosphoryl donors. We also demonstrate that TGBp1 is efficiently phosphorylated by recombinant tobacco CK2 alpha subunit and by partially purified tobacco CK2. Phosphopeptide mass mapping reveals that TGBp1 is phosphorylated in Ser-165, which is localized within a CK2 consensus sequence. Our results strongly suggest that a N. tabacum kinase of the CK2 family is involved in TBGp1 phosphorylation during the course of viral infection.

Efficient expression of a Toxoplasma gondii dense granule Gra4 antigen in tobacco leaves.

A His-tagged truncated version of Toxoplasma gondii dense granule 4 protein (Gra4(163-345)) was transiently expressed in tobacco leaves. Two genetic constructions were used to accomplish this goal. In one of them, based in a Potato virus X (PVX) amplicon, the sequence encoding His-Gra4(163-345) was placed under control of an additional PVX coat protein subgenomic promoter. In the other, the same sequence was fused to an apoplastic transport signal and placed under the direction of the cauliflower mosaic virus 35S promoter. His-Gra4(163-345) accumulation in agroinfiltrated tobacco leaves was estimated by Western blot analysis using mouse anti-Gra4 antibody and a seropositive human serum. Here, we demonstrated the feasibility of producing a Gra4 antigen using transient expression methods in plants.

Plant cell proliferation inside an inorganic host.

In recent years, much attention has been paid to plant cell culture as a tool for the production of secondary metabolites and the expression of recombinant proteins. Plant cell immobilization offers many advantages for biotechnological processes. However, the most extended matrices employed, such as calcium-alginate, cannot fully protect entrapped cells. Sol-gel chemistry of silicates has emerged as an outstanding strategy to obtain biomaterials in which living cells are truly protected. This field of research is rapidly developing and a large number of bacteria and yeast-entrapping ceramics have already been designed for different applications. But even mild thermal and chemical conditions employed in sol-gel synthesis may result harmful to cells of higher organisms. Here we present a method for the immobilization of plant cells that allows cell growth at cavities created inside a silica matrix. Plant cell proliferation was monitored for a 6-month period, at the end of which plant calli of more than 1 mm in diameter were observed inside the inorganic host. The resulting hybrid device had good mechanical stability and proved to be an effective barrier against biological contamination, suggesting that it could be employed for long-term plant cell entrapment applications.

Accumulation of hEGF and hEGF-fusion proteins in chloroplast-transformed tobacco plants is higher in the dark than in the light.

Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it has been reported that heterologous protein accumulation in chloroplasts could be hindered by post-transcriptional mechanisms not yet characterized. Here, we describe the development and characterization of transplastomic tobacco plants transformed with four different transformation vectors for the expression of human epidermal growth factor (hEGF). We showed that, although the corresponding transcript was present in all of the analyzed plants, hEGF could only be detected when fused to the first 186 amino acids of bacterial beta-glucuronidase (GUS). In addition, we observed that the expression levels of recombinant protein increased when plants were placed in the dark or when leaves were incubated in the presence of electron transport inhibitors, such as methyl viologen (MV) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These results suggest that the mechanism responsible for hEGF instability in chloroplasts is regulated by light.

Expression of tuberculosis antigen ESAT-6 in Nicotiana tabacum using a potato virus X-based vector.

A good candidate antigen to create a therapeutic vaccine against TB is the ESAT-6 protein. Antigens produced in plants have already been successfully used as experimental vaccines, and small single-stranded RNA plant viruses have emerged as promising tools to rapidly express large amounts of foreign proteins in susceptible host plants. Here, we present the expression of ESAT-6 protein in Nicotiana tabacum using a vector based on potato virus X (PVX). The complete ESAT-6 open reading frame is expressed as a fusion protein with the 2A peptide of Foot and Mouth Disease Virus and the amino terminal of the PVX coat protein (CP) (PVXESAT-6). This strategy allows the production of free CP and ESAT-6 as well as fused ESAT-2A-CP to obtain recombinant chimaeric virions expressing ESAT-6 at the surface to be used as particulate antigen in vaccination. ESAT-6 expression was tested in agroinfiltrated tobacco leaves and products of the expected molecular masses corresponding to cleaved CP and ESAT-2A-CP fusion protein were observed, with ESAT-6 yields ranging from 0.5% to 1% of total soluble protein. Our study describes for the first time the expression of the ESAT-6 protein in tobacco plants using a PVX-derived vector. This strategy should serve as a convenient, rapid, low-cost expression system and can also be used for the assessment of ESAT-6 production and function prior to stable plant transformation.

Production of the main surface antigen of Toxoplasma gondii in tobacco leaves and analysis of its antigenicity and immunogenicity.

We adapted a previously described Agrobacterium-mediated transient expression system to test the expression level of three constructs carrying the surface antigen 1 (SAG1) of Toxoplasma gondii. Two constructs were based in a Potato virus X (PVX) amplicon. In one of them, the PVX movement protein genes were replaced by the sag1 gene. In the other, the sag1 gene was placed under the control of an additional coat protein subgenomic promoter. In the third construct, the sag1 gene was fused to an apoplastic peptide signal under the CaMV 35S promoter. Western blot analysis of leaf extracts infiltrated with each construct revealed a protein of 35 kDa. SAG1 accumulation in leaves ranged from 0.1 to 0.06% of total soluble protein (equivalent to 10 microg and 6 microg of SAG1 per gram of fresh leaf tissue, respectively). Three of five human seropositive samples reacted with tobacco-expressed SAG1 in Western blot analysis. The C3H mice were immunized with SAG-expressing leaf extracts and perorally challenged with a nonlethal dose of the T. gondii Me49 strain. Mice vaccinated with SAG1 showed significantly lower brain cyst burdens compared to those from the control group. Immunization with SAG1-expressing leaves elicited a specific humoral response with predominant participation of type IgG2a. In conclusion, a functional SAG1 version could be transiently expressed in tobacco leaves.

Over-expression of potato virus X TGBp1 movement protein in transgenic tobacco plants causes developmental and metabolic alterations.

Transgenic Nicotiana tabacum plants expressing the TGBp1 movement protein of potato virus X (PVX) were studied to investigate the effects caused by this protein on plant physiology and development. TGBp1 caused consistent reductions of size and weight in different organs of these plants; however shoot-to-root ratios were similar to those of control plants. Transgenic seedlings showed smaller root meristems and calli derived from TGBp1 leaves grew at a slower rate through successive subcultures. Microscopic observations of TGBp1 plants revealed flattened chloroplasts containing plastoglobuli-like bodies. Further analyses showed a considerable reduction in photosynthetic rate, lower starch levels in leaves and roots, higher nitrate accumulation in leaves and induction of pathogenesis-related (PR) protein genes. Since these changes were not observed when other PVX sequences were expressed in tobacco, we postulate that TGBp1 is an important symptom contributor in PVX infections.

Immunological Detection of a GarV-Type Virus in Argentine Garlic Cultivars.

A cDNA library representing the genomes of several garlic viruses was generated using a viral RNA mixture as template. Analysis of randomly selected clones allowed the identification of different viral genomic sequences. On the basis of amino acid and nucleotide sequence comparisons, one of them was assigned to garlic virus A (GarV-A), a novel flexuous, rod-shaped virus recently reported in Japan. The coat protein (CP) of this virus was expressed in Escherichia coli cells and used as immunogen to produce polyclonal antibodies. The expression protein was recognized by an antiserum detecting garlic mite-borne filamentous virus, but did not react with antibodies specific for other garlic viruses. Antibodies raised against the viral CP reacted with extracts infected with garlic mite-borne viruses and fail to recognize preparations of onion yellow dwarf potyvirus, leek yellow stripe potyvirus, and carnation latent carlavirus. The same antibodies decorated viral particles exhibiting a modal length of 586 nm in immuno electron microscopy with decoration assays. Double-antibody enzyme-linked immunosorbent assays and tissue printing assays performed with these antibodies allowed detection of GarV-A in most garlic cultivars used in Argentina.