Alejandro Mentaberry

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.

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

BackgroundLegumes 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.ResultsWe 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.ConclusionsThe 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.

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.

A phosphatase acting on dolichyl phosphate in membranes from neuronal perikarya

Since the isolation of Del-P-Glc in 1970 [I] significant progress has been made in the elucidation ofhow the carbohydrate moieties of the asparagine-type glycoproteins are synthesized. Clycosylation occurs via sugar lipid intermediates [2]. Some of the properties of most of the enzymes in this pathway have been studied [2]. There is however scant amount of information* available about Dol-P and Dol-P-P phosphatases. The control mechanism operating in the dolichol pathway is yet to be elucidated. It has been proposed that the endogenous level of Dol-P could play a regulatory role in protein glycosylation [3]. By analogy with procaryotic systems [4-61, Dol-P, Dol.P-P phosphatases and dolichoi kinase may be implicated in the control bf the endogenous level of Dol-P. Enzymes that split the phosphate bonds from prenyl phosphates have been described in mammalian systems [7--91, although in these cases Dol-P was not used as substrate and Mgzf were always included in the incubation mixture. Here we report an enzymatic activity present in membranes from neuronal perikarya which removes the pl~osphate from Dol-P and requires no divalent cations.

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.

Coat protein gene sequences of garlic and onion isolates of the onion yellow dwarf potyvirus (OYDV)

SummaryPartial genomic sequences from an unknown garlic potyvirus and from an onion isolate of the onion yellow dwarf potyvirus (OYDV) were obtained. Comparison of the deduced amino acid sequences showed a similarity of 88% between the respective viral coat proteins. The garlic potyvirus coat protein was expressed inE. coli cells, purified, and subjected to Western blot analysis using antibodies raised against different garlic-infecting viruses. The expression protein was consistently recognised by anti-OYDV antibodies and did not react with antibodies specific for leek yellow stripe potyvirus (LYSV), garlic common latent carlavirus (GCLV) and shallot latent carlavirus (SLV). Besides, the garlic potyvirus coat protein was obtained as a fusion protein and used as antigen to produce polyclonal antibodies. These antibodies reacted with purified OYDV virions, but failed to recognise LYSV particles. In the light of this evidence the garlic potyvirus was identified as the garlic strain of OYDV.

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.

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

BackgroundSoil 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.ResultsWe 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.ConclusionThis 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.

cDNA sequence of the pepper severe mosaic virus coat protein gene.

PeSMV was originally isolated in Argentina from diseased Capsicum annuum plants and classed as a possible member of the potyvirus group on the basis of morphological, biological, and immunological evidence (Feldman and Gracia, 1977). Although information concerning its economic significance is still imprecise, the virus is present in most pepperproducing areas and yields are drastically reduced in chronically infected plants. As part of a project to obtain resistant transgenic plants by means of coat protein-mediated protection, we cloned the PeSMV gRNA and obtained a partia1 nucleotide sequence of the PeSMV genome including the 3’ end of the replicase gene, the complete CP gene, and the 3’ noncoding region (Table I). PeSMV gRNA from strain 5-163 was used for cDNA synthesis. Clones located in the genome 3‘ end were identified by low-stringency hybridization with a radioactive PVY CP probe and dideoxy sequenced. A sequence spanning 1329 nucleotides from the 3‘ end was obtained, the first 998 encoding a continuous open reading frame. Comparison of the sequence encoding the putative PeSMV CP with that of PVY revealed a similarity of 71.6%. The 3’ noncoding sequences are different lengths (PeSMV, 471 nucleotides; PVY, 331 nucleotides) and were not directly comparable. The translated amino acid sequence corresponding to the putative PeSMV CP showed similarities of 80.9 and 62.1% with the PVY (Bravo-Almonacid and Mentaberry, 1989) and tobacco etch virus (Allison et al., 1985) CPs, respectively. Most of the sequence changes are clustered at the N-terminal region. The PeSMV sequence contains two additional codons between nucleotides 142 and 147 of the PVY CP sequence. On the other hand, severa1 sequence motifs strongly conserved in most potyvirus are present in the C-terminal threequarters of the sequence. Remarkably, the PeSMV sequence does not contain the amino acid motif DAG that was associated with aphid transmissibility (Atreya et al., 1991). This fact agrees with previous data showing that PeSMV is not aphid transmitted (Feldman and Gracia, 1977).

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.