A Core AFLP Map of Aposporic Tetraploid \u3cem\u3ePaspalum Notatum\u3c/em\u3e (Bahiagrass)

Paspalum notatum (Bahiagrass) is a perennial rhizomatous species that reproduces by aposporous apomixis. Tetraploid races (2n=4x=40) are widely distributed from Central to South America and constitute one of the most valuable natural forage grasses for the subtropical areas of Paraguay, southern Brazil and north-eastern Argentina. Apospory in the species is controlled by a single locus, which exhibits a distorted segregation ratio. The objectives of this work were to develop a core genetic linkage map of the species by using AFLP markers and characterize the genomic region related to apospory

A Comprehensive Analysis of Gene Expression and Genomic Alterations in a Newly Formed Autotetraploid of \u3cem\u3ePaspalum Notatum\u3c/em\u3e

The proportion of angiosperms that have experienced one or more episodes of chromosome doubling is estimated to be of the order of 50-70%. This prominence of polyploidy probably implies some adaptive significance. The emergence of novel phenotypes could allow polyploids to enter new niches or enhance their chances of being selected for use in agriculture. Although the causes of novel variation in polyploids are not well understood, they could involve changes in gene expression through dosage-regulation, altered regulatory interactions and rapid genetic/epigenetic changes. The objective of this work was to carry out an extensive transcript profiling and genome analysis of a diploid genotype of the pasture grass Paspalum notatum and its tetraploid derivative obtained after colchicine treatment

Novel Genotypes of the Subtropical Grass \u3cem\u3eEragrostis Curvula\u3c/em\u3e for the Analysis of Apomixis (Diplospory)

Eragrostis curvula (Schrad.) Nees is a variable grass native to Southern Africa. Its several forms, known as lovegrasses, were introduced to Australia, USA and Argentina as forage perennial grasses. Apomixis is a common trait in the genus Eragrostis, with diplospory being the most frequent type. Sexual reproduction also occurs in Eragrostis, although not frequently. Since most tetraploid Eragrostis lines are apomictic, the generation of a sexual tetraploid strain is a requirement for linkage analysis of the gene(s) governing the apomictic character. Furthermore, isogenic lines of the same ploidy, reproducing alternatively by sexuality or apomixes, represent an ideal system for comparative transcriptome analysis. The aim of this work was the generation and characterization of two novel genotypes of E. curvula: a dihaploid strain obtained in vitro from an apomictic cultivar and a tetraploid plant derived from the dihaploid after chromosome duplication

Discovery and Functional Categorisation of Expressed Sequence Tags from Flowers of \u3cem\u3eEragrostis Curvula\u3c/em\u3e Genotypes Showing Different Ploidy Levels and Reproductive Modes

Two novel genotypes of weeping lovegrass (Eragrostis curvula) - a dihaploid strain obtained in vitro from an apomictic cultivar and a tetraploid plant derived from the dihaploid after chromosome duplication – have recently been developed. These materials represent an excellent system for the identification, through transcriptional profiling, of genes involved in diplospory and/or ploidy level gene regulation. The aim of this work was the discovery and functional classification of expressed sequence tags (ESTs) from immature inflorescences of the apomictic E. curvula cultivar Tanganyika (2n=4x=40), a dihaploid sexual strain derived from it (2n=2x=20) and a tetraploid sexual strain (2n=4x=40) obtained by colchicine duplication of the dihaploid

PN_SCD1, VESICLE TRAFFICKING REGULATOR IS DEMETHYLATED AND OVEREXPRESSED IN FLORETS OF APOMICTIC PASPALUM NOTATUM GENOTYPES

Apomixis (asexual reproduction through seeds) is considered a deviation of the sexual reproductive pathway leading to the formation of clonal progenies genetically identical to the mother plant. It has been suggested that apomixis might be a consequence of epigenetic alterations, such as interspecific hybridization and polyploidization, resulting in a wide deregulation of reproductive development. Studies on epigenetic are transforming our actual idea of the structural variation and diversity that prevails at key steps of plant female gametogenesis, with deep implications for understanding the evolutionary trends that model innovation in reproductive development and adaptation. Recent results have provided evidences indicating that epigenetic mechanisms are crucial to control events that distinguish sexual from apomictic development. Therefore, the epigenetic regulation of apomixis is an attractive theory as it potentially accounts for the facultative nature of apomixis as well as the ability of apomictic to revert back to sexuality. In this work we used the Methylation-Sensitive Amplification Polymorphism (MSAP) technique to characterize floral genome cytosine methylation patterns occurring in sexual and aposporous Paspalum notatum genotypes, in order to identify epigenetically-controlled genes putatively involved in apomixis development. A partial and rather divergent methylation reprogramming was detected in apomictic genotypes. From twelve polymorphic MSAP-derived sequences, one (PN_6.6, renamed PN_SCD1) was selected due to its relevant annotation and differential representation in 454 floral transcriptome libraries of sexual and apomictic P. notatum. PN_6.6 encodes the DENN domain/WD repeat-containing protein SCD1, which interacts with RAB GTPases- and/or MAPKs to promote specialized cell division, functions in clathrin-mediated membrane transport and was defined as potential substrate receptor of CUL4 E3 ubiquitin ligases. Quantitative RT-PCR and comparative RNAseq analyses of laser microdissected nucellar cells confirmed PN_SCD1 upregulation in florets of apomictic plants and revealed that overexpression takes place just before the onset of apospory initials. Moreover, we found that several SCD1 molecular partners are upregulated in florets of P. notatum apomictic plants. Our results revealed a specific vesicle trafficking molecular pathway epigenetically modulated during apomixis. Results will be presented and critically discussed

Expression Analysis of the Ligands for the Natural Killer Cell Receptors NKp30 and NKp44

BACKGROUND: The natural cytotoxicity receptors (NCR) are important to stimulate the activity of Natural Killer (NK) cells against transformed cells. Identification of NCR ligands and their level of expression on normal and neoplastic cells has important implications for the rational design of immunotherapy strategies for cancer. METHODOLOGY/PRINCIPAL FINDINGS: Here we analyze the expression of NKp30 ligand and NKp44 ligand on 30 transformed or non-transformed cell lines of different origin. We find intracellular and surface expression of these two ligands on almost all cell lines tested. Expression of NKp30 and NKp44 ligands was variable and did not correlate with the origin of the cell line. Expression of NKp30 and NKp44 ligand correlated with NKp30 and NKp44-mediated NK cell lysis of tumor cells, respectively. The surface expression of NKp30 ligand and NKp44 ligand was sensitive to trypsin treatment and was reduced in cells arrested in G(2)/M phase. CONCLUSION/SIGNIFICANCE: These data demonstrate the ubiquitous expression of the ligands for NKp30 and NKp44 and give an important insight into the regulation of these ligands

Natural Killer Lysis Receptor (NKLR)/NKLR-Ligand Matching as a Novel Approach for Enhancing Anti-Tumor Activity of Allogeneic NK Cells

NK cells are key players in anti tumor immune response, which can be employed in cell-based therapeutic modalities. One of the suggested ways to amplify their anti tumor effect, especially in the field of stem cell transplantation, is by selecting donor/recipient mismatches in specific HLA, to reduce the inhibitory effect of killer Ig-like receptors (KIRs). Here we suggest an alternative approach for augmentation of anti tumor effect of allogeneic NK cells, which is founded on profile matching of donor NK lysis receptors (NKLR) phenotype with tumor lysis-ligands.We show that an NKLR-mediated killing directly correlates with the NKLR expression intensity on NK cells. Considerable donor variability in the expression of CD16, NKp46, NKG2D and NKp30 on circulating NK cells, combined with the stability of phenotype in several independently performed tests over two months, indicates that NKLR-guided selection of donors is feasible. As a proof of concept, we show that melanoma cells are dominantly recognized by three NKLRs: NKG2D, NKp30 and NKp44. Notably, the expression of NKp30 on circulating NK cells among metastatic melanoma patients was significantly decreased, which diminishes their ability to kill melanoma cells. Ex vivo expansion of NK cells results not only in increased amount of cells but also in a consistently superior and predictable expression of NKG2D, NKp30 and NKp44. Moreover, expanded NK cultures with high expression of NKG2D or NKp30 were mostly derived from the corresponding NKG2D(high) or NK30(high) donors. These NK cultures subsequently displayed an improved cytotoxic activity against melanoma in a HLA/KIR-ligand mismatched setup, which was NKLR-dependent, as demonstrated with blocking anti-NKG2D antibodies.NKLR/NKLR-ligand matching reproducibly elicits enhanced NK anti-tumor response. Common NKLR recognition patterns of tumors, as demonstrated here in melanoma, would allow implementation of this approach in solid malignancies and potentially in hematological malignancies, either independently or in adjunction to other modalities

Modular assembly of proteins on nanoparticles

Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum, for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold–sulfur bonds (Au–S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes, which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond