Foxtail mosaic virus: a new viral vector for protein expression in cereals

Rapid and cost-effective virus-derived transient expression systems for plants are invaluable in elucidating gene function. These are particularly useful in the case of plant species for which transformation-based methods are either not yet developed, or are too time- and labor-demanding, such as wheat and maize. The Virus-mediated overexpression (VOX) vectors based on Barley stripe mosaic virus (BSMV) or Wheat streak mosaic virus (WSMV) previously described for these species are incapable of expressing free recombinant proteins >150-250 amino-acids (aa), not suited for high throughput screens, and have other limitations. In this study, we report the development of a new VOX vector based on a monopartite single-stranded positive sense RNA virus, Foxtail mosaic virus (FoMV, genus Potexvirus). The gene of interest is inserted downstream of a duplicated sub-genomic promoter of the viral coat protein gene and the corresponding protein is expressed in its free form. This new vector, PV101, allowed expression of a 239 aa-long green fluorescent protein (GFP) in both virus inoculated and upper uninoculated (systemic) leaves of wheat and maize, and directed systemic expression of a larger ca. 600 aa protein GUSPlus in maize. Moreover, we demonstrated that PV101 can be used for in planta expression and functional analysis of apoplastic pathogen effector proteins such as host-specific toxin ToxA of Parastagonospora nodorum. Therefore, this new VOX vector opens new possibilities for functional genomics studies in two of the most important cereal crops

Transcriptomic Characterization of a Synergistic Genetic Interaction during Carpel Margin Meristem Development in Arabidopsis thaliana

In flowering plants the gynoecium is the female reproductive structure. In Arabidopsis thaliana ovules initiate within the developing gynoecium from meristematic tissue located along the margins of the floral carpels. When fertilized the ovules will develop into seeds. SEUSS (SEU) and AINTEGUMENTA (ANT) encode transcriptional regulators that are critical for the proper formation of ovules from the carpel margin meristem (CMM). The synergistic loss of ovule initiation observed in the seu ant double mutant suggests that SEU and ANT share overlapping functions during CMM development. However the molecular mechanism underlying this synergistic interaction is unknown. Using the ATH1 transcriptomics platform we identified transcripts that were differentially expressed in seu ant double mutant relative to wild type and single mutant gynoecia. In particular we sought to identify transcripts whose expression was dependent on the coordinated activities of the SEU and ANT gene products. Our analysis identifies a diverse set of transcripts that display altered expression in the seu ant double mutant tissues. The analysis of overrepresented Gene Ontology classifications suggests a preponderance of transcriptional regulators including multiple members of the REPRODUCTIVE MERISTEMS (REM) and GROWTH-REGULATING FACTOR (GRF) families are mis-regulated in the seu ant gynoecia. Our in situ hybridization analyses indicate that many of these genes are preferentially expressed within the developing CMM. This study is the first step toward a detailed description of the transcriptional regulatory hierarchies that control the development of the CMM and ovule initiation. Understanding the regulatory hierarchy controlled by SEU and ANT will clarify the molecular mechanism of the functional redundancy of these two genes and illuminate the developmental and molecular events required for CMM development and ovule initiation

Ultracentrifuge methods for the analysis of polysaccharides, glycoconjugates, and lignins

Although like proteins, polysaccharides are synthesized by enzymes, unlike proteins there is no template. This means that they are polydisperse, do not generally have compact folded structures, and are often very large with greater nonideality behavior in solution. This chapter considers the relevant analytical ultracentrifuge methodology available for characterizing these and related carbohydrate-based systems and information this methodology supplies, in terms of sizes, shapes, and interactions using a comprehensive range of examples, including glycoconjugates and lignins. The relevance and potential of recent software developments such as SEDFIT-MSTAR, the Extended Fujita algorithm, and HYDFIT are considered

Agrobacterium-mediated rice (Oryza sativa L.) transformation

SIGLEAvailable from British Library Document Supply Centre-DSC:DXN025846 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

SEUSS and SEUSS-LIKE Transcriptional Adaptors Regulate Floral and Embryonic Development in Arabidopsis1[C][W][OA]

Multimeric protein complexes are required during development to regulate transcription and orchestrate cellular proliferation and differentiation. The Arabidopsis (Arabidopsis thaliana) SEUSS (SEU) gene encodes a transcriptional adaptor that shares sequence similarity with metazoan Lim domain-binding transcriptional adaptors. In Arabidopsis, SEU forms a physical complex with the LEUNIG transcriptional coregulator. This complex regulates a number of diverse developmental events, including proper specification of floral organ identity and number and the development of female reproductive tissues derived from the carpel margin meristem. In addition to SEU, there are three Arabidopsis SEUSS-LIKE (SLK) genes that encode putative transcriptional adaptors. To determine the functions of the SLK genes and to investigate the degree of functional redundancy between SEU and SLK genes, we characterized available slk mutant lines in Arabidopsis. Here, we show that mutations in any single SLK gene failed to condition an obvious morphological abnormality. However, by generating higher order mutant plants, we uncovered a degree of redundancy between the SLK genes and between SLK genes and SEU. We report a novel role for SEU and the SLK genes during embryonic development and show that the concomitant loss of both SEU and SLK2 activities conditions severe embryonic and seedling defects characterized by a loss of the shoot apical meristem. Furthermore, we demonstrate that SLK gene function is required for proper development of vital female reproductive tissues derived from the carpel margin. We propose a model that posits that SEU and SLK genes support organ development from meristematic regions through two different pathways: one that facilitates auxin response and thus organ initiation and a second that sustains meristematic potential through the maintenance of SHOOTMERISTEM-LESS and PHABULOSA expression