A split green fluorescent protein system to enhance spatial and temporal sensitivity of translating ribosome affinity purification.

Translating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity co-purification of ribosomes and the mRNAs that they are translating. This population of actively translated mRNAs (translatome) can be interrogated by quantitative PCR or RNA sequencing. Condition- or cell-specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell-specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: One is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split green fluorescent protein (GFP); the second is controlled by a stimulus-specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi-molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre-existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant-pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies.

Genetic transformation technologies for the common dandelion, Taraxacum officinale.

BACKGROUND: Taraxacum officinale, or the common dandelion, is a widespread perennial species recognized worldwide as a common lawn and garden weed. Common dandelion is also cultivated for use in teas, as edible greens, and for use in traditional medicine. It produces latex and is closely related to the Russian dandelion, T. kok-saghyz, which is being developed as a rubber crop. Additionally, the vast majority of extant common dandelions reproduce asexually through apomictically derived seeds- an important goal for many major crops in modern agriculture. As such, there is increasing interest in the molecular control of important pathways as well as basic molecular biology and reproduction of common dandelion. RESULTS: Here we present an improved Agrobacterium-based genetic transformation and regeneration protocol, a protocol for generation and transformation of protoplasts using free DNA, and a protocol for leaf Agrobacterium infiltration for transient gene expression. These protocols use easily obtainable leaf explants from soil-grown plants and reagents common to most molecular plant laboratories. We show that common markers used in many plant transformation systems function as expected in common dandelion including fluorescent proteins, GUS, and anthocyanin regulation, as well as resistance to kanamycin, Basta, and hygromycin. CONCLUSION: Reproducible, stable and transient transformation methods are presented that will allow for needed molecular structure and function studies of genes and proteins in T. officinale.

Review: Functional linkages between amino acid transporters and plant responses to pathogens.

Upon infection, plant pathogens become dependent on their hosts for nutrition. Therefore, the interaction between the two organisms is tightly linked to the availability and flux of nutrients in the plant. The plant's nitrogen metabolism is reprogrammed during pathogen attack, likely reflecting plant's response to invasion by the pathogen and active modification by the pathogen to promote feeding. Several lines of evidence indicate that plant-derived amino acids are an important source of nitrogen for diverse pathogens. Moreover, amino acid homeostasis is interconnected with the plant's immune signaling pathways. Here, we critically examine the knowns and unknowns about connections between plant-encoded amino acid transporters and resistance or susceptibility to pathogens and pests. We use recent insights into sugar transporters to frame a perspective with potential applicability to amino acids and other nutrients. We emphasize different approaches that have provided insight in this topic and we conclude with suggestions to fill gaps in foundational knowledge and explore new avenues for disease control.

Update on amino acid transporter functions and on possible amino acid sensing mechanisms in plants.

Amino acids are essential components of plant metabolism, not only as constituents of proteins, but also as precursors of important secondary metabolites and as carriers of organic nitrogen between the organs of the plant. Transport across intracellular membranes and translocation of amino acids within the plant is mediated by membrane amino acid transporters. The past few years have seen the identification of a new family of amino acid transporters in Arabidopsis, the characterization of intracellular amino acid transporters, and the discovery of new roles for already known proteins. While amino acid metabolism needs to be tightly coordinated with amino acid transport activity and carbohydrate metabolism, no gene involved in amino acid sensing in plants has been unequivocally identified to date. This review aims at summarizing the recent data accumulated on the identity and function of amino acid transporters in plants, and discussing the possible identity of amino acid sensors based on data from other organisms.