Plant Synthetic Biology requires robust and efficient methods for assembling multigene constructs. Golden Gate cloning provides a precision module-based cloning technique for facile assembly of multiple genes in one construct. We present here a versatile resource for plant biologists comprising a set of cloning vectors and 96 standardized parts to enable Golden Gate construction of multigene constructs for plant transformation. Parts include promoters, untranslated sequences, reporters, antigenic tags, localization signals, selectable markers, and terminators. The comparative performance of parts in the model plant Nicotiana benthamiana is discussed.

SummaryInventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

The polypeptide growth factor, hepatocyte growth factor͞scatter factor (HGF͞SF), shares the multidomain structure and proteolytic mechanism of activation of plasminogen and other complex serine proteinases. HGF͞SF, however, has no enzymatic activity. Instead, it controls the growth, morphogenesis, or migration of epithelial, endothelial, and muscle progenitor cells through the receptor tyrosine kinase MET. Using small-angle x-ray scattering and cryoelectron microscopy, we show that conversion of pro(single-chain)-HGF͞SF into the active two-chain form is associated with a major structural transition from a compact, closed conformation to an elongated, open one. We also report the structure of a complex between two-chain HGF͞SF and the MET ectodomain (MET928) with 1:1 stoichiometry in which the N-terminal and first kringle domain of HGF͞SF contact the face of the seven-blade ␤-propeller domain of MET harboring the loops connecting the ␤-strands b-c and d-a, whereas the C-terminal serine proteinase homology domain binds the opposite ''b'' face. Finally, we describe a complex with 2:2 stoichiometry between two-chain HGF͞SF and a truncated form of the MET ectodomain (MET567), which is assembled around the dimerization interface seen in the crystal structure of the NK1 fragment of HGF͞SF and displays the features of a functional, signaling unit. The study shows how the proteolytic mechanism of activation of the complex proteinases has been adapted to cell signaling in vertebrate organisms, offers a description of monomeric and dimeric ligand-receptor complexes, and provides a foundation to the structural basis of HGF͞SF-MET signaling.cell signaling ͉ plasminogen ͉ serine proteinases ͉ kringle ͉ x-ray scattering H epatocyte growth factor͞scatter factor (HGF͞SF) (1-6) are vertebrate-specific polypeptide growth factors with a domain structure related to that of plasminogen (7). Interest in HGF͞SF and its receptor MET (8) stems from unique biological roles in embryogenesis (9-11), tissue regeneration (12, 13), and cancer (14). These activities have led to a strong interest in the structure of the molecules as this knowledge may underpin the development of MET-based therapeutics.HGF͞SF consists of six domains: an N-terminal domain (n), four copies of the kringle domain (k1-k4), and a C-terminal domain (sp) structurally related to the catalytic domain of serine proteinases (Fig. 1A). The factor is synthesized as a precursor protein (pro-or single-chain HGF͞SF) and is proteolytically processed to a two-chain form by cleavage of the linker connecting the k4 and sp domains ( Fig. 1 A and B). Single-chain HGF͞SF binds MET (15, 16) but is unable to induce biological responses, for example, dispersion of MDCK cell colonies, even at concentrations 100-fold higher than two-chain HGF͞SF (Fig. 1 C-E).MET is also synthesized as a single-chain precursor that is cleaved by furin yielding an N-terminal ␣-chain and a C-terminal ␤-chain. The MET ectodomain consists of two moieties: the large, N-terminal sema domain, which is responsible f...