The development of sensitive and versatile techniques to detect protein-protein interactions in vivo is important for understanding protein functions. The previously described techniques, fluorescence resonance energy transfer and bimolecular fluorescence complementation, which are used widely for protein-protein interaction studies in plants, require extensive instrumentation. To facilitate protein-protein interaction studies in plants, we adopted the luciferase complementation imaging assay. The amino-terminal and carboxyl-terminal halves of the firefly luciferase reconstitute active luciferase enzyme only when fused to two interacting proteins, and that can be visualized with a low-light imaging system. A series of plasmid constructs were made to enable the transient expression of fusion proteins or generation of stable transgenic plants. We tested nine pairs of proteins known to interact in plants, including Pseudomonas syringae bacterial effector proteins and their protein targets in the plant, proteins of the SKP1-Cullin-F-box protein E3 ligase complex, the HSP90 chaperone complex, components of disease resistance protein complex, and transcription factors. In each case, strong luciferase complementation was observed for positive interactions. Mutants that are known to compromise protein-protein interactions showed little or much reduced luciferase activity. Thus, the assay is simple, reliable, and quantitative in detection of protein-protein interactions in plants.Noncovalent interactions among proteins are vital for all aspects of cellular processes. Thus, the identification and characterization of interacting proteins are key to our understanding of protein functions. A plethora of techniques have been developed to detect protein-protein interactions in vitro and in vivo (Piehler, 2005). The most widely used among these techniques is the yeast two-hybrid assay, which is ideal for largescale screening for interacting proteins and the construction of protein interactomes (Fields and Song, 1989;Li et al., 2004). However, the yeast two-hybrid assay detects protein-protein interactions under heterologous conditions, and results must be validated by assays under physiological conditions. Examination of protein-protein interactions under physiological conditions is often technically demanding and requires tedious procedures. For example, the co-immunoprecipitation assay requires specific antibodies; lengthy procedures that are influenced by parameters such as schemes for protein extraction, binding, and washing; and expertise of individuals performing the experiment. Thus, the results are often variable from laboratory to laboratory. Tandem affinity purification represents a more advanced technique primarily designed to identify new proteins in a protein complex in a native state (Puig et al., 2001;Rohila et al., 2006).The development of reporter-based in vivo proteinprotein interaction assays, such as fluorescence resonance energy transfer (FRET;Ha et al., 1996;Heim and Tsien, 1996;Mahajan et al., 1998), the ...

Pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) trigger plant immunity that forms the first line inducible defenses in plants. The regulatory mechanism of MAMP-triggered immunity, however, is poorly understood. Here, we show that Arabidopsis thaliana transcription factors ETHYLENE INSENSITIVE3 (EIN3) and ETHYLENE INSENSITIVE3-LIKE1 (EIL1), previously known to mediate ethylene signaling, also negatively regulate PAMP-triggered immunity. Plants lacking EIN3 and EIL1 display enhanced PAMP defenses and heightened resistance to Pseudomonas syringae bacteria. Conversely, plants overaccumulating EIN3 are compromised in PAMP defenses and exhibit enhanced disease susceptibility to Pseudomonas syringae. Microarray analysis revealed that EIN3 and EIL1 negatively control PAMP response genes. Further analyses indicated that SALICYLIC ACID INDUCTION DEFICIENT2 (SID2), which encodes isochorismate synthase required for pathogen-induced biosynthesis of salicylic acid (SA), is a key target of EIN3 and EIL1. Consistent with this, the ein3-1 eil1-1 double mutant constitutively accumulates SA in the absence of pathogen attack, and a mutation in SID2 restores normal susceptibility in the ein3 eil1 double mutant. EIN3 can specifically bind SID2 promoter sequence in vitro and in vivo. Taken together, our data provide evidence that EIN3/EIL1 directly target SID2 to downregulate PAMP defenses.

Structural variations (SVs) represent a major source of genetic diversity. However, the functional impact and formation mechanisms of SVs in plant genomes remain largely unexplored. Here, we report a nucleotide-resolution SV map of cucumber (Cucumis sativas) that comprises 26,788 SVs based on deep resequencing of 115 diverse accessions. The largest proportion of cucumber SVs was formed through nonhomologous end-joining rearrangements, and the occurrence of SVs is closely associated with regions of high nucleotide diversity. These SVs affect the coding regions of 1676 genes, some of which are associated with cucumber domestication. Based on the map, we discovered a copy number variation (CNV) involving four genes that defines the Female (F) locus and gives rise to gynoecious cucumber plants, which bear only female flowers and set fruit at almost every node. The CNV arose from a recent 30.2-kb duplication at a meiotically unstable region, likely via microhomology-mediated breakinduced replication. The SV set provides a snapshot of structural variations in plants and will serve as an important resource for exploring genes underlying key traits and for facilitating practical breeding in cucumber.

Sex determination in plants involves a variety of mechanisms. Here, we report the map-based cloning and characterization of the unisexual-flower-controlling gene M. M was identified as a previously characterized putative 1-aminocyclopropane-1-carboxylic acid synthase gene, while the m allele that mutated at a conserved site (Gly33Cys) lost activity in the original enzymatically active allele.

Plant trichomes serve as a highly suitable model for investigating cell differentiation at the single-cell level. The regulatory genes involved in unicellular trichome development in Arabidopsis thaliana have been intensively studied, but genes regulating multicellular trichome development in plants remain unclear. Here, we characterized Cucumis sativus (cucumber) trichomes as representative multicellular and unbranched structures, and identified Micro-trichome (Mict), using map-based cloning in an F 2 segregating population of 7,936 individuals generated from a spontaneous mict mutant. In mict plants, trichomes in both leaves and fruits, are small, poorly developed, and denser than in the wild type. Sequence analysis revealed that a 2,649-bp genomic deletion, spanning the first and second exons, occurred in a plant-specific class I homeodomain-leucine zipper gene. Tissue-specific expression analysis indicated that Mict is strongly expressed in the trichome cells. Transcriptome profiling identified potential targets of Mict including putative homologs of genes known in other systems to regulate trichome development, meristem determinacy, and hormone responsiveness. Phylogenic analysis charted the relationships among putative homologs in angiosperms. Our paper represents initial steps toward understanding the development of multicellular trichomes.