Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.

The activities of transcription factors (TFs) require interactions with specific DNA sequences and other regulatory proteins. To detect such interactions in Arabidopsis, we developed a high-throughput screening system with a Gateway-compatible Gal4-AD-TF library of 1589 Arabidopsis TFs, which can be easily screened by mating-based yeast-one-hybrid (Y1H) and yeast-two-hybrid (Y2H) methods. The efficiency of the system was validated by examining two well-characterized TF-DNA and TF-protein interactions: the CHE-CCA1 promoter interaction by Y1H and NPR1-TGAs interactions by Y2H. We used this system to identify eight TFs that interact with a Mediator subunit, Med25, a key regulator in JA signaling. We identified five TFs that interacted with the GCC-box cis-element in the promoter of PDF1.2, a downstream gene of Med25. We found that three of these TFs, all from the AP2-EREBP family, interact directly both with Med25 and the GCC-box of PDF1.2, suggesting that Med25 regulates PDF1.2 expression through these three TFs. These results demonstrate that this high-throughput Y1H/Y2H screening system is an efficient tool for studying transcriptional regulation networks in Arabidopsis. This system will be available for other Arabidopsis researchers, and thus it provides a vital resource for the Arabidopsis community.

These authors contributed equally to this work. SUMMARYIron is an essential micronutrient for plants and animals, and plants are a major source of iron for humans. Therefore, understanding the regulation of iron homeostasis in plants is critical. We identified a T-DNA insertion mutant, yellow and sensitive to iron-deficiency 1 (yid1), that was hypersensitive to iron deficiency, containing a reduced amount of iron. YID1 encodes the Arabidopsis Mediator complex subunit MED16. We demonstrated that YID1/MED16 interacted with another subunit, MED25. MED25 played an important role in regulation of iron homeostasis by interacting with EIN3 and EIL1, two transcription factors in ethylene signaling associated with regulation of iron homeostasis. We found that the transcriptome in yid1 and med25 mutants was significantly affected by iron deficiency. In particular, the transcription levels of FIT, IRT1 and FRO2 were reduced in the yid1 and med25 mutants under iron-deficient conditions. The finding that YID1/MED16 and MED25 positively regulate iron homeostasis in Arabidopsis increases our understanding of the complex transcriptional regulation of iron homeostasis in plants.

One of the most fascinating aspects of plant morphology is the regular geometric arrangement of leaves and flowers, called phyllotaxy. The shoot apical meristem (SAM) determines these patterns, which vary depending on species and developmental stage. Auxin acts as an instructive signal in leaf initiation, and its transport has been implicated in phyllotaxy regulation in Arabidopsis (Arabidopsis thaliana). Altered phyllotactic patterns are observed in a maize (Zea mays) mutant, aberrant phyllotaxy1 (abph1, also known as abphyl1), and ABPH1 encodes a cytokinin-inducible type A response regulator, suggesting that cytokinin signals are also involved in the mechanism by which phyllotactic patterns are established. Therefore, we investigated the interaction between auxin and cytokinin signaling in phyllotaxy. Treatment of maize shoots with a polar auxin transport inhibitor, 1-naphthylphthalamic acid, strongly reduced ABPH1 expression, suggesting that auxin or its polar transport is required for ABPH1 expression. Immunolocalization of the PINFORMED1 (PIN1) polar auxin transporter revealed that PIN1 expression marks leaf primordia in maize, similarly to Arabidopsis. Interestingly, maize PIN1 expression at the incipient leaf primordium was greatly reduced in abph1 mutants. Consistently, auxin levels were reduced in abph1, and the maize PIN1 homolog was induced not only by auxin but also by cytokinin treatments. Our results indicate distinct roles for ABPH1 as a negative regulator of SAM size and a positive regulator of PIN1 expression. These studies highlight a complex interaction between auxin and cytokinin signaling in the specification of phyllotactic patterns and suggest an alternative model for the generation of altered phyllotactic patterns in abph1 mutants. We propose that reduced auxin levels and PIN1 expression in abph1 mutant SAMs delay leaf initiation, contributing to the enlarged SAM and altered phyllotaxy of these mutants.

Genomic resources have significantly impacted plant biology research in recent years. Cell biology has been further enabled by an ongoing revolution in visualization technologies. Using fluorescent proteins (FPs), we now have unprecedented views of cellular architecture, and we can study real-time dynamics of cell structure, function, and protein localization. To date, these technologies have been most widely used in Arabidopsis (Arabidopsis thaliana); however, the grasses provide a unique opportunity to study the underlying mechanisms and inter-related controls of cell growth, morphogenesis, and physiology in leading crop models.Here, we present a resource that leverages the emerging maize (Zea mays) genome sequence to develop tools to study protein structure and function in a cellular context. Traditionally, such studies relied on fixed tissue or FP fusions driven by constitutive promoters, which can lead to significant artifacts. The maize genome sequence now provides access to regulatory regions that can be used to drive native expression. We have developed streamlined methods to generate maize FP-tagged lines using these regulatory elements, allowing analysis of tissue-specific expression and localized function. Identification of diverse proteins that function in specific subcellular compartments will provide the tools for understanding basic developmental, biochemical, and physiological processes in maize, with direct application potential for crop improvement. METHODOLOGYWe developed a protocol to generate fusion proteins with yellow (YFP), cyan (CFP), or red (RFP) color variants of FPs driven by native regulatory elements, based on our previous work in Arabidopsis (Tian et al., 2004). In brief, the method uses triple template PCR to generate products of the full genomic sequence with the FP insert, which is flanked by linker peptides to minimize folding interference between the FP and tagged protein. The product is cloned using the Gateway system (Invitrogen) into the donor vector, pDONR207. The tagged gene is transferred into binary destination vectors and ultimately transformed into maize. Full details of the protocols are available at http://maize.jcvi.org/cellgenomics/protocol/maizeTT protocolGFP_111405.shtml.Candidate genes were selected for tagging based on several criteria, including, as first priority, the availability of full genomic sequence plus regulatory regions that included 3 kb upstream and 1 kb downstream of the coding region. A size limit of 8 to 9 kb for the full genomic region with the FP insertion is imposed to ensure good cloning efficiency. Given these size constraints, we next prioritized genes that encoded proteins with robust predicted functions. These decisions were based on homology to other well-studied proteins, known localizations to specific compartments, and/or corroborating antibody or expression data. Genes with available mutations were also given high priority so as to provide a means of functional complementation. Our final criterion was to include candidates that wou...

A total of 149 porcine Staphylococcus isolates with florfenicol MICs of >16 g/ml were screened for the presence of the multiresistance gene cfr, its location on plasmids, and its genetic environment. In total, 125 isolates carried either cfr (16 isolates), fexA (92 isolates), or both genes (17 isolates). The 33 cfr-carrying staphylococci, which included isolates of the species Staphylococcus cohnii, S. arlettae, and S. saprophyticus in which the cfr gene has not been described before, exhibited a wide variety of SmaI pulsed-field gel electrophoresis patterns. In 18 cases, the cfr gene was located on plasmids. Four different types of cfr-carrying plasmids-pSS-01 (n ‫؍‬ 2; 40 kb), pSS-02 (n ‫؍‬ 3; 35.4 kb), pSS-03 (n ‫؍‬ 10; 7.1 kb), and pBS-01 (n ‫؍‬ 3; 16.4 kb)-were differentiated on the basis of their sizes, restriction patterns, and additional resistance genes. Sequence analysis revealed that in plasmid pSS-01, the cfr gene was flanked in the upstream part by a complete aacA-aphD-carrying Tn4001-like transposon and in the downstream part by a complete fexA-carrying transposon Tn558. In plasmid pSS-02, an insertion sequence IS21-558 and the cfr gene were integrated into transposon Tn558 and thereby truncated the tnpA and tnpB genes. The smallest cfr-carrying plasmid pSS-03 carried the macrolide-lincosamide-streptogramin B resistance gene erm(C). Plasmid pBS-01, previously described in Bacillus spp., harbored a Tn917-like transposon, including the macrolide-lincosamide-streptogramin B resistance gene erm(B) in the cfr downstream region. Plasmids, which in part carry additional resistance genes, seem to play an important role in the dissemination of the gene cfr among porcine staphylococci.

Summary Plants display thermomorphogenesis in response to high temperature (HT). PHYTOCHROME INTERACTING FACTOR 4 (PIF4) is a central integrator regulated by numerous negative regulators. However, the mechanisms underpinning PIF4 positive regulation are largely unknown. Here, we find that TEOSINTE BRANCHED 1/CYCLOIDEA/PCF 5 (TCP5), TCP13, and TCP17 transcription factors promote the activity of PIF4 at transcriptional and post-transcriptional levels. TCP5 is rapidly induced by HT treatment, and TCP5 protein stability increases under HT. The overexpression of TCP5 causes constitutive thermomorphogenic phenotypes, whereas the tcp5 tcp13 tcp17 triple mutant exhibits aberrant thermomorphogenesis. We demonstrate that TCP5 not only physically interacts with PIF4 to enhance its activity but also directly binds to the promoter of PIF4 to increase its transcript. TCP5 and PIF4 share common downstream targets. The tcp5 tcp13 tcp17 mutant partially restores the long hypocotyls caused by PIF4 overexpression. Our findings provide a layer of understanding about the fine-scale regulation of PIF4 and plant thermomorphogenesis.

BackgroundColistin acts as the last line of defense against severe infections caused by carbapenem-resistant Enterobacteriaceae. Infections caused by extensively drug-resistant isolates coproducing MCR and carbapenemases have posed a serious public health concern.PurposeIn this study, we reported the first clinical colistin and carbapenem-resistant Enterobacter hormaechei isolate SCNJ07 coharboring blaNDM-1 and mcr-9 from a patient with bloodstream infection in China.MethodsBacterial antimicrobial susceptibility testing was performed using the broth microdilution method. Conjugation assay was carried out to investigate the transferability of mcr-9 and blaNDM-1. Whole-genome sequencing of strain SCNJ07 was performed using an Illumina HiSeq system and the genetic characteristics of the mcr-9- and blaNDM-1-harboring plasmids were analyzed.ResultsConjugation assays revealed that both blaNDM-1 and mcr-9 genes could successfully transfer their resistance phenotype to Escherichia coli strain J53. Whole genome sequencing showed that SCNJ07 possessed an FIB36:FIIY4 type self-transmissible plasmid bearing blaNDM-1, which possessed high similarity to previously reported pRJF866 in China. mcr-9 was located on a ~28-kb self-transmissible plasmid pMCR-SCNJ07 with both IncHI2 and IncR replicons. Two copies of intact IS903 that bracketed a ~8-kb region containing the mcr-9 gene were identified in pMCR-SCNJ07. BLASTn analysis revealed that a number of mcr-9-positive plasmids have been around for a while among Enterobacteriaceae worldwide.ConclusionThis study reveals the likelihood of a wide dissemination of this newly identified colistin resistance gene mcr-9 among Enterobacteriaceae. Further surveillance is urgently needed to understand the prevalence and dissemination of mcr-9, thereby facilitating establishment of measures to control its spread.