Plasticity of plasma membrane compartmentalization during plant immune responses

Plasma membranes require high levels of plasticity to modulate the perception and transduction of extracellular and intracellular signals. Dynamic lateral assembly of protein complexes combined with an independent compositional lipid patterning in both membrane leaflets provide cells the opportunity to decorate this interface with specific proteins in an organized but dynamic manner. Such ability to dynamically reorganize the protein content of the plasma membrane is essential for the regulation of processes such as polarity of transport, development, and microbial infection. While the plant cell wall represents the first physical and mostly unspecific barrier for invading microbes, the plasma membrane is at the forefront of microbial recognition and initiation of defense responses. Accumulating evidence indicating dynamic compartmentalization of plasma membranes in response to environmental cues has increased the interest in the compositional heterogeneity of this bilayer. Here, we elucidate the recruitment of specific proteins into defined membrane structures that ensure functional compartmentalization of the bilayer during infection processes

Tagging of MADS domain proteins for chromatin immunoprecipitation

<p>Abstract</p> <p>Background</p> <p>Most transcription factors fulfill their role in complexes and regulate their target genes upon binding to DNA motifs located in upstream regions or introns. To date, knowledge about transcription factor target genes and their corresponding transcription factor binding sites are still very limited. Two related methods that allow <it>in vivo </it>identification of transcription factor binding sites are chromatin immunoprecipitation (ChIP) and chromatin affinity purification (ChAP). For ChAP, the protein of interest is tagged with a peptide or protein, which can be used for affinity purification of the protein-DNA complex and hence, the identification of the target gene.</p> <p>Results</p> <p>Here, we present the results of experiments aiming at the development of a generic tagging approach for the Arabidopsis MADS domain proteins AGAMOUS, SEPALLATA3, and FRUITFULL. For this, Arabidopsis wild type plants were transformed with constructs containing a MADS-box gene fused to either a double <it>Strep</it>-tag^®II-FLAG-tag, a triple HA-tag, or an eGFP-tag, all under the control of the constitutive double 35S Cauliflower Mosaic Virus (CaMV) promoter. Strikingly, in all cases, the number of transformants with loss-of-function phenotypes was much larger than those with an overexpression phenotype. Using endogenous promoters in stead of the 35S CaMV resulted in a dramatic reduction in the frequency of loss-of-function phenotypes. Furthermore, pleiotropic defects occasionally caused by an overexpression strategy can be overcome by using the native promoter of the gene. Finally, a ChAP result is presented using GFP antibody on plants carrying a genomic fragment of a MADS-box gene fused to GFP.</p> <p>Conclusion</p> <p>This study revealed that MADS-box proteins are very sensitive to fusions with small peptide tags and GFP tags. Furthermore, for the expression of chimeric versions of MADS-box genes it is favorable to use the entire genomic region in frame to the tag of choice. Interestingly, though unexpected, it appears that the use of chimeric versions of MADS-box genes under the control of the strong 35S CaMV promoter is a very efficient method to obtain dominant-negative mutants, either caused by cosuppression or by alteration of the activity of the recombinant protein. Finally, we were able to demonstrate AGAMOUS binding to one of its targets by ChAP.</p

In planta localisation patterns of MADS domain proteins during floral development in Arabidopsis thaliana

BACKGROUND: MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). RESULTS: We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. CONCLUSION: This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during development, points to mechanisms other than transcriptional control. Together this information is essential to understand the role of these proteins in the regulatory processes that drive floral development and leads to new hypotheses

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The current assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n=14) containing 32,928 and 36,697 protein-coding genes, respectively. The Petunia lineage has experienced at least two rounds of paleohexaploidization, the older gamma hexaploidy event, which is shared with other Eudicots, and the more recent Solanaceae paleohexaploidy event that is shared with tomato and other Solanaceae species. Transcription factors that were targets of selection during the shift from bee- to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral color patterns and pollination systems. The high quality genome sequences will enhance the value of Petunia as a model system for basic and applied research on a variety of unique biological phenomena

Follow-up of contacts of middle east respiratory syndrome coronavirus–infected returning travelers, the Netherlands, 2014

Notification of 2 imported cases of infection with Middle East respiratory syndrome coronavirus in the Netherlands triggered comprehensive monitoring of contacts. Observed low rates of virus transmission and the psychological effect of contact monitoring indicate that thoughtful assessment of close contacts is prudent and must be guided by clinical and epidemiologic risk factors

Investigation of MADS domain transcription factor dynamics in the floral meristem

To study the importance of intercellular transport for MADS domain transcription factor functioning during floral development, we analyzed the dynamic behavior of fluorescently-tagged MADS domain proteins in transgenic plants by Confocal Laser Scanning Microscopy. These analyses, described in a recent paper in The Plant Journal, provided proof for previous suggestions that the Arabidopsis thaliana C-type protein AGAMOUS has a non-cell-autonomous role in floral meristem integrity. Furthermore, it indicated a possible non-cell-autonomous role for the B-type proteins APETALA3 and PISTILLATA and the E-type protein SEPALLATA3, through lateral intercellular movement in the floral meristem. In this addendum we compare some of the available fluorescent protein-based technologies for the investigation of transcription factor movements and dynamics

In planta localisation patterns of MADS domain proteins during floral development in <it>Arabidopsis thaliana</it>

Abstract Background MADS domain transcription factors play important roles in various developmental processes in flowering plants. Members of this family play a prominent role in the transition to flowering and the specification of floral organ identity. Several studies reported mRNA expression patterns of the genes encoding these MADS domain proteins, however, these studies do not provide the necessary information on the temporal and spatial localisation of the proteins. We have made GREEN FLUORESCENT PROTEIN (GFP) translational fusions with the four MADS domain proteins SEPALLATA3, AGAMOUS, FRUITFULL and APETALA1 from the model plant Arabidopsis thaliana and analysed the protein localisation patterns in living plant tissues by confocal laser scanning microscopy (CLSM). Results We unravelled the protein localisation patterns of the four MADS domain proteins at a cellular and subcellular level in inflorescence and floral meristems, during development of the early flower bud stages, and during further differentiation of the floral organs. The protein localisation patterns revealed a few deviations from known mRNA expression patterns, suggesting a non-cell autonomous action of these factors or alternative control mechanisms. In addition, we observed a change in the subcellular localisation of SEPALLATA3 from a predominantly nuclear localisation to a more cytoplasmic localisation, occurring specifically during petal and stamen development. Furthermore, we show that the down-regulation of the homeodomain transcription factor WUSCHEL in ovular tissues is preceded by the occurrence of both AGAMOUS and SEPALLATA3 proteins, supporting the hypothesis that both proteins together suppress WUSCHEL expression in the ovule. Conclusion This approach provides a highly detailed in situ map of MADS domain protein presence during early and later stages of floral development. The subcellular localisation of the transcription factors in the cytoplasm, as observed at certain stages during development, points to mechanisms other than transcriptional control. Together this information is essential to understand the role of these proteins in the regulatory processes that drive floral development and leads to new hypotheses.</p

Northern blot analysis of leaf tissue of different Arabidopsis lines containing various tagging constructs

<p><b>Copyright information:</b></p><p>Taken from "Tagging of MADS domain proteins for chromatin immunoprecipitation"</p><p>http://www.biomedcentral.com/1471-2229/7/47</p><p>BMC Plant Biology 2007;7():47-47.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2071916.</p><p></p> (A) Expression analysis of (pARC276) lines. (B) Expression analysis of (pARC277) lines. (C) Expression analysis of --tagII-FLAG-tag (pARC117) lines, -like plants are indicated with 'm' and WT-like plants with 'n'. WT, wild-type; +, line with an overexpression phenotype