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Mitogen‐activated protein kinase (MAPK) cascades play key roles in plant immune signalling, and elucidating their regulatory functions requires the identification of the pathway‐specific substrates. We used yeast two‐hybrid interaction screens, in vitro kinase assays and mass spectrometry‐based phosphosite mapping to study a family of MAPK substrates. Site‐directed mutagenesis and promoter‐reporter fusion studies were performed to evaluate the impact of substrate phosphorylation on downstream signalling. A subset of the Arabidopsis thaliana VQ‐motif‐containing proteins (VQPs) were phosphorylated by the MAPKs MPK3 and MPK6, and renamed MPK3/6‐targeted VQPs (MVQs). When plant protoplasts (expressing these MVQs) were treated with the flagellin‐derived peptide flg22, several MVQs were destabilized in vivo. The MVQs interact with specific WRKY transcription factors. Detailed analysis of a representative member of the MVQ subset, MVQ1, indicated a negative role in WRKY‐mediated defence gene expression – with mutation of the VQ‐motif abrogating WRKY binding and causing mis‐regulation of defence gene expression. We postulate the existence of a variety of WRKY‐VQP‐containing transcriptional regulatory protein complexes that depend on spatio‐temporal VQP and WRKY expression patterns. Defence gene transcription can be modulated by changing the composition of these complexes – in part – through MAPK‐mediated VQP degradation.

PurposeStarting from the status quo, the paper outlines perspectives and challenges for the connection and interlinking of digitised and digital archival data. The following topics are addressed: Where are fields of action and what are the means of archives? Which functional and technical requirements are to be considered, and what is the role of portal infrastructures linking together various different institutions?Design/methodology/approachConsidering needs of users and general framework conditions, the paper examines new approaches emerging in Germany. It outlines recent projects and considerations aiming to improve services and visibility of archives within the national data infrastructure in Germany.FindingsCross-connections are no new phenomenon, but change their appearance significantly in a digital context. In this respect, both smaller and bigger archives profit from participation in larger digital networks. Furthermore, archives need to keep in mind to reflect the quality of their digital (meta)data regularly and to offer or join systems that functionally and technically support cross-connection and interlinking of data.Originality/valueThe paper endeavours to show the importance of digital cross-connections and the role of portal infrastructures for visibility, online-distribution and use of digital archival metadata and data.

Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6), a previously reported DBP1 interactor, and MAP kinase (MAPK) MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV), and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

Toll-like receptor (TLR) 3 agonists emerged as attractive candidates for vaccination strategies against tumors and pathogens. An important mechanism of action of such agonists is based on the activation of TLR3-expressing dendritic cells (DCs), which display a unique capacity to induce and stimulate T-cell responses. In this context, it has been demonstrated that targeting of TLR3 by double-stranded RNA such as poly(I:C) results in potent activation of DCs. Major disadvantages of poly(I:C) comprise its undefined chemical structure and very poor homogeneity, with subsequent unpredictable pharmacokinetics and high toxicity. In the present study, we evaluated the physicochemical properties and biological activity of the novel TLR3 agonist RGC100. RGC100 has a defined chemical structure, with a defined length (100 bp) and molecular weight (64.9 KDa) and a good solubility. RGC100 is stable in serum and activates myeloid DCs through TLR3 targeting, as evidenced by gene silencing experiments. Activation of mouse and human myeloid CD1c+ DCs by RGC100 leads to secretion of several proinflammatory cytokines. In addition, RGC100 improves the ability of CD1c+ DCs to stimulate T-cell proliferation. Due to its physicochemical properties and its immunostimulatory properties, RGC100 may represent a promising adjuvant for prophylactic and therapeutic vaccination strategies.

Phosphorylation is an important post-translational protein modification with regulatory roles in diverse cellular signaling pathways. Despite recent advances in mass spectrometry, the detection of phosphoproteins involved in signaling is still challenging, as protein phosphorylation is typically transient and/or occurs at low levels. In green plant tissues, the presence of highly abundant proteins, such as the subunits of the RuBisCO complex, further complicates phosphoprotein analysis. Here, we describe a simple, but powerful, method, which we named prefractionation-assisted phosphoprotein enrichment (PAPE), to increase the yield of phosphoproteins from Arabidopsis thaliana leaf material. The first step, a prefractionation via ammonium sulfate precipitation, not only depleted RuBisCO almost completely, but, serendipitously, also served as an efficient phosphoprotein enrichment step. When coupled with a subsequent metal oxide affinity chromatography (MOAC) step, the phosphoprotein content was highly enriched. The reproducibility and efficiency of phosphoprotein enrichment was verified by phospho-specific staining and, further, by mass spectrometry, where it could be shown that the final PAPE fraction contained a significant number of known and additionally novel (potential) phosphoproteins. Hence, this facile two-step procedure is a good prerequisite to probe the phosphoproteome and gain deeper insight into plant phosphorylation-based signaling events.

Mitogen‐activated protein kinase ( MAPK ) cascades play key roles in plant immune signalling, and elucidating their regulatory functions requires the identification of the pathway‐specific substrates. We used yeast two‐hybrid interaction screens, in vitro kinase assays and mass spectrometry‐based phosphosite mapping to study a family of MAPK substrates. Site‐directed mutagenesis and promoter‐reporter fusion studies were performed to evaluate the impact of substrate phosphorylation on downstream signalling. A subset of the A rabidopsis thaliana VQ ‐motif‐containing proteins ( VQP s) were phosphorylated by the MAPK s MPK 3 and MPK 6, and renamed MPK 3/6‐targeted VQP s ( MVQ s). When plant protoplasts (expressing these MVQ s) were treated with the flagellin‐derived peptide flg22, several MVQ s were destabilized in vivo . The MVQ s interact with specific WRKY transcription factors. Detailed analysis of a representative member of the MVQ subset, MVQ 1, indicated a negative role in WRKY ‐mediated defence gene expression – with mutation of the VQ ‐motif abrogating WRKY binding and causing mis‐regulation of defence gene expression. We postulate the existence of a variety of WRKY ‐ VQP ‐containing transcriptional regulatory protein complexes that depend on spatio‐temporal VQP and WRKY expression patterns. Defence gene transcription can be modulated by changing the composition of these complexes – in part – through MAPK ‐mediated VQP degradation.

Three-dimensional DNA nanoscaffolds such as supramolecular tetrahedra can self-assemble from tris-oligonucleotidyls -- synthetic three-armed building blocks in which three identical or non-identical short DNA sequences are connected by a tris-linking backbone. Here we show that the connectivity information contained in these building blocks can be copied by using template-directed tris-linking. This finding is a crucial step towards the replication of nanoarchitectures that are based on tris-oligonucleotidyls and to the realization of artificially self-replicating systems on a nanometre scale.

Three-dimensional DNA nanoscaffolds such as supramolecular tetrahedra can self-assemble from tris-oligonucleotidyls — synthetic three-armed building blocks in which three identical or non-identical short DNA sequences are connected by a tris-linking backbone 1 , 2 . Here we show that the connectivity information contained in these building blocks can be copied by using template-directed tris-linking. This finding is a crucial step towards the replication of nanoarchitectures that are based on tris-oligonucleotidyls and to the realization of artificially self-replicating systems on a nanometre scale.