Pou5f1 protein expression and posttranslational modification during early zebrafish development.

BACKGROUND: Pou5f1/Oct4 is a transcription factor essential for maintenance of pluripotency in mammals and for control of blastula and gastrula stage gene regulatory networks in zebrafish. Information on Pou5f1 protein distribution was before this study not available for zebrafish. Therefore, we generated polyclonal antibodies that selectively recognize Pou5f1 and analyzed its protein distribution and modification during development. RESULTS: Pou5f1 protein is present in unfertilized oocytes, and persists in all embryonic and enveloping layer cell nuclei until the end of gastrulation, but is absent from yolk syncytial nuclei. Pou5f1 is subject to multiple developmentally regulated phosphorylations, with the higher phosphorylated forms prevailing in the oocyte and during late gastrulation. CONCLUSIONS: The developmental protein profile correlates with the stages during which deep cells are not committed to a specific germ layer. The posttranslational modification by phosphorylation opens the possibility that Pou5f1 may be subject to temporal or region specific modulation of its activity or stability by embryonic signaling mechanisms.

Expression of AtWRKY33 encoding a pathogen- or PAMP-responsive WRKY transcription factor is regulated by a composite DNA motif containing W box elements.

WRKY transcription factors regulate distinct parts of the plant defense transcriptome. Expression of many WRKY genes themselves is induced by pathogens or pathogen-mimicking molecules. Here, we demonstrate that Arabidopsis WRKY33 responds to various stimuli associated with plant defense as well as to different kinds of phytopathogens. Although rapid pathogen-induced AtWRKY33 expression does not require salicylic acid (SA) signaling, it is dependent on PAD4, a key regulator upstream of SA. Activation of AtWRKY33 is independent of de novo protein synthesis, suggesting that it is at least partly under negative regulatory control. We show that a set of three WRKY-specific cis-acting DNA elements (W boxes) within the AtWRKY33 promoter is required for efficient pathogen- or PAMP-triggered gene activation. This strongly indicates that WRKY transcription factors are major components of the regulatory machinery modulating immediate to early expression of this gene in response to pathogen attack.

Members of the Arabidopsis WRKY group III transcription factors are part of different plant defense signaling pathways.

WRKY proteins are a large group of transcription factors restricted to the plant kingdom. In Arabidopsis thaliana, the gene family consists of 74 members. Here, we analyzed the expression of all 13 members of one main WRKY subgroup and found that the majority are responsive both to pathogen infection and to salicylic acid. Temporal expression studies during compatible, incompatible, and nonhost interactions and employing plant defense-signaling mutants allowed us to define four distinct WRKY subsets responding to different signaling queues along defense pathways. These subsets did not reflect phylogenetic relationships. Promoter studies of one member, AtWRKY54, using a reporter gene construct in transgenic Arabidopsis plants, revealed that regulatory regions mediating pathogen and SA inducibility are clearly separable. In an AtWRKY54 knockout line, resistance to Peronospora parasitica was not compromised, but the transient expression kinetics of several WRKY genes was affected, suggesting both the existence of functional redundancy and intense cross-talk between signaling networks.

Two immediate-early pathogen-responsive members of the AtCMPG gene family in Arabidopsis thaliana and the W-box-containing elicitor-response element of AtCMPG1.

The Arabidopsis thaliana genome contains at least 50 predicted AtCMPG genes. The encoded protein family is defined by a common domain possessing four strictly conserved amino acid residues [Cys, Met, Pro, and Gly (CMPG)] that designate the family. Two members, AtCMPG1 and AtCMPG2, with high sequence similarity to the previously described, immediate-early pathogen-responsive PcCMPG1 gene from Petroselinum crispum were selected for analysis of their expression modes and defense-related promoter elements. Among the most striking similarities with PcCMPG1 were immediate-early transcriptional activation on infection or treatment with a pathogen-derived elicitor and the functional importance of a W-box-containing AtCMPG1 promoter element. Remarkably, this strongly pathogen/elicitor-responsive element, F, did not respond to wounding, in contrast to the AtCMPG1 promoter itself. Comparative analysis, both within the A. thaliana genome and across species, provided further insight into the large structural diversity of W-box-containing elements. Possible roles of AtCMPG proteins in regulatory processes are discussed with reference to a large variety of family members, partly with assigned functions, from plants as well as animals.

Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling.

Pathogen-inducible plant promoters contain multiple cis-acting elements, only some of which may contribute to pathogen inducibility. Therefore, we made defined synthetic promoters containing tetramers of only a single type of element and present evidence that a range of cis-acting elements (boxes W1, W2, GCC, JERE, S, Gst1, and D) can mediate local gene expression in planta after pathogen attack. The expression patterns of the promoters were monitored during interactions with a number of pathogens, including compatible, incompatible, and nonhost interactions. Interestingly, there were major differences in the inducibilities of the various promoters with the pathogens tested as well as differences in the speed of induction and in the basal expression levels. We also show that defense signaling is largely conserved across species boundaries at the cis-acting element level. Many of these promoters also direct local wound-induced expression, and this provides evidence for the convergence of resistance gene, nonhost, and wound responses at the level of the promoter elements. We have used these cis-acting elements to construct improved synthetic promoters and show the effects of varying the number, order, and spacing of such elements. These promoters are valuable additions to the study of signaling and transcriptional activation during plant-pathogen interactions.