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Primary cilia are specialized organelles supporting the development and function of cells and organisms. Intraflagellar transport (IFT) is essential for cilia formation, maintenance, and function. In C. elegans ciliated sensory neurons, IFT interacts with signaling molecules to generate distinct morphological and function features and also to maintain the integrity of cilia. Here, we report an IFT-dependent feedback control on the conserved MAPKKK DLK-1 in the ciliated sensory neurons. DLK proteins are widely known to act in synapse formation, axon regeneration, and degeneration, but their roles in other neuronal compartments are understudied. By forward genetic screening for altered expression of the endogenously tagged DLK-1 we identified multiple ift mutants showing increased DLK-1 accumulation in the defective sensory endings. We show that in response to acute IFT disruption, DLK-1 accumulates rapidly and reversibly. The expression levels of the transcription factor CEBP-1, known to act downstream of DLK-1 in the development and maintenance of synapses and axons, are also increased in the ciliated sensory neurons of ift mutants. Interestingly, the regulation of CEBP-1 expression shows sensory neuron-type dependency on DLK-1. Moreover, in the sensory neuron AWC, which has elaborate cilia morphology, up-regulated CEBP-1 represses DLK-1 at the transcription level, thereby dampening DLK-1 accumulation. Last, the IFT-dependent regulatory loop of DLK-1 and CEBP-1 offers neuroprotection in a cilia degeneration model. These findings uncover a surveillance mechanism in which tight control on the DLK-1 signaling protects cilia integrity in a context-specific manner.

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The Activator Protein-1 transcription factor family (AP-1) transcriptional complex is historically defined as an early response group of transcription factors formed by dimeric complexes of the Jun, Fos, Atf, and Maf bZIP proteins that control cell proliferation and differentiation by regulating gene expression. It has been greatly investigated in many model organisms across metazoan evolution. Nevertheless, its complexity and variability of action made its multiple functions difficult to be defined. Here, we place the foundations for understanding the complexity of AP-1 transcriptional members in tunicates. We investigated the gene members of this family in the ascidian Ciona robusta and identified single copies of Jun, Fos, Atf3, Atf2/7, and Maf bZIP-related factors that could have a role in the formation of the AP-1 complex. We highlight that mesenchyme is a common cellular population where all these factors are expressed during embryonic development, and that, moreover, Fos shows a wider pattern of expression including also notochord and neural cells. By ectopic expression in transgenic embryos of Jun and Fos genes alone or in combination, we investigated the phenotypic alterations induced by these factors and highlighted a degree of functional conservation of the AP-1 complex between Ciona and vertebrates. The lack of gene redundancy and the first pieces of evidence of conserved functions in the control of cell movements and structural organization exerted by these factors open the way for using Ciona as a helpful model system to uncover the multiple potentialities of this highly complex family of bZIP transcription factors.

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We have characterized a rice bZIP protein-coding gene OsbZIP62/OsFD7 that is expressed preferentially in the shoot apical meristem and during early panicle developmental stages in comparison with other OsFD genes characterized to date. Surprisingly, unlike OsFD1, OsFD7 interacts directly and more efficiently with OsFTLs; the interaction is strongest with OsFTL1 followed by Hd3a and RFT1, as confirmed by fluorescence lifetime imaging-Förster resonant energy transfer (FLIM-FRET) analysis. In addition, OsFD7 is phosphorylated at its C-terminal end by OsCDPK41 and OsCDPK49 in vitro, and this phosphorylated moiety is recognized by OsGF14 proteins. OsFD7 RNAi transgenics were late flowering; the transcript levels of some floral meristem identity genes (e.g. OsMADS14, OsMADS15, and OsMADS18) were also down-regulated. RNAi lines also exhibited dense panicle morphology with an increase in the number of primary and secondary branches resulting in longer panicles and more seeds, probably due to down-regulation of SEPALLATA family genes. In comparison with other FD-like proteins previously characterized in rice, it appears that OsFD7 may have undergone diversification during evolution, resulting in the acquisition of newer functions and thus playing a dual role in floral transition and panicle development in rice.

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Seed dormancy is closely related to pre-harvest sprouting resistance. Both plant hormone abscisic acid (ABA) and DELAY OF GERMINATION 1 (DOG1) protein are key regulators of seed dormancy. Their relationship is well reported in Arabidopsis, but little is known in rice. Here, we show that a quantitative trait locus, qSd-1-1 contributes significantly to seed dormancy differences between the strongly dormant indica variety N22 and non-dormant japonica variety Nanjing35. It encodes a DOG1-like protein named OsDOG1L-3 with homology to Arabidopsis DOG1. There were evident promoter and expression differences in OsDOG1L-3 between N22 and Nanjing35, and overexpression or introduction of the N22 OsDOG1L-3 allele in Nanjing35 enhanced its seed dormancy. OsDOG1L-3 expression was positively correlated with seed dormancy and induced by ABA. OsbZIP75 and OsbZIP78 bound directly with the promoter of OsDOG1L-3 to induce its expression. Overexpression of OsbZIP75 increased OsDOG1L-3 protein abundance and promoted seed dormancy. OsDOG1L-3 upregulated expression of ABA-related genes and increased ABA content. We propose that the N22 OsDOG1L-3 allele is a candidate gene for the seed dormancy in QTL qSd-1-1, and that it participates in the ABA pathway to establish seed dormancy in rice.

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BACKGROUND: bZIP proteins participate in the regulation of gene expression, playing crucial roles in various biological processes in plants, including response to environmental changes. Luminosity is an environmental factor of extreme importance for plant metabolism, acting as a regulator of its growth and development. Despite advances in the identification of bZIP proteins in several plant species, studies on these transcription factors in cassava are lacking. Cassava (Manihot esculenta Crantz) is one of the most important food crops in tropical and subtropical regions, mainly in developing countries, where its storage root is a major source of calories for low-income people. OBJECTIVES: Our main aim was the isolation of a cDNA sequence encoding a bZIP protein from cassava (MebZIP) as well as the in silico characterization of its nucleotide and deduced amino acid sequences. In addition, we evaluated the expression pattern of the MebZIP gene in response to light, and its possible relationship with regulation of the chalcone synthase (MeCHS) gene. METHODS: RT-PCR and 3' and 5' RACE assays were used to isolate the full-length cDNA sequence of MebZIP. Bioinformatics tools were used to characterize the nucleotide and amino acid sequences of MebZIP. Semiquantitative RT-PCR assays were used to evaluate the expression levels of MebZIP and MeCHS genes. RESULTS: We isolated the full-length cDNA sequence of MebZIP with a 1320-bp ORF encoding a deduced protein with a predicted molecular weight and isoelectric point of 47 kDa and 5.85, respectively. Comparative analyses with GenBank sequences showed high identity of MebZIP with bZIP CPRF-2 of Hevea brasiliensis (XP_021650934) and Petroselinum crispum (Q99090.2). Besides the basic region and leucine zipper domains, MebZIP contains putative conserved domains (D1- D4), found in parsley CPRF-2 and bZIP proteins closely related to this protein. Since CPRF proteins are known for their function in regulation of the CHS gene by light, we evaluated the expression levels of the MebZIP gene and the possible target gene to be regulated by MebZIP (the MeCHS gene) in cassava under light conditions. Semi-quantitative RT-PCR assays revealed that MebZIP transcription increased in response to white light, with maximum expression levels at 6 h of light exposure. On the other hand, the expression levels of the MeCHS gene were statistically constant in all samples, indicating that they were not influenced by the experimental conditions used here. CONCLUSION: The putative MebZIP protein identified in this work contains the conserved domains (bZIP, D1-D4) that indicate its functionality, thus allowing it to be considered a new member of the bZIP transcription factor CPRF-2 family. The expression levels of the MebZIP gene increased during white light exposure, indicating a potential function in light-response in cassava.

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Vaccines play a crucial role in the protection of animals and humans from deadly pathogens. The first vaccine that also protected against cancer was developed against the highly oncogenic herpesvirus Marek's disease virus (MDV). MDV infects chickens and causes severe immunosuppression, neurological signs, and fatal lymphomas, a process that requires the viral oncogene, meq The most frequently used Marek's disease vaccine is the live-attenuated CVI988/Rispens (CVI) strain, which efficiently protects chickens and prevents tumorigenesis. Intriguingly, CVI expresses at least two isoforms of meq; however, it remains unknown to what extent these isoforms contribute to virus attenuation. In this study, we individually examined the contribution of the two CVI-meq isoforms to the attenuation of the vaccine. We inserted the respective isoforms into a very virulent MDV (strain RB-1B), thereby replacing its original meq gene. Surprisingly, we could demonstrate that the longer isoform of meq strongly enhanced virus-induced pathogenesis and tumorigenesis, indicating that other mutations in the CVI genome contribute to virus attenuation. On the contrary, the shorter isoform completely abrogated pathogenesis, demonstrating that changes in the meq gene can indeed play a key role in virus attenuation. Taken together, our study provides important evidence on attenuation of one of the most frequently used veterinary vaccines worldwide.IMPORTANCE Marek's disease virus (MDV) is one of several oncogenic herpesviruses and causes fatal lymphomas in chickens. The current "gold standard" vaccine is the live-attenuated MDV strain CVI988/Rispens (CVI), which is widely used and efficiently prevents tumor formation. Intriguingly, CVI expresses two predominant isoforms of the major MDV oncogene meq: one variant with a regular size of meq (Smeq) and one long isoform (Lmeq) harboring an insertion of 180 bp in the transactivation domain. In our study, we could break the long-standing assumption that the Lmeq isoform is an indicator for virus attenuation. Using recombinant viruses that express the different CVI-meq isoforms, we could demonstrate that both isoforms drastically differ in their abilities to promote pathogenesis and tumor formation in infected chickens.

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The pathogenesis of Epstein-Barr virus (EBV) infection, including development of lymphomas and carcinomas, is dependent on the ability of the virus to transit from latency to the lytic phase. This conversion, and ultimately disease development, depends on the molecular switch protein, ZEBRA, a viral bZIP transcription factor that initiates transcription from promoters of viral lytic genes. By binding to the origin of viral replication, ZEBRA is also an essential replication protein. Here, we identified a novel DNA-binding motif of ZEBRA, N terminal to the canonical bZIP domain. This RRTRK motif is important for high-affinity binding to DNA and is essential for recognizing the methylation state of viral promoters. Mutations in this motif lead to deficiencies in DNA binding, recognition of DNA methylation, lytic cycle DNA replication, and viral late gene expression. This work advances our understanding of ZEBRA-dependent activation of the viral lytic cascade.IMPORTANCE The binding of ZEBRA to methylated and unmethylated viral DNA triggers activation of the EBV lytic cycle, leading to viral replication and, in some patients, cancer development. Our work thoroughly examines how ZEBRA uses a previously unrecognized basic motif to bind nonmethylated and methylated DNA targets, leading to viral lytic activation. Our findings show that two different positively charged motifs, including the canonical BZIP domain and a newly identified RRTRK motif, contribute to the mechanism of DNA recognition by a viral AP-1 protein. This work contributes to the assessment of ZEBRA as a potential therapeutic target for antiviral and oncolytic treatments.

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Alcelaphine herpesvirus-1 (AlHV-1) causes malignant catarrhal fever (MCF). The A2 gene of AlHV-1 is a member of the bZIP transcription factor family. We wished to determine whether A2 is a virulence gene or not and whether it is involved in pathogenesis by interference with host transcription pathways. An A2 gene knockout (A2ΔAlHV-1) virus, revertant (A2revAlHV-1) virus, and wild-type virus (wtAlHV-1) were used to infect three groups of rabbits. A2ΔAlHV-1-infected rabbits succumbed to MCF, albeit with a delayed onset compared to the control groups, so A2 is not a critical virulence factor. Differential gene transcription analysis by RNAseq and qRT-PCR validation of a selection of these was performed in infected large granular lymphocyte (LGL) T cells obtained in culture from the MCF-affected animals. A2 was involved in the transcriptional regulation of immunological, cell cycle and apoptosis pathways. In particular, there was a bias towards γδ T cell receptor (TCR) expression and downregulation of αß TCR. TCR signalling, apoptosis, cell cycle, IFN-γ and NFAT pathways were affected. Of particular interest was partial inhibition of the cytotoxicity-associated pathways involving perforin and the granzymes A and B in the A2ΔAlHV-1-infected LGLs compared to controls. In functional assays, A2ΔAlHV-1-infected LGLs were significantly less cytotoxic than wtAlHV-1- and A2revAlHV-1-infected LGLs using rabbit corneal epithelial cells (SIRC) as targets. This implies that A2 is involved in a pathway enhancing the expression of LGL cytotoxicity. This is important as virus-infected T cell cytotoxicity in vivo has been suggested as a potential mechanism of disease induction in MCF.

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