Stimulating the autophagic-lysosomal axis enhances host defense against fungal infection in a zebrafish model of invasive Aspergillosis.

The increasing prevalence of antifungal-resistant human pathogenic fungi, particularly azole-resistant Aspergillus fumigatus, is a life-threatening challenge to the immunocompromised population. Autophagy-related processes such as LC3-associated phagocytosis have been shown to be activated in the host response against fungal infection, but their overall effect on host resistance remains uncertain. To analyze the relevance of these processes in vivo, we used a zebrafish animal model of invasive Aspergillosis. To confirm the validity of this model to test potential treatments for this disease, we confirmed that immunosuppressive treatments or neutropenia rendered zebrafish embryos more susceptible to A. fumigatus. We used GFP-Lc3 transgenic zebrafish to visualize the autophagy-related processes in innate immune phagocytes shortly after phagocytosis of A. fumigatus conidia, and found that both wild-type and melanin-deficient conidia elicited Lc3 recruitment. In macrophages, we observed GFP-Lc3 accumulation in puncta after phagocytosis, as well as short, rapid events of GFP-Lc3 decoration of single and multiple conidia-containing vesicles, while neutrophils covered single conidia-containing vesicles with bright and long-lasting GFP-Lc3 signal. Next, using genetic and pharmacological stimulation of three independent autophagy-inducing pathways, we showed that the antifungal autophagy response improves the host survival against A. fumigatus infection, but only in the presence of phagocytes. Therefore, we provide proof-of-concept that stimulating the (auto)phagolysosomal pathways is a promising approach to develop host-directed therapies against invasive Aspergillosis, and should be explored further either as adjunctive or stand-alone therapy for drug-resistant Aspergillus infections.Abbreviations: DMSO: dimethyl sulfoxide; HR: hazard ratio; HDT: host-directed therapy; Hpf: hours post fertilization; IA: invasive Aspergillosis; LAP: LC3-associated phagocytosis; MTZ: metronidazole; PTU: N-phenylthiourea; ROS: reactive oxygen species.

Correction: Variation of virulence of five Aspergillus fumigatus isolates in four different infection models.

[This corrects the article DOI: 10.1371/journal.pone.0252948.].

Variation of virulence of five Aspergillus fumigatus isolates in four different infection models.

Conidia of Aspergillus fumigatus are inhaled by humans on daily basis. As a consequence, these conidia can cause infections that differ in severity ranging from allergic bronchopulmonary aspergillosis to invasive aspergillosis. In this study we compared virulence of five A. fumigatus isolates in four different infection models to address the predictive value of different model systems. Two of the A. fumigatus strains were isolated from dogs with a non-invasive sino-nasal aspergillosis (DTO271-B5 and DTO303-F3), while three strains were isolated from human patients with invasive aspergillosis (Af293, ATCC46645 and CEA10). Infection models used encompassed cultured type II A549 lung epithelial cells, Protostelium aurantium amoeba, Galleria melonella larvae and zebrafish embryos. No major differences in virulence between these five strains were observed in the lung epithelial cell model. In contrast, strain ATCC46645 was most virulent in the amoeba and zebrafish model, whereas it was much less virulent in the Galleria infection model. DTO303-F3 was most virulent in the latter model. In general, reference strain Af293 was less virulent as compared to the other strains. Genome sequence analysis showed that this latter strain differed from the other four strains in 136 SNPs in virulence-related genes. Together, our results show that virulence of individual A. fumigatus strains show significant differences between infection models. We conclude that the predictive value of different model systems varies since the relative virulence across fungal strains does not hold up across different infection model systems.

CXCR4 signaling regulates metastatic onset by controlling neutrophil motility and response to malignant cells.

Developing tumors interact with the surrounding microenvironment. Myeloid cells exert both anti- and pro-tumor functions and chemokines are known to drive immune cell migration towards cancer cells. It is documented that CXCR4 signaling supports tumor metastasis formation in tissues where CXCL12, its cognate ligand, is abundant. On the other hand, the role of the neutrophilic CXCR4 signaling in driving cancer invasion and metastasis formation is poorly understood. Here, we use the zebrafish xenotransplantation model to study the role of CXCR4 signaling in driving the interaction between invasive human tumor cells and host neutrophils, supporting early metastasis formation. We found that zebrafish cxcr4 (cxcr4b) is highly expressed in neutrophils and experimental micrometastases fail to form in mutant larvae lacking a functional Cxcr4b. We demonstrated that Cxcr4b controls neutrophil number and motility and showed that Cxcr4b transcriptomic signature relates to motility and adhesion regulation in neutrophils in tumor-naïve larvae. Finally, Cxcr4b deficient neutrophils failed to interact with cancer cells initiating early metastatic events. In conclusion, we propose that CXCR4 signaling supports the interaction between tumor cells and host neutrophils in developing tumor metastases. Therefore, targeting CXCR4 on tumor cells and neutrophils could serve as a double bladed razor to limit cancer progression.

Transcriptomic Approaches in the Zebrafish Model for Tuberculosis-Insights Into Host- and Pathogen-specific Determinants of the Innate Immune Response.

Mycobacterium marinum infection in zebrafish has become a well-established model of tuberculosis. Both embryonic and adult zebrafish infection studies have contributed to our knowledge of the development and function of tuberculous granulomas, which are typical of mycobacterial pathogenesis. In this review we discuss how transcriptome profiling studies have helped to characterize this infection process. We illustrate this using new RNA sequencing (RNA-Seq) data that reveals three main phases in the host response to M. marinum during the early stages of granuloma development in zebrafish embryos and larvae. The early phase shows induction of complement and transcription factors, followed by a relatively minor induction of pro-inflammatory cytokines within hours following phagocytosis of M. marinum. A minimal response is observed in the mid-phase, between 6 hours and 1day post infection, when the tissue dissemination of M. marinum begins. During subsequent larval development the granulomas expand and a late-phase response is apparent, which is characterized by progressively increasing induction of complement, transcription factors, pro-inflammatory cytokines, matrix metalloproteinases, and other defense and inflammation-related gene groups. This late-phase response shares common components with the strong and acute host transcriptome response that has previously been reported for Salmonella typhimurium infection in zebrafish embryos. In contrast, the early/mid-phase response to M. marinum infection, characterized by suppressed pro-inflammatory signaling, is strikingly different from the acute response to S. typhimurium infection. Furthermore, M. marinum infection shows a collective and strongly fluctuating regulation of lipoproteins, while S. typhimurium infection has pronounced effects on amino acid metabolism and glycolysis.

The embryonic expression patterns of zebrafish genes encoding LysM-domains.

The function and structure of LysM-domain containing proteins are very diverse. Although some LysM domains are able to bind peptidoglycan or chitin type carbohydrates in bacteria, in fungi and in plants, the function(s) of vertebrate LysM domains and proteins remains largely unknown. In this study we have identified and annotated the six zebrafish genes of this family, which encode at least ten conceptual LysM-domain containing proteins. Two distinct sub-families called LysMD and OXR were identified and shown to be highly conserved across vertebrates. The detailed characterization of LysMD and OXR gene expression in zebrafish embryos showed that all the members of these sub-families are strongly expressed maternally and zygotically from the earliest stages of a vertebrate embryonic development. Moreover, the analysis of the spatio-temporal expression patterns, by whole mount and fluorescent in situ hybridizations, demonstrates pronounced LysMD and OXR gene expression in the zebrafish brain and nervous system during stages of larval development. None of the zebrafish LysMD or OXR genes was responsive to challenge with bacterial pathogens in embryo models of Salmonella and Mycobacterium infections. In addition, the expression patterns of the OXR genes were mapped in a zebrafish brain atlas.

Genomic annotation and transcriptome analysis of the zebrafish (Danio rerio) hox complex with description of a novel member, hox b 13a.

The zebrafish (Danio rerio) is an important model in evolutionary developmental biology, and its study is being revolutionized by the zebrafish genome project. Sequencing is at an advanced stage, but annotation is largely the result of in silico analyses. We have performed genomic annotation, comparative genomics, and transcriptional analysis using microarrays of the hox homeobox-containing transcription factors. These genes have important roles in specifying the body plan. Candidate sequences were located in version Z v 4 of the Ensembl genome database by TBLASTN searching with Danio and other vertebrate published Hox protein sequences. Homologies were confirmed by alignment with reference sequences, and by the relative position of genes along each cluster. RT-PCR using adult Tübingen cDNA was used to confirm annotations, to check the genomic sequence and to confirm expression in vivo. Our RT-PCR and microarray data show that all 49 hox genes are expressed in adult zebrafish. Significant expression for all known hox genes could be detected in our microarray analysis. We also find significant expression of hox 8 paralogs and hox b 7 a in the anti-sense direction. A novel gene, D. rerio hox b 13 a, was identified, and a preliminary characterization by in situ hybridization showed expression at 24 hpf at the tip of the developing tail. We are currently characterizing this gene at the functional level. We argue that the oligo design for microarrays can be greatly enhanced by the availability of genomic sequences.

Transcription and somatic transposition of the maize En/Spm transposon system in rice.

Transposition of the maize En/Spm system in rice was investigated using a two-component construct consisting of an immobilised transposase source driven by the CaMV 35S-promoter, and a modified I/dSpm transposon. Mobilization of I/dSpm in somatic sectors was demonstrated by sequencing of excision products and isolation of flanking genomic sequences in T0 and T1 progeny plants. Since the transposition efficiency appeared to be considerably lower than that observed in maize or in other heterologous systems like Arabidopsis, we examined En/Spm transcription and splicing in the transgenic rice plants. Northern analysis revealed the presence of transcripts encoding the active TnpA and TnpD transposases, with the latter predominating; this is the reverse of what is seen in maize and Arabidopsis. RT-PCR analysis confirmed the occurrence of correct splicing and the formation of the two other alternatively spliced transcripts (TnpB and TnpC), as previously described for maize. Two alternative splice donor sites at the end of exon 1 were identified in maize at positions 578 and 704. We observe that rice is similar to maize in that TnpA is preferentially spliced at position 578. We also show that in Arabidopsis splicing occurs preferentially at position 704, as in other dicots like tobacco. These observations indicate differences in the splicing of transcripts of the maize En/Spm element between dicot and monocot hosts. Nevertheless, the ratio in which the transcripts for the active transposases are produced seems to determine the efficiency of transposition, irrespective of the host considered. A limiting amount of TnpA might therefore be responsible for the lower transposition activity of En/Spm in rice. Alternatively, reduced mobility of the modified I/dSpm element used may have resulted from the absence of critical sequences necessary for transposition. The influence of endogenous, autonomous, En/Spm -related elements present in the rice genome on the transposition behaviour of the exogenous maize element is also considered.

Transpositional behaviour of an Ac/Ds system for reverse genetics in rice.

A collection of transposon Ac/ Ds enhancer trap lines is being developed in rice that will contribute to the development of a rice mutation machine for the functional analysis of rice genes. Molecular analyses revealed high transpositional activity in early generations, with 62% of the T0 primary transformants and more than 90% of their T1 progeny lines showing ongoing active transposition. About 10% of the lines displayed amplification of the Ds copy number. However, inactivation of Ds seemed to occur in about 70% of the T2 families and in the T3 generation. Southern blot analyses revealed a high frequency of germinal insertions inherited in the T1 progeny plants, and transmitted preferentially over the many other somatic inserts to later generations. The sequencing of Ds flanking sites in subsets of T1 plants indicated the independence of insertions in different T1 families originating from the same T0 line. Almost 80% of the insertion sites isolated showing homology to the sequenced genome, resided in genes or within a range at which neighbouring genes could be revealed by enhancer trapping. A strategy involving the propagation of a large number of T0 and T1 independent lines is being pursued to ensure the recovery of a maximum number of independent insertions in later generations. The inactive T2 and T3 lines produced will then provide a collection of stable insertions to be used in reverse genetics experiments. The preferential insertion of Ds in gene-rich regions and the use of lines containing multiple Ds transposons will enable the production of a large population of inserts in a smaller number of plants. Additional features provided by the presence of lox sites for site-specific recombination, or the use of different transposase sources and selectable markers, are discussed.

Subdural hygroma: a rare complication of spinal anesthesia.

IMPLICATIONS: We describe a patient with frontal hygroma after spinal anesthesia--a rare complication. This condition should be considered in patients after spinal anesthesia with persisting orthostatic headache. Risk factors include ventriculo-peritoneal shunt or brain atrophy caused by old age.

Glucocorticoid-inducible gene expression in rice.

We have studied the use of a glucocorticoid receptor-based inducible gene expression system in the monocotyledonous model plant rice (Oryza sativa L.). This system, originally developed by T. Aoyama and N.-H. Chua [(1997) Plant J 11: 605-612], is based on the chimaeric transcriptional activator GVG, consisting of the yeast Gal4 DNA-binding domain, the VP16 activation domain and the glucocorticoid receptor domain. For application in rice, we designed an optimized binary vector series (pINDEX) and tested this with the beta-glucuronidase (gusA) reporter gene. GUS expression was tightly controlled and relatively low concentrations (1-10 microM) of the glucocorticoid hormone dexamethasone (DEX) were able to induce GUS activities to levels comparable to those conferred by the strong cauliflower mosaic virus (CaMV) 35S promoter. DEX was taken up efficiently by the roots of tissue-cultured plantlets or mature plants in hydroponic culture, and induced GUS activity throughout the whole plant. DEX-induced GUS expression patterns were consistent in all lines and their T1 progeny. The phenotype of tissue-cultured rice plantlets was not affected when inductions with 10-100 microM DEX were limited to 1-4 days or when 2-week inductions were performed with 1 microM DEX, which was already sufficient to reach near-maximal GUS activity. However, 2-week inductions with 10 microM DEX caused growth retardation and developmental defects. As the severity of these effects varied between different lines, we could select lines with a mild phenotype for future use as activator lines in crosses with 'target' plants.

Yeast one-hybrid screening for DNA-protein interactions.

One-hybrid screening in yeast is a powerful method to rapidly identify heterologous transcription factors that can interact with a specific regulatory DNA sequence of interest (the bait sequence). In this technique, the interaction between two proteins (bait and prey) is detected via in vivo reconstitution of a transcriptional activator that turns on expression of a reporter gene. Detection is based on the interaction of a transcription factor (prey) with a bait DNA sequence upstream of a reporter gene. To ensure that DNA binding results in reporter-gene activation, cDNA expression libraries are used to produce hybrids between the prey and a strong trans-activating domain. The advantage of cloning transcription factors or other DNA-binding proteins via one-hybrid screenings, compared to biochemical techniques, is that the procedure does not require specific optimization of in vitro conditions.

A role for the rice homeobox gene Oshox1 in provascular cell fate commitment.

The vascular tissues of plants form a network of interconnected cell files throughout the plant body. The transition from a genetically totipotent meristematic precursor to different stages of a committed procambial cell, and its subsequent differentiation into a mature vascular element, involves developmental events whose molecular nature is still mostly unknown. The rice protein Oshox1 is a member of the homeodomain leucine zipper family of transcription factors. Here we show that the strikingly precise onset of Oshox1 gene expression marks critical, early stages of provascular ontogenesis in which the developmental fate of procambial cells is specified but not yet stably determined. This suggests that the Oshox1 gene may be involved in the establishment of the conditions required to restrict the developmental potential of procambial cells. In support of this hypothesis, ectopic expression of Oshox1 in provascular cells that normally do not yet express this gene results in anticipation of procambial cell fate commitment, eventually culminating in premature vascular differentiation. Oshox1 represents the first example of a transcription factor whose function can be linked to specification events mediating provascular cell fate commitment.

HD-Zip proteins of families I and II from rice: interactions and functional properties.

Proteins of the closely related homeodomain-leucine zipper (HD-Zip) families I and II in plants are putative transcription factors that interact with similar pseudopalindromic DNA recognition sites. We have previously described the Oshox1 gene from rice, which encodes an HD-Zip II protein. To identify further rice HD-Zip proteins, one-hybrid screens were performed in yeast strains containing a HIS3 reporter gene with upstream HD-Zip recognition sites. This resulted in the isolation of six new cDNAs encoding HD-Zip proteins belonging to family I (Oshox4, -5, -6) or family II (Oshox2, -3, -7). In transient assays, using rice suspension-cultured cells transformed by particle bombardment, we showed previously that Oshox1 can transcriptionally repress the activity of reporter gene constructs with upstream HD-Zip binding sites. Here, we confirm the repression properties of Oshox1 by showing that the repression function can be conferred on a heterologous DNA-binding domain. This portable functional domain (residues 1-155) is located proximal to the HD-Zip domain. In yeast, the same region of the Oshox1 protein was found to confer transcriptional activation instead of repression, pointing to the possibility that cell type-specific factors may determine the functional properties of the Oshox1 protein in rice. Like Oshox1, another HD-Zip family II protein (Oshox3) was also found to function as a transcriptional repressor in rice cells. In contrast, two HD-Zip I family proteins (Oshox4 and -5) appeared to act as activators in both rice and yeast cells. Results of two-hybrid assays and electrophoretic mobility shift assays strongly suggest that all HD-Zip proteins of families I and II can form homodimers and also heterodimers with all HD-Zip proteins of the same family. Heterodimerization across the HD-Zip families I and II apparently does not to occur.

Characterization of the KNOX class homeobox genes Oskn2 and Oskn3 identified in a collection of cDNA libraries covering the early stages of rice embryogenesis.

For identification of genes involved in embryogenesis in the model cereal rice, we have constructed a collection of cDNA libraries of well-defined stages of embryo development before, during and after organ differentiation. Here, we focus on the possible role of KNOX (maize Knotted1-like) class homeobox genes in regulation of rice embryogenesis. Three types of KNOX clones were identified in libraries of early zygotic embryos. Two of these, Oskn2 and Oskn3, encode newly described KNOX genes, whereas the third (Oskn1) corresponds to the previously described OSH1 gene. In situ hybridizations showed that during the early stages of embryo development, all three KNOX genes are expressed in the region where the shoot apical meristem (SAM) is organizing, suggesting that these genes are involved in regulating SAM formation. Whereas OSH1 was previously proposed to function also in SAM maintenance, Oskn3 may be involved in patterning organ positions, as its expression was found to mark the boundaries of different embryonic organs following SAM formation. The expression pattern of Oskn2 suggested an additional role in scutellum and epiblast development. Transgenic expression of Oskn2 and Oskn3 in tobacco further supported their involvement in cell fate determination, like previously reported for Knotted1 and OSH1 ectopic expression. Whereas Oskn3 transformants showed the most pronounced phenotypic effects during vegetative development, Oskn2 transformants showed relatively mild alterations in the vegetative phase but a more severely affected flower morphology. The observation that the KNOX genes produce similar though distinct phenotypic reponses in tobacco, indicates that their gene products act on overlapping but different sets of target genes, or that cell-type specific factors determine their precise action.

Vectors for transcription factor cloning and target site identification by means of genetic selection in yeast.

We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage lambda vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector.

Transcriptional repression by Oshox1, a novel homeodomain leucine zipper protein from rice.

This paper describes the characterization of Oshox1, a cDNA clone from rice encoding a member of the homeodomain-leucine zipper (HD-Zip) class of putative transcription factors. Oshox1 maps to chromosome 10 and belongs to a family of related rice genes. Two-hybrid assays showed that Oshox1 protein can homodimerize, but can also form heterodimers with an Arabidopsis HD-Zip protein. This suggests that protein-protein interactions may also occur between different HD-Zip proteins in rice, which would provide enormous versatility for generating specific gene-control mechanisms. Oshox1 mRNA could be detected in various rice tissues at different developmental stages, with highest levels in embryos, shoots of seedlings, and leaves of mature plants. Transgenic expression of Oshox1 in Arabidopsis retarded growth and affected leaf size and shape, indicative of a role as developmental regulator. In vitro and in vivo DNA-binding studies revealed that Oshox1 interacts with the pseudopalindromic sequence CAAT(C/G)ATTG, confirming that the protein represents a transcription factor. Oshox1 was found to repress reporter gene activity in rice suspension cells, most likely by a mechanism of active transcriptional repression. Repression was strictly dependent on the presence of upstream Oshox1 binding sites in the reporter gene constructs and a function of the N-terminal region of Oshox1, preceding the homeodomain.

Agrobacterium-mediated transformation of the terpenoid indole alkaloid-producing plant species Tabernaemontana pandacaqui.

Plants of the Apocynaceae family produce a wide range of terpenoid indole alkaloids (TIAs) which have important pharmaceutical applications. Studies of the molecular mechanisms controlling TIA biosynthesis may eventually provide possibilities to improve product yield by genetic modification of plants or cell cultures. However, these studies suffer from the lack of transformation/regeneration protocols for Apocynaceae plants. We chose to study the feasibility of Agrobacterium tumefaciens-mediated transformation of Tabernaemontana pandacaqui, because of the availability of an efficient regeneration procedure for this member of the Apocynaceae family. A procedure to produce transgenic T. pandacaqui plants was established, albeit with low efficiency. Transgenic expression was demonstrated of an intron-containing ß-glucuronidase reporter gene and of a gene coding for the TIA biosynthetic enzyme strictosidine synthase from Catharanthus roseus, another Apocynaceae species.

A promoter region that controls basal and elicitor-inducible expression levels of the NADPH:cytochrome P450 reductase gene (Cpr) from Catharanthus roseus binds nuclear factor GT-1.

NADPH:cytochrome P450 reductase (CPR) is essential for the activation of cytochrome P450 enzymes, which are involved in a wide variety of metabolic pathways in plants, including those related to defence responses. In the subtropical plant Catharanthus roseus several cytochrome P450 enzymes operate in the biosynthesis of defence-related terpenoid indole alkaloids (TIAs). In agreement with the importance of CPR in defence, Cpr mRNA levels in C. roseus were found to be enhanced by fungal elicitor preparations that also induce TIA biosynthesis and P450 gene expression. Here we describe the isolation of a C. roseus genomic DNA clone covering the 5' part of the Cpr gene and 1.6-kb of upstream sequences. Mapping of the transcription start site showed the untranslated leader sequence is approximately 280 bp long. To study the control of gene expression by the Cpr promoter, transcriptional fusions between Cpr promoter fragments and the gusA reporter gene were generated and their expression was analyzed in stably transformed tobacco plants. The Cpr promoter fragment extending from -1510 to -8, with respect to the ATG start codon, conferred basal and elicitor-inducible expression on the gusA reporter gene, strongly indicating that the Cpr gene of C. roseus is indeed controlled by this promoter region. Progressive deletion from the 5' end of the promoter to position -632 had little effect on gusA expression. However, deletion to position -366 resulted in a complete loss of basal activity and largely eliminated elicitor-induced expression, indicating that the region from -632 to -366 contains the main transcription-enhancing cis-regulatory sequences. Electrophoretic mobility shift assays with tobacco nuclear extracts showed that binding sites for nuclear factor GT-1 are redundant in the Cpr promoter, but absent from the downstream part of the leader sequence. The presence of strong GT-1 binding sites in the main enhancer region (-632 to -366), is suggestive of a functional role for this factor in basal expression and elicitor responsiveness of the Cpr promoter.

Novel members of a family of AT hook-containing DNA-binding proteins from rice are identified through their in vitro interaction with consensus target sites of plant and animal homeodomain proteins.

The AT hook is an AT-rich DNA-binding domain that occurs three times in mammalian high-mobility-group I/Y chromosomal proteins and has recently also been identified in DNA-binding proteins from plants. We unexpectedly isolated three rice cDNA clones encoding AT hook-containing proteins in an attempt to isolate homeobox cDNA clones by south-western screening of an expression library with known binding sites for Arabidopsis and animal homeodomain proteins. One of these clones (Os-PF1) has previously been identified due to the binding of its encoded protein to PE1, a cis-acting element from the oat phytochrome promoter. The other two clones represent newly described cDNA clones, designated Os-AT1 and Os-AT2. The Os-AT1 and Os-AT2 proteins were found to have the same specificities as Os-PF1 with respect to in vitro binding of wild-type and mutant PE1 versions. However, all three proteins appeared to bind much stronger in south-western assays to two of the rather AT-rich sequences used in our screening than to the PE1 element. In none of the AT hook proteins clear homologies to transcriptional activation domains could be identified, but the N-terminal regions of Os-AT1 and Os-PF1 were found to show similarity to histone H1 chromosomal proteins. Given their structural characteristics it is conceivable that the rice AT hook proteins bind to gene promoter regions as accessory proteins that may alter the accessibility of chromatin to other nuclear factors. Their predominant expression in young and meristematic tissues suggests that the presence of the AT hook proteins may affect the expression of genes that determine the differentiation status of cells.