LTR retrotransposons in the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and <Emphasis Type="Italic">A. nidulans</Emphasis> genomes

Fungi of the genus Aspergillus can infect all tissues and organs, causing invasive mycosis (aspergillosis). This disease can be fatal, especially in immunocompromised patients. Microbiological monitoring of these infectious agents is obligatory in modern medical facilities. Mobile elements can be used as markers to identify the Aspergillus species and strains found indoors as well as to diagnose aspergillosis. Genomic sequences of two Aspergillus species, A. fumigatus and A. nidulans, were analyzed in silico in order to detect LTR retrotransposons. These species were found to considerably differ in the composition of retrotransposon families. One of the families, present in both Aspergillus species, was phylogenetically quite different from all known fungal retrotransposons. The majority of its elements were damaged copies. Nevertheless, allegedly undamaged LTR retrotransposon copies were described that contained intact ORFs and might be active.     

Study of the Interaction between HP1 Family Proteins and Untranslated Regulatory Regions of the <Emphasis Type="Italic">Gypsy</Emphasis> Retrotransposons in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

In vitro interaction between recombinant protein paralogs (HP1a, HP1b, and HP1c) of the HP1 family and 5′-untranslated regulatory regions of the gypsy retrotransposon group in Drosophila melanogaster—gypsy, Springer, Tirant, ZAM, Rover, and 17.6—was studied. Using competitive DNA, the conditions that enable specific binding with a matrix were identified. It was found that HP1 family proteins efficiently bind to the 5′-untranslated regulatory region of retrotransposons with tandem repeats. It was found that repeats are absolutely necessary for HP1a to bind to the 5′-untranslated region of the Tirant and ZAM mobile genetic elements. The absence of repeats (ZAM) or the presence of fewer than two repeats (Tirant) makes such interaction impossible. Thus, the presence of tandem repeats in the 5′-untranslated region of the gypsy retrotransposons is an important tool for regulating their transposition by heterochromatin proteins.     

The first complete <Emphasis Type="Italic">Mag</Emphasis> family retrotransposons discovered in <Emphasis Type="Italic">Drosophila</Emphasis>

A retrotransposon of the Mag family was found in the Drosophila simulans genome for the first time. We also identified novel transposable elements representing the Mag family in seven Drosophila species. The high similarity between the 3’ and 5’ long terminal repeats in the found copies of transposable elements indicates that their retrotransposition has occurred relatively recently. Thus, the Mag family of retrotransposons is quite common for the genus Drosophila.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Induction of two cyclotide-like genes <Emphasis Type="Italic">Zmcyc1</Emphasis> and <Emphasis Type="Italic">Zmcyc5</Emphasis> by abiotic and biotic stresses in <Emphasis Type="Italic">Zea mays</Emphasis>

Cyclotides are small plant disulfide-rich and cyclic proteins with a diverse range of biological activities. Cyclotide-like genes show key sequence features of cyclotides and are present in the Poaceae. In this study the cDNA of the nine cyclotide-like genes were cloned and sequenced using 3′RACE from Zea mays. The gene expression of two of these genes (Zmcyc1 and Zmcyc5) were analyzed by real-time PCR in response to biotic (Fusarium graminearum, Ustilago maydis and Rhopalosiphum maydis) and abiotic (mechanical wounding, water deficit and salinity) stresses, as well as in response to salicylic acid and methyl jasmonate elicitors to mimic biotic stresses. All isolated genes showed significant similarity to other cyclotide-like genes and were classified in two separate clusters. Both Zmcyc1 and Zmcyc5 were expressed in all studied tissues with the highest expression in leaves and lowest expression in roots. Wounding, methyl jasmonate and salicylic acid significantly induced the expression of Zmcyc1 and Zmcyc5 genes, but the higher expression was observed for Zmcyc1 as compared with Zmcyc5. Expression levels of these two genes were also induced in inoculated leaves with F. graminearum, U. maydis and also in response to insect infestation. In addition, the 1000-base-pairs (bp) upstream of the promoter of Zmcyc1 and Zmcyc5 genes were identified and analyzed using the PlantCARE database and consequently a large number of similar biotic and abiotic cis-regulatory elements were identified for these two genes.     

Nuclear DHT-Receptor in <Emphasis Type="Italic">Tfm</Emphasis>/<Emphasis Type="Italic">Y</Emphasis> Kidney Cell

OUR previous studies on the X-linked testicular feminization (Tfm) mutation¹ of the mouse^2–4 showed that the so-called cytosol and nuclear 5αx-dihydrotestosterone (DHT) receptor protein^5–7 might be a regulatory protein specified by the Tfm locus. The dual role of being a translational repressor in the cytoplasma and a mediator of hypertrophy in the nucleus was envisaged⁸. We found, however, another class of androgen-receptor in the polysome fraction of kidney proximal tubule cells which seems better qualified to be a translational regulator. Since a single gene locus specifies only one kind of polypeptide chain, we re-examined whether the cytosol and nuclear DHT-receptor protein underwent a true mutational change in Tfm/Y individuals.     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.

     

The role of putrescine against the long terminal repeat (LTR) retrotransposon polymorphisms induced by salinity stress in <Emphasis Type="Italic">Triticum aestivum</Emphasis>

This study aimed to research the impact of putrescine against the long terminal repeat (LTR) retrotransposon polymorphisms (Nikita-E2647, Sukkula, Stowaway, WLTR2105 and 5′LTR) induced by salinity stress in Triticum aestivum using inter-retrotransposon amplified polymorphism (IRAP) assay. The results showed that the LTR retrotransposon polymorphisms can be induced by all treated sodium chloride (NaCl) doses (0, 50, 100, 200 and 300 mM NaCl). On the other hand, the LTR retrotransposons polymorphisms were decreased effectively by treatment with putrescine (0, 0.01, 0.1 and 1 mM) together with NaCl. These results suggest that putrescine could effectively inhibit salt-induced LTR retrotransposon polymorphisms, and putrescine positively contributed to salt stress tolerance.     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in situ</Emphasis> characterization of the zebrafish (<Emphasis Type="Italic">Danio rerio</Emphasis>) <Emphasis Type="Italic">gnrh3</Emphasis> (sGnRH) gene

Background

Gonadotropin releasing hormone (GnRH) is responsible for stimulation of gonadotropic hormone (GtH) in the hypothalamus-pituitary-gonadal axis (HPG). The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio).

Results

We have characterized a zebrafish [Trp⁷, Leu⁸] or salmon (s) GnRH variant, gnrh 3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH), was shown capable of driving cell specific reporter gene expression in transgenic zebrafish.

Conclusions

The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar) GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

     

Mildew Resistance Locus O Gene Cloning,Characterization, and Expression Pattern in Mulberry (<Emphasis Type="Italic">Morus multicaulis</Emphasis>) and Its Prokaryotic Expression in <Emphasis Type="Italic">E. coli</Emphasis>

MLO (mildew resistance locus O), which encodes a transmembrane protein 7TM, is considered to be a model plant gene suitable for studying broad-spectrum resistance. It is a negative regulator of powdery mildew resistance and thus has potential applications in plant breeding. In the present paper, a full cDNA sequence encoding MLO was cloned from the leaves of mulberry (Morus multicaulis) based on mulberry expressed sequence tags (EST), homologous cloning technology, and 5′-RLM-RACE using RT-PCR;the sequence was designated MMLO (GenBank accession no. KX683296). The full cDNA was 1943 bp in length with 5′-untranslated region (UTR) of 106 bp, 3′-UTR of 160 bp, and an open reading frame (ORF) of 1677 bp encoding a protein of 558 amino acids. The estimated molecular weight and isoelectric point (pI) of the putative protein were 62.48 kDa and 9.03, respectively. The MMLO protein had Mlo domain and belonged to the Mlo superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the MLO gene from various species showed that mulberry was closely related to Eucalyptus grandis, Ziziphus jujube, and Juglansregia. Quantitative real-time PCR (qRT-PCR) analysis revealed that MMLO was expressed in all the tissues tested, including leaf, bud, fruit, stem, phloem, and xylem in mulberry with the highest expression in the phloem. The expression level of the mRNA increased and significantly changed under drought, cold, and salt stress treatments compared to the normal growth environment. The ORF segment of the MMLO was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. SDS-PAGE result revealed that fusion protein was successfully expressed. Overall, these results provide a better understanding of the molecular basis for the signal transduction mechanism during the stress responses in mulberry trees.