Down-regulation of the sucrose transporters HvSUT1 and HvSUT2 affects sucrose homeostasis along its delivery path in barley grains.

Sucrose transport and partitioning are crucial for seed filling. While many plasma-membrane-localised sucrose transporters (SUT1 family members) have been analysed in seeds, the functions of vacuolar SUT2 members are still obscure. In barley grains, expression of HvSUT1 and HvSUT2 overlap temporally and spatially, suggesting concerted functions to regulate sucrose homeostasis.

Gene Targeting Without DSB Induction Is Inefficient in Barley.

Double strand-break (DSB) induction allowed efficient gene targeting in barley (), but little is known about efficiencies in its absence. To obtain such data, an assay system based on the () gene was established, a target gene which had been used previously in rice and Expression of recombinases RAD51 and RAD54 had been shown to improve gene targeting in and positive-negative (P-N) selection allows the routine production of targeted mutants without DSB induction in rice. We implemented these approaches in barley and analysed gene targeting with the ALS gene in wild type and RAD51 and RAD54 transgenic lines.

The promoter of ZmMRP-1, a maize transfer cell-specific transcriptional activator, is induced at solute exchange surfaces and responds to transport demands.

Transfer cells have specializations that facilitate the transport of solutes across plant exchange surfaces. ZmMRP-1 is a maize (Zea mays) endosperm transfer cell-specific transcriptional activator that plays a central role in the regulatory pathways controlling transfer cell differentiation and function. The present work investigates the signals controlling the expression of ZmMRP-1 through the production of transgenic lines of maize, Arabidopsis, tobacco and barley containing ZmMRP-1promoter:GUS reporter constructs.

Differential requirements for RAD51 in Physcomitrella patens and Arabidopsis thaliana development and DNA damage repair.

RAD51, the eukaryotic homolog of the bacterial RecA recombinase, plays a central role in homologous recombination (HR) in yeast and animals. Loss of RAD51 function causes lethality in vertebrates but not in other animals or in the flowering plant Arabidopsis thaliana, suggesting that RAD51 is vital for highly developed organisms but not for others. Here, we found that loss of RAD51 function in the moss Physcomitrella patens, a plant of less complexity, caused a significant vegetative phenotype, indicating an important function for RAD51 in this organism.

Jekyll encodes a novel protein involved in the sexual reproduction of barley.

Cereal seed development depends on the intimate interaction of filial and maternal tissues, ensuring nourishment of the new generation. The gene jekyll, which was identified in barley (Hordeum vulgare), is preferentially expressed in the nurse tissues. JEKYLL shares partial similarity with the scorpion Cn4 toxin and is toxic when ectopically expressed in Escherichia coli and tobacco (Nicotiana tabacum).

RAR1 positively controls steady state levels of barley MLA resistance proteins and enables sufficient MLA6 accumulation for effective resistance.

The polymorphic barley (Hordeum vulgare) Mla locus harbors allelic race-specific resistance (R) genes to the powdery mildew fungus Blumeria graminis f sp hordei. The highly sequence-related MLA proteins contain an N-terminal coiled-coil structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR region. Using transgenic barley lines expressing epitope-tagged MLA1 and MLA6 derivatives driven by native regulatory sequences, we show a reversible and salt concentration-dependent distribution of the intracellular MLA proteins in soluble and membrane-associated pools.

Alteration of GCN5 levels in maize reveals dynamic responses to manipulating histone acetylation.

The role played by histone acetyltransferase (HAT), GCN5, in transcriptional co-activation has been analysed in detail in yeast and mammals. Here, we present the cloning and expression pattern of Zmgcn5, the maize homologue. The enzymatic activity of the recombinant ZmGCN5 was analysed with histone and nucleosome substrates.

Complete nucleotide sequence and analysis of the putative polyprotein of maize dwarf mosaic virus genomic RNA (Bulgarian isolate).

The complete nucleotide sequence of maize dwarf mosaic virus Bulgarian isolate (MDMV-Bg) was determined. The viral genome was 9515 nt and contained an open reading frame encoding 3042 amino acids, flanked by 3'- and 5'-UTRs of 139 and 250 nucleotides, respectively. MDMV-Bg was more conserved in the coding region (52.

Complete RNA1 sequences of two UK isolates of barley mild mosaic virus: a wild-type fungus-transmissible isolate and a non-fungus-transmissible derivative.

The complete RNA1 sequences of two isolates (fungus transmissible and non-fungus transmissible) of barley mild mosaic virus (BaMMV) were obtained. The two isolates' RNA1 sequences had very high sequence identity (99.3%), and of the 15 amino acid differences (out of 2258) between the putative polyproteins, 11 were conservative and unlikely to affect the structure or function of the protein.

A large duplication in the 3'-untranslated region of a subpopulation of RNA2 of the UK-M isolate of barley mild mosaic bymovirus.

The UK-M isolate of the bipartite barley mild mosaic bymovirus (BaMMV UK-M) cannot be fungally transmitted, and has previously been shown to have a 1092 nt deletion in the coding region of RNA2. We now report, using sequence and reverse transcriptase-polymerase chain reaction (RT-PCR) data, that a subpopulation of BaMMV UK-M RNA2 contains a direct imperfect sequence repeat of 552 nt in the 3' untranslated region. The secondary structure of the 3' end of RNA2, and its possible effects on replication of the virus, are also discussed.

Cooperative binding to nucleic acids by barley yellow mosaic bymovirus coat protein and characterization of a nucleic acid-binding domain.

The capacity of several coat protein (CP) mutants of a German isolate of barley yellow mosaic bymovirus (BaYMV) to bind of nucleic acids was studied in vitro. Recombinant CP, produced by overexpression in Escherichia coli, was purified from inclusion bodies and subsequently renatured. Binding to single-stranded (ss) RNA and ssDNA oligonucleotides was found to be cooperative and sequence non-specific.

The complete nucleotide sequence of RNA-2 of a fungally-transmitted UK isolate of barley mild mosaic bymovirus and identification of amino acid combinations possibly involved in fungus transmission.

The complete nucleotide sequence of RNA-2 of a fungally-transmitted UK isolate of barley mild mosaic bymovirus (BaMMV isolate UK-F) was determined and compared with other published sequences, particularly UK-M, an isolate derived from the same source but which has been mechanically passaged for several years, has a deletion of about 1 kb and cannot be fungally transmitted. From an alignment of the BaMMV RNA-2 encoded protein with that for barley yellow mosaic bymovirus (BaYMV), several regions of consistent homology were identified and extensive searches made for similarities with the proteins of other fungally-transmitted viruses, especially amongst the furovirus capsid readthrough proteins which seem especially prone to deletion and which have already been implicated in fungus transmission. The amino acid combinations ER (glutamic acid-arginine) or QR (glutamine-arginine) were found consistently in all of the viruses.

Inefficient expression of the DNA-binding domain of GAL4 in transgenic plants.

The DNA-binding domain of the yeast transcriptional activator GAL4 was expressed in transgenic tobacco plants in order to attempt to obtain specific repression of reporter genes. Expression of the GUS and the NPTII gene was essentially the same regardless of the presence of a gene encoding a functional GAL4 DNA-binding domain or the presence of GAL4 recognition sequences in the promoter region of the reporter genes. Despite high levels of GAL4 mRNA, no translation product was detectable in transgenic plants indicating that the failure to detect functional GAL4 in plants is due to the inefficiency of GAL4 mRNA translation in plants.

Cloning and sequence analysis of RNA-2 of a mechanically transmitted UK isolate of barley mild mosaic bymovirus (BaMMV).

A mutant of the 'Streatley' isolate of barley mild mosaic bymovirus was selected from the original field isolate by repeated mechanical inoculation. Unlike the wild-type barley mild mosaic virus, which is transmitted by the soilborne fungus Polymyxa graminis, the mutant could not be transmitted by this vector. RNA-2 of the mutant virus was shorter than that of the wild-type virus suggesting that a deletion of part of the genome segment had occurred.

Transient expression of a lysine-rich vicilin gene ofVicia faba in barley endosperm detected by immunological tissue printing after particle bombardment.

Using immunological tissue printing we detected transient expression of a faba bean vicilin gene with or without introns driven by the B1 hordein promoter in barley endosperm after particle bombardment. The described method generally allows the analysis of transient expression of genes without depending on reporter gene constructs and specifically suggests correct splicing of dicot introns by a monocot splicing machinery.

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

Virulence proteins VirD1 and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirD1-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conjugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant cell nuclei.

Sequence analysis of the 3'-terminal half of RNA 1 of wheat spindle streak mosaic virus.

cDNA complementary to the 3'-terminal half of RNA 1 of wheat spindle streak mosaic virus (WSSMV) from Southern France has been cloned and sequenced. One large open reading frame (ORF) of 4410 nucleotides and a nontranslated region (NTR) of 213 nucleotides at the 3'-end excluding the poly(A)-tail were found. Because of the amino acid sequence homology to the polyprotein of barley yellow mosaic virus (BaYMV) RNA 1, the encoded polyprotein of the sequenced region of WSSMV is supposed to comprise the C-terminal part of the putative cytoplasmic inclusion (CI) protein, the nuclear inclusion a (NIa) proteinase, the (NIb) RNA-polymerase and the capsid protein.

Molecular analysis of the capsid protein gene of a german isolate of barley mild mosaic virus.

Barley mild mosaic virus (BaMMV) is one of the agents causing the barley yellow mosaic disease. The sequence corresponding to the 3'end of the BaMMV RNA1 of a German isolate was sequenced and the coding sequence for the 251 amino acid containing capsid protein was determined. Comparison of this sequence to other potyviral sequences and to the corresponding sequence of two Japanese isolates of BaMMV was done.

The complete nucleotide sequence of RNA 1 of a German isolate of barley yellow mosaic virus and its comparison with a Japanese isolate.

The nucleotide sequence of RNA 1 of a German isolate of barley yellow mosaic virus has been determined and compared with a Japanese isolate of the same virus. The sequence identity is 93.6% at the nucleotide level and 96% at the amino acid level.

Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake.

Efficient regeneration (80%) and high frequency genetic transformation (10-33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes.

The nucleotide sequence of RNA 2 of barley yellow mosaic virus.

The complete nucleotide sequence of RNA 2 of a German isolate of barley yellow mosaic virus (BaYMV) has been determined. The RNA is 3585 nucleotides in length excluding a 3'-terminal poly(A) tail. The viral plus and minus strands in all three reading frames contained only one large open reading frame which started at positions 156 to 158 and terminated with a UAG codon at positions 2828 to 2830, thus encoding an Mr 98,000 polypeptide.