Hans-Henning Steinbiss

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. A posttranscriptional process directs fourfold greater accumulation of MLA1 over MLA6. Unexpectedly, in rar1 mutant plants that are compromised for MLA6 but not MLA1 resistance, the steady state level of both MLA isoforms is reduced. Furthermore, differential steady state levels of MLA1/MLA6 hybrid proteins correlate with their requirement for RAR1; the RAR1-independent hybrid protein accumulates to higher levels and the RAR1-dependent one to lower levels. Interestingly, yeast two-hybrid studies reveal that the LRR domains of RAR1-independent but not RAR1-dependent MLA isoforms interact with SGT1, a RAR1 interacting protein required for the function of many NB-LRR type R proteins. Our findings implicate the existence of a conserved mechanism to reach minimal NB-LRR R protein thresholds that are needed to trigger effective resistance responses.

Protoplast derived tobacco cells can survive capillary microinjection of the fluorescent dye Lucifer Yellow

SummaryThe first steps to successful microinjection in plant cells of different types of macromolecules (RNA, DNA and protein) are reported. Evidence is also given that plant cells can survive the mechanical injury of the injection procedure. By attaching the cells to coverslips with polylysine, the progeny of an individual cell can be followed microscopically. Using the fluorescent dye Lucifer Yellow, the injection procedure can be monitored and the injected volume calculated.

[6] Expression vectors for high-level gene expression in dicotyledonous and monocotyledonous plants

Publisher Summary This chapter describes the sets of vectors that are derivatives of the expression vector cassette pRT100, which uses the CaMV 35S RNA promoter in combination with various reporter and selectable marker genes. It introduces a set of expression vectors for enhanced gene expression in monocotyledonous plants, especially agronomically important cereals. These vectors have been constructed as basic tools applicable for transient gene expression, as well as for stable integration of foreign genes into plant genomes. Improvement of the cassette pRT101 cat to produce pRT-ex/s-int/s-cat gives a construct, leading to a high level of gene expression in monocotyledonous plants and in particular in agronomically important cereals. The construct pRT-ex/s-int/s-cat has been constructed to facilitate the replacement of the CAT-coding region, such as a Bam HI or Xba I fragment, or an exchange of the promoter region, such as a Hinc II /Xho I or Hinc II /Kpn I fragment. The entire chimeric gene can be transferred to other vectors using Pst I or Sph I. Moreover, the construct pRT-ex/s-int/scat might be helpful for the analysis of plant promoters or for the achievement of high levels of gene product in transgenic plants.

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). In barley, jekyll is upregulated in cells destined for autolysis. The gene generates a gradient of expression in the nucellar projection, which mediates the maternal–filial interaction during seed filling. Downregulation of jekyll by the RNA interference technique in barley decelerates autolysis and cell differentiation within the nurse tissues. Flower development and seed filling are thereby extended, and the nucellar projection no longer functions as the main transport route for assimilates. A slowing down in the proliferation of endosperm nuclei and a severely impaired ability to accumulate starch in the endosperm leads to the formation of irregular and small-sized seeds at maturity. Overall, JEKYLL plays a decisive role in the differentiation of the nucellar projection and drives the programmed cell death necessary for its proper function. We further suggest that cell autolysis during the differentiation of the nucellar projection allows the optimal provision of basic nutrients for biosynthesis in endosperm and embryo.

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. Similar values have been found for the polyproteins of the RNA 2 of both isolates (95%). Both isolates show an RNA 1-encoded protein arrangement similar to that of potyviruses such as tobacco etch virus. In contrast, the polyproteins of the small RNAs (RNA 2) do not show such a similarity to the polyproteins of other potyviruses. However, there is a striking difference between the two isolates in the generally highly conserved active site of the RNA-dependent RNA polymerase. The German isolate exactly matches the consensus sequences for previously described potyviral RNA-dependent RNA polymerases, whereas the Japanese isolate does not.

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. Moreover, loss of RAD51 caused marked hypersensitivity to the double-strand break-inducing agent bleomycin in P. patens but not in Arabidopsis. Therefore, HR is used for somatic DNA damage repair in P. patens but not in Arabidopsis. These data imply fundamental differences in the use of recombination pathways between plants. Moreover, these data demonstrate that the importance of RAD51 for viability is independent of taxonomic position or complexity of an organism. The involvement of HR in DNA damage repair in the slowly evolving species P. patens but not in fast-evolving Arabidopsis suggests that the choice of the recombination pathway is related to the speed of evolution in plants.

Functional complementation analysis in potato via biolistic transformation with BAC large DNA fragments

Gene isolation from plants by positional cloning frequently requires several rounds of transformation. To reduce the resources invested and to accelerate the process, we have used large DNA fragments in transformation experiments, followed by analysis of transgenic plants to assess functional complementation. Specifically, the transformation of potato with DNA from the 106 kb BAC plasmid BA87d17 is described. The large fragment was introduced into the potato genome by biolistic transformation, while attempting to clone the R1 gene conferring a race specific resistance to Phytophthora infestans. Thirty-one kanamycin resistant plants were regenerated of which thirteen showed the necrotic lesions typical for the hypersensitive response after infection with the incompatible P. infestans race 4, which carries the avirulence gene Avr1. The successful complementation supported the location of the R1 gene in the BAC insertion of the BA87d17 plasmid. Based on PCR and Southern gel blot analysis, both complete and incomplete integrations of the large construct into the recipient genome were demonstrated.

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. They occurred in positions predicted to be on the outside of the protein, and therefore available for interaction with the fungus vector, and were also within the regions prone to spontaneous deletion. In view of the lack of other structural or sequence homologies, it is suggested that these motifs are strong candidates for involvement in fungus transmission.

Preparation and transformation of monocot protoplasts.

Publisher Summary This chapter discusses the preparation and transformation of monocot protoplasts. Efficient introduction of chimeric genes by direct DNA-transfer techniques is crucial in the manipulation of monocot cells. Several conceptually and technically different methods for the introduction of DNA into plant cells have been developed. Microprojectile bombardment has successfully been used for stable transformation and transient expression of chimeric genes but turned out to have limited efficiency. This is mainly because only a small fraction of the competent cells of a culture are targeted. The development of efficient systems for protoplast isolation and plant regeneration tends to favor the use of PEG-mediated gene transfer or electroporation, with DNA-uptake frequencies of about 50%. Data by Maas and Werr showed that extracellular DNA is precipitated efficiently by the combined action of PEG together with divalent cations. The DNA seems to be taken up by the plant protoplast in the precipitated form. The particle size of the precipitate is strongly affected by the pH of the PEG solution.

Plant regeneration from cultured fertilized barley ovules

Abstract A method for the culture of barley ( Hordeum vulgare ) ovules isolated directly after fertilization is described. Up to 28% of these ovules contained a poorly developed embryo after 10–12 days of culture. After dissection these embryos were cultured and about 20% of them yielded mature plants. Our observations indicate that plant regeneration from cultured ovules, isolated 1–2 h after pollination, is possible. Similar methods also allow culture and regeneration of wheat plants from ovules isolated after 2 days post anthesis.