Pattern recognition receptors (PRRs) belonging to the multigene family of receptor-like kinases (RLKs) are the sensing devices of plants for microbe- or pathogen-associated molecular patterns ...released from microbial organisms. Here we describe
(for
) encoding HvLEMK1, a LRR-malectin domain-containing transmembrane RLK that mediates non-host resistance of barley to the non-adapted wheat powdery mildew fungus
f.sp.
. Transgenic barley lines with silenced
allow entry and colony growth of the non-adapted pathogen, although sporulation was reduced and final colony size did not reach that of the adapted barley powdery mildew fungus
f.sp.
. Transient expression of the barley or wheat
genes enhanced resistance in wheat to the adapted wheat powdery mildew fungus while expression of the same genes did not protect barley from attack by the barley powdery mildew fungus. The results suggest that
is a factor mediating non-host resistance in barley and quantitative host resistance in wheat to the wheat powdery mildew fungus.
Gene pairs resulting from whole genome duplication (WGD), so-called ohnologous genes, are retained if at least one member of the pair undergoes neo- or sub-functionalization. Phylogenetic analyses of ...the ohnologous genes
ALBOSTRIANS
(
HvAST/HvCMF7
) and
A
LBO
S
TRIANS-
L
IKE
(
HvASL
/
HvCMF3
) of barley (
Hordeum vulgare
) revealed them as members of a subfamily of genes coding for CCT motif (
C
ONSTANS,
C
ONSTANS-LIKE and
T
IMING OF CAB1) proteins characterized by a single CCT domain and a putative N-terminal chloroplast transit peptide. Recently, we showed that
HvCMF7
is needed for chloroplast ribosome biogenesis. Here we demonstrate that mutations in
HvCMF3
lead to seedlings delayed in development. They exhibit a yellowish/light green –
xantha –
phenotype and successively develop pale green leaves. Compared to wild type, plastids of mutant seedlings show a decreased PSII efficiency, impaired processing and reduced amounts of ribosomal RNAs; they contain less thylakoids and grana with a higher number of more loosely stacked thylakoid membranes. Site-directed mutagenesis of
HvCMF3
identified a previously unknown functional domain, which is highly conserved within this subfamily of CCT domain containing proteins. HvCMF3:GFP fusion constructs were localized to plastids and nucleus.
Hvcmf3Hvcmf7
double mutants exhibited a
xantha
-albino or albino phenotype depending on the strength of molecular lesion of the
HvCMF7
allele. The chloroplast ribosome deficiency is discussed as the primary observed defect of the
Hvcmf3
mutants. Based on our observations, the genes
HvCMF3
and
HvCMF7
have similar but not identical functions in chloroplast development of barley supporting our hypothesis of neo-/sub-functionalization between both ohnologous genes.
Barley (Hordeum vulgare) spikes are developmentally switched from two-rowed to six-rowed by a single recessive gene, six-rowed spike 1 (vrs1), which encodes a homeodomain-leucine zipper I class ...transcription factor. Vrs1 is a paralog of HvHox2 and both were generated by duplication of an ancestral gene. HvHox2 is conserved among cereals, whereas Vrs1 acquired its current function during the evolution of barley. It was unclear whether divergence of expression pattern or protein function accounted for the functionalization of Vrs1.
Here, we conducted a comparative analysis of protein functions and gene expression between HvHox2 and Vrs1 to clarify the functionalization mechanism.
We revealed that the transcriptional activation activity of HvHOX2 and VRS1 was conserved. In situ hybridization analysis showed that HvHox2 is localized in vascular bundles in developing spikes, whereas Vrs1 is expressed exclusively in the pistil, lemma, palea and lodicule of lateral spikelets. The transcript abundance of Vrs1 was > 10-fold greater than that of HvHox2 during the pistil developmental stage, suggesting that the essential function of Vrs1 is to inhibit gynoecial development. We demonstrated the quantitative function of Vrs1 using RNAi transgenic plants and Vrs1 expression variants. Expression analysis of six-rowed spike mutants that are nonallelic to vrs1 showed that Vrs1 expression was up-regulated by Vrs4, whereas HvHox2 expression was not.
These data demonstrate that the divergence of gene expression pattern contributed to the neofunctionalization of Vrs1.
The Arabidopsis gene
(
) encodes a transcription factor that positively affects the activity of nuclear genes for chloroplast ribosomal proteins and chloroplast protein import machineries.
(
) is the ...paralogous gene of
. We generated a
mutant by site-directed mutagenesis and compared it with
and
double mutant. Phenotype of the
mutant did not differ from the wild type under our growth conditions, except faster growth and earlier time to flowering. Compared to
, the
mutant showed more impaired chloroplast functions and reduced amounts of plastid ribosomal RNAs.
analyses predict for CIA2 and CIL a C-terminal CCT domain and an N-terminal chloroplast transit peptide (cTP). Chloroplast (and potentially nuclear) localization was previously shown for HvCMF3 and HvCMF7, the homologs of CIA2 and CIL in barley. We observed nuclear localization of CIL after transient expression in Arabidopsis protoplasts. Surprisingly, transformation of
with
,
, or with a truncated
lacking the predicted cTP could partially rescue the pale-green phenotype of
. These data are discussed with respect to potentially overlapping functions between CIA2, CIL, and their barley homologs and to the function of the putative cTPs of CIA2 and CIL.
Stable genetic transformation represents the gold standard approach to the detailed elucidation of plant gene functions. This is particularly relevant in barley, an important experimental model ...widely employed in applied molecular, genetic and cell biological research, and biotechnology. Presented are details of the establishment of a protocol for
Agrobacterium-mediated gene transfer to immature embryos, which enables the highly efficient generation of transgenic barley. Advancements were achieved through comparative experiments on the influence of various explant treatments and co-cultivation conditions. The analysis of representative numbers of transgenic lines revealed that the obtained T-DNA copy numbers are typically low, the generative transmission of the recombinant DNA is in accordance with the Mendelian rules and the vast majority of the primary transgenics produce progeny that expresses the respective transgene product. Moreover, the newly established protocol turned out to be useful to transform not only the highly amenable cultivar (cv.) ‘Golden Promise’ but also other spring and winter barley genotypes, albeit with substantially lower efficiency. As a major result of this study, a very useful tool is now available for future functional gene analyses as well as genetic engineering approaches. With the aim to modify the expression of barley genes putatively involved in plant–fungus interactions, numerous transgenic plants have been generated using diverse expression cassettes. These plants represent an example of how transformation technology may contribute to further our understanding of important biological processes.
Chloroplasts, the sites of photosynthesis in higher plants, have evolved several means to tolerate short episodes of drought stress through biosynthesis of diverse metabolites essential for plant ...function, but these become ineffective when the duration of the stress is prolonged. Cyanobacteria are the closest bacterial homologs of plastids with two photosystems to perform photosynthesis and to evolve oxygen as a byproduct. The presence of
genes encoding flavodiiron proteins has been shown to enhance stress tolerance in cyanobacteria. In an attempt to support the growth of plants exposed to drought, the
genes
and
were expressed in barley with their products being targeted to the chloroplasts. The heterologous expression of both
accelerated days to heading, increased biomass, promoted the number of spikes and grains per plant, and improved the total grain weight per plant of transgenic lines exposed to drought. Improved growth correlated with enhanced availability of soluble sugars, a higher turnover of amino acids and the accumulation of lower levels of proline in the leaf.
and
maintained the energy status of the leaves in the stressed plants by converting sucrose to glucose and fructose, immediate precursors for energy production to support plant growth under drought. The results suggest that sugars and amino acids play a fundamental role in the maintenance of the energy status and metabolic activity to ensure growth and survival under stress conditions, that is, water limitation in this particular case. Engineering chloroplasts by
genes into the plant genome, therefore, has the potential to improve plant productivity wherever drought stress represents a significant production constraint.
Barley is an attractive vehicle for producing recombinant protein, since it is a readily transformable diploid crop species in which doubled haploids can be routinely generated. High amounts of ...protein are naturally accumulated in the grain, but optimal endosperm-specific promoters have yet to be perfected. Here, the oat GLOBULIN1 promoter was combined with the legumin B4 (LeB4) signal peptide and the endoplasmic reticulum (ER) retention signal (SE)KDEL. Transgenic barley grain accumulated up to 1.2 g/kg dry weight of recombinant protein (GFP), deposited in small roundish compartments assumed to be ER-derived protein bodies. The molecular farming potential of the system was tested by generating doubled haploid transgenic lines engineered to synthesize the anti-HIV-1 monoclonal antibody 2G12 with up to 160 μg recombinant protein per g grain. The recombinant protein was deposited at the periphery of protein bodies in the form of a mixture of various N-glycans (notably those lacking terminal N-acetylglucosamine residues), consistent with their vacuolar localization. Inspection of protein-A purified antibodies using surface plasmon resonance spectroscopy showed that their equilibrium and kinetic rate constants were comparable to those associated with recombinant 2G12 synthesized in Chinese hamster ovary cells.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The crop species barley is a genetic model for the small grain temperate cereals. Thanks to the availability of whole genome sequence and the development of customizable endonucleases, site-directed ...genome modification has recently revolutionized genetic engineering. Several platforms have been established in plants, with the most flexible one offered by the clustered regularly interspaced short palindromic repeats (CRISPR) technology. In this protocol, commercially available synthetic guide RNAs (gRNAs), Cas enzymes, or custom-generated reagents are used for targeted mutagenesis in barley. The protocol has been successfully used with immature embryo explants to generate site-specific mutations in regenerants. As the double-strand break-inducing reagents are customizable and can be efficiently delivered, pre-assembled ribonucleoprotein (RNP) complexes allow efficient generation of genome-modified plants.
Varietal differences within a species with agronomic importance are often based on minor changes in the genomic sequence. For example, fungus-resistant and fungus-susceptible wheat varieties may vary ...in only one amino acid. The situation is similar with the reporter genes Gfp and Yfp where two base pairs cause a shift in the emission spectrum from green to yellow. Methods of targeted double-strand break induction now allow this exchange precisely with the simultaneous transfer of the desired repair template. However, these changes rarely lead to a selective advantage that can be used in generating such mutant plants. The protocol presented here allows a corresponding allele replacement at the cellular level using ribonucleoprotein complexes in combination with an appropriate repair template. The efficiencies achieved are comparable to other methods with direct DNA transfer or integration of the corresponding building blocks in the host genome. They are in the range of 35 percent, considering one allele in a diploid organism as barley and using Cas9 RNP complexes.
Cystatins have been largely used for pest control against phytophagous species. However, cystatins have not been commonly overexpressed in its cognate plant species to test their pesticide capacity. ...Since the inhibitory role of barley HvCPI-6 cystatin against the phytophagous mite
has been previously demonstrated, the purpose of our study was to determine if barley transgenic lines overexpressing its own
gene were more resistant against this phytophagous infestation. Besides, a transcriptomic analysis was done to find differential expressed genes among wild-type and transformed barley plants. Barley plants overexpressing
cystatin gene remained less susceptible to
attack when compared to wild-type plants, with a significant lesser foliar damaged area and a lower presence of the mite. Transcriptomic analysis revealed a certain reprogramming of cellular metabolism and a lower expression of several genes related to photosynthetic activity. Therefore, although caution should be taken to discard potential deleterious pleiotropic effects, cystatins may be used as transgenes with impact on agricultural crops by conferring enhanced levels of resistance to phytophagous pests.
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