Promoters of the barley germin-like GER4 gene cluster enable strong transgene expression in response to pathogen attack.

Immunity of plants triggered by pathogen-associated molecular patterns (PAMPs) is based on the execution of an evolutionarily conserved defense response that includes the accumulation of pathogenesis-related (PR) proteins as well as multiple other defenses. The most abundant PR transcript of barley (Hordeum vulgare) leaf epidermis attacked by the powdery mildew fungus Blumeria graminis f. sp hordei encodes the germin-like protein GER4, which has superoxide dismutase activity and functions in PAMP-triggered immunity. Here, we show that barley GER4 is encoded by a dense cluster of tandemly duplicated genes (GER4a-h) that underwent several cycles of duplication. The genomic organization of the GER4 locus also provides evidence for repeated gene birth and death cycles. The GER4 promoters contain multiple WRKY factor binding sites (W-boxes) preferentially located in promoter fragments that were exchanged between subfamily members by gene conversion. Mutational analysis of TATA-box proximal W-boxes used GER4c promoter-beta-glucuronidase fusions to reveal their enhancing effects and functional redundancy on pathogen-induced promoter activity. The data suggest enhanced transcript dosage as an evolutionary driving force for the local expansion and functional redundancy of the GER4 locus. In addition, the GER4c promoter provides a tool to study signal transduction of PAMP-triggered immunity and to engineer strictly localized and pathogen-regulated disease resistance in transgenic cereal crops.

A set of modular binary vectors for transformation of cereals.

Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination. Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley.

Transcriptome analysis of mlo-mediated resistance in the epidermis of barley.

SUMMARY The shoot epidermis of plants is of prime importance for host and non-host defence against a large number of fungal diseases, including powdery mildew of barley, caused by Blumeria graminis (DC.) E.O. Speer f.sp. hordei. In order to understand better the mechanisms within the epidermis leading to susceptibility as well as durable host resistance, we characterized the transcriptome of two Blumeria-attacked, near-isogenic barley lines of cv Ingrid differing in the presence or absence of the mlo5 resistance gene. A cDNA array was established containing 3128 unique sequences from pathogen-attacked, resistant barley epidermis, which was hybridized with cDNA probes from pathogen-challenged epidermis. Expression analysis resulted in the identification of 311 candidate genes that were differentially expressed in a reproducible manner between control and inoculated epidermis. Among the up-regulated genes were 18 of fungal origin. The amplitude of differential host gene expression was generally higher in the presence of the mlo5 resistance gene as compared with a susceptible interaction. This suggests that mlo-mediated resistance is based on multiple defence compounds and mechanisms once pathogen-derived molecular patterns have been recognized, which yields a possible explanation for the durability of the phenomenon in the field.

Large-scale analysis of the barley transcriptome based on expressed sequence tags.

To provide resources for barley genomics, 110,981 expressed sequence tags (ESTs) were generated from 22 cDNA libraries representing tissues at various developmental stages. This EST collection corresponds to approximately one-third of the 380,000 publicly available barley ESTs. Clustering and assembly resulted in 14,151 tentative consensi (TCs) and 11 073 singletons, altogether representing 25 224 putatively unique sequences. Of these, 17.5% showed no significant similarity to other barley ESTs present in dbEST. More than 41% of all barley genes are supposed to belong to multigene families and approximately 4% of the barley genes undergo alternative splicing. Based on the functional annotation of the set of unique sequences, the functional category 'Energy' was further analysed to reveal tissue- and stage-specific differences in gene expression. Hierarchical clustering of 362 differentially expressed TCs resulted in the identification of seven major clusters. The clusters reflect biochemical pathways predominantly activated in specific tissues and at various developmental stages. During seed germination glycolysis could be identified as the most predominant biochemical pathway. Germination-specific glycolysis is characterized by the coordinated expression of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase, whose antagonistic actions possibly regulate the flux of amino acids into protein biosynthesis and gluconeogenesis respectively. The expression of defence-related and antioxidant genes during germination might be controlled by the ethylene-signalling pathway as concluded from the coordinated expression of those genes and the transcription factors (TF) EIN3 and EREBPG. Moreover, because of their predominant expression in germinating seeds, TF of the AP2 and MYB type are presumably major regulators of germination.

The allene oxide cyclase of barley (Hordeum vulgare L.)--cloning and organ-specific expression.

The naturally occurring enantiomer of the various octadecanoids and jasmonates is established in a biosynthetic step catalyzed by the allene oxide cyclase (AOC). The AOC converts an allene oxide formed by an allene oxide synthase (AOS). Here, we show cloning and characterization of cDNAs encoding the AOC and a third AOS, respectively, in addition to the two AOSs previously published (Plant J. 21, 199-213, 2000). The ORF of the AOC-cDNA of 717 bp codes for a protein of 238 amino acid residues carrying a putative chloroplast target sequence. Overexpression without chloroplast target sequence revealed AOC activity. The AOC was found to be a single copy gene which mapped on chromosome 6H. AOC mRNA accumulation appeared in leaf segments upon treatment with various jasmonates, octadecanoids and ABA or during stress such as treatment with sorbitol or glucose solutions. Infection with powdery mildew activated AOC expression in susceptible and resistant lines of barley which correlated with PR1b expression. Among different tissues of barley seedlings, the scutellar node and leaf base accumulated AOC mRNA preferentially which correlated with accumulation of mRNAs for other biosynthetic enzymes (lipoxygenases, AOSs). AOC mRNA accumulation appeared also abundantly in parts of the root containing the tip and correlated with elevated levels of jasmonates. The data suggest a link of AOC expression and JA formation and support role of JA in stress responses and development of barley.