A high-throughput <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation system for the grass model species <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> L.

In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA((R)), while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T(0) plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.     

The multivesicular body-localized GTPase ARFA1b/1c is important for callose deposition and ROR2 syntaxin-dependent preinvasive basal defense in barley

Host cell vesicle traffic is essential for the interplay between plants and microbes. ADP-ribosylation factor (ARF) GTPases are required for vesicle budding, and we studied the role of these enzymes to identify important vesicle transport pathways in the plant-powdery mildew interaction. A combination of transient-induced gene silencing and transient expression of inactive forms of ARF GTPases provided evidence that barley (Hordeum vulgare) ARFA1b/1c function is important for preinvasive penetration resistance against powdery mildew, manifested by formation of a cell wall apposition, named a papilla. Mutant studies indicated that the plasma membrane-localized REQUIRED FOR MLO-SPECIFIED RESISTANCE2 (ROR2) syntaxin, also important for penetration resistance, and ARFA1b/1c function in the same vesicle transport pathway. This was substantiated by a requirement of ARFA1b/1c for ROR2 accumulation in the papilla. ARFA1b/1c is localized to multivesicular bodies, providing a functional link between ROR2 and these organelles in penetration resistance. During Blumeria graminis f sp hordei penetration attempts, ARFA1b/1c-positive multivesicular bodies assemble near the penetration site hours prior to the earliest detection of callose in papillae. Moreover, we showed that ARFA1b/1c is required for callose deposition in papillae and that the papilla structure is established independently of ARFA1b/1c. This raises the possibility that callose is loaded into papillae via multivesicular bodies, rather than being synthesized directly into this cell wall apposition.     

Genetics of avirulence genes in Blumeria graminis f.sp. hordei and physical mapping of AVR(a22) and AVR(a12).

Powdery mildew fungi are parasites that cause disease on a wide range of important crops. Plant resistance (R) genes, which induce host defences against powdery mildews, encode proteins that recognise avirulence (AVR) molecules from the parasite in a gene-for-gene manner. To gain insight into how virulence evolves in Blumeria graminis f.sp. hordei, associations between segregating AVR genes were established. As a prerequisite to the isolation of AVR genes, two loci were selected for further analysis. AVR(a22) is located in a tightly linked cluster comprising AVR(a10) and AVR(k1) as well as up to five other AVR genes. The ratio between physical and genetic distance in the cluster ranged between 0.7 and 35 kB/cM. The AVR(a22) locus was delimited by the previously isolated gene AVR(a10) and two cleaved amplified polymorphic sequence (CAPS) markers, 19H12R and 74E9L. By contrast, AVR(a12) was not linked to other AVR genes in two crosses. Bulk segregant analysis of over 100,000 AFLP fragments yielded two markers, ETAMTG-285 and PAAMACT-473, mapping 10 and 2cM from AVR(a12), respectively, thus delimiting AVR(a12) on one side. All markers obtained for AVR(a12) mapped proximal to it, indicating that the gene is located at the end of a chromosome. Three more AVR(a10) paralogues were identified at the locus interspersed among genes for metabolic enzymes and abundant repetitive elements, especially those homologous to the CgT1 class of retrotransposons. The flanking and close markers obtained will facilitate the isolation of AVR(a22) and AVR(a12) and provide useful tools for studies of the evolution of powdery mildew fungi in agriculture and nature.     

Physical association of Arabidopsis hypersensitive induced reaction proteins (HIRs) with the immune receptor RPS2

Arabidopsis RPS2 is a typical nucleotide-binding leucine-rich repeat resistance protein, which indirectly recognizes the bacterial effector protein AvrRpt2 and thereby activates effector-triggered immunity (ETI). Previously, we identified two hypersensitive induced reaction (AtHIR) proteins, AtHIR1 (At1g09840) and AtHIR2 (At3g01290), as potential RPS2 complex components. AtHIR proteins contain the stomatin/prohibitin/flotillin/HflK/C domain (also known as the prohibitin domain or band 7 domain). In this study, we confirmed that AtHIR1 and AtHIR2 form complexes with RPS2 in Arabidopsis and Nicotiana benthamiana using a pulldown assay and fluorescence resonance energy transfer (FRET) analysis. Arabidopsis has four HIR family genes (AtHIR1-4). All AtHIR proteins could form homo- and hetero-oligomers in vivo and were enriched in membrane microdomains of the plasma membrane. The mRNA levels of all except AtHIR4 were significantly induced by microbe-associated molecular patterns, such as the bacterial flagellin fragment flg22. Athir2-1 and Athir3-1 mutants allowed more growth of Pto DC3000 AvrRpt2, but not Pto DC3000, indicating that these mutations reduce RPS2-mediated ETI but do not affect basal resistance to the virulent strain. Overexpression of AtHIR1 and AtHIR2 reduced growth of Pto DC3000. Taken together, the results show that the AtHIR proteins are physically associated with RPS2, are localized in membrane microdomains, and quantitatively contribute to RPS2-mediated ETI.     

Ethanol increases sensitivity of oxalate oxidase assays and facilitates direct activity staining in SDS gels

Oxalate oxidase, and H₂O₂-generating enzyme, has been characterized from several plants, and is widely used for clinical detection of oxalate. Using a germin-like oxalate oxidase from barley leaves, we have developed and optimized novel methods for measuring oxalate oxidase activity. As oxalate oxidase is SDS-tolerant, its activity can be detected directly in SDS-PAGE gels in the presence of ethanol. This ethanol-dependent method is a hundred times more sensitive than the current methods. Furthermore, ethanol also improves the sensitivity of oxalate oxidase assays performed in solution. We found at least a 10-fold increase in sensitivity in comparison to a current method. The assay in solution is, in addition, useful for detection of oxalate. This elevation in sensitivity may be due to the immobilization of the enzyme in protein precipitates as a result of the treatment with ethanol.     

Coevolution between a Family of Parasite Virulence Effectors and a Class of LINE-1 Retrotransposons

Parasites are able to evolve rapidly and overcome host defense mechanisms, but the molecular basis of this adaptation is poorly understood. Powdery mildew fungi (Erysiphales, Ascomycota) are obligate biotrophic parasites infecting nearly 10,000 plant genera. They obtain their nutrients from host plants through specialized feeding structures known as haustoria. We previously identified the AVR _k1 powdery mildew-specific gene family encoding effectors that contribute to the successful establishment of haustoria. Here, we report the extensive proliferation of the AVR _k1 gene family throughout the genome of B. graminis, with sequences diverging in formae speciales adapted to infect different hosts. Also, importantly, we have discovered that the effectors have coevolved with a particular family of LINE-1 retrotransposons, named TE1a. The coevolution of these two entities indicates a mutual benefit to the association, which could ultimately contribute to parasite adaptation and success. We propose that the association would benefit 1) the powdery mildew fungus, by providing a mechanism for amplifying and diversifying effectors and 2) the associated retrotransposons, by providing a basis for their maintenance through selection in the fungal genome.