John P. Vogel

Unique aspects of the grass cell wall

Grasses are amongst the most important crops worldwide, and the composition of their cell walls is critical for uses as food, feed, and energy crops. Grass cell walls differ dramatically from dicot cell walls in terms of the major structural polysaccharides present, how those polysaccharides are linked together, and the abundance and importance of pectins, proteins and phenolic compounds. Recent advances, spurred by the availability of genomic resources for several plant species, include the characterization of cellulose synthase like (Csl) gene families that are unique to the grasses and the demonstration that members of one of those gene families, CslF, are responsible for making the mixed linkage glucans that are unique to the order Poales.

Conserved requirement for a plant host cell protein in powdery mildew pathogenesis

In the fungal phylum Ascomycota, the ability to cause disease in plants and animals has been gained and lost repeatedly during phylogenesis. In monocotyledonous barley, loss-of-function mlo alleles result in effective immunity against the Ascomycete Blumeria graminis f. sp. hordei, the causal agent of powdery mildew disease. However, mlo-based disease resistance has been considered a barley-specific phenomenon to date. Here, we demonstrate a conserved requirement for MLO proteins in powdery mildew pathogenesis in the dicotyledonous plant species Arabidopsis thaliana. Epistasis analysis showed that mlo resistance in A. thaliana does not involve the signaling molecules ethylene, jasmonic acid or salicylic acid, but requires a syntaxin, glycosyl hydrolase and ABC transporter. These findings imply that a common host cell entry mechanism of powdery mildew fungi evolved once and at least 200 million years ago, suggesting that within the Erysiphales (powdery mildews) the ability to cause disease has been a stable trait throughout phylogenesis.

PMR6 , a Pectate Lyase–Like Gene Required for Powdery Mildew Susceptibility in Arabidopsis

The plant genes required for the growth and reproduction of plant pathogens are largely unknown. In an effort to identify these genes, we isolated Arabidopsis mutants that do not support the normal growth of the powdery mildew pathogen Erysiphe cichoracearum. Here, we report on the cloning and characterization of one of these genes, PMR6. PMR6 encodes a pectate lyase–like protein with a novel C-terminal domain. Consistent with its predicted gene function, mutations in PMR6 alter the composition of the plant cell wall, as shown by Fourier transform infrared spectroscopy. pmr6-mediated resistance requires neither salicylic acid nor the ability to perceive jasmonic acid or ethylene, indicating that the resistance mechanism does not require the activation of well-described defense pathways. Thus, pmr6 resistance represents a novel form of disease resistance based on the loss of a gene required during a compatible interaction rather than the activation of known host defense pathways.

High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3

Brachypodium distachyon (Brachypodium) is a small grass with biological attributes (rapid generation time, small genome, diploid accessions, small stature and simple growth requirements) that make it suitable for use as a model system. In addition, a growing list of genomic resources have been developed or are currently under development including: cDNA libraries, BAC libraries, EST sequences, BAC end sequences, a physical map, genetic markers, a linkage map and, most importantly, the complete genome sequence. To maximize the utility of Brachypodium as a model grass it is necessary to develop an efficient Agrobacterium-mediated transformation system. In this report we describe the identification of a transformable inbred diploid line, Bd21-3, and the development of a transformation method with transformation efficiencies as high as 41% of co-cultivated calluses producing transgenic plants. Conducting the co-cultivation step under desiccating conditions produced the greatest improvement in transformation efficiency.

Brachypodium as a model for the grasses: Today and the future

Over the past several years, Brachypodium distachyon (Brachypodium) has emerged as a tractable model system to study biological questions relevant to the grasses. To place its relevance in the larger context of plant biology, we outline here the expanding adoption of Brachypodium as a model grass and compare this to the early history of another plant model, Arabidopsis thaliana. In this context, Brachypodium has followed an accelerated path in which the development of genomic resources, most notably a whole genome sequence, occurred concurrently with the generation of other experimental tools (e.g. highly efficient transformation and large collections of natural accessions). This update provides a snapshot of available and upcoming Brachypodium resources and an overview of the community including the trajectory of Brachypodium as a model grass.

Agrobacterium-mediated transformation and inbred line development in the model grass Brachypodium distachyon

Brachypodium distachyon (Brachypodium) has been proposed as a model temperate grass because its physical, genetic, and genome attributes (small stature, simple growth requirements, small genome size, availability of diploid ecotypes, annual lifecycle and self fertility) are suitable for a model plant system. Two additional requirements that are necessary before Brachypodium can be widely accepted as a model system are an efficient transformation system and homogeneous inbred reference genotypes. Here we describe the development of inbred lines from 27 accessions of Brachypodium. Determination of c-values indicated that five of the source accessions were diploid. These diploid lines exhibit variation for a variety of morphological traits. Conditions were identified that allow generation times as fast as two months in the diploids. An Agrobacterium-mediated transformation protocol was developed and used to successfully transform 10 of the 19 lines tested with efficiencies ranging from 0.4% to 15%. The diploid accession Bd21 was readily transformed. Segregation of transgenes in the T1 generation indicated that most of the lines contained an insertion at a single genetic locus. The new resources and methodologies reported here will advance the development and utilization of Brachypodium as a new model system for grass genomics.

Closing the ranks to attack by powdery mildew

Powdery mildews are among the most common plant diseases, infecting over 650 monocot and over 9000 dicot species. Analysis in domesticated barley and wild Arabidopsis has begun to unravel the genetic and molecular frameworks underlying the mechanisms of susceptibility and resistance to these biotrophic fungal pathogens. This has revealed multiple pathways regulating host defense, some of which are also involved in determining the host range of the pathogen. Host-cell death and rapid cell-wall remodeling have emerged as frequent themes in powdery-mildew resistance. Several mutants have been isolated that might shed light on the enigma of susceptibility determinants in plants.

Overexpression of the maize Corngrass1 microRNA prevents flowering, improves digestibility, and increases starch content of switchgrass

Biofuels developed from biomass crops have the potential to supply a significant portion of our transportation fuel needs. To achieve this potential, however, it will be necessary to develop improved plant germplasm specifically tailored to serve as energy crops. Liquid transportation fuel can be created from the sugars locked inside plant cell walls. Unfortunately, these sugars are inherently resistant to hydrolytic release because they are contained in polysaccharides embedded in lignin. Overcoming this obstacle is a major objective toward developing sustainable bioenergy crop plants. The maize Corngrass1 (Cg1) gene encodes a microRNA that promotes juvenile cell wall identities and morphology. To test the hypothesis that juvenile biomass has superior qualities as a potential biofuel feedstock, the Cg1 gene was transferred into several other plants, including the bioenergy crop Panicum virgatum (switchgrass). Such plants were found to have up to 250% more starch, resulting in higher glucose release from saccharification assays with or without biomass pretreatment. In addition, a complete inhibition of flowering was observed in both greenhouse and field grown plants. These results point to the potential utility of this approach, both for the domestication of new biofuel crops, and for the limitation of transgene flow into native plant species.

Novel microRNAs uncovered by deep sequencing of small RNA transcriptomes in bread wheat (Triticum aestivum L.) and Brachypodium distachyon (L.) Beauv

The small RNA transcriptomes of bread wheat and its emerging model Brachypodium distachyon were obtained by using deep sequencing technology. Small RNA compositions were analyzed in these two species. In addition to 70 conserved microRNAs (miRNAs) from 25 families, 23 novel wheat miRNAs were identified. For Brachypodium, 12 putative miRNAs were predicted from a limited number of expressed sequence tags, of which one was a potential novel miRNA. Also, 94 conserved miRNAs from 28 families were identified in this species. Expression validation was performed for several novel wheat miRNAs. RNA ligase-mediated 5′ rapid amplification of complementary DNA ends experiments demonstrated their capability to cleave predicted target genes including three disease-resistant gene analogs. Differential expression of miRNAs was observed between Brachypodium vegetative and reproductive tissues, suggesting their different roles at the two growth stages. Our work significantly increases the novel miRNA numbers in wheat and provides the first set of small RNAs in B. distachyon.

Development of SSR markers and analysis of diversity in Turkish populations of Brachypodium distachyon

BackgroundBrachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to facilitate research aimed at improving grass crops for grain, forage and energy production. To characterize the natural diversity of Brachypodium and provide a valuable new tool to the growing list of resources available to Brachypodium researchers, we created and characterized a large, diverse collection of inbred lines.ResultsWe developed 84 inbred lines from eight locations in Turkey. To enable genotypic characterization of this collection, we created 398 SSR markers from BAC end and EST sequences. An analysis of 187 diploid lines from 56 locations with 43 SSR markers showed considerable genotypic diversity. There was some correlation between SSR genotypes and broad geographic regions, but there was also a high level of genotypic diversity at individual locations. Phenotypic analysis of this new germplasm resource revealed considerable variation in flowering time, seed size, and plant architecture. The inbreeding nature of Brachypodium was confirmed by an extremely high level of homozygosity in wild plants and a lack of cross-pollination under laboratory conditions.ConclusionTaken together, the inbreeding nature and genotypic diversity observed at individual locations suggest a significant amount of long-distance seed dispersal. The resources developed in this study are freely available to the research community and will facilitate experimental applications based on natural diversity.