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Cereal transformation and gene editing can be a complex and costly undertaking. It is therefore important to validate and understand the performance of the components to achieve high rates of transformation and gene editing. Here, we have made a direct comparison of different CRISPR/Cas9 guide systems to target the genome in three cereal species. We show that the guide sequences driven by the same pol II promoter in rice, wheat and barley show large differences in editing efficiency. The differences seen were based on the way the guides were presented and factors outside of the guide sequence itself. While both the tRNA system and ribozyme system performed well in rice, their effectiveness varied in wheat and barley. Specifically, the tRNA system outperformed the ribozyme system, achieving higher rates of editing in stable transformed plants. Overall, high levels of editing are observed in all three species when strong expression of the SpCas9 is coupled with the CmYLCV promoter to drive a tRNA array of guide RNAs. Stable inheritance is also achievable in all three species when plants are sampled shortly after the tissue culture concludes. Overall, inheritance rates were above 85% in all three species, particularly when mutations are detected early after plants emerge from tissue culture. [ABSTRACT FROM AUTHOR]

The mutualistic arbuscular mycorrhizal (AM) symbiosis arose in land plants more than 450 million years ago and is still widely found in all major land plant lineages. Despite its broad taxonomic distribution, little is known about the molecular components underpinning symbiosis outside of flowering plants. The ARBUSCULAR RECEPTOR-LIKE KINASE (ARK) is required for sustaining AM symbiosis in distantly related angio-sperms. Here, we demonstrate that ARK has an equivalent role in symbiosis maintenance in the bryophyte Marchantia paleacea and is part of a broad AM genetic program conserved among land plants. In addition, our comparative transcriptome analysis identified evolutionarily conserved expression patterns for several genes in the core symbiotic program required for presymbiotic signaling, intracellular colonization, and nutrient exchange. This study provides insights into the molecular pathways that consistently associate with AM symbiosis across land plants and identifies an ancestral role for ARK in governing symbiotic balance. [ABSTRACT FROM AUTHOR]

There is a large demand to reduce inputs for current crop production, particularly phosphate and nitrogen inputs which are the two most frequently added supplements to agricultural production. Gene characterization is often limited to the native species from which it was identified, but may offer benefits to other species. To understand if the rice gene Phosphate Starvation Tolerance 1 (PSTOL) OsPSTOL, a gene identified from rice which improves tolerance to low P growth conditions, might improve performance and provide the same benefit in wheat, OsPSTOL was transformed into wheat and expressed from a constitutive promoter. The ability of OsPSTOL to improve nutrient acquisition under low phosphate or low nitrogen was evaluated. Here we show that OsPSTOL works through a conserved pathway in wheat and rice to improve yields under both low phosphate and low nitrogen. This increase is yield is mainly driven by improved uptake from the soil driving increased biomass and ultimately increased seed number, but does not change the concentration of N in the straw or grain. Overexpression of OsPSTOL in wheat modifies N regulated genes to aid in this uptake whereas the putative homolog TaPSTOL does not suggesting that expression of OsPSTOL in wheat can help to improve yields under low input agriculture. [ABSTRACT FROM AUTHOR]

The regulation of lipid metabolism in oil seeds is still not fully understood and increasing our knowledge in this regard is of great economic, as well as intellectual, importance. Oilseed rape (Brassica napus) is a major global oil crop where increases in triacylglycerol (TAG) accumulation have been achieved by overexpression of relevant biosynthetic enzymes. In this study, we expressed Arabidopsis phospholipid: diacylglycerol acyltransferase (PDAT1), one of the two major TAG-forming plant enzymes in B. napus DH12075 to evaluate its effect on lipid metabolism in developing seeds and to estimate its flux control coefficient. Despite several-fold increase in PDAT activity, seeds of three independently generated PDAT transgenic events showed a small but consistent decrease in seed oil content and had altered fatty acid composition of phosphoglycerides and TAG, towards less unsaturation. Mass spectrometry imaging of seed sections confirmed the shift in lipid compositions and indicated that PDAT overexpression altered the distinct heterogeneous distributions of phosphatidylcholine (PC) molecular species. Similar, but less pronounced, changes in TAG molecular species distributions were observed. Our data indicate that PDAT exerts a small, negative, flux control on TAG biosynthesis and could have under-appreciated effects in fine-tuning of B. napus seed lipid composition in a tissue-specific manner. This has important implications for efforts to increase oil accumulation in similar crops. [ABSTRACT FROM AUTHOR]

The application of CRISPR/Cas9 technologies has transformed our ability to target and edit designated regions of a genome. It's broad adaptability to any organism has led to countless advancements in our understanding of many biological processes. Many current tools are designed for simple plant systems such as diploid species, however, efficient deployment in crop species requires a greater efficiency of editing as these often contain polyploid genomes. Here, we examined the role of temperature to understand if CRISPR/Cas9 editing efficiency can be improved in wheat. The recent finding that plant growth under higher temperatures could increase mutation rates was tested with Cas9 expressed from two different promoters in wheat. Increasing the temperature of the tissue culture or of the seed germination and early growth phase increases the frequency of mutation in wheat when the Cas9 enzyme is driven by the ZmUbi promoter but not OsActin. In contrast, Cas9 expression driven by the OsActin promoter did not increase the mutations detected in either transformed lines or during the transformation process itself. These results demonstrate that CRISPR/Cas9 editing efficiency can be significantly increased in a polyploid cereal species with a simple change in growth conditions to facilitate increased mutations for the creation of homozygous or null knock-outs. [ABSTRACT FROM AUTHOR]

Wheat is grown on more land than any other crop in the world. Current estimates suggest that yields will have to increase sixty percent by 2050 to meet the demand of an ever‐increasing human population; however, recent wheat yield gains have lagged behind other major crops such as rice and maize. One of the reasons suggested for the lag in yield potential is the lack of a robust hybrid system to harness the potential yield gains associated with heterosis, also known as hybrid vigor. Here, we set out to identify candidate genes for a genic hybrid system in wheat and characterize their function in wheat using RNASeq on stamens and carpels undergoing meiosis. Twelve genes were identified as potentially playing a role in pollen viability. CalS5‐ and RPG1‐like genes were identified as pre‐ and post‐meiotic genes for further characterization and to determine their role in pollen viability. It appears that all three homoeologues of both CalS5 and RPG1 are functional in wheat as all three homoeologues need to be knocked out in order to cause male sterility. However, one functional homoeologue is sufficient to maintain male fertility in wheat. [ABSTRACT FROM AUTHOR]

The biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals and grasses. We present the first crystal structure of a B. graminis effector of pathogenicity (CSEP0064/BEC1054), demonstrating it has a ribonuclease (RNase)-like fold. This effector is part of a group of RNase-like proteins (termed RALPHs) which comprise the largest set of secreted effector candidates within the B. graminis genomes. Their exceptional abundance suggests they play crucial functions during pathogenesis. We show that transgenic expression of RALPH CSEP0064/BEC1054 increases susceptibility to infection in both monocotyledonous and dicotyledonous plants. CSEP0064/BEC1054 interacts in planta with the pathogenesis-related protein PR10. The effector protein associates with total RNA and weakly with DNA. Methyl jasmonate (MeJA) levels modulate susceptibility to aniline-induced host RNA fragmentation. In planta expression of CSEP0064/BEC1054 reduces the formation of this RNA fragment. We propose CSEP0064/BEC1054 is a pseudoenzyme that binds to host ribosomes, thereby inhibiting the action of plant ribosome-inactivating proteins (RIPs) that would otherwise lead to host cell death, an unviable interaction and demise of the fungus. [ABSTRACT FROM AUTHOR]

Examines the heat shock response of Rhizobium leguminosarum. Induction of protein synthesis after a ten minute heat shock at 37 degrees celsius; Cross-reaction of a monoclonal antibody raised against Escherichia coli Cpn60 with proteins of Mr 60000 and 58000 in R; Hybridization of an E. coli cpn60 probe to total deoxyribonucleic acid from Rhizobium leguminosarum.

Background: Phosphorus (P) is an essential macronutrient for plant growth, and is required in large quantities by elite varieties of crops to maintain yields. Approximately 70% of global cultivated land suffers from P deficiency, and it has recently been estimated that worldwide P resources will be exhausted by the end of this century, increasing the demand for crops more efficient in their P usage. A greater understanding of how plants are able to maintain yield with lower P inputs is, therefore, highly desirable to both breeders and farmers. Here, we clone the wheat (Triticum aestivum L.) homologue of the rice PSTOL gene (OsPSTOL), and characterize its role in phosphate nutrition plus other agronomically important traits. Results: TaPSTOL is a single copy gene located on the short arm of chromosome 5A, encoding a putative kinase protein, and shares a high level of sequence similarity to OsPSTOL. We re-sequenced TaPSTOL from 24 different wheat accessions and (3) three T. durum varieties. No sequence differences were detected in 26 of the accessions, whereas two indels were identified in the promoter region of one of the durum wheats. We characterised the expression of TaPSTOL under different P concentrations and demonstrated that the promoter was induced in root tips and hairs under P limiting conditions. Overexpression and RNAi silencing of TaPSTOL in transgenic wheat lines showed that there was a significant effect upon root biomass, flowering time independent of P treatment, tiller number and seed yield, correlating with the expression of TaPSTOL. However this did not increase PUE as elevated P concentration in the grain did not correspond to increased yields. Conclusions: Manipulation of TaPSTOL expression in wheat shows it is responsible for many of the previously described phenotypic advantages as OsPSTOL except yield. Furthermore, we show TaPSTOL contributes to additional agronomically important traits including flowering time and grain size. Analysis of TaPSTOL sequences from a broad selection of wheat varieties, encompassing 91% of the genetic diversity in UK bread wheat, showed that there is very little genetic variation in this gene, which would suggest that this locus may have been under high selection pressure. [ABSTRACT FROM AUTHOR]