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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 angiosperms. 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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Milner, Bowden, Craze and Wallington.)

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., (© 2022 The Author(s).)

There is a strong pressure to reduce nitrogen (N) fertilizer inputs while maintaining or increasing current cereal crop yields. We show that overexpression of TaDWF4-B, the dominant shoot expressed homoeologue of OsDWF4, in wheat can increase plant productivity by up to 105% under a range of N levels on marginal soils, resulting in increased N use efficiency (NUE). We show that a two to four-fold increase in TaDWF4 transcript levels enhances the responsiveness of genes regulated by N. The productivity increases seen were primarily due to the maintenance of photosystem II operating efficiency and carbon assimilation in plants when grown under limiting N conditions and not an overall increase in photosynthesis capacity. The increased biomass production and yield per plant in TaDWF4 OE lines could be linked to modified carbon partitioning and changes in expression pattern of the growth regulator Target Of Rapamycin, offering a route towards breeding for sustained yield and lower N inputs., (© 2022. The Author(s).)

Background: Grain size is thought to be a major component of yield in many plant species. Here we set out to understand if knowledge from other cereals such as rice could translate to increased yield gains in wheat and lead to increased nitrogen use efficiency. Previous findings that the overexpression of OsBG1 in rice increased yields while increasing seed size suggest translating gains from rice to other cereals may help to increase yields., Results: The orthologous genes of OsBG1 were identified in wheat. One homoeologous wheat gene was cloned and overexpressed in wheat to understand its role in controlling seed size. Potential alteration in the nutritional profile of the grains were also analyzed in wheat overexpressing TaBG1. It was found that increased TaBG1-A expression could indeed lead to larger seed size but was linked to a reduction in seed number per plant leading to no significant overall increase in yield. Other important components of yield such as biomass or tillering did not change significantly with increased TaBG1-A expression. The nutritional profile of the grain was altered, with a significant decrease in the Zn levels in the grain associated with increased seed size, but Fe and Mn concentrations were unchanged. Protein content of the wheat grain also fell under moderate N fertilization levels but not under deficient or adequate levels of N., Conclusions: TaBG1 does control seed size in wheat but increasing the seed size per se does not increase yield and may come at the cost of lower concentrations of essential elements as well as potentially lower protein content. Nevertheless, TaBG1 could be a useful target for further breeding efforts in combination with other genes for increased biomass., (© 2021. The Author(s).)

Wheat is the most widely grown crop globally, providing 20% of all human calories and protein. Achieving step changes in genetic yield potential is crucial to ensure food security, but efforts are thwarted by an apparent trade-off between grain size and number. Expansins are proteins that play important roles in plant growth by enhancing stress relaxation in the cell wall, which constrains cell expansion. Here, we describe how targeted overexpression of an α-expansin in early developing wheat seeds leads to a significant increase in grain size without a negative effect on grain number, resulting in a yield boost under field conditions. The best-performing transgenic line yielded 12.3% higher average grain weight than the control, and this translated to an increase in grain yield of 11.3% in field experiments using an agronomically appropriate plant density. This targeted transgenic approach provides an opportunity to overcome a common bottleneck to yield improvement across many crops., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Foundation.)

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., Competing Interests: Elsoms Developments Ltd., funded the initial wheat transformation experiment with construct pMM20 and analysis of AK38 and AK39 T0 wheat plants. MM is co-inventor on a patent application with Anthony Keeling, Elsoms Developments Ltd., on the AK38 and AK39 lines described in this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Milner, Craze, Hope and Wallington.)

Stem solidness is an important agronomic trait of durum ( Triticum turgidum L. var. durum ) and bread ( Triticum aestivum L.) wheat that provides resistance to the wheat stem sawfly. This dominant trait is conferred by the SSt1 locus on chromosome 3B. However, the molecular identity and mechanisms underpinning stem solidness have not been identified. Here, we demonstrate that copy number variation of TdDof , a gene encoding a putative DNA binding with one finger protein, controls the stem solidness trait in wheat. Using map-based cloning, we localized TdDof to within a physical interval of 2.1 Mb inside the SSt1 locus. Molecular analysis revealed that hollow-stemmed wheat cultivars such as Kronos carry a single copy of TdDof , whereas solid-stemmed cultivars such as CDC Fortitude carry multiple identical copies of the gene. Deletion of all TdDof copies from CDC Fortitude resulted in the loss of stem solidness, whereas the transgenic overexpression of TdDof restored stem solidness in the TdDof deletion mutant pithless1 and conferred stem solidness in Kronos. In solid-stemmed cultivars, increased TdDof expression was correlated with the down-regulation of genes whose orthologs have been implicated in programmed cell death (PCD) in other species. Anatomical and histochemical analyses revealed that hollow-stemmed lines had stronger PCD-associated signals in the pith cells compared to solid-stemmed lines, which suggests copy number-dependent expression of TdDof could be directly or indirectly involved in the negative regulation of PCD. These findings provide opportunities to manipulate stem development in wheat and other monocots for agricultural or industrial purposes., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Most plants associate with beneficial arbuscular mycorrhizal (AM) fungi that facilitate soil nutrient acquisition. Prior to contact, partner recognition triggers reciprocal genetic remodelling to enable colonisation. The plant Dwarf14-Like (D14L) receptor conditions pre-symbiotic perception of AM fungi, and also detects the smoke constituent karrikin. D14L-dependent signalling mechanisms, underpinning AM symbiosis are unknown. Here, we present the identification of a negative regulator from rice, which operates downstream of the D14L receptor, corresponding to the homologue of the Arabidopsis thaliana Suppressor of MAX2-1 (AtSMAX1) that functions in karrikin signalling. We demonstrate that rice SMAX1 is a suppressor of AM symbiosis, negatively regulating fungal colonisation and transcription of crucial signalling components and conserved symbiosis genes. Similarly, rice SMAX1 negatively controls strigolactone biosynthesis, demonstrating an unexpected crosstalk between the strigolactone and karrikin signalling pathways. We conclude that removal of SMAX1, resulting from D14L signalling activation, de-represses essential symbiotic programmes and increases strigolactone hormone production.

Ingestion of gluten proteins (gliadins and glutenins) from wheat, barley and rye can cause coeliac disease (CD) in genetically predisposed individuals. The only remedy is a strict and lifelong gluten-free diet. There is a growing desire for coeliac-safe, whole-grain wheat-based products, as consumption of whole-grain foods reduces the risk of chronic diseases. However, due to the large number of gluten genes and the complexity of the wheat genome, wheat that is coeliac-safe but retains baking quality cannot be produced by conventional breeding alone. CD is triggered by immunogenic epitopes, notably those present in α-, γ-, and ω-gliadins. RNA interference (RNAi) silencing has been used to down-regulate gliadin families. Recently, targeted gene editing using CRISPR/Cas9 has been applied to gliadins. These methods produce offspring with silenced, deleted, and/or edited gliadins, that overall may reduce the exposure of patients to CD epitopes. Here we review methods to efficiently screen and select the lines from gliadin gene editing programs for CD epitopes at the DNA and protein level, for baking quality, and ultimately in clinical trials. The application of gene editing for the production of coeliac-safe wheat is further considered within the context of food production and in view of current national and international regulatory frameworks., (Copyright © 2020 Jouanin, Gilissen, Schaart, Leigh, Cockram, Wallington, Boyd, van den Broeck, van der Meer, America, Visser and Smulders.)

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., Competing Interests: M.M. and A.K. have filed a patent application based on the results reported in this paper. All other authors declare no competing financial interests., (© 2020 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Background: Wheat grains contain gluten proteins, which harbour immunogenic epitopes that trigger Coeliac disease in 1-2% of the human population. Wheat varieties or accessions containing only safe gluten have not been identified and conventional breeding alone struggles to achieve such a goal, as the epitopes occur in gluten proteins encoded by five multigene families, these genes are partly located in tandem arrays, and bread wheat is allohexaploid. Gluten immunogenicity can be reduced by modification or deletion of epitopes. Mutagenesis technologies, including CRISPR/Cas9, provide a route to obtain bread wheat containing gluten proteins with fewer immunogenic epitopes., Results: In this study, we analysed the genetic diversity of over 600 α- and γ-gliadin gene sequences to design six sgRNA sequences on relatively conserved domains that we identified near coeliac disease epitopes. They were combined in four CRISPR/Cas9 constructs to target the α- or γ-gliadins, or both simultaneously, in the hexaploid bread wheat cultivar Fielder. We compared the results with those obtained with random mutagenesis in cultivar Paragon by γ-irradiation. For this, Acid-PAGE was used to identify T1 grains with altered gliadin protein profiles compared to the wild-type endosperm. We first optimised the interpretation of Acid-PAGE gels using Chinese Spring deletion lines. We then analysed the changes generated in 360 Paragon γ-irradiated lines and in 117 Fielder CRISPR/Cas9 lines. Similar gliadin profile alterations, with missing protein bands, could be observed in grains produced by both methods., Conclusions: The results demonstrate the feasibility and efficacy of using CRISPR/Cas9 to simultaneously edit multiple genes in the large α- and γ-gliadin gene families in polyploid bread wheat. Additional methods, generating genomics and proteomics data, will be necessary to determine the exact nature of the mutations generated with both methods.

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., Competing Interests: The authors have declared that no competing interests exist.

In terrestrial ecosystems most plant species live in mutualistic symbioses with nutrient-delivering arbuscular mycorrhizal (AM) fungi. Establishment of AM symbioses includes transient, intracellular formation of fungal feeding structures, the arbuscules. A plant-derived peri-arbuscular membrane (PAM) surrounds the arbuscules, mediating reciprocal nutrient exchange. Signaling at the PAM must be well coordinated to achieve this dynamic cellular intimacy. Here, we identify the PAM-specific Arbuscular Receptor-like Kinase 1 (ARK1) from maize and rice to condition sustained AM symbiosis. Mutation of rice ARK1 causes a significant reduction in vesicles, the fungal storage structures, and a concomitant reduction in overall root colonization by the AM fungus Rhizophagus irregularis. Arbuscules, although less frequent in the ark1 mutant, are morphologically normal. Co-cultivation with wild-type plants restores vesicle and spore formation, suggesting ARK1 function is required for the completion of the fungal life-cycle, thereby defining a functional stage, post arbuscule development.

Background: The use of CRISPR/Cas9 systems could prove to be a valuable tool in crop research, providing the ability to fully knockout gene function in complex genomes or to precisely adjust gene function by knockout of individual alleles., Results: We compare gene editing in hexaploid wheat (Triticum aestivum) with diploid barley (Hordeum vulgare), using a combination of single genome and tri-genome targeting. High efficiency gene editing, 11-17% for single genome targeted guides and 5% for tri-genome targeted guides, was achieved in wheat using stable Agrobacterium-mediated transformation. Gene editing in wheat was shown to be predominantly heterozygous, edits were inherited in a Mendelian fashion over multiple generations and no off-target effects were observed. Comparison of editing between the two species demonstrated that more stable, heritable edits were produced in wheat, whilst barley exhibited continued and somatic editing., Conclusion: Our work shows the potential to obtain stable edited transgene-free wheat lines in 36 weeks through only two generations and that targeted mutagenesis of individual homeologues within the wheat genome is achievable with a modest amount of effort, and without off-target mutations or the need for lengthy crossing strategies.

A strict gluten-free diet is currently the only treatment for the 1-2% of the world population who suffer from coeliac disease (CD). However, due to the presence of wheat and wheat derivatives in many food products, avoiding gluten consumption is difficult. Gluten-free products, made without wheat, barley or rye, typically require the inclusion of numerous additives, resulting in products that are often less healthy than gluten-based equivalents. Here, we present and discuss two broad approaches to decrease wheat gluten immunogenicity for CD patients. The first approach is based on food processing strategies, which aim to remove gliadins or all gluten from edible products. We find that several of the candidate food processing techniques to produce low gluten-immunogenic products from wheat already exist. The second approach focuses on wheat breeding strategies to remove immunogenic epitopes from the gluten proteins, while maintaining their food-processing properties. A combination of breeding strategies, including mutation breeding and possibly genome editing, will be necessary to produce coeliac-safe wheat. Individuals suffering from CD and people genetically susceptible who may develop CD after prolonged gluten consumption would benefit from reduced CD-immunogenic wheat. Although the production of healthy and less CD-toxic wheat varieties and food products will be challenging, increasing global demand may require these issues to be addressed in the near future by food processing and cereal breeding companies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

The following method enables the rapid production of transgenic potato plants and microtubers for gene validation and expression, or promoter studies. The method is highly efficient, with reproducible transformation efficiencies of at least 50% to 60% with potato cv. Desiree, and can produce transgenic microtubers within 6 months of initiation of the experiment. Microtubers are produced in the absence of hormones, giving an in vitro gene testing system broadly analogous to the natural state. © 2018 by John Wiley & Sons, Inc., (© 2018 John Wiley & Sons, Inc.)

Oilseed rape (Brassica napus) is a commercially important member of the Brassicacea family. It is grown for its edible and industrial oils as well as for animal feed. Genetic transformation technology has been used to study gene function and produce oilseed rape with improved agronomic characteristics. This protocol describes a method for the Agrobacterium tumefaciens-mediated transformation of oilseed rape cotyledonary petioles. The method is reproducible and has been used to transform both spring and winter cultivars. Modifications have been made to the rooting stage, which have reduced the vitrification of shoots. This has not only increased the number of phenotypically normal shoots but has also resulted in an increase in transformation efficiency, concomitant with a dramatic reduction in the number of escapes regenerated. Transformation frequencies typically range from 5% to 10%, with an average of 12% using doubled haploid model varieties, but a maximum efficiency of 20% has been achieved. © 2017 by John Wiley & Sons, Inc., (© 2017 John Wiley & Sons, Inc.)

High erucic acid rapeseed (HEAR) oil is of interest for industrial purposes because erucic acid (22:1) and its derivatives are important renewable raw materials for the oleochemical industry. Currently available cultivars contain only about 50% erucic acid in the seed oil. A substantial increase in erucic acid content would significantly reduce processing costs and could increase market prospects of HEAR oil. It has been proposed that erucic acid content in rapeseed is limited because of insufficient fatty acid elongation, lack of insertion of erucic acid into the central sn-2 position of the triaclyglycerol backbone and due to competitive desaturation of the precursor oleic acid (18:1) to linoleic acid (18:2). The objective of the present study was to increase erucic content of HEAR winter rapeseed through over expression of the rapeseed fatty acid elongase gene (fae1) in combination with expression of the lysophosphatidic acid acyltransferase gene from Limnanthes douglasii (Ld-LPAAT), which enables insertion of erucic acid into the sn-2 glycerol position. Furthermore, mutant alleles for low contents of polyunsaturated fatty acids (18:2 + 18:3) were combined with the transgenic material. Selected transgenic lines showed up to 63% erucic acid in the seed oil in comparison to a mean of 54% erucic acid of segregating non-transgenic HEAR plants. Amongst 220 F(2) plants derived from the cross between a transgenic HEAR line and a non-transgenic HEAR line with a low content of polyunsaturated fatty acids, recombinant F(2) plants were identified with an erucic acid content of up to 72% and a polyunsaturated fatty acid content as low as 6%. Regression analysis revealed that a reduction of 10% in polyunsaturated fatty acids content led to a 6.5% increase in erucic acid content. Results from selected F(2) plants were confirmed in the next generation by analysing F(4) seeds harvested from five F(3) plants per selected F(2) plant. F(3) lines contained up to 72% erucic acid and as little as 4% polyunsaturated fatty acids content in the seed oil. The 72% erucic acid content of rapeseed oil achieved in the present study represents a major breakthrough in breeding high erucic acid rapeseed.

Although many bacteria contain only a single groE operon encoding the essential chaperones GroES and GroEL, examples of bacteria containing more than one groE operon are common. The root-nodulating bacterium Rhizobium leguminosarum contains at least three operons encoding homologues to Escherichia coli GroEL, referred to as Cpn60.1, Cpn60.2 and Cpn60.3, respectively. We report here a detailed analysis of the requirement for and relative levels of these three proteins. Cpn60.1 is present at higher levels than Cpn60.2, and Cpn60.3 protein could not be detected under any conditions although the cpn60.3 gene is transcribed under anaerobic conditions. Insertion mutations could not be constructed in cpn60.1 unless a complementing copy was present, showing that this gene is essential for growth under the conditions used here. Both cpn60.2 and cpn60.3 could be inactivated with no loss of viability, and a double cpn60.2 cpn60.3 mutant was also constructed which was fully viable. Thus only Cpn60.1 is required for growth of this organism.

Homologous recombination was used to construct a series of hybrid chaperonin genes, containing various lengths of Escherichia coli groEL replaced by the equivalent region from the homologous cpn60-1 gene of Rhizobium leguminosarum. Analysis of proteins produced by these hybrids showed that many of them formed structures with properties consistent with their being single heptameric rings under some conditions, as opposed to the double ring form in which both the GroEL and the Cpn60-1 proteins are found. By determining precise cross-over points, two regions in Cpn60-1 were defined which appeared to be critical for ring-ring interactions. Within one of these regions is a highly conserved arginine residue (Arg101), which we hypothesised to interact with a residue or residues toward the C terminus of the protein, this contact being required for double rings to form. To test this hypothesis, we mutagenised this residue from arginine to threonine in chaperonin genes from two different species of Rhizobium. In both cases, proteins which ran on non-denaturing gels as single rings were produced. Conversion of Arg101 to serine also had the same effect, whereas conversion of Arg101 to lysine did not. Two different single rings created by homologous recombination could be converted back to double rings by changing the threonine, which naturally occurs at this position in E. coli GroEL, back to arginine. The in vivo properties of the proteins were investigated by complementation following deletion of the chromosomal copy of the groEL gene, and by monitoring the ability of cells expressing the hybrid proteins to plate bacteriophage. Most of the hybrid and mutant proteins were functional in these assays, despite their altered properties compared to wild-type GroEL., (Copyright 1998 Academic Press.)

Bacterial Cpn60 proteins (homologues to the Escherichia coli GroEL protein) are often examined for function by testing their ability to complement a temperature sensitive mutation in the E. coli groEL gene. Such tests suffer from two drawbacks: the Cpn600 protein may come from a strain with a lower optimum growth temperature than E. coli, and the requirements for successful complementation in E. coli are likely to be more stringent at 43 degrees C than at lower temperatures. Here we describe the construction of a strain of E. coli where the chromosomal gene for the essential molecular chaperone GroEL has been deleted, with GroEL being expressed from a tightly regulated plasmid borne copy of the gene. The deletion can be transduced into strains expressing heterologous Cpn60 proteins, to test for complementation at any temperature. We show that a Cpn60 protein from the bacterium Rhizobium leguminosarum can function to allow E. coli growth at 37 degrees C but not at 43 degrees C. By switching off the plasmid borne groEL gene, the effects of progressive depletion of GroEL protein from E. coli cells can also be monitored at any temperature.

We have examined the heat shock response of Rhizobium leguminosarum. After normal growth at 28 degrees C, a 10 min heat shock at 37 degrees C induced the synthesis of proteins with approximate M(r) values of 90,000, 70,000, 60,000, 58,000, 19,000, 17,000 and 13,000. A monoclonal antibody raised against the E. coli Cpn60 cross-reacted with proteins of M(r) 60,000 and 58,000 in R. leguminosarum, suggesting that both were Cpn60 homologues. Hybridization of an E. coli cpn60 probe to total DNA from Rhizobium leguminosarum also showed evidence for at least two cpn60 homologues. One of these was cloned and completely sequenced, and showed close homology to cpn60 sequences from other prokaryotes. The expression of this gene in E. coli failed to complement a cpn60 mutation, either for growth at high temperature or for growth of bacteriophage lambda. Hybridization of total R. leguminosarum DNA with a probe from this gene revealed the presence of a third putative cpn60 gene. Two further hybridizing clones were analysed and found to consist of two additional cpn60 sequences plus upstream regions containing putative cpn10 genes.