Enhancing crop yields through improvements in the efficiency of photosynthesis and respiration.

The rate with which crop yields per hectare increase each year is plateauing at the same time that human population growth and other factors increase food demand. Increasing yield potential ( Y p ) of crops is vital to address these challenges. In this review, we explore a component of Y p that has yet to be optimised - that being improvements in the efficiency with which light energy is converted into biomass ( ε c ) via modifications to CO2 fixed per unit quantum of light (α), efficiency of respiratory ATP production ( ε prod ) and efficiency of ATP use ( ε use ). For α, targets include changes in photoprotective machinery, ribulose bisphosphate carboxylase/oxygenase kinetics and photorespiratory pathways. There is also potential for ε prod to be increased via targeted changes to the expression of the alternative oxidase and mitochondrial uncoupling pathways. Similarly, there are possibilities to improve ε use via changes to the ATP costs of phloem loading, nutrient uptake, futile cycles and/or protein/membrane turnover. Recently developed high-throughput measurements of respiration can serve as a proxy for the cumulative energy cost of these processes. There are thus exciting opportunities to use our growing knowledge of factors influencing the efficiency of photosynthesis and respiration to create a step-change in yield potential of globally important crops.

Escherichia coli expressing chloroplast chaperones as a proxy to test heterologous Rubisco production in leaves.

Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Escherichia coli. While this enhances opportunities for catalytic improvement, there remain limitations in the utility of the expression system. Here we generate, optimize, and test a robust Golden Gate cloning E. coli expression system incorporating the protein folding machinery of tobacco chloroplasts. By comparing the expression of different plant Rubiscos in both E. coli and plastome-transformed tobacco, we show that the E. coli expression system can accurately predict high level Rubisco production in chloroplasts but poorly forecasts the biogenesis potential of isoforms with impaired production in planta. We reveal that heterologous Rubisco production in E. coli and tobacco plastids poorly correlates with Rubisco large subunit phylogeny. Our findings highlight the need to fully understand the factors governing Rubisco biogenesis if we are to deliver an efficient, low-cost screening tool that can accurately emulate chloroplast expression.

Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast rbcL gene and nuclear RbcS genes. Here we develop an RNAi-RbcS tobacco (Nicotiana tabacum) master-line, tobRrΔS, for producing homogenous plant Rubisco by rbcL-rbcS operon chloroplast transformation. Four genotypes encoding alternative rbcS genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a rbcS gene whose codon use and 5' untranslated-region matched rbcL Additional tobacco genotypes produced here incorporated differing potato (Solanum tuberosum) rbcL-rbcS operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pST-subunit. The pS3-subunit caused impairment of potato Rubisco production by ∼15% relative to the lines producing pS1, pS2, or pST However, the ßA-ßB loop Asn-55-His and Lys-57-Ser substitutions in the pS3-subunit improved carboxylation rates by 13% and carboxylation efficiency (CE) by 17%, relative to potato Rubisco incorporating pS1 or pS2-subunits. Tobacco photosynthesis and growth were most impaired in lines producing potato Rubisco incorporating the pST-subunit, which reduced CE and CO2/O2 specificity 40% and 15%, respectively. Returning the rbcS gene to the plant plastome provides an effective bioengineering chassis for introduction and evaluation of novel homogeneous Rubisco complexes in a whole plant context.

Transcriptome analysis of the fungal pathogen Fusarium oxysporum f. sp. medicaginis during colonisation of resistant and susceptible Medicago truncatula hosts identifies differential pathogenicity profiles and novel candidate effectors.

BACKGROUND: Pathogenic members of the Fusarium oxysporum species complex are responsible for vascular wilt disease on many important crops including legumes, where they can be one of the most destructive disease causing necrotrophic fungi. We previously developed a model legume-infecting pathosystem based on the reference legume Medicago truncatula and a pathogenic F. oxysporum forma specialis (f. sp.) medicaginis (Fom). To dissect the molecular pathogenicity arsenal used by this root-infecting pathogen, we sequenced its transcriptome during infection of a susceptible and resistant host accession. RESULTS: High coverage RNA-Seq of Fom infected root samples harvested from susceptible (DZA315) or resistant (A17) M. truncatula seedlings at early or later stages of infection (2 or 7 days post infection (dpi)) and from vegetative (in vitro) samples facilitated the identification of unique and overlapping sets of in planta differentially expressed genes. This included enrichment, particularly in DZA315 in planta up-regulated datasets, for proteins associated with sugar, protein and plant cell wall metabolism, membrane transport, nutrient uptake and oxidative processes. Genes encoding effector-like proteins were identified, including homologues of the F. oxysporum f. sp. lycopersici Secreted In Xylem (SIX) proteins, and several novel candidate effectors based on predicted secretion, small protein size and high in-planta induced expression. The majority of the effector candidates contain no known protein domains but do share high similarity to predicted proteins predominantly from other F. oxysporum ff. spp. as well as other Fusaria (F. solani, F. fujikori, F. verticilloides, F. graminearum and F. pseudograminearum), and from another wilt pathogen of the same class, a Verticillium species. Overall, this suggests these novel effector candidates may play important roles in Fusaria and wilt pathogen virulence. CONCLUSION: Combining high coverage in planta RNA-Seq with knowledge of fungal pathogenicity protein features facilitated the identification of differentially expressed pathogenicity associated genes and novel effector candidates expressed during infection of a resistant or susceptible M. truncatula host. The knowledge from this first in depth in planta transcriptome sequencing of any F. oxysporum ff. spp. pathogenic on legumes will facilitate the dissection of Fusarium wilt pathogenicity mechanisms on many important legume crops.

Comparative genomics and prediction of conditionally dispensable sequences in legume-infecting Fusarium oxysporum formae speciales facilitates identification of candidate effectors.

BACKGROUND: Soil-borne fungi of the Fusarium oxysporum species complex cause devastating wilt disease on many crops including legumes that supply human dietary protein needs across many parts of the globe. We present and compare draft genome assemblies for three legume-infecting formae speciales (ff. spp.): F. oxysporum f. sp. ciceris (Foc-38-1) and f. sp. pisi (Fop-37622), significant pathogens of chickpea and pea respectively, the world's second and third most important grain legumes, and lastly f. sp. medicaginis (Fom-5190a) for which we developed a model legume pathosystem utilising Medicago truncatula. RESULTS: Focusing on the identification of pathogenicity gene content, we leveraged the reference genomes of Fusarium pathogens F. oxysporum f. sp. lycopersici (tomato-infecting) and F. solani (pea-infecting) and their well-characterised core and dispensable chromosomes to predict genomic organisation in the newly sequenced legume-infecting isolates. Dispensable chromosomes are not essential for growth and in Fusarium species are known to be enriched in host-specificity and pathogenicity-associated genes. Comparative genomics of the publicly available Fusarium species revealed differential patterns of sequence conservation across F. oxysporum formae speciales, with legume-pathogenic formae speciales not exhibiting greater sequence conservation between them relative to non-legume-infecting formae speciales, possibly indicating the lack of a common ancestral source for legume pathogenicity. Combining predicted dispensable gene content with in planta expression in the model legume-infecting isolate, we identified small conserved regions and candidate effectors, four of which shared greatest similarity to proteins from another legume-infecting ff. spp. CONCLUSIONS: We demonstrate that distinction of core and potential dispensable genomic regions of novel F. oxysporum genomes is an effective tool to facilitate effector discovery and the identification of gene content possibly linked to host specificity. While the legume-infecting isolates didn't share large genomic regions of pathogenicity-related content, smaller regions and candidate effector proteins were highly conserved, suggesting that they may play specific roles in inducing disease on legume hosts.

Evaluation of the preserving efficacy of lubricant eye drops with a novel preservative system.

Products provided in multidose containers must be adequately preserved in order to prevent contamination during repeated use. The aim of this study was to evaluate the ability of a lubricant eye drop formulation without traditional preservatives to control and prevent contamination under extreme microbial conditions. Bottles of a new lubricant eye drop with a novel preservative system (SYSTANE FREE Lubricant Eye Drops, Alcon Laboratories, Inc., Fort Worth, TX) were stored at elevated (40 degrees C) and room temperature (25 degrees C) for up to 1 year. The formulation was inoculated with microorganisms to contain high levels (approximately 1x10(6) colony forming units [cfu]/mL) of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Aspergillus niger. The inoculated samples were stored at 20-25 degrees C during testing. Three types of tests were conducted: (1) tests with 1 inoculation, (2) tests with 2 inoculations over 28-day test periods, and (3) tests with 6 inoculations over a 6-month test period. Additionally, a comparison of the preservative efficacy of the SYSTANE FREE formulation to marketed products, including sodium perborate, stabilized oxychloro complex, and polyhexamethylene biguanide eye drops, was performed. In all studies, the number of survivors was determined using standard microbiological dilution pour-plate techniques. The results consistently showed that the SYSTANE FREE formulation meets preservative efficacy standards equally well, or better, than, marketed eye drop products. Even with multiple inoculations, the formulation repeatedly showed no recovery of the organisms. Though the SYSTANE FREE formulation contains no traditional preservatives, its unique blend of ions and buffers was capable of preventing contamination of the contents even after exposure to extreme conditions.

Minnesota physician workforce analysis: rural supply and demand.

Considering the physician supply in 2002, the potential number of new physicians, and the number of vacancies that year, rural Minnesota has shortages in the following specialties: family practice, internal medicine, obstetrics/gynecology, pediatrics, general surgery, gastroenterology, ophthalmology, orthopedic surgery, otolaryngology, psychiatry, and urology. Metropolitan areas have fewer shortages; however, there is a gap between supply and demand in obstetrics/gynecology, gastroenterology, general surgery, otolaryngology, and urology. Future areas of concern for all parts of the state include family practice, internal medicine, obstetrics/gynecology, pediatrics, cardiology, general surgery, psychiatry, ophthalmology, orthopedic surgery, and urology. In Minnesota, with several specialties facing a substantial number of retirements in the next 10 years, the number of new physicians completing graduate medical education remaining steady or declining, and the population growing--especially among people ages 65 to 84, the current shortages may increase, and additional specialties may experience a gap if the supply is not increased.

Amoebicidal activity of multipurpose contact lens solutions.

PURPOSE: To compare the amoebicidal activity of two commercially available multipurpose contact lens care systems, by using cyst and trophozoite growth of two strains each of Acanthamoeba castellanii and Acanthamoeba polyphaga. METHODS: Trophozoites and cysts of Acanthamoeba species, isolated from corneal ulcers and an ATCC strain, were exposed to two commonly used multipurpose solutions and a saline control for 4 or 6 hours at ambient temperature. After neutralization of the disinfecting solution, track forming units were enumerated on Escherichia coli-seeded nutrient agar plates. RESULTS: There were significant differences (P<0.01) between solutions, strains, and cysts or trophozoites, but not between the different periods. Solution 2, containing polyquaternium-1 and myristamidopropyl dimethylamine (among other ingredients), gave greater mean log reductions than did solution 1, containing polyaminopropyl biguanide and poloxamine (among other ingredients). Both solutions generally reduced more trophozoites than cysts. CONCLUSIONS: The multipurpose solution containing polyquaternium-1 and myristamidopropyl dimethylamine reduced more cysts and trophozoites than did the alternative solution tested. Because there were differences in the effect of the multipurpose solutions on strains, it is recommended that more than one strain or species of Acanthamoeba be tested to determine efficacy of disinfecting solutions. The lack of difference between disinfection times may indicate that 4 hours could be considered as an adequate disinfecting time to ensure sufficient kill of Acanthamoeba species.