David E. Cook

Copy Number Variation of Multiple Genes at Rhg1 Mediates Nematode Resistance in Soybean

Resistance to a damaging disease of soybean is conferred by a cluster of linked genes present in multiple copies. The rhg1-b allele of soybean is widely used for resistance against soybean cyst nematode (SCN), the most economically damaging pathogen of soybeans in the United States. Gene silencing showed that genes in a 31-kilobase segment at rhg1-b, encoding an amino acid transporter, an α-SNAP protein, and a WI12 (wound-inducible domain) protein, each contribute to resistance. There is one copy of the 31-kilobase segment per haploid genome in susceptible varieties, but 10 tandem copies are present in an rhg1-b haplotype. Overexpression of the individual genes in roots was ineffective, but overexpression of the genes together conferred enhanced SCN resistance. Hence, SCN resistance mediated by the soybean quantitative trait locus Rhg1 is conferred by copy number variation that increases the expression of a set of dissimilar genes in a repeated multigene segment. Soybean crops, which supply valuable protein, oil, and renewable fuel, are under attack by a nematode for which there is no effective pesticide. Instead, agriculture relies on resistance derived from a genetic locus, which is now represented in most of the soybean crops cultivated in the United States. Cook et al. (p. 1206, published online 11 October) elucidated the mechanisms by which this rhg1-b allele protects against disease. The region carries several genes, none of which resemble other known immune receptor genes. Experiments silencing one or another of the genes showed that the genes work as a cluster. However, one set of the genes is not enough: Plants need multiple repeats of the locus to acquire resistance.

A nematode demographics assay in transgenic roots reveals no significant impacts of the Rhg1 locus LRR-Kinase on soybean cyst nematode resistance

BackgroundSoybean cyst nematode (Heterodera glycines, SCN) is the most economically damaging pathogen of soybean (Glycine max) in the U.S. The Rhg1 locus is repeatedly observed as the quantitative trait locus with the greatest impact on SCN resistance. The Glyma18g02680.1 gene at the Rhg1 locus that encodes an apparent leucine-rich repeat transmembrane receptor-kinase (LRR-kinase) has been proposed to be the SCN resistance gene, but its function has not been confirmed. Generation of fertile transgenic soybean lines is difficult but methods have been published that test SCN resistance in transgenic roots generated with Agrobacterium rhizogenes.ResultsWe report use of artificial microRNA (amiRNA) for gene silencing in soybean, refinements to transgenic root SCN resistance assays, and functional tests of the Rhg1 locus LRR-kinase gene. A nematode demographics assay monitored infecting nematode populations for their progress through developmental stages two weeks after inoculation, as a metric for SCN resistance. Significant differences were observed between resistant and susceptible control genotypes. Introduction of the Rhg1 locus LRR-kinase gene (genomic promoter/coding region/terminator; Peking/PI 437654-derived SCN-resistant source), into rhg1- SCN-susceptible plant lines carrying the resistant-source Rhg4+locus, provided no significant increases in SCN resistance. Use of amiRNA to reduce expression of the LRR-kinase gene from the Rhg1 locus of Fayette (PI 88788 source of Rhg1) also did not detectably alter resistance to SCN. However, silencing of the LRR-kinase gene did have impacts on root development.ConclusionThe nematode demographics assay can expedite testing of transgenic roots for SCN resistance. amiRNAs and the pSM103 vector that drives interchangeable amiRNA constructs through a soybean polyubiqutin promoter (Gmubi), with an intron-GFP marker for detection of transgenic roots, may have widespread use in legume biology. Studies in which expression of the Rhg1 locus LRR-kinase gene from different resistance sources was either reduced or complemented did not reveal significant impacts on SCN resistance.

Distinct copy number, coding sequence and locus methylation patterns underlie Rhg1-mediated soybean resistance to soybean cyst nematode

A multiple-gene locus in soybean conferring disease resistance has evolved three locus types defined by copy number, protein coding, expression, and DNA methylation differences. Copy number variation of kilobase-scale genomic DNA segments, beyond presence/absence polymorphisms, can be an important driver of adaptive traits. Resistance to Heterodera glycines (Rhg1) is a widely utilized quantitative trait locus that makes the strongest known contribution to resistance against soybean cyst nematode (SCN), Heterodera glycines, the most damaging pathogen of soybean (Glycine max). Rhg1 was recently discovered to be a complex locus at which resistance-conferring haplotypes carry up to 10 tandem repeat copies of a 31-kb DNA segment, and three disparate genes present on each repeat contribute to SCN resistance. Here, we use whole-genome sequencing, fiber-FISH (fluorescence in situ hybridization), and other methods to discover the genetic variation at Rhg1 across 41 diverse soybean accessions. Based on copy number variation, transcript abundance, nucleic acid polymorphisms, and differentially methylated DNA regions, we find that SCN resistance is associated with multicopy Rhg1 haplotypes that form two distinct groups. The tested high-copy-number Rhg1 accessions, including plant introduction (PI) 88788, contain a flexible number of copies (seven to 10) of the 31-kb Rhg1 repeat. The identified low-copy-number Rhg1 group, including PI 548402 (Peking) and PI 437654, contains three copies of the Rhg1 repeat and a newly identified allele of Glyma18g02590 (a predicted α-SNAP [α-soluble N-ethylmaleimide–sensitive factor attachment protein]). There is strong evidence for a shared origin of the two resistance-conferring multicopy Rhg1 groups and subsequent independent evolution. Differentially methylated DNA regions also were identified within Rhg1 that correlate with SCN resistance. These data provide insights into copy number variation of multigene segments, using as the example a disease resistance trait of high economic importance.

Economics of Alternative Corn Stover Logistics Systems

Eight different corn stover logistics systems were modeled for the purpose of evaluating their sensitivity to key system parameters to identify the lowest cost feedstock system. Reducing field operations and maintaining harvester productivity were vital to reducing harvest costs. Storage costs were lowest with systems that had low losses and transport costs were minimized when truck weight limit was attained. Deconstructing and size-reducing bales added considerably to processing costs compared to bulk system where size-reduction took place at harvest. For these reasons, bulk harvest and storage systems consistently provided lower cost feedstock than bale systems. Of the systems considered where the stover and grain were separated at harvest, the two-pass chopped, bulk system where moist stover was conserved by anaerobic storage and fermentation produced the lowest feedstock cost at

Mode of action of hypoglycemic agents—IV: Control of the hypoglycemic activity of phenethylbiguanide in rats and guinea-pigs☆

84/Mg, a difference of

Whole-plant Corn as Biomass Feedstock: Harvest, Storage and Pretreatment

26.7/Mg or 24% lower than the conventional three-pass LSB, covered storage scenario. Storage and transport costs made up a significant fraction of total costs with this bulk system, but these greater costs were offset by much lower harvest and processing costs compared to other systems. A sensitivity analysis showed that yield and the size of the processor had the most impact on feedstock cost. They do so, primarily by the impact on transportation distance, which was found to be the largest cost. A new approach was considered in which very mature corn grain and stover would be harvested, stored and shipped together. The grain and stover fractions would then be separated for further processing. The stover feedstock costs were 55% less than conventional stover harvest systems and 34% less than the lowest cost two-pass system. This system offers the best opportunity to significantly reduce corn stover feedstock costs.

Influence of Glucagon on the Metabolism of Xylitol and Dihydroxyacetone in the Isolated Perfused Rat Liver

Abstract Phenethylbiguanide (PEBG) is a more active hypoglycemic agent in guinea-pigs than in rats. The rat metabolizes the compound to the hypoglycemically inactive p-hydroxyphenethylbiguanide derivative to a greater extent than the guinea-pig. To determine if para-hydroxylation is the controlling factor in the differential response of rats and guinea-pigs to phenethylbiguanide, guinea-pigs were treated with 3,4-benzpyrene to induce microsomal hydroxylation. Phenethylbiguanide lowered blood glucose to 73 per cent of control in these guinea-pigs as compared to 45 per cent of control in those receiving only phenethylbiguanide. Rats given phenethylbiguanide and 2,4-dichloro-6-phenylphenoxy-ethyl-diethylamine HBr (Lilly (18947) to inhibit microsomal hydroxylation had blood glucose levels 65 per cent of control compared to 90 per cent of control for those receiving only phenethylbiguanide. Isolated perfused rat livers produced glucose from lactate at only 46 per cent of the rate of controls when phenethylbiguanide and Lilly 18947 were added to the perfusate as compared to 95 per cent of control when only phenethylbiguanide was added. The presence of Lilly 18947 in the perfusate had no effect on glucose production. It inhibited the para-hydroxylation of phenethylbiguanide. In the perfused rat liver system, Lilly 18947 had no effect on the inhibition of the rate of glucose production from lactate by the non-metabolized hypoglycemic biguanide, 1,1-dimethylbiguanide. Also, the para-substituted compound, p-fluorophenethylbiguanide, was a more effective inhibitor of gluconeogenesis in the perfused system than phenethylbiguanide. The data are consistent with the hypothesis that the hydroxylation of phenethylbiguanide is a major controlling factor in the differential response of rats and guinea-pigs to this drug.

Interaction of Propionate and Lactate in the Perfused Rat Liver EFFECTS OF GLUCAGON AND OLEATE

This research investigated the harvest, ambient pre-treatment, and storage of whole-plant corn as an alternative to conventional systems where corn grain and stover are fractionated at harvest. Harvesting the whole-plant, both grain and most of the above ground stover, after physiological maturity can reduce the intense logistics challenges typically associated with corn harvest and expand the harvest window. To determine the feasibility of the proposed system, corn was harvested at 65 to 16% whole-plant moisture (w.b.) using a forage harvester and then ensiled in pilot scale silos. Ambient pretreatment during storage was investigated using both dilute acid and lime. Both pretreated and control whole-plant silages were well conserved during anaerobic storage with dry matter (DM) losses generally less than 4%. Hydrodynamic separation of the grain and stover fractions after storage was found to be more effective at fractionating starch and fiber than conventional dry grain harvest, and both fractions had desirable composition. Pretreatment at storage with sulfuric acid significantly enhanced stover cell wall enzymatic degradability and subsequent fermentation to ethanol, increasing the cellulose conversion efficiency by 19, 11, and 4 percentage units for sulfuric acid pretreatments of 100, 30, and 10 g(kg DM)-1, respectively. The whole-plant harvest and storage system shows promise as a viable alternative to conventional corn grain and stover systems for producing feedstocks for biochemical conversion.