Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Discovery of a seventh Rpp soybean rust resistance locus in soybean accession PI 605823.

KEY MESSAGE: A novel Rpp gene from PI 605823 for resistance to Phakopsora pachyrhizi was mapped on chromosome 19. Soybean rust, caused by the obligate biotrophic fungal pathogen Phakopsora pachyrhizi Syd. & P. Syd, is a disease threat to soybean production in regions of the world with mild winters. Host plant resistance conditioned by resistance to P. pachyrhizi (Rpp) genes has been found in numerous soybean accessions, and at least 10 Rpp genes or alleles have been mapped to six genetic loci. Identifying additional disease-resistance genes will facilitate development of soybean cultivars with durable resistance. PI 605823, a plant introduction from Vietnam, was previously identified as resistant to US populations of P. pachyrhizi in greenhouse and field trials. In this study, bulked segregant analysis using an F2 population derived from 'Williams 82' × PI 605823 identified a genomic region associated with resistance to P. pachyrhizi isolate GA12, which had been collected in the US State of Georgia in 2012. To further map the resistance locus, linkage mapping was carried out using single-nucleotide polymorphism markers and phenotypic data from greenhouse assays with an F2:3 population derived from Williams 82 × PI 605823 and an F4:5 population derived from '5601T' × PI 605823. A novel resistance gene, Rpp7, was mapped to a 154-kb interval (Gm19: 39,462,291-39,616,643 Glyma.Wm82.a2) on chromosome 19 that is different from the genomic locations of any previously reported Rpp genes. This new gene could be incorporated into elite breeding lines to help provide more durable resistance to soybean rust.

What you see and what you are told: an action-specific effect that is unaffected by explicit feedback.

A critical question for theories of spatial vision concerns the nature of the inputs to perception. The action-specific account asserts that information related to action, specifically a perceiver's ability to perform the intended action, is one of these sources of information. This claim challenges assumptions about the mind in general and perception in particular, and not surprisingly, has been met with much resistance. Alternative explanations include that these effects are due to response bias, rather than genuine differences in perception. Using a paradigm in which ease to block a ball impacts estimated speed of the ball, participants were given explicit feedback about their perceptual judgements to test the response bias alternative. Despite the feedback, the action-specific effect still persisted, thus ruling out a response-bias interpretation. Coupled with other research ruling out additional alternative explanations, the current findings offer an important step towards the claim that a person's ability to act truly influences spatial perception.

A novel Phakopsora pachyrhizi resistance allele (Rpp) contributed by PI 567068A.

KEY MESSAGE: The Rpp6 locus of PI 567102B was mapped from 5,953,237 to 5,998,461 bp (chromosome 18); and a novel allele at the Rpp6 locus or tightly linked gene Rpp[PI567068A] of PI 567068A was mapped from 5,998,461 to 6,160,481 bp. Soybean rust (SBR), caused by the obligate, fungal pathogen Phakopsora pachyrhizi is an economic threat to soybean production, especially in the Americas. Host plant resistance is an important management strategy for SBR. The most recently described resistance to P. pachyrhizi (Rpp) gene is Rpp6 contributed by PI 567102B. Rpp6 was previously mapped to an interval of over four million base pairs on chromosome 18. PI 567068A was recently demonstrated to possess a resistance gene near the Rpp6 locus, yet PI 567068A gave a differential isolate reaction to several international isolates of P. pachyrhizi. The goals of this research were to fine map the Rpp6 locus of PI 567102B and PI 567068A and determine whether or not PI 567068A harbors a novel Rpp6 allele or another allele at a tightly linked resistance locus. Linkage mapping in this study mapped Rpp6 from 5,953,237 to 5,998,461 bp (LOD score of 58.3) and the resistance from PI 567068A from 5,998,461 to 6,160,481 bp (LOD score of 4.4) (Wm82.a1 genome sequence). QTL peaks were 139,033 bp apart from one another as determined by the most significant SNPs in QTL mapping. The results of haplotype analysis demonstrated that PI 567102B and PI 567068A share the same haplotype in the resistance locus containing both Rpp alleles, which was designated as the Rpp6/Rpp[PI567068A] haplotype. The Rpp6/Rpp[PI567068A] haplotype identified in this study can be used as a tool to rapidly screen other genotypes that possess a Rpp gene(s) and detect resistance at the Rpp6 locus in diverse germplasm.

An action-specific effect on perception that avoids all pitfalls.

The visual system is influenced by action. Objects that are easier to reach or catch look closer and slower, respectively. Here, we describe evidence for one action-specific effect, and show that none of the six pitfalls can account for the results. Vision is not an isolate module, as shown by this top-down effect of action on perception.

Fine mapping of the Asian soybean rust resistance gene Rpp2 from soybean PI 230970.

KEY MESSAGE: Asian soybean rust (ASR) resistance gene Rpp2 has been fine mapped into a 188.1 kb interval on Glyma.Wm82.a2, which contains a series of plant resistance ( R ) genes. Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrihizi Syd. & P. Syd., is a serious disease in major soybean [Glycine max (L.) Merr.] production countries worldwide and causes yield losses up to 75 %. Defining the exact chromosomal position of ASR resistance genes is critical for improving the effectiveness of marker-assisted selection (MAS) for resistance and for cloning these genes. The objective of this study was to fine map the ASR resistance gene Rpp2 from the plant introduction (PI) 230970. Rpp2 was previously mapped within a 12.9-cM interval on soybean chromosome 16. The fine mapping was initiated by identifying recombination events in F2 and F3 plants using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers that flank the gene. Seventeen recombinant plants were identified and then tested with additional genetic markers saturating the gene region to localize the positions of each recombination. The progeny of these selected plants were tested for resistance to ASR and with SSR markers resulting in the mapping of Rpp2 to a 188.1 kb interval on the Williams 82 reference genome (Glyma.Wm82.a2). Twelve genes including ten toll/interleukin-1 receptor (TIR)-nucleotide-binding site (NBS)-leucine-rich repeat (LRR) genes were predicted to exist in this interval on the Glyma.Wm82.a2.v1 gene model map. Eight of these ten genes were homologous to the Arabidopsis TIR-NBS-LRR gene AT5G17680.1. The identified SSR and SNP markers close to Rpp2 and the candidate gene information presented in this study will be significant resources for MAS and gene cloning research.

Biolistic transformation of elite genotypes of switchgrass (Panicum virgatum L.).

Transformation of elite switchgrass (Panicum virgatum L.) genotypes would facilitate the characterization of genes related to cell wall recalcitrance to saccharification. However, transformation of explants from switchgrass plants has remained difficult. Therefore, the objective of this study was to develop a biolistic transformation protocol for elite genotypes. Three switchgrass genotypes (ST1, ST2, and AL2) were previously selected for tissue culture responsiveness. One genotype, SA37, was selected for further use due to its improved formation of callus amenable to transformation. Various medium sets were compared and a previously published medium set provided cultures with >96 % embryogenic callus, and data on transient and stable gene expression of RFP were used to optimize biolistic parameters, and further validate the switchgrass (PvUbi1) promoter. SA37 proved to be the most transformable, whereas eight transgenic calli on average were recovered per bombardment of 20 calli (40 % efficiency) when using a three-day day preculture step, 0.6 M osmotic adjustment medium, 4,482 kPa rupture disks and 0.4 µm gold particles which traveled 9 cm before hitting the target callus tissue. Regenerability was high, especially for ST2, for which it is possible to recover on average over 400 plants per half-gram callus tissue. It is now possible to routinely and efficiently engineer elite switchgrass genotypes using biolistic transformation.

Gateway-compatible vectors for high-throughput gene functional analysis in switchgrass (Panicum virgatum L.) and other monocot species.

Switchgrass (Panicum virgatum L.) is a C4 perennial grass and has been identified as a potential bioenergy crop for cellulosic ethanol because of its rapid growth rate, nutrient use efficiency and widespread distribution throughout North America. The improvement of bioenergy feedstocks is needed to make cellulosic ethanol economically feasible, and genetic engineering of switchgrass is a promising approach towards this goal. A crucial component of creating transgenic switchgrass is having the capability of transforming the explants with DNA sequences of interest using vector constructs. However, there are limited options with the monocot plant vectors currently available. With this in mind, a versatile set of Gateway-compatible destination vectors (termed pANIC) was constructed to be used in monocot plants for transgenic crop improvement. The pANIC vectors can be used for transgene overexpression or RNAi-mediated gene suppression. The pANIC vector set includes vectors that can be utilized for particle bombardment or Agrobacterium-mediated transformation. All the vectors contain (i) a Gateway cassette for overexpression or silencing of the target sequence, (ii) a plant selection cassette and (iii) a visual reporter cassette. The pANIC vector set was functionally validated in switchgrass and rice and allows for high-throughput screening of sequences of interest in other monocot species as well.

Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation.

BACKGROUND: The ubiquitin protein is present in all eukaryotic cells and promoters from ubiquitin genes are good candidates to regulate the constitutive expression of transgenes in plants. Therefore, two switchgrass (Panicum virgatum L.) ubiquitin genes (PvUbi1 and PvUbi2) were cloned and characterized. Reporter constructs were produced containing the isolated 5' upstream regulatory regions of the coding sequences (i.e. PvUbi1 and PvUbi2 promoters) fused to the uidA coding region (GUS) and tested for transient and stable expression in a variety of plant species and tissues. RESULTS: PvUbi1 consists of 607 bp containing cis-acting regulatory elements, a 5' untranslated region (UTR) containing a 93 bp non-coding exon and a 1291 bp intron, and a 918 bp open reading frame (ORF) that encodes four tandem, head -to-tail ubiquitin monomer repeats followed by a 191 bp 3' UTR. PvUbi2 consists of 692 bp containing cis-acting regulatory elements, a 5' UTR containing a 97 bp non-coding exon and a 1072 bp intron, a 1146 bp ORF that encodes five tandem ubiquitin monomer repeats and a 183 bp 3' UTR. PvUbi1 and PvUbi2 were expressed in all examined switchgrass tissues as measured by qRT-PCR. Using biolistic bombardment, PvUbi1 and PvUbi2 promoters showed strong expression in switchgrass and rice callus, equaling or surpassing the expression levels of the CaMV 35S, 2x35S, ZmUbi1, and OsAct1 promoters. GUS staining following stable transformation in rice demonstrated that the PvUbi1 and PvUbi2 promoters drove expression in all examined tissues. When stably transformed into tobacco (Nicotiana tabacum), the PvUbi2+3 and PvUbi2+9 promoter fusion variants showed expression in vascular and reproductive tissues. CONCLUSIONS: The PvUbi1 and PvUbi2 promoters drive expression in switchgrass, rice and tobacco and are strong constitutive promoter candidates that will be useful in genetic transformation of monocots and dicots.

Bacterial communities associated with tuberculate ectomycorrhizae of Rhizopogon spp.

We have previously reported the design of a new PCR primer pair that allows amplification of a broad range of eubacterial 16S rDNA sequences from ectomycorrhizae (ECM) without co-amplification of plastid or mitochondrial sequences. Here, we report using a similar primer combination to generate three small 16S rDNA libraries from tuberculate ECM of Rhizopogon spp., two from R. vinicolor ECM (libraries Rvi18 and Rvi24) and one from R. vesiculosus ECM (library Rve13). At the class level, libraries were dominated by sequences from the Alphaproteobacteria, Gammaproteobacteria, and Acidobacteria, with some Sphingobacteria, Actinobacteria, Planctomycetacia, and Verrucomicrobiae present as well. Based on the parsimony test implemented in TreeClimber, libraries Rvi18 and Rvi24 were significantly different from Rve13 at the alpha = 0.05 level, while they were only borderline significantly different from each other (p = 0.07). Differences between Rvi and Rve libraries were primarily due to differences in the number of Alphaproteobacteria sequences and specifically sequences from the Rhizobiales, which were more common in the Rve13 library. It is currently unknown what drives these differences between eubacterial communities. Amplification success for eubacterial 16S rDNA sequences was generally low in this study indicating low abundance of bacteria on tuberculate ECM. Attempts to amplify nitrogenase reductase (nifH) sequences were unsuccessful.