A dense linkage map of hybrid cottonwood (Populus fremontii x P. angustifolia) contributes to long-term ecological research and comparison mapping in a model forest tree

Cottonwoods are foundation riparian species, and hybridization among species is known to produce ecological effects at levels higher than the population, including effects on dependent species, communities and ecosystems. Because these patterns result from increased genetic variation in key cottonwood traits, novel applications of genetic tools (for example, QTL mapping) could be used to place broad-scale ecological research into a genomic perspective. In addition, linkage maps have been produced for numerous species within the genus, and, coupled with the recent publication of the Populus genome sequence, these maps present a unique opportunity for genome comparisons in a model system. Here, we conducted linkage analyses in order to (1) create a platform for QTL and candidate gene studies of ecologically important traits, (2) create a framework for chromosomal-scale perspectives of introgression in a natural population, and (3) enhance genome-wide comparisons using two previously unmapped species. We produced 246 backcross mapping (BC(1)) progeny by crossing a naturally occurring F(1) hybrid (Populus fremontii x P. angustifolia) to a pure P. angustifolia from the same population. Linkage analysis resulted in a dense linkage map of 541 AFLP and 111 SSR markers distributed across 19 linkage groups. These results compared favorably with other Populus linkage studies, and addition of SSR loci from the poplar genome project provided coarse alignment with the genome sequence. Preliminary applications of the data suggest that our map represents a useful framework for applying genomic research to ecological questions in a well-studied system, and has enhanced genome-wide comparisons in a model tree.     

Herbivory differentially alters plant litter dynamics of evergreen and deciduous trees

Here we propose that herbivore-induced changes in leaf litter quality can modify aboveground litterfall dynamics differentially in evergreen and deciduous trees. Because aboveground plant litterfall is an important source of nutrients in terrestrial ecosystems, any factor that alters plant litter quality can have large "afterlife" effects on the decomposition rate of that litter and the subsequent rate of nutrient release. Two contrasting patterns emerge from the literature and are corroborated by our two experimental case studies. First, in evergreens, herbivory commonly results in premature leaf abscission, improved litter "quality" and an acceleration of litter decomposition. Second, in deciduous trees, herbivory commonly results in the induction of secondary compounds that decelerates decomposition. We argue that these broad patterns reflect predictable differential responses to herbivores that can have important consequences for terrestrial nutrient cycling and productivity and that warrant more attention in the literature.     

Prediction of the in planta Phakopsora pachyrhizi secretome and potential effector families

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales‐ or P. pachyrhizi‐specific families (tribes). Members of some families were strongly up‐regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.     

Temporal and spatial Bean pod mottle virus‐induced gene silencing in soybean

Virus‐induced gene silencing (VIGS) is a powerful reverse genetics tool in plant science. In this study, we investigated the temporal and spatial silencing patterns achieved by Bean pod mottle virus (BPMV)‐based VIGS in soybean using virus constructs targeting green fluorescence protein (GFP). Silencing GFP enabled an in‐depth analysis of silencing in soybean tissues over time in a transgenic line constitutively expressing GFP. We discovered evidence for variable GFP silencing based on insert orientation and targeted region in the coding sequence. A 3′ sequence in reverse orientation produced the strongest silencing phenotypes. Furthermore, we documented that BPMV VIGS can achieve widespread silencing in a broad range of tissues, including leaves, stems, flowers and roots. Near‐complete silencing was attained in leaves and flowers. Although weaker than in shoots, the observed gene silencing in soybean roots will also allow reverse genetics studies in this tissue. When GFP fluorescence was assayed in cross‐sections of stems and leaf petioles, near‐complete and uniform silencing was observed in all cell types. Silencing was observed from as early as 2 weeks post‐virus inoculation in leaves to 7 weeks post‐virus inoculation in flowers, suggesting that this system can induce and maintain silencing for significant durations.     

Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) can be induced in SJL/J mice by immunization with spinal cord homogenate and adjuvant. The specific Ag(s) responsible for acute disease and subsequent relapses in this model is unknown. Myelin basic protein (BP), an encephalitogenic peptide of BP (BP 87-99), and proteolipid protein (PLP) can each induce R-EAE in SJL/J mice, and a peptide of PLP (PLP 139-151) has been reported to induce acute EAE. To determine the encephalitogens in cord-immunized mice with R-EAE, the in vitro proliferative responses of lymph node cells (LNC) and central nervous system mononuclear cells to BP, BP peptides, and PLP peptides were examined during acute EAE and during relapses. LNC responded only to PLP peptides 139-151 and 141-151 and did not respond to BP or its peptides during acute or chronic disease. Central nervous system mononuclear cells also preferentially responded to PLP 139-151 and 141-151 during acute and relapsing disease. A PLP 139-151 peptide-specific Th cell line was selected from LNC of cord-immunized donors. Five million peptide-specific line cells transferred severe relapsing demyelinating EAE to naive recipients. We conclude that PLP peptide 139-151 is the major encephalitogen for R-EAE in cord-immunized SJL/J mice. We demonstrate for the first time that Th cells specific for this peptide are sufficient to transfer relapsing demyelinating EAE. The predominance of a PLP immune response rather than a BP response in SJL/J mice suggests that genetic background may determine the predominant myelin Ag response in human demyelinating diseases such as multiple sclerosis.     

Discrimination among pinyon pine trees by Clark's Nutcrackers: effects of cone crop size and cone characters

Summary The influences of Colorado pinyon pine (Pinus edulis) cone crop size, cone and seed weight, cone length, number of seeds per cone, number of viable seeds, and percent viable seeds on the foraging behavior of avian seed dispersal agents were examined in field and laboratory settings. In the field, there was a significant positive relationship between cone number per tree and both the absolute number of cones and the percentage of the cone crop from which seeds were harvested. Cone weight and the number of viable seeds were also significantly related to seed harvest intensity. Laboratory experiments examined the relationship between crop size and cone characters on seed harvest by 18 Clark's Nutcrackers (Nucifraga columbiana). Nutcrackers were offered a choice of two tree types: one with 20 cones attached, and another with 10 cones attached. Significantly more birds chose to remove seeds first from the tree with 20 cones than the tree with 10 cones. In timed trials, they also harvested seeds from significantly more cones on the tree with the higher cone density. In the laboratory, cones chosen for seed removal by the nutcrackers had significantly more viable seeds, more seeds, and were longer compared to cones that were not chosen. Such discriminatory foraging behavior may increase avian foraging efficiency and result in differential reproductive success of pinyon pines. This behavior may therefore influence the evolution of pinyon pine reproductive traits.     

Genetically based trait in a dominant tree affects ecosystem processes

Fundamental links between genes and ecosystem processes have remained elusive, although they have the potential to place ecosystem sciences within a genetic and evolutionary framework. Utilizing common gardens with cottonwood trees of known genotype, we found that the concentration of condensed tannins is genetically based and is the best predictor of ecosystem‐level processes. Condensed tannin inputs from foliage explained 55–65% of the variation in soil net nitrogen (N) mineralization under both field and laboratory conditions. Alternative associations with litter lignin, soil moisture or soil temperature were relatively poor predictors of litter decomposition and net N mineralization. In contrast to the paradigm that the effects of genes are too diffuse to be important at the ecosystem‐level, here we show that plant genes had strong, immediate effects on ecosystem function via a tight coupling of plant polyphenols to rates of nitrogen cycling.     

Transforming growth factor-beta enhances the in vivo effector function and memory phenotype of antigen-specific T helper cells in experimental autoimmune encephalomyelitis.

Transforming growth factor-beta (TGF-beta) had a profound effect on the in vitro phenotypic development of Ag-activated Th cells and enhanced the in vivo effector function of these cells upon adoptive transfer. Previous studies have shown that there are two types of Th cell populations found in unimmunized animals, naive helper cells, which are short-lived and express low levels of CD44 and high levels of CD45R and Mel-14, and memory helper cells, which have a long life span and express high levels of CD44 and low levels of CD45R and Mel-14. Culturing of Ag-specific murine Th cell lines and clones in the presence of TGF-beta greatly enhanced both the memory phenotype of the cultured cells and the effector function upon adoptive transfer in experimental autoimmune encephalomyelitis. Histologic evaluation of spinal cords from recipients receiving passively transferred murine T cell lines cultured with TGF-beta revealed large demyelinated plaques (multiple sclerosis-like) that were not present in animals receiving cells cultured with Ag alone. TGF-beta also enhanced the capability of myelin basic protein-specific Lewis rat T cell lines to transfer experimental autoimmune encephalomyelitis and potentiated a purified protein derivative-specific rat helper cell line to transfer delayed type hypersensitivity. Thus, the effects of TGF-beta did not appear to be limited by species specificity, Ag specificity, or in vivo T cell function. This is the first study showing that TGF-beta can potentiate the development and maintenance of the Th cell memory phenotype in vitro and enhance their in vivo effector function in an animal disease model.     

Chronic herbivory negatively impacts cone and seed production,seed quality and seedling growth of susceptible pinyon pines

Although herbivory often reduces the reproduction of attacked trees, few studies have examined how naturally occurring insect-resistant and susceptible trees differ in their reproduction, nor have these effects been experimentally examined through long-term herbivore removals. In addition, few studies have examined the effects of herbivory on the quality of seeds produced and the implications of reduced seed quality on seedling establishment. We evaluated the impact of chronic herbivory by the stem-boring moth, Dioryctria albovittella, on cone and seed production of the pinyon pine (Pinus edulis) during two mast years. Three patterns emerged. First, moth herbivory was associated with reductions in cone production, viable seed production and seed mass. Specifically, pinyons susceptible to moth attack had 93–95% lower cone production, and surviving cones produced 31–37% fewer viable seeds, resulting in a 96–97% reduction in whole tree viable seed production. In addition, surviving seeds from susceptible trees had 18% lower mass than resistant trees. Second, long-term experimental removal of the herbivore resulted in increased rates of cone and seed production and quality, indicating that moth herbivory was the driver of these reductions. Third, seed size was positively associated with seed germination and seedling biomass and height, suggesting that trees suffering chronic herbivory produce poorer quality offspring. Thus, the resistance traits of pinyons can affect the quality of offspring, which in turn may affect subsequent seedling establishment and population dynamics.