Opposing functions of the plant TOPLESS gene family during SNC1-mediated autoimmunity.

Regulation of the plant immune system is important for controlling the specificity and amplitude of responses to pathogens and in preventing growth-inhibiting autoimmunity that leads to reductions in plant fitness. In previous work, we reported that SRFR1, a negative regulator of effector-triggered immunity, interacts with SNC1 and EDS1. When SRFR1 is non-functional in the Arabidopsis accession Col-0, SNC1 levels increase, causing a cascade of events that lead to autoimmunity phenotypes. Previous work showed that some members of the transcriptional co-repressor family TOPLESS interact with SNC1 to repress negative regulators of immunity. Therefore, to explore potential connections between SRFR1 and TOPLESS family members, we took a genetic approach that examined the effect of each TOPLESS member in the srfr1 mutant background. The data indicated that an additive genetic interaction exists between SRFR1 and two members of the TOPLESS family, TPR2 and TPR3, as demonstrated by increased stunting and elevated PR2 expression in srfr1 tpr2 and srfr1 tpr2 tpr3 mutants. Furthermore, the tpr2 mutation intensifies autoimmunity in the auto-active snc1-1 mutant, indicating a novel role of these TOPLESS family members in negatively regulating SNC1-dependent phenotypes. This negative regulation can also be reversed by overexpressing TPR2 in the srfr1 tpr2 background. Similar to TPR1 that positively regulates snc1-1 phenotypes by interacting with SNC1, we show here that TPR2 directly binds the N-terminal domain of SNC1. In addition, TPR2 interacts with TPR1 in vivo, suggesting that the opposite functions of TPR2 and TPR1 are based on titration of SNC1-TPR1 complexes by TPR2 or altered functions of a SNC1-TPR1-TPR2 complex. Thus, this work uncovers diverse functions of individual members of the TOPLESS family in Arabidopsis and provides evidence for the additive effect of transcriptional and post-transcriptional regulation of SNC1.

Class I TCP transcription factor AtTCP8 modulates key brassinosteroid-responsive genes.

The plant-specific TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factor family is most closely associated with regulating plant developmental programs. Recently, TCPs were also shown to mediate host immune signaling, both as targets of pathogen virulence factors and as regulators of plant defense genes. However, comprehensive characterization of TCP gene targets is still lacking. Loss of function of the class I TCP gene AtTCP8 attenuates early immune signaling and, when combined with mutations in AtTCP14 and AtTCP15, additional layers of defense signaling in Arabidopsis (Arabidopsis thaliana). Here, we focus on TCP8, the most poorly characterized of the three to date. We used chromatin immunoprecipitation and RNA sequencing to identify TCP8-bound gene promoters and differentially regulated genes in the tcp8 mutant; these datasets were heavily enriched in signaling components for multiple phytohormone pathways, including brassinosteroids (BRs), auxin, and jasmonic acid. Using BR signaling as a representative example, we showed that TCP8 directly binds and activates the promoters of the key BR transcriptional regulatory genes BRASSINAZOLE-RESISTANT1 (BZR1) and BRASSINAZOLE-RESISTANT2 (BZR2/BES1). Furthermore, tcp8 mutant seedlings exhibited altered BR-responsive growth patterns and complementary reductions in BZR2 transcript levels, while TCP8 protein demonstrated BR-responsive changes in subnuclear localization and transcriptional activity. We conclude that one explanation for the substantial targeting of TCP8 alongside other TCP family members by pathogen effectors may lie in its role as a modulator of BR and other plant hormone signaling pathways.

Chemical Diversity, Biological Activity, and Genetic Aspects of Three Ocotea Species from the Amazon.

Ocotea species present economic importance and biological activities attributed to their essential oils (EOs) and extracts. For this reason, various strategies have been developed for their conservation. The chemical compositions of the essential oils and matK DNA sequences of O. caudata, O. cujumary, and O. caniculata were subjected to comparison with data from O. floribunda, O. veraguensis, and O. whitei, previously reported. The multivariate analysis of chemical composition classified the EOs into two main clusters. Group I was characterized by the presence of α-pinene (9.8-22.5%) and ß-pinene (9.7-21.3%) and it includes O. caudata, O. whitei, and O. floribunda. In group II, the oils of O. cujumary and O. caniculata showed high similarity due amounts of ß-caryophyllene (22.2% and 18.9%, respectively). The EO of O. veraguensis, rich in p-cymene (19.8%), showed minor similarity among all samples. The oils displayed promising antimicrobial and cytotoxic activities against Escherichia coli (minimum inhibitory concentration (MIC) < 19.5 µg·mL-1) and MCF-7 cells (median inhibitory concentration (IC50) ≅ 65.0 µg·mL-1), respectively. The analysis of matK gene displayed a good correlation with the main class of chemical compounds present in the EOs. However, the matK gene data did not show correlation with specific compounds.

Genetic manipulation of putrescine biosynthesis reprograms the cellular transcriptome and the metabolome.

BACKGROUND: With the increasing interest in metabolic engineering of plants using genetic manipulation and gene editing technologies to enhance growth, nutritional value and environmental adaptation, a major concern is the potential of undesirable broad and distant effects of manipulating the target gene or metabolic step in the resulting plant. A comprehensive transcriptomic and metabolomic analysis of the product may shed some useful light in this regard. The present study used these two techniques with plant cell cultures to analyze the effects of genetic manipulation of a single step in the biosynthesis of polyamines because of their well-known roles in plant growth, development and stress responses. RESULTS: The transcriptomes and metabolomes of a control and a high putrescine (HP) producing cell line of poplar (Populus nigra x maximowiczii) were compared using microarrays and GC/MS. The HP cells expressed an ornithine decarboxylase transgene and accumulated several-fold higher concentrations of putrescine, with only small changes in spermidine and spermine. The results show that up-regulation of a single step in the polyamine biosynthetic pathway (i.e. ornithine → putrescine) altered the expression of a broad spectrum of genes; many of which were involved in transcription, translation, membrane transport, osmoregulation, shock/stress/wounding, and cell wall metabolism. More than half of the 200 detected metabolites were significantly altered (p ≤ 0.05) in the HP cells irrespective of sampling date. The most noteworthy differences were in organic acids, carbohydrates and nitrogen-containing metabolites. CONCLUSIONS: The results provide valuable information about the role of polyamines in regulating nitrogen and carbon use pathways in cell cultures of high putrescine producing transgenic cells of poplar vs. their low putrescine counterparts. The results underscore the complexity of cellular responses to genetic perturbation of a single metabolic step related to nitrogen metabolism in plants. Combined with recent studies from our lab, where we showed that higher putrescine production caused an increased flux of glutamate into ornithine concurrent with enhancement in glutamate production via additional nitrogen and carbon assimilation, the results from this study provide guidance in designing transgenic plants with increased nitrogen use efficiency, especially in plants intended for non-food/feed applications (e.g. increased biomass production for biofuels).

Global agricultural intensification during climate change: a role for genomics.

Agriculture is now facing the 'perfect storm' of climate change, increasing costs of fertilizer and rising food demands from a larger and wealthier human population. These factors point to a global food deficit unless the efficiency and resilience of crop production is increased. The intensification of agriculture has focused on improving production under optimized conditions, with significant agronomic inputs. Furthermore, the intensive cultivation of a limited number of crops has drastically narrowed the number of plant species humans rely on. A new agricultural paradigm is required, reducing dependence on high inputs and increasing crop diversity, yield stability and environmental resilience. Genomics offers unprecedented opportunities to increase crop yield, quality and stability of production through advanced breeding strategies, enhancing the resilience of major crops to climate variability, and increasing the productivity and range of minor crops to diversify the food supply. Here we review the state of the art of genomic-assisted breeding for the most important staples that feed the world, and how to use and adapt such genomic tools to accelerate development of both major and minor crops with desired traits that enhance adaptation to, or mitigate the effects of climate change.

Modeling forest ecosystem responses to elevated carbon dioxide and ozone using artificial neural networks.

Rising atmospheric levels of carbon dioxide and ozone will impact productivity and carbon sequestration in forest ecosystems. The scale of this process and the potential economic consequences provide an incentive for the development of models to predict the types and rates of ecosystem responses and feedbacks that result from and influence of climate change. In this paper, we use phenotypic and molecular data derived from the Aspen Free Air CO2 Enrichment site (Aspen-FACE) to evaluate modeling approaches for ecosystem responses to changing conditions. At FACE, it was observed that different aspen clones exhibit clone-specific responses to elevated atmospheric levels of carbon dioxide and ozone. To identify the molecular basis for these observations, we used artificial neural networks (ANN) to examine above and below-ground community phenotype responses to elevated carbon dioxide, elevated ozone and gene expression profiles. The aspen community models generated using this approach identified specific genes and subnetworks of genes associated with variable sensitivities for aspen clones. The ANN model also predicts specific co-regulated gene clusters associated with differential sensitivity to elevated carbon dioxide and ozone in aspen species. The results suggest ANN is an effective approach to predict relevant gene expression changes resulting from environmental perturbation and provides useful information for the rational design of future biological experiments.

Cell-specific polymerization-driven biomolecular condensate formation fine-tunes root tissue morphogenesis.

Formation of biomolecular condensates can be driven by weak multivalent interactions and emergent polymerization. However, the mechanism of polymerization-mediated condensate formation is less studied. We found lateral root cap cell (LRC)-specific SUPPRESSOR OF RPS4-RLD1 (SRFR1) condensates fine-tune primary root development. Polymerization of the SRFR1 N-terminal domain is required for both LRC condensate formation and optimal root growth. Surprisingly, the first intrinsically disordered region (IDR1) of SRFR1 can be functionally substituted by a specific group of intrinsically disordered proteins known as dehydrins. This finding facilitated the identification of functional segments in the IDR1 of SRFR1, a generalizable strategy to decode unknown IDRs. With this functional information we further improved root growth by modifying the SRFR1 condensation module, providing a strategy to improve plant growth and resilience.

A splicing variant of EDS1 from Vitis vinifera forms homodimers but no heterodimers with PAD4.

Enhanced Disease Susceptibility 1 (EDS1), a key component of microbe-triggered immunity and effector-triggered immunity in most higher plants, forms functional heterodimeric complexes with its homologs Phytoalexin Deficient 4 (PAD4) or Senescence-associated Gene 101 (SAG101). Here, the crystal structure of VvEDS1Nterm , the N-terminal domain of EDS1 from Vitis vinifera, is reported, representing the first structure of an EDS1 entity beyond the model plant Arabidopsis thaliana. VvEDS1Nterm has an α/ß-hydrolase fold, is similar to the N-terminal domain of A. thaliana EDS1 and forms stable homodimers in solution as well as in crystals. These VvEDS1Nterm homodimers are spatially incompatible with heterodimers with PAD4 or SAG101, they explain why VvEDS1Nterm does not interact with V. vinifera PAD4 according to gel filtration, and they serve as a guide to develop a plausible, albeit experimentally not verified model of full-length EDS1. VvEDS1Nterm is a splicing variant comprising two of three exons of the VvEDS1 gene. It originates from a naturally occurring mRNA, in which the first of two introns was removed while the second one containing a stop codon close to the exon/intron border was retained. This is a potential case of intron retention and the first report of this phenomenon in the context of EDS1. Its biological significance has not yet been clarified, nor has the question if a VvEDS1Nterm protein with a specific function can occur under physiological conditions.

Efficient CRISPR-Cas9 based cytosine base editors for phytopathogenic bacteria.

Phytopathogenic bacteria play important roles in plant productivity, and developments in gene editing have potential for enhancing the genetic tools for the identification of critical genes in the pathogenesis process. CRISPR-based genome editing variants have been developed for a wide range of applications in eukaryotes and prokaryotes. However, the unique mechanisms of different hosts restrict the wide adaptation for specific applications. Here, CRISPR-dCas9 (dead Cas9) and nCas9 (Cas9 nickase) deaminase vectors were developed for a broad range of phytopathogenic bacteria. A gene for a dCas9 or nCas9, cytosine deaminase CDA1, and glycosylase inhibitor fusion protein (cytosine base editor, or CBE) was applied to base editing under the control of different promoters. Results showed that the RecA promoter led to nearly 100% modification of the target region. When residing on the broad host range plasmid pHM1, CBERecAp is efficient in creating base edits in strains of Xanthomonas, Pseudomonas, Erwinia and Agrobacterium. CBE based on nCas9 extended the editing window and produced a significantly higher editing rate in Pseudomonas. Strains with nonsynonymous mutations in test genes displayed expected phenotypes. By multiplexing guide RNA genes, the vectors can modify up to four genes in a single round of editing. Whole-genome sequencing of base-edited isolates of Xanthomonas oryzae pv. oryzae revealed guide RNA-independent off-target mutations. Further modifications of the CBE, using a CDA1 variant (CBERecAp-A) reduced off-target effects, providing an improved editing tool for a broad group of phytopathogenic bacteria.

Transcriptomic comparison in the leaves of two aspen genotypes having similar carbon assimilation rates but different partitioning patterns under elevated [CO2].

This study compared the leaf transcription profiles, physiological characteristics and primary metabolites of two Populus tremuloides genotypes (clones 216 and 271) known to differ in their responses to long-term elevated [CO2] (e[CO2]) at the Aspen free-air CO2 enrichment site near Rhinelander, WI, USA. The physiological responses of these clones were similar in terms of photosynthesis, stomatal conductance and leaf area index under e[CO2], yet very different in terms of growth enhancement (0-10% in clone 216; 40-50% in clone 271). Although few genes responded to long-term exposure to e[CO2], the transcriptional activity of leaf e[CO2]-responsive genes was distinctly different between the clones, differentially impacting multiple pathways during both early and late growing seasons. An analysis of transcript abundance and carbon/nitrogen biochemistry suggested that the CO2-responsive clone (271) partitions carbon into pathways associated with active defense/response to stress, carbohydrate/starch biosynthesis and subsequent growth. The CO2-unresponsive clone (216) partitions carbon into pathways associated with passive defense (e.g. lignin, phenylpropanoid) and cell wall thickening. This study indicates that there is significant variation in expression patterns between different tree genotypes in response to long-term exposure to e[CO2]. Consequently, future efforts to improve productivity or other advantageous traits for carbon sequestration should include an examination of genetic variability in CO2 responsiveness.

Identification of PTM5 protein interaction partners, a MADS-box gene involved in aspen tree vegetative development.

In a past article, our lab described the identification and characterization of a novel vegetative MADS-box gene from quaking aspen trees, Populus tremuloides MADS-box 5 (PTM5). PTM5 was shown to be a member of the SOC1/TM3 class of MADS-box genes with a seasonal expression pattern specific to developing vascular tissues including the vascular cambium, the precursor to all woody branches, stems, and roots. Since the proper function of MADS-box proteins is dependent on specific interactions with other regulatory proteins, we further examined PTM5 protein-protein interactions as a means to better understand its function. Through yeast two-hybrid analyses, it was demonstrated that, like other SOC1/TM3 class proteins, PTM5 is capable of interacting with itself as well as other MADS-box proteins from aspen. In addition, yeast two-hybrid library screening revealed that PTM5 interacts with two non-MADS proteins, an actin depolymerizing factor (PtADF) and a novel leucine-rich repeat protein (PtLRR). In situ RNA localization was used to verify the overlapping expression patterns of these genes, and transgenic studies showed that over-expression of PTM5 in aspen causes alterations in root vasculature and root biomass development consistent with the cell growth and expansion functions of related ADF and LRR genes. These results suggest that the interaction of vegetative MADS-box genes with specific protein cofactors is a key step in the mechanisms that control woody tissue development in trees.

SEP-class genes in Populus tremuloides and their likely role in reproductive survival of poplar trees.

One of the most important processes to the survival of a species is its ability to reproduce. In plants, SEPALLATA-class MADS-box genes have been found to control the development of the inner whorls of flowers. However, while much is known about floral development in herbaceous plants, similar systems in woody trees remain poorly understood. Populus tremuloides (trembling aspen) is a widespread North American tree having important economic value, and its floral development differs from that of well-studied species in that the flowers have only two whorls and are truly unisexual. Sequence based analyses indicate that PTM3 (Populus tremuloides MADS-box 3), and a duplicate gene PTM4, are related to the SEPALLATA1-and 2-class of MADS-box genes. Another gene, PTM6, is related to SEP3, and each of these genes has a counterpart in the poplar genomic database along with additional members of the A, B, C, D, and E-classes of MADS-box genes. PTM3/4 and 6 are expressed in all stages of male and female aspen floral development. However, PTM3/4 is also expressed in the terminal buds, young leaves, and young stems. In situ RNA localization identified PTM3/4 and 6 transcripts predominantly in the inner, sexual whorl, within developing ovules of female flowers and anther primordia of male flowers. Tree researchers often use heterologous systems to help study tree floral development due to the long juvenile periods found in most trees. We found that the participation of PTM3/4 in floral development is supported by transgenic experiments in both P. tremuloides and heterologous systems such as tobacco and Arabidopsis. However, phenotypic artifacts were observed in the heterologous systems. Together the results suggest a role for poplar SEP-class genes in reproductive viability.

Multi-Omics Approach Identifies Molecular Mechanisms of Plant-Fungus Mycorrhizal Interaction.

In mycorrhizal symbiosis, plant roots form close, mutually beneficial interactions with soil fungi. Before this mycorrhizal interaction can be established however, plant roots must be capable of detecting potential beneficial fungal partners and initiating the gene expression patterns necessary to begin symbiosis. To predict a plant root-mycorrhizal fungi sensor systems, we analyzed in vitro experiments of Populus tremuloides (aspen tree) and Laccaria bicolor (mycorrhizal fungi) interaction and leveraged over 200 previously published transcriptomic experimental data sets, 159 experimentally validated plant transcription factor binding motifs, and more than 120-thousand experimentally validated protein-protein interactions to generate models of pre-mycorrhizal sensor systems in aspen root. These sensor mechanisms link extracellular signaling molecules with gene regulation through a network comprised of membrane receptors, signal cascade proteins, transcription factors, and transcription factor biding DNA motifs. Modeling predicted four pre-mycorrhizal sensor complexes in aspen that interact with 15 transcription factors to regulate the expression of 1184 genes in response to extracellular signals synthesized by Laccaria. Predicted extracellular signaling molecules include common signaling molecules such as phenylpropanoids, salicylate, and jasmonic acid. This multi-omic computational modeling approach for predicting the complex sensory networks yielded specific, testable biological hypotheses for mycorrhizal interaction signaling compounds, sensor complexes, and mechanisms of gene regulation.

Application of genomics-assisted breeding for generation of climate resilient crops: progress and prospects.

Climate change affects agricultural productivity worldwide. Increased prices of food commodities are the initial indication of drastic edible yield loss, which is expected to increase further due to global warming. This situation has compelled plant scientists to develop climate change-resilient crops, which can withstand broad-spectrum stresses such as drought, heat, cold, salinity, flood, submergence and pests, thus helping to deliver increased productivity. Genomics appears to be a promising tool for deciphering the stress responsiveness of crop species with adaptation traits or in wild relatives toward identifying underlying genes, alleles or quantitative trait loci. Molecular breeding approaches have proven helpful in enhancing the stress adaptation of crop plants, and recent advances in high-throughput sequencing and phenotyping platforms have transformed molecular breeding to genomics-assisted breeding (GAB). In view of this, the present review elaborates the progress and prospects of GAB for improving climate change resilience in crops, which is likely to play an ever increasing role in the effort to ensure global food security.

Characterization of PTM5 in aspen trees: a MADS-box gene expressed during woody vascular development.

The vascular component of trees possesses some of the most specialized processes active in the formation of roots, stems, and branches, and its wood component continues to be of primary importance to our daily lives. The molecular mechanisms of wood development, however, remain poorly understood with few well-characterized regulatory genes. We have identified a vascular tissue-specific MADS-box gene, Populus tremuloides MADS-box 5 (PTM5) that is expressed in differentiating primary and secondary xylem and phloem. Phylogenetic analysis has shown that PTM5 is a member of the SOC1/TM3 class of MADS-box genes. Temporal expression analysis of PTM5 in staged vascular cambium and other tissues indicated that PTM5 expression is seasonal and is limited to spring wood formation and rapidly expanding floral catkins. Spatial expression analysis using in situ hybridization revealed that PTM5 expression is localized within a few layers of differentiating vascular cambium and xylem tissues as well as the vascular bundles of expanding catkins. Since many MADS-box genes are known to act as transcription factors, these results suggest that the coordinated expression of PTM5 with other vascular developmental genes may be a hallmark of the complex events that lead to the formation of the woody plant body.

A PCR-based genotyping method to distinguish between wild-type and ornamental varieties of Imperata cylindrica.

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas(1-2). Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety(4) (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed. Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances(5-7). JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass(8-10), and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential(3,7,11). The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

Using next generation transcriptome sequencing to predict an ectomycorrhizal metabolome.

BACKGROUND: Mycorrhizae, symbiotic interactions between soil fungi and tree roots, are ubiquitous in terrestrial ecosystems. The fungi contribute phosphorous, nitrogen and mobilized nutrients from organic matter in the soil and in return the fungus receives photosynthetically-derived carbohydrates. This union of plant and fungal metabolisms is the mycorrhizal metabolome. Understanding this symbiotic relationship at a molecular level provides important contributions to the understanding of forest ecosystems and global carbon cycling. RESULTS: We generated next generation short-read transcriptomic sequencing data from fully-formed ectomycorrhizae between Laccaria bicolor and aspen (Populus tremuloides) roots. The transcriptomic data was used to identify statistically significantly expressed gene models using a bootstrap-style approach, and these expressed genes were mapped to specific metabolic pathways. Integration of expressed genes that code for metabolic enzymes and the set of expressed membrane transporters generates a predictive model of the ectomycorrhizal metabolome. The generated model of mycorrhizal metabolome predicts that the specific compounds glycine, glutamate, and allantoin are synthesized by L. bicolor and that these compounds or their metabolites may be used for the benefit of aspen in exchange for the photosynthetically-derived sugars fructose and glucose. CONCLUSIONS: The analysis illustrates an approach to generate testable biological hypotheses to investigate the complex molecular interactions that drive ectomycorrhizal symbiosis. These models are consistent with experimental environmental data and provide insight into the molecular exchange processes for organisms in this complex ecosystem. The method used here for predicting metabolomic models of mycorrhizal systems from deep RNA sequencing data can be generalized and is broadly applicable to transcriptomic data derived from complex systems.

High efficiency poplar transformation.

With the completion of the poplar tree genome database, Populus species have become one of the most useful model systems for the study of woody plant biology. Populus tremuloides (quaking aspen) is the most wide-spread tree species in North America, and its rapid growth generates the most abundant wood-based biomass out of any other plant species. To study such beneficial traits, there is a need for easier and more efficient transformation procedures that will allow the study of large numbers of tree genes. We have developed transformation procedures that are suitable for high-throughput format transformations using either Agrobacterium tumefaciens to produce transformed trees or Agrobacterium rhizogenes to generate hairy roots. Our method uses Agrobacterium inoculated aspen seedling hypocotyls followed by direct thidiazuron (TDZ)-mediated shoot regeneration on selective media. Transformation was verified through beta-glucuronidase (GUS) reporter gene expression in all tree tissues, PCR amplification of appropriate vector products from isolated genomic DNA, and northern hybridization of incorporated and expressed transgenes. The hairy root protocol follows the same inoculation procedures and was tested using GUS reporter gene integration and antibiotic selection. The benefit of these procedures is that they are simple and efficient, requiring no maintenance of starting materials and allowing fully formed transgenic trees (or hairy roots) to be generated in only 3-4 months, rather than the 6-12 months required by more traditional methods. Likewise, the fact that the protocols are amenable to high-throughput formats makes them better suited for large-scale functional genomics studies in poplars.