Paired Medicago receptors mediate broad-spectrum resistance to nodulation by Sinorhizobium meliloti carrying a species-specific gene.

Plants have evolved the ability to distinguish between symbiotic and pathogenic microbial signals. However, potentially cooperative plant-microbe interactions often abort due to incompatible signaling. The Nodulation Specificity 1 (NS1) locus in the legume Medicago truncatula blocks tissue invasion and root nodule induction by many strains of the nitrogen-fixing symbiont Sinorhizobium meliloti. Controlling this strain-specific nodulation blockade are two genes at the NS1 locus, designated NS1a and NS1b, which encode malectin-like leucine-rich repeat receptor kinases. Expression of NS1a and NS1b is induced upon inoculation by both compatible and incompatible Sinorhizobium strains and is dependent on host perception of bacterial nodulation (Nod) factors. Both presence/absence and sequence polymorphisms of the paired receptors contribute to the evolution and functional diversification of the NS1 locus. A bacterial gene, designated rns1, is required for activation of NS1-mediated nodulation restriction. rns1 encodes a type I-secreted protein and is present in approximately 50% of the nearly 250 sequenced S. meliloti strains but not found in over 60 sequenced strains from the closely related species Sinorhizobium medicae. S. meliloti strains lacking functional rns1 are able to evade NS1-mediated nodulation blockade.

Expression and Variation of the Genes Involved in Rhizobium Nodulation in Red Clover.

Red clover (Trifolium pratense L.) is an important forage crop and serves as a major contributor of nitrogen input in pasture settings because of its ability to fix atmospheric nitrogen. During the legume-rhizobial symbiosis, the host plant undergoes a large number of gene expression changes, leading to development of root nodules that house the rhizobium bacteria as they are converted into nitrogen-fixing bacteroids. Many of the genes involved in symbiosis are conserved across legume species, while others are species-specific with little or no homology across species and likely regulate the specific plant genotype/symbiont strain interactions. Red clover has not been widely used for studying symbiotic nitrogen fixation, primarily due to its outcrossing nature, making genetic analysis rather complicated. With the addition of recent annotated genomic resources and use of RNA-seq tools, we annotated and characterized a number of genes that are expressed only in nodule forming roots. These genes include those encoding nodule-specific cysteine rich peptides (NCRs) and nodule-specific Polycystin-1, Lipoxygenase, Alpha toxic (PLAT) domain proteins (NPDs). Our results show that red clover encodes one of the highest number of NCRs and ATS3-like/NPDs, which are postulated to increase nitrogen fixation efficiency, in the Inverted-Repeat Lacking Clade (IRLC) of legumes. Knowledge of the variation and expression of these genes in red clover will provide more insights into the function of these genes in regulating legume-rhizobial symbiosis and aid in breeding of red clover genotypes with increased nitrogen fixation efficiency.

Transcriptome analysis of Epichloë strains in tall fescue in response to drought stress.

Epichloë coenophiala, a systemic fungal symbiont (endophyte) of tall fescue (Lolium arundinaceum), has been documented to confer to this grass better persistence than plants lacking the endophyte, especially under stress conditions such as drought. The response, if any, of the endophyte to imposition of stress on the host plant has not been characterized previously. Therefore, we investigated effects on gene expression by E. coenophiala and a related endophyte when plant-endophyte symbiota were subjected to acute water-deficit stress. Plants harboring different endophyte strains were grown in sand in the greenhouse, then half were deprived of water for 48 h and the other half were watered controls. RNA was isolated from different plant tissues, and mRNA sequencing (RNA-seq) was conducted to identify genes that were differentially expressed comparing stress treatment with control. We compared two different plants harboring the common toxic E. coenophiala strain (CTE) and two non-ergot-alkaloid-producing Epichloë strains in tall fescue pseudostems, and in a second experiment we compared responses of E. coenophiala CTE in plant pseudostem and crown tissues. The endophytes responded to the stress with increased expression of genes involved in oxidative stress response, oxygen radical detoxification, C-compound carbohydrate metabolism, heat shock, and cellular transport pathways. The magnitude of fungal gene responses during stress varied among plant-endophyte symbiota. Responses in pseudostems and crowns involved some common pathways as well as some tissue-specific pathways. The fungal response to water-deficit stress involved gene expression changes in similar pathways that have been documented for plant stress responses, indicating that Epichloë spp. and their host plants either coordinate stress responses or separately activate similar stress response mechanisms that work together for mutual protection.

Differential gene expression in tall fescue tissues in response to water deficit.

Tall fescue (Festuca arundinacea Schreb.) is a popular pasture and turf grass particularly known for drought resistance, allowing for its persistence in locations that are unfavorable for other cool-season grasses. Also, its seed-borne fungal symbiont (endophyte) Epichloë coenophiala, which resides in the crown and pseudostem, can be a contributing factor in its drought tolerance. Because it contains the apical meristems, crown survival under drought stress is critical to plant survival as well as the endophyte. In this study, we subjected tall fescue plants with their endophyte to water-deficit stress or, as controls with normal watering, then compared plant transcriptome responses in four vegetative tissues: leaf blades, pseudostem, crown, and roots. A transcript was designated a differentially expressed gene (DEG) if it exhibited at least a twofold expression difference between stress and control samples with an adjusted p value of .001. Pathway analysis of the DEGs across all tissue types included photosynthesis, carbohydrate metabolism, phytohormone biosynthesis and signaling, cellular organization, and a transcriptional regulation. While no specific pathway was observed to be differentially expressed in the crown, genes encoding auxin response factors, nuclear pore anchors, structural maintenance of chromosomes, and class XI myosin proteins were more highly differentially expressed in crown than in the other vegetative tissues, suggesting that regulation in expression of these genes in the crown may aid in survival of the meristems in the crown.

Isoflavone levels, nodulation and gene expression profiles of a CRISPR/Cas9 deletion mutant in the isoflavone synthase gene of red clover.

KEY MESSAGE: Isoflavones are not involved in rhizobial signaling in red clover, but likely play a role in defense in the rhizosphere. Red clover (Trifolium pratense) is a high-quality forage legume, well suited for grazing and hay production in the temperate regions of the world. Like many legumes, red clover produces a number of phenylpropanoid compounds including anthocyanidins, flavan-3-ols, flavanols, flavanones, flavones, and isoflavones. The study of isoflavone biosynthesis and accumulation in legumes has come into the forefront of biomedical and agricultural research due to potential for medicinal, antimicrobial, and environmental implications. CRISPR/Cas9 was used to knock out the function of a key enzyme in the biosynthesis of isoflavones, isoflavone synthase (IFS1). A hemizygous plant carrying a 9-bp deletion in the IFS1 gene was recovered and was intercrossed to obtain homozygous mutant plants. Levels of the isoflavones formononetin, biochanin A and genistein were significantly reduced in the mutant plants. Wild-type and mutant plants were inoculated with rhizobia to test the effect of the mutation on nodulation, but no significant differences were observed, suggesting that these isoflavones do not play important roles in nodulation. Gene expression profiling revealed an increase in expression of the upstream genes producing the precursors for IFS1, namely, phenylalanine ammonium lyase and chalcone synthase, but there were no significant differences in IFS1 gene expression or in the downstream genes in the production of specific isoflavones. Higher expression in genes involved in ethylene response was observed in the mutant plants. This response is normally associated with biotic stress, suggesting that the plants may have been responding to cues in the surrounding rhizosphere due to lower levels of isoflavones.

The Impacts of Domestication and Breeding on Nitrogen Fixation Symbiosis in Legumes.

Legumes are the second most important family of crop plants. One defining feature of legumes is their unique ability to establish a nitrogen-fixing root nodule symbiosis with soil bacteria known as rhizobia. Since domestication from their wild relatives, crop legumes have been under intensive breeding to improve yield and other agronomic traits but with little attention paid to the belowground symbiosis traits. Theoretical models predict that domestication and breeding processes, coupled with high-input agricultural practices, might have reduced the capacity of crop legumes to achieve their full potential of nitrogen fixation symbiosis. Testing this prediction requires characterizing symbiosis traits in wild and breeding populations under both natural and cultivated environments using genetic, genomic, and ecological approaches. However, very few experimental studies have been dedicated to this area of research. Here, we review how legumes regulate their interactions with soil rhizobia and how domestication, breeding and agricultural practices might have affected nodulation capacity, nitrogen fixation efficiency, and the composition and function of rhizobial community. We also provide a perspective on how to improve legume-rhizobial symbiosis in sustainable agricultural systems.

Tall Fescue and E. coenophiala Genetics Influence Root-Associated Soil Fungi in a Temperate Grassland.

A constitutive, host-specific symbiosis exists between the aboveground fungal endophyte Epichloë coenophiala (Morgan-Jones & W. Gams) and the cool-season grass tall fescue (Lolium arundinaceum (Schreb.) Darbysh.), which is a common forage grass in the United States, Australia, New Zealand, and temperate European grasslands. New cultivars of tall fescue are continually developed to improve pasture productivity and animal health by manipulating both grass and E. coenophiala genetics, yet how these selected grass-endophyte combinations impact other microbial symbionts such as mycorrhizal and dark septate fungi remains unclear. Without better characterizing how genetically distinct grass-endophyte combinations interact with belowground microorganisms, we cannot determine how adoption of new E. coenophiala-symbiotic cultivars in pasture systems will influence long-term soil characteristics and ecosystem function. Here, we examined how E. coenophiala presence and host × endophyte genetic combinations control root colonization by belowground symbiotic fungi and associated plant nutrient concentrations and soil properties in a 2-year manipulative field experiment. We used four vegetative clone pairs of tall fescue that consisted of one endophyte-free (E-) and one E. coenophiala-symbiotic (E+) clone each, where E+ clones within each pair contained one of four endophyte genotypes: CTE14, CTE45, NTE16, or NTE19. After 2 years of growth in field plots, we measured root colonization of arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE), extraradical AMF hyphae in soil, total C, N, and P in root and shoot samples, as well as C and N in associated soils. Although we observed no effects of E. coenophiala presence or symbiotic genotype on total AMF or DSE colonization rates in roots, different grass-endophyte combinations altered AMF arbuscule presence and extraradical hyphal length in soil. The CTE45 genotype hosted the fewest AMF arbuscules regardless of endophyte presence, and E+ clones within NTE19 supported significantly greater soil extraradical hyphae compared to E- clones. Because AMF are often associated with improved soil physical characteristics and C sequestration, our results suggest that development and use of unique grass-endophyte combinations may cause divergent effects on long-term ecosystem properties.

Transcriptome Analysis and Differential Expression in Tall Fescue Harboring Different Endophyte Strains in Response to Water Deficit.

Two tall fescue [Lolium arundinaceum (Schreb.) Darbysh. = Schedonorus arundinaceus (Schreb.) Dumort. = Festuca arundinacea var. arundinacea Schreb.] plant genotypes with an Epichloë coenophiala (Morgan-Jones & W. Gams) C.W. Bacon & Schardl common toxic endophyte (CTE), one with a nontoxic strain (NTE19) and one with another Epichloë species (FaTG-4) were evaluated and compared with their respective endophyte-free clones for responses to water-deficit stress in the greenhouse. One of the plant genotypes (P27) showed a positive effect of its CTE strain on tiller production after stress and resumed watering. In transcriptome analysis of the pseudostems (leaf sheath whorls), differentially expressed genes (DEGs) were defined as having at least twofold expression difference and false discovery rate (FDR) < 0.05 in comparisons of water treatment (stressed or watered), endophyte presence or absence, or both. Stress affected 38% of the plant transcripts including those for the expected stress-response pathways. The DEGs affected by endophyte in stressed plants were unique to individual plant genotypes. In unstressed plants, endophyte presence tended to reduce expression of genes putatively for defense against fungi, but in unstressed P27 endophyte presence there was enhanced expression of dehydrin and heat shock protein genes. Our results indicated subtle and variable effects of endophytes on tall fescue gene expression; where the endophyte confers protection, its effects on plant gene expression may help prime the plant for stress resistance.

Transcriptomics of Epichloë-Grass Symbioses in Host Vegetative and Reproductive Stages.

Epichloë species are fungal symbionts (endophytes) of cool-season grasses that transmit vertically via inflorescence primordia (IP), ovaries (OV), and ultimately, embryos. Epichloë coenophiala, an endophyte of tall fescue (Schedonorus arundinaceus), provides multiple protective benefits to the grass. We conducted transcriptome analysis of the tall fescue-E. coenophiala symbiosis, comparing IP, OV, vegetative pseudostems (PS), and the lemma and palea (LP) (bracts) of the young floret. Transcriptomes of host OV and PS exhibited almost no significant differences attributable to endophyte presence or absence. Comparison of endophyte gene expression in different plant parts revealed numerous differentially expressed genes (DEGs). The 150 endophyte DEGs significantly higher in PS over OV included genes for alkaloid biosynthesis and sugar or amino acid transport. The 277 endophyte DEGs significantly higher in OV over PS included genes for protein chaperones (including most heat-shock proteins), trehalose synthesis complex, a bax inhibitor-1 protein homolog, the CLC chloride ion channel, catalase, and superoxide dismutase. Similar trends were apparent in the Brachypodium sylvaticum-Epichloë sylvatica symbiosis. Gene expression profiles in tall fescue IP and LP indicated that the endophyte transcriptome shift began early in host floral development. We discuss possible roles of the endophyte DEGs in colonization of reproductive grass tissues.

Genome-wide atlas of alternative polyadenylation in the forage legume red clover.

Studies on prevalence and significance of alternative polyadenylation (APA) in plants have been so far limited mostly to the model plants. Here, a genome-wide analysis of APA was carried out in different tissue types in the non-model forage legume red clover (Trifolium pratense L). A profile of poly(A) sites in different tissue types was generated using so-called 'poly(A)-tag sequencing' (PATseq) approach. Our analysis revealed tissue-wise dynamics of usage of poly(A) sites located at different genomic locations. We also identified poly(A) sites and underlying genes displaying APA in different tissues. Functional categories enriched in groups of genes manifesting APA between tissue types were determined. Analysis of spatial expression of genes encoding different poly(A) factors showed significant differential expression of genes encoding orthologs of FIP1(V) and PCFS4, suggesting that these two factors may play a role in regulating spatial APA in red clover. Our analysis also revealed a high degree of conservation in diverse plant species of APA events in mRNAs encoding two key polyadenylation factors, CPSF30 and FIP1(V). Together with our previously reported study of spatial gene expression in red clover, this study will provide a comprehensive account of transcriptome dynamics in this non-model forage legume.

Transcriptome response of Lolium arundinaceum to its fungal endophyte Epichloë coenophiala.

Tall fescue (Lolium arundinaceum) is one of the primary forage and turf grasses in temperate regions of the world. A number of favourable characteristics of tall fescue are enhanced by its seed-transmissible fungal symbiont (endophyte) Epichloë coenophiala. Our approach was to assemble the tall fescue transcriptome, then identify differentially expressed genes (DEGs) for endophyte-symbiotic (E+) vs endophyte-free (E-) clones in leaf blades, pseudostems, crowns and roots. RNA-seq reads were used to construct a tall fescue reference transcriptome and compare gene expression profiles. Over all tissues examined, 478 DEGs were identified between the E+ and E- clones for at least one tissue (more than two-fold; P < 0.0001, 238 E+ > E- and 240 E- > E+), although no genes were differentially expressed in all four tissues. Gene ontology (GO) terms, GO:0010200 (response to chitin), GO:0002679 (respiratory burst during defence response) and GO:0035556 (intracellular signal transduction) were significantly overrepresented among 25 E- > E+ DEGs in leaf blade, and a number of other DEGs were associated with defence and abiotic response. In particular, endophyte effects on various WRKY transcription factors may have implications for symbiotic stability, endophyte distribution in the plant, or defence against pathogens.

De novo Transcriptome Assembly and Dynamic Spatial Gene Expression Analysis in Red Clover.

Red clover ( L.) is a cool-season forage legume grown throughout the northeastern United States and is the most widely planted forage legume after alfalfa ( L.). Red clover provides high-value feed to the livestock because of high protein content and easy digestibility. To date, genomic resources for red clover are scarce. In the current study, a de novo transcriptome assembly of red clover was constructed representing different tissue types. The draft assembly consists of 37,565 contigs with N50 and average contig length of 1707 and 1262 bp, respectively. A comparative study with three other legume species displayed a high degree of sequence conservation between red clover and other legumes. The assembled transcriptome was annotated to allow identification of desirable genes. In particular, a genome-wide identification of red clover transcripts encoding putative transcription factors was performed. A comparative gene expression analysis between different tissue types was performed using the assembled transcriptome as the reference, which revealed dynamic gene expression patterns across different tissue types and also identified spatially dynamic gene coexpression clusters. Genes representing tissue-enriched clusters were subjected to gene ontology (GO) enrichment analysis to identify over-represented functional groups. Identification of these tissue-enriched gene coexpression clusters can help in future research focusing on developmental studies across tissues or in biotechnological improvement of red clover.

Tall fescue endophyte effects on tolerance to water-deficit stress.

BACKGROUND: The endophytic fungus, Neotyphodium coenophialum, can enhance drought tolerance of its host grass, tall fescue. To investigate endophyte effects on plant responses to acute water deficit stress, we did comprehensive profiling of plant metabolite levels in both shoot and root tissues of genetically identical clone pairs of tall fescue with endophyte (E+) and without endophyte (E-) in response to direct water deficit stress. The E- clones were generated by treating E+ plants with fungicide and selectively propagating single tillers. In time course studies on the E+ and E- clones, water was withheld from 0 to 5 days, during which levels of free sugars, sugar alcohols, and amino acids were determined, as were levels of some major fungal metabolites. RESULTS: After 2-3 days of withholding water, survival and tillering of re-watered plants was significantly greater for E+ than E- clones. Within two to three days of withholding water, significant endophyte effects on metabolites manifested as higher levels of free glucose, fructose, trehalose, sugar alcohols, proline and glutamic acid in shoots and roots. The fungal metabolites, mannitol and loline alkaloids, also significantly increased with water deficit. CONCLUSIONS: Our results suggest that symbiotic N. coenophialum aids in survival and recovery of tall fescue plants from water deficit, and acts in part by inducing rapid accumulation of these compatible solutes soon after imposition of stress.

An Arabidopsis ATP-dependent, DEAD-box RNA helicase loses activity upon IsoAsp formation but is restored by PROTEIN ISOASPARTYL METHYLTRANSFERASE.

Orthodox seeds are capable of withstanding severe dehydration. However, in the dehydrated state, Asn and Asp residues in proteins can convert to succinimide residues that can further react to predominantly form isomerized isoAsp residues upon rehydration (imbibition). IsoAsp residues can impair protein function and can render seeds nonviable, but PROTEIN ISOASPARTYL METHYLTRANSFERASE (PIMT) can initiate isoAsp conversion to Asp residues. The proteins necessary for translation upon imbibition in orthodox seeds may be particularly important to maintain in an active state. One such protein is the large, multidomain protein, Arabidopsis thaliana PLANT RNA HELICASE75 (PRH75), a DEAD-box helicase known to be susceptible to isoAsp residue accumulation. However, the consequences of such isomerization on PRH75 catalysis and for the plant are unknown. Here, it is demonstrated that PRH75 is necessary for successful seed development. It acquires isoAsp rapidly during heat stress, which eliminates RNA unwinding (but not rewinding) competence. The repair by PIMT is able to restore PRH75's complex biochemical activity provided isoAsp formation has not led to subsequent, destabilizing conformational alterations. For PRH75, an important enzymatic activity associated with translation would be eliminated unless rapidly repaired by PIMT prior to additional, deleterious conformational changes that would compromise seed vitality and germination.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

The alleles at the E1 locus impact the expression pattern of two soybean FT-like genes shown to induce flowering in Arabidopsis.

A small gene family of phosphatidyl ethanolamine-binding proteins (PEBP) has been shown to function as key regulators in flowering; in Arabidopsis thaliana the FT protein promotes flowering whilst the closely related TFL1 protein represses flowering. Control of flowering time in soybean [Glycine max (L.) Merrill] is important for geographic adaptation and maximizing yield. Soybean breeders have identified a series of loci, the E-genes, that control photoperiod-mediated flowering time, yet how these loci control flowering is poorly understood. The objectives of this study were to evaluate the expression of GmFT-like genes in the E1 near-isogenic line (NIL) background. Of the 20 closely related PEBP proteins in the soybean genome, ten are similar to the Arabidopsis FT protein. Expression analysis of these ten GmFT-like genes confirmed that only two are detectable in the conditions tested. Further analysis of these two genes in the E1 NILs grown under short-day (SD) and long-day (LD) conditions showed a diurnal expression and tissue specificity expression commensurate with soybean flowering time under SD and LD conditions, suggesting that these were good candidates for flowering induction in soybean. Arabidopsis ft mutant lines flowered early when transformed with the two soybean genes, suggesting that the soybean genes can complement the Arabidopsis FT function. Flowering time in E1 NILs is consistent with the differential expression of the two GmFT-like genes under SD and LD conditions, suggesting that the E1 locus, at least in part, impacts time to flowering through the regulation of soybean FT expression.

Microarray analysis of endophyte-infected and endophyte-free tall fescue.

Many grasses have mutualistic symbioses with fungi of the family Clavicipitaceae. Tall fescue can harbor the obligate endophyte, Neotyphodium coenophialum that is asexually propagated and transmitted via host seeds. Total RNA was isolated from pseudostems of known endophyte-infected (E+) and endophyte-free (E-) plants and tested in triplicate on the Affymetrix Wheat Genome Array GeneChip and Barley1 Genome Array GeneChip. Overall 14-15% and 17-18% of the probe sets were called present on the wheat and barley chips, respectively. In order to identify genes that were specifically differentially expressed between the E+ and E- tall fescue, a combination of both barley and wheat target sequences that were differentially expressed (greater than twofold) that were similar on both chips on both barley and wheat arrays yielded 32 probe set (genes) that were differentially expressed. Tall fescue ESTs were identified for a number of the probe sets that were differentially expressed on the barley and wheat arrays. PCR primers were designed to fescue ESTs and tested to verify the expression profile observed in the microarray experiments. Some primers confirmed the expected results, although in other cases no differences were observed between the E+ and E- plants, or the results were contrary to what was expected. Our results suggest that while some differentially expressed genes were identified by this method, the cross-species hybridization appears to have significant limitations for the transcriptome analysis of tall fescue.

Expression of flowering-time genes in soybean E1 near-isogenic lines under short and long day conditions.

Control of soybean flowering time is important for geographic adaptation and maximizing yield. Plant breeders have identified a series of genes (E genes) that condition time to flowering; however, the molecular basis in the control of flowering by these E genes, in conjunction with canonical flowering-time genes, has not been studied. Time to flowering in near-isogenic lines (NILs) at the E1 locus was tested using a reciprocal transfer experiment under short day (SD) and long day (LD) conditions. Beginning 8 days after planting, three plant samples were harvested every 3 h for a 48-h period. RNA was isolated from these plants, and RNA samples were pooled for each line and each time period for cDNA synthesis. RT-PCR analysis was performed using primers synthesized for a number of putative flowering-time genes based on homology of soybean EST and genomic sequences to Arabidopsis genes. The results of the reciprocal transfer experiment suggest that the pre-inductive photoperiod-sensitive phase of the E1 NILs responsible for inducing flowering is perceived as early as 5-7-day post-planting. No gene expression differences were found between the E1 and e1 NILs, suggesting that the E1 gene does not directly affect the flowering-time genes during the time period tested; however, differences were observed in gene expression between SD and LD treatments for the putative soybean TOC1, CO, and FT genes. The gene expression results in this study were similar to those of flowering-time genes found in other SD species, suggesting that the selected genes correspond to the soybean flowering-time orthologs.

Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits.

BACKGROUND: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to further explore the functions of AtCPSF30, the subcellular distribution of the protein was examined by over-expressing fusion proteins containing fluorescent reporters linked to different CPSF subunits. RESULTS: It was found that AtCPSF30 by itself localizes, not to the nucleus, but to the cytoplasm. AtCPSF30 could be found in the nucleus when co-expressed with AtCPSF160 or AtCPSF73(I), one of the two Arabidopsis orthologs of CPSF73. This re-directing of AtCPSF30 indicates that AtCPSF30 is retained in the nucleus via interactions with either or both of these other CPSF subunits. Co-expression of AtCSPF30 with AtCPSF100 altered the location, not of AtCPSF30, but rather of AtCPSF100, with these proteins residing in the cytoplasm. Deletion of plant-specific N- or C-terminal domains of AtCPSF30 abolished various of the interactions between AtCPSF30 and other CPSF subunits, suggesting that the plant CPSF complex assembles via novel protein-protein interactions. CONCLUSION: These results suggest that the nuclear CPSF complex in plants is a dynamic one, and that the interactions between AtCPSF30 and other CPSF subunits are different from those existing in other eukaryotes.

Improvement of a monopartite ecdysone receptor gene switch and demonstration of its utility in regulation of transgene expression in plants.

In plants, regulation of transgene expression is typically accomplished through the use of inducible promoter systems. The ecdysone receptor (EcR) gene switch is one of the best inducible systems available to regulate transgene expression in plants. However, the monopartite EcR gene switches developed to date require micromolar concentrations of ligand for activation. We tested several EcR mutants that were generated by changing one or two amino acid residues in the highly flexible ligand-binding domain of Choristoneura fumiferana EcR (CfEcR). Based on the transient expression assays, we selected a double mutant, V395I + Y415E (VY), of CfEcR (CfEcR(VY)) for further testing in stable transformation experiments. The CfEcR(VY) mutant only slightly improved the induction characteristics of the two-hybrid gene switch, whereas the CfEcR(VY) mutant significantly improved the induction characteristics of the monopartite gene switch (VGCfE(VY)). The ligand sensitivity of the VGCfE(VY) switch was improved by 125-15 625-fold in different transgenic lines analyzed, compared to the VGCfE(Wt) switch. The utility of the VGCfE(VY) switch was tested by regulating the expression of an Arabidopsis zinc finger protein gene (AtZFP11) in both tobacco and Arabidopsis plants. These data showed that the VGCfE(VY) switch efficiently regulated the expression of AtZFP11 and that the phenotype of AtZFP11 could be induced by the application of ligand. In addition, the affected plants recovered after withdrawal of the ligand, demonstrating the utility of this gene switch in regulating the expression of critical transgenes in plants.