MsSPL9 Modulates Nodulation under Nitrate Sufficiency Condition in Medicago sativa.

Nodulation in Leguminous spp. is induced by common environmental cues, such as low nitrogen availability conditions, in the presence of the specific Rhizobium spp. in the rhizosphere. Medicago sativa (alfalfa) is an important nitrogen-fixing forage crop that is widely cultivated around the world and relied upon as a staple source of forage in livestock feed. Although alfalfa's relationship with these bacteria is one of the most efficient between rhizobia and legume plants, breeding for nitrogen-related traits in this crop has received little attention. In this report, we investigate the role of Squamosa-Promoter Binding Protein-Like 9 (SPL9), a target of miR156, in nodulation in alfalfa. Transgenic alfalfa plants with SPL9-silenced (SPL9-RNAi) and overexpressed (35S::SPL9) were compared to wild-type (WT) alfalfa for phenotypic changes in nodulation in the presence and absence of nitrogen. Phenotypic analyses showed that silencing of MsSPL9 in alfalfa caused an increase in the number of nodules. Moreover, the characterization of phenotypic and molecular parameters revealed that MsSPL9 regulates nodulation under a high concentration of nitrate (10 mM KNO3) by regulating the transcription levels of the nitrate-responsive genes Nitrate Reductase1 (NR1), NR2, Nitrate transporter 2.5 (NRT2.5), and a shoot-controlled autoregulation of nodulation (AON) gene, Super numeric nodules (SUNN). While MsSPL9-overexpressing transgenic plants have dramatically increased transcript levels of SUNN, NR1, NR2, and NRT2.5, reducing MsSPL9 caused downregulation of these genes and displayed a nitrogen-starved phenotype, as downregulation of the MsSPL9 transcript levels caused a nitrate-tolerant nodulation phenotype. Taken together, our results suggest that MsSPL9 regulates nodulation in alfalfa in response to nitrate.

Genome-wide occupancy of Arabidopsis SWI/SNF chromatin remodeler SPLAYED provides insights into its interplay with its close homolog BRAHMA and Polycomb proteins.

SPLAYED (SYD) is a SWItch/Sucrose Non-Fermentable (SWI/SNF)-type chromatin remodeler identified in Arabidopsis thaliana (Arabidopsis). It is believed to play both redundant and differential roles with its closest homolog BRAHMA (BRM) in diverse plant growth and development processes. To better understand how SYD functions, we profiled the genome-wide occupancy of SYD and its impact on the global transcriptome and trimethylation of histone H3 on lysine 27 (H3K27me3). To map the global occupancy of SYD, we generated a GFP-tagged transgenic line and used it for chromatin immunoprecipitation experiments followed by next-generation sequencing, by which more than 6000 SYD target genes were identified. Through integrating SYD occupancy and transcriptome profiles, we found that SYD preferentially targets to nucleosome-free regions of expressed genes. Further analysis revealed that SYD occupancy peaks exhibit five distinct patterns, which were also shared by BRM and BAF60, a conserved SWI/SNF complex component, indicating the common target sites of these SWI/SNF chromatin remodelers and the functional relevance of such distinct patterns. To investigate the interplay between SYD and Polycomb-group (PcG) proteins, we performed a genome-wide analysis of H3K27me3 in syd-5. We observed both increases and decreases in H3K27me3 levels at a few hundred genes in syd-5 compared to wild type. Our results imply that SYD can act antagonistically or synergistically with PcG at specific genes. Together, our SYD genome-wide occupancy data and the transcriptome and H3K27me3 profiles provide a much-needed resource for dissecting SYD's crucial roles in the regulation of plant growth and development.

Deciphering the role of SPL12 and AGL6 from a genetic module that functions in nodulation and root regeneration in Medicago sativa.

KEY MESSAGE: Our results show that SPL12 plays a crucial role in regulating nodule development in Medicago sativa L. (alfalfa), and that AGL6 is targeted and downregulated by SPL12. Root architecture in plants is critical because of its role in controlling nutrient cycling, water use efficiency and response to biotic and abiotic stress factors. The small RNA, microRNA156 (miR156), is highly conserved in plants, where it functions by silencing a group of SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. We previously showed that transgenic Medicago sativa (alfalfa) plants overexpressing miR156 display increased nodulation, improved nitrogen fixation and enhanced root regenerative capacity during vegetative propagation. In alfalfa, transcripts of eleven SPLs, including SPL12, are targeted for cleavage by miR156. In this study, we characterized the role of SPL12 in root architecture and nodulation by investigating the transcriptomic and phenotypic changes associated with altered transcript levels of SPL12, and by determining SPL12 regulatory targets using SPL12-silencing and -overexpressing alfalfa plants. Phenotypic analyses showed that silencing of SPL12 in alfalfa caused an increase in root regeneration, nodulation, and nitrogen fixation. In addition, AGL6 which encodes AGAMOUS-like MADS box transcription factor, was identified as being directly targeted for silencing by SPL12, based on Next Generation Sequencing-mediated transcriptome analysis and chromatin immunoprecipitation assays. Taken together, our results suggest that SPL12 and AGL6 form a genetic module that regulates root development and nodulation in alfalfa.

RNA polymerase II-independent recruitment of SPT6L at transcription start sites in Arabidopsis.

SPT6 is a conserved elongation factor that is associated with phosphorylated RNA polymerase II (RNAPII) during transcription. Recent transcriptome analysis in yeast mutants revealed its potential role in the control of transcription initiation at genic promoters. However, the mechanism by which this is achieved and how this is linked to elongation remains to be elucidated. Here, we present the genome-wide occupancy of Arabidopsis SPT6-like (SPT6L) and demonstrate its conserved role in facilitating RNAPII occupancy across transcribed genes. We also further demonstrate that SPT6L enrichment is unexpectedly shifted, from gene body to transcription start site (TSS), when its association with RNAPII is disrupted. Protein domains, required for proper function and enrichment of SPT6L on chromatin, are subsequently identified. Finally, our results suggest that recruitment of SPT6L at TSS is indispensable for its spreading along the gene body during transcription. These findings provide new insights into the mechanisms underlying SPT6L recruitment in transcription and shed light on the coordination between transcription initiation and elongation.

Transcriptome-IPMS analysis reveals a tissue-dependent miR156/SPL13 regulatory mechanism in alfalfa drought tolerance.

BACKGROUND: We previously reported on the interplay between miR156/SPL13 and WD40-1/DFR to improve response to drought stress in alfalfa (Medicago sativa L.). Here we aimed to investigate whether the role of miR156/SPL13 module in drought response is tissue-specific, and to identify SPL13-interacting proteins. We analyzed the global transcript profiles of leaf, stem, and root tissues of one-month old RNAi-silenced SPL13 (SPL13RNAi) alfalfa plants exposed to drought stress and conducted protein-protein interaction analysis to identify SPL13 interacting partners. RESULT: Transcript analysis combined with weighted gene co-expression network analysis showed tissue and genotype-specific gene expression patterns. Moreover, pathway analysis of stem-derived differentially expressed genes (DEG) revealed upregulation of genes associated with stress mitigating primary and specialized metabolites, whereas genes associated with photosynthesis light reactions were silenced in SPL13RNAi plants. Leaf-derived DEG were attributed to enhanced light reactions, largely photosystem I, II, and electron transport chains, while roots of SPL13RNAi plants upregulated transcripts associated with metal ion transport, carbohydrate, and primary metabolism. Using immunoprecipitation combined with mass spectrometry (IPMS) we showed that SPL13 interacts with proteins involved in photosynthesis, specialized metabolite biosynthesis, and stress tolerance. CONCLUSIONS: We conclude that the miR156/SPL13 module mitigates drought stress in alfalfa by regulating molecular and physiological processes in a tissue-dependent manner.

The interplay between miR156/SPL13 and DFR/WD40-1 regulate drought tolerance in alfalfa.

BACKGROUND: Developing Medicago sativa L. (alfalfa) cultivars tolerant to drought is critical for the crop's sustainable production. miR156 regulates various plant biological functions by silencing SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors. RESULTS: To understand the mechanism of miR156-modulated drought stress tolerance in alfalfa we used genotypes with altered expression levels of miR156, miR156-regulated SPL13, and DIHYDROFLAVONOL-4-REDUCTASE (DFR) regulating WD40-1. Previously we reported the involvement of miR156 in drought tolerance, but the mechanism and downstream genes involved in this process were not fully studied. Here we illustrate the interplay between miR156/SPL13 and WD40-1/DFR to regulate drought stress by coordinating gene expression with metabolite and physiological strategies. Low to moderate levels of miR156 overexpression suppressed SPL13 and increased WD40-1 to fine-tune DFR expression for enhanced anthocyanin biosynthesis. This, in combination with other accumulated stress mitigating metabolites and physiological responses, improved drought tolerance. We also demonstrated that SPL13 binds in vivo to the DFR promoter to regulate its expression. CONCLUSIONS: Taken together, our results reveal that moderate relative miR156 transcript levels are sufficient to enhance drought resilience in alfalfa by silencing SPL13 and increasing WD40-1 expression, whereas higher miR156 overexpression results in drought susceptibility.

Analysis of a novel mutant allele of GSL8 reveals its key roles in cytokinesis and symplastic trafficking in Arabidopsis.

BACKGROUND: Plant cell walls are mainly composed of polysaccharides such as cellulose and callose. Callose exists at a very low level in the cell wall; however, it plays critical roles at different stages of plant development as well as in defence against unfavorable conditions. Callose is accumulated at the cell plate, at plasmodesmata and in male and female gametophytes. Despite the important roles of callose in plants, the mechanisms of its synthesis and regulatory properties are not well understood. RESULTS: CALLOSE SYNTHASE (CALS) genes, also known as GLUCAN SYNTHASE-LIKE (GSL), comprise a family of 12 members in Arabidopsis thaliana. Here, we describe a new allele of GSL8 (named essp8) that exhibits pleiotropic seedling defects. Reduction of callose deposition at the cell plates and plasmodesmata in essp8 leads to ectopic endomitosis and an increase in the size exclusion limit of plasmodesmata during early seedling development. Movement of two non-cell-autonomous factors, SHORT ROOT and microRNA165/6, both required for root radial patterning during embryonic root development, are dysregulated in the primary root of essp8. This observation provides evidence for a molecular mechanism explaining the gsl8 root phenotype. We demonstrated that GSL8 interacts with PLASMODESMATA-LOCALIZED PROTEIN 5, a ß-1,3-glucanase, and GSL10. We propose that they all might be part of a putative callose synthase complex, allowing a concerted regulation of callose deposition at plasmodesmata. CONCLUSION: Analysis of a novel mutant allele of GSL8 reveals that GSL8 is a key player in early seedling development in Arabidopsis. GSL8 is required for maintaining the basic ploidy level and regulating the symplastic trafficking. Callose deposition at plasmodesmata is highly regulated and occurs through interaction of different components, likely to be incorporated into a callose biosynthesis complex. We are providing new evidence supporting an earlier hypothesis that GSL8 might have regulatory roles apart from its enzymatic function in plasmodesmata regulation.

The Arabidopsis SWI2/SNF2 chromatin Remodeler BRAHMA regulates polycomb function during vegetative development and directly activates the flowering repressor gene SVP.

The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.

Acetolactate synthase regulatory subunits play divergent and overlapping roles in branched-chain amino acid synthesis and Arabidopsis development.

BACKGROUND: Branched-chain amino acids (BCAAs) are synthesized by plants, fungi, bacteria, and archaea with plants being the major source of these amino acids in animal diets. Acetolactate synthase (ALS) is the first enzyme in the BCAA synthesis pathway. Although the functional contribution of ALS to BCAA biosynthesis has been extensively characterized, a comprehensive understanding of the regulation of this pathway at the molecular level is still lacking. RESULTS: To characterize the regulatory processes governing ALS activity we utilized several complementary approaches. Using the ALS catalytic protein subunit as bait we performed a yeast two-hybrid (Y2H) screen which resulted in the identification of a set of interacting proteins, two of which (denoted as ALS-INTERACTING PROTEIN1 and 3 [AIP1 and AIP3, respectively]) were found to be evolutionarily conserved orthologues of bacterial feedback-regulatory proteins and therefore implicated in the regulation of ALS activity. To investigate the molecular role AIPs might play in BCAA synthesis in Arabidopsis thaliana, we examined the functional contribution of aip1 and aip3 knockout alleles to plant patterning and development and BCAA synthesis under various growth conditions. Loss-of-function genetic backgrounds involving these two genes exhibited differential aberrant growth responses in valine-, isoleucine-, and sodium chloride-supplemented media. While BCAA synthesis is believed to be localized to the chloroplast, both AIP1 and AIP3 were found to localize to the peroxisome in addition to the chloroplast. Analysis of free amino acid pools in the mutant backgrounds revealed that they differ in the absolute amount of individual BCAAs accumulated and exhibit elevated levels of BCAAs in leaf tissues. Despite the phenotypic differences observed in aip1 and aip3 backgrounds, functional redundancy between these loci was suggested by the finding that aip1/aip3 double knockout mutants are severely developmentally compromised. CONCLUSIONS: Taken together the data suggests that the two regulatory proteins, in conjunction with ALS, have overlapping but distinct functions in BCAA synthesis, and also play a role in pathways unrelated to BCAA synthesis such as sodium-ion homeostasis, extending to broader aspects of patterning and development.

Subcellular localization of Arabidopsis arogenate dehydratases suggests novel and non-enzymatic roles.

Arogenate dehydratases (ADTs) catalyze the final step in phenylalanine biosynthesis in plants. The Arabidopsis thaliana genome encodes a family of six ADTs capable of decarboxylating/dehydrating arogenate into phenylalanine. Using cyan fluorescent protein (CFP)-tagged proteins, the subcellular localization patterns of all six A. thaliana ADTs were investigated in intact Nicotiana benthamiana and A. thaliana leaf cells. We show that A. thaliana ADTs localize to stroma and stromules (stroma-filled tubules) of chloroplasts. This localization pattern is consistent with the enzymatic function of ADTs as many enzymes required for amino acid biosynthesis are primarily localized to chloroplasts, and stromules are thought to increase metabolite transport from chloroplasts to other cellular compartments. Furthermore, we provide evidence that ADTs have additional, non-enzymatic roles. ADT2 localizes in a ring around the equatorial plane of chloroplasts or to a chloroplast pole, which suggests that ADT2 is a component of the chloroplast division machinery. In addition to chloroplasts, ADT5 was also found in nuclei, again suggesting a non-enzymatic role for ADT5. We also show evidence that ADT5 is transported to the nucleus via stromules. We propose that ADT2 and ADT5 are moonlighting proteins that play an enzymatic role in phenylalanine biosynthesis and a second role in chloroplast division or transcriptional regulation, respectively.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 2 controls floral organ development and plant fertility by activating ASYMMETRIC LEAVES 2 in Arabidopsis thaliana.

A network of genes is coordinately expressed to ensure proper development of floral organs and fruits, which are essential for generating new offspring in flowering plants. In Arabidopsis thaliana, microRNA156 (miR156) plays a role in regulating the development of flowers and siliques by targeting members of the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) gene family. Despite the important roles of the miR156/SPL network, our understanding of its downstream genes that are involved in floral organ and silique growth is still incomplete. Here, we report that the miR156/SPL2 regulatory pathway regulates pollen production, fertility rate, and the elongation of floral organs, including petals, sepals, and siliques in Arabidopsis. Transgenic plants exhibiting both overexpression of miR156 and dominant-negative alleles of SPL2 had reduced ASYMMETRIC LEAVES 2 (AS2) transcript levels in their siliques. Furthermore, their fertility phenotype was similar to that of the AS2 loss-of-function mutant. We also demonstrate that the SPL2 protein binds to the 5'UTR of the AS2 gene in vivo, indicating that AS2 is directly regulated by SPL2. Our results suggest that the miR156/SPL2 pathway affects floral organs, silique development and plant fertility, as well as directly regulates AS2 expression.

Soybean AROGENATE DEHYDRATASES (GmADTs): involvement in the cytosolic isoflavonoid metabolon or trans-organelle continuity?

Soybean (Glycine max) produces a class of phenylalanine (Phe) derived specialized metabolites, isoflavonoids. Isoflavonoids are unique to legumes and are involved in defense responses in planta, and they are also necessary for nodule formation with nitrogen-fixing bacteria. Since Phe is a precursor of isoflavonoids, it stands to reason that the synthesis of Phe is coordinated with isoflavonoid production. Two putative AROGENATE DEHYDRATASE (ADT) isoforms were previously co-purified with the soybean isoflavonoid metabolon anchor ISOFLAVONE SYNTHASE2 (GmIFS2), however the GmADT family had not been characterized. Here, we present the identification of the nine member GmADT family. We determined that the GmADTs share sequences required for enzymatic activity and allosteric regulation with other characterized plant ADTs. Furthermore, the GmADTs are differentially expressed, and multiple members have dual substrate specificity, also acting as PREPHENATE DEHYDRATASES. All GmADT isoforms were detected in the stromules of chloroplasts, and they all interact with GmIFS2 in the cytosol. In addition, GmADT12A interacts with multiple other isoflavonoid metabolon members. These data substantiate the involvement of GmADT isoforms in the isoflavonoid metabolon.

Protein body formation in stable transgenic tobacco expressing elastin-like polypeptide and hydrophobin fusion proteins.

BACKGROUND: Plants are recognized as an efficient and inexpensive system to produce valuable recombinant proteins. Two different strategies have been commonly used for the expression of recombinant proteins in plants: transient expression mediated by Agrobacterium; or stable transformation of the plant genome. However, the use of plants as bioreactors still faces two main limitations: low accumulation levels of some recombinant proteins and lack of efficient purification methods. Elastin-like polypeptide (ELP), hydrophobin I (HFBI) and Zera® are three fusion partners found to increase the accumulation levels of recombinant proteins and induce the formation of protein bodies (PBs) in leaves when targeted to the endoplasmic reticulum (ER) in transient expression assays. In this study the effects of ELP and HFBI fusion tags on recombinant protein accumulation levels and PB formation was examined in stable transgenic Nicotiana tabacum. RESULTS: The accumulation of recombinant protein and PB formation was evaluated in two cultivars of Nicotiana tabacum transformed with green fluorescent protein (GFP) fused to ELP or HFBI, both targeted and retrieved to the ER. The ELP and HFBI tags increased the accumulation of the recombinant protein and induced the formation of PBs in leaves of stable transgenic plants from both cultivars. Furthermore, these tags induced the formation of PBs in a concentration-dependent manner, where a specific level of recombinant protein accumulation was required for PBs to appear. Moreover, agro-infiltration of plants accumulating low levels of recombinant protein with p19, a suppressor of post-transcriptional gene silencing (PTGS), increased accumulation levels in four independent transgenic lines, suggesting that PTGS might have caused the low accumulation levels in these plants. CONCLUSION: The use of ELP and HFBI tags as fusion partners in stable transgenic plants of tobacco is feasible and promising. In a constitutive environment, these tags increase the accumulation levels of the recombinant protein and induce the formation of PBs regardless of the cultivar used. However, a specific level of recombinant protein accumulation needs to be reached for PBs to form.

High-level production of human interleukin-10 fusions in tobacco cell suspension cultures.

The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER.

Localization of Arabidopsis Glucan Synthase-Like 5, 8, and 12 to plasmodesmata and the GSL8-dependent role of PDLP5 in regulating plasmodesmal permeability.

Cell-to-cell communication via membranous channels called plasmodesmata (PD) plays critical roles during plant development and in response to biotic and abiotic stresses. Several enzymes and receptor-like proteins (RLPs), including Arabidopsis thaliana glucan synthase-likes (GSLs), also known as callose synthases (CALSs), and PD-located proteins (PDLPs), have been implicated in plasmodesmal permeability regulation and intercellular communication. Localization of PDLPs to punctate structures at the cell periphery and their receptor-like identity have raised the hypothesis that PDLPs are involved in the regulation of symplastic trafficking during plant development and in response to endogenous and exogenous signals. Indeed, it was shown that PDLP5 could limit plasmodesmal permeability through inducing an increase in callose accumulation at PD. However, mechanistically, how this is achieved remains to be elucidated. To address this key issue in understanding the regulation of PD, physical and functional interactions between PDLPs and GSLs (using the PDLP5-GSL8/CALS10 pair as a model) were investigated. Our results show that GSL8/CALS10 plays essential roles and is required for the function and plasmodesmal localization of PDLP5. Furthermore, it was demonstrated that the localization of PDLP5 to PD and its function in inducing callose deposition are GSL8-dependent. Importantly, our transgenic study shows that three key members of the GSL family, i.e., GSL5/CALS12, GSL8/CALS10, and GSL12/CALS3, localize to PD and co-localize with PDLP5, suggesting that GSL8/CALS10 might not be the only callose synthase with the determining role in PD regulation. These findings, together with our previous observation showing the direct interaction of GSL8/CALS10 with PDLP5, indicate the pivotal role of the GSL8/CALS10-PDLP5 interplay in regulating PD permeability. Future work is needed to investigate whether the PDLP5 functionality and localization are also disrupted in gsl5 and gsl12, or it is just gsl8-specific.

Plant production of a virus-like particle-based vaccine candidate against porcine reproductive and respiratory syndrome.

Porcine reproductive and respiratory syndrome (PRRS) is a disease leading to spontaneous abortions and stillbirths in sows and lowered life quality and expectancy in growing pigs. PRRS is prevalent worldwide and has significant economic impacts to swine industries around the globe. Co-expression of the two most abundant proteins in the viral envelope, the matrix protein (M) and glycosylated protein 5 (GP5), can produce a neutralizing immune response for the virus providing a potentially effective subunit vaccine against the disease, but these proteins are difficult to express. The goal of this research was to display antigenic portions of the M and GP5 proteins on the surface of tobacco mosaic virus-like particles. A modified tobacco mosaic virus coat protein (TMVc) was transiently expressed in Nicotiana benthamiana leaves and targeted to three subcellular compartments along the secretory pathway to introduce glycosylation patterns important for M-GP5 epitope immunogenicity. We found that accumulation levels in the apoplast were similar to the ER and the vacuole. Because glycans present on plant apoplastic proteins are closest to those present on PRRSV proteins, a TMVc-M-GP5 fusion construct was targeted to the apoplast and accumulated at over 0.5 mg/g of plant fresh weight. TMVc virus-like particles self-assembled in plant cells and surface-displayed the M-GP5 epitope, as visualized by transmission electron microscopy and immunogold localization. These promising findings lay the foundation for immunogenicity and protective-immunity studies in animals to examine the efficacy of this vaccine candidate as a measure to control PRRS.

A novel, semi-dominant allele of MONOPTEROS provides insight into leaf initiation and vein pattern formation.

Leaf vein pattern is proposed to be specified by directional auxin transport through presumptive vein cells. Activation of auxin response, which induces downstream genes that entrain auxin transport and lead to vascular differentiation, occurs through a set of transcription factors, the auxin response factors. In the absence of auxin, auxin response factors are inactive because they interact with repressor proteins, the Aux/IAA proteins. One member of the auxin response factor protein family, Auxin Response Factor 5/MONOPTEROS (MP), is critical to vein formation as indicated by reduced vein formation in loss-of-function MP alleles. We have identified a semi-dominant, gain-of-function allele of MP, autobahn or mp ( abn ), which results in vein proliferation in leaves and cotyledons. mp ( abn ) is predicted to encode a truncated product that lacks domain IV required for interaction with its Aux/IAA repressor BODENLOS (BDL). We show that the truncated product fails to interact with BDL in yeast two-hybrid assays. Ectopic expression of MP targets including the auxin efflux protein PINFORMED1 (PIN1) further supports the irrepressible nature of mp ( abn ). Asymmetric PIN1:GFP cellular localization does not occur within the enlarged PIN1:GFP expression domains, suggesting the asymmetry requires differential auxin response in neighbouring cells. Organ initiation from mp ( abn ) meristems is altered, consistent with disruption to source/sink relationships within the meristem and possible changes in gene expression. Finally, mp ( abn ) anthers fail to dehisce and their indehiscence can be relieved by jasmonic acid treatment, suggesting a specific role for MP in late anther development.

Purification and Proteomics Analysis of Phloem Tissues from Virus-Infected Plants.

The plant phloem vasculature is crucial for plant growth and development, and is essential for the systemic movement (SM) of plant viruses. Recent transcriptomic studies of the phloem during virus infection have shown the importance of this tissue, yet transcript levels do not provide definitive answers how virus-host interactions favour successful viral SM. Proteomic analyses have been used to identify host-virus protein interactions, uncovering a variety of ways by which viruses utilize host cellular machinery for completion of the viral infection cycle. Despite this new evidence through proteomics, very few phloem centric studies during viral infection have been performed. Here, we describe a protocol for the isolation of phloem tissues and proteins and the subsequent label-free quantitation (LFQ), for identification of proteomic alterations caused by viral infection.

SPL12 Regulates AGL6 and AGL21 to Modulate Nodulation and Root Regeneration under Osmotic Stress and Nitrate Sufficiency Conditions in Medicago sativa.

The highly conserved plant microRNA, miR156, affects root architecture, nodulation, symbiotic nitrogen fixation, and stress response. In Medicago sativa, transcripts of eleven SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE, SPLs, including SPL12, are targeted for cleavage by miR156. Our previous research revealed the role of SPL12 and its target gene, AGL6, in nodulation in alfalfa. Here, we investigated the involvement of SPL12, AGL6 and AGL21 in nodulation under osmotic stress and different nitrate availability conditions. Characterization of phenotypic and molecular parameters revealed that the SPL12/AGL6 module plays a negative role in maintaining nodulation under osmotic stress. While there was a decrease in the nodule numbers in WT plants under osmotic stress, the SPL12-RNAi and AGL6-RNAi genotypes maintained nodulation under osmotic stress. Moreover, the results showed that SPL12 regulates nodulation under a high concentration of nitrate by silencing AGL21. AGL21 transcript levels were increased under nitrate treatment in WT plants, but SPL12 was not affected throughout the treatment period. Given that AGL21 was significantly upregulated in SPL12-RNAi plants, we conclude that SPL12 may be involved in regulating nitrate inhibition of nodulation in alfalfa by targeting AGL21. Taken together, our results suggest that SPL12, AGL6, and AGL21 form a genetic module that regulates nodulation in alfalfa under osmotic stress and in response to nitrate.

Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin.

Nuclear pore complexes (NPCs) are vital to nuclear-cytoplasmic communication in eukaryotes. The yeast NPC-associated TREX-2 complex, also known as the Thp1-Sac3-Cdc31-Sus1 complex, is anchored on the NPC via the nucleoporin Nup1, and is essential for mRNA export. Here we report the identification and characterization of the putative Arabidopsis thaliana TREX-2 complex and its anchoring nucleoporin. Physical and functional evidence support the identification of the Arabidopsis orthologs of yeast Thp1 and Nup1. Of three Arabidopsis homologs of yeast Sac3, two are putative TREX-2 components, but, surprisingly, none are required for mRNA export as they are in yeast. Physical association of the two Cdc31 homologs, but not the Sus1 homolog, with the TREX-2 complex was observed. In addition to identification of these TREX-2 components, direct interactions of the Arabidopsis homolog of DSS1, which is an established proteasome component in yeast and animals, with both the TREX-2 complex and the proteasome were observed. This suggests the possibility of a link between the two complexes. Thus this work has identified the putative Arabidopsis TREX-2 complex and provides a foundation for future studies of nuclear export in Arabidopsis.