SPINDLY Research Articles

O-glycosylation of the transcription factor SPATULA promotes style development in Arabidopsis.

O-linked β-N-acetylglucosamine (O-GlcNAc) and O-fucose are two sugar-based post-translational modifications whose mechanistic role in plant signalling and transcriptional regulation is still largely unknown. Here we investigated how two O-glycosyltransferase enzymes of Arabidopsis thaliana, SPINDLY (SPY) and SECRET AGENT (SEC), promote the activity of the basic helix-loop-helix transcription factor SPATULA (SPT) during morphogenesis of the plant female reproductive organ apex, the style. SPY and SEC modify amino-terminal residues of SPT in vivo and in vitro by attaching O-fucose and O-GlcNAc, respectively. This post-translational regulation does not impact SPT homo- and heterodimerization events, although it enhances the affinity of SPT for the kinase PINOID gene locus and its transcriptional repression. Our findings offer a mechanistic example of the effect of O-GlcNAc and O-fucose on the activity of a plant transcription factor and reveal previously unrecognized roles for SEC and SPY in orchestrating style elongation and shape.

E. coli-expressed SECRET AGENT O-GlcNAc modifies threonine 829 of GIGANTEA.

The Arabidopsis thaliana glycosyl transferases SPINDLY (SPY) and SECRET AGENT (SEC) modify nuclear and cytosolic proteins with O-linked fucose or O-linked N-acetylglucosamine (O-GlcNAc), respectively. O-fucose and O-GlcNAc modifications can occur at the same sites. SPY interacts physically and genetically with GIGANTEA (GI), suggesting that it could be modified by both enzymes. Previously, we found that, when co-expressed in Escherichia coli, SEC modifies GI; however, the modification site was not determined. By analyzing the overlapping sub-fragments of GI, we identified a region that was modified by SEC in E. coli. Modification was undetectable when threonine 829 (T829) was mutated to alanine, while the T834A and T837A mutations reduced the modification, suggesting that T829 was the primary or the only modification site. Mapping using mass spectrometry detected only the modification of T829. Previous studies have shown that the positions modified by SEC in E. coli are modified in planta, suggesting that T829 is O-GlcNAc modified in planta.

Structure-based virtual screening identifies small-molecule inhibitors of O-fucosyltransferase SPINDLY in Arabidopsis.

Protein O-glycosylation is a nutrient signaling mechanism that plays an essential role in maintaining cellular homeostasis across different species. In plants, SPINDLY (SPY) and SECRET AGENT (SEC) posttranslationally modify hundreds of intracellular proteins with O-fucose and O-linked N-acetylglucosamine, respectively. SPY and SEC play overlapping roles in cellular regulation, and loss of both SPY and SEC causes embryo lethality in Arabidopsis (Arabidopsis thaliana). Using structure-based virtual screening of chemical libraries followed by in vitro and in planta assays, we identified a SPY O-fucosyltransferase inhibitor (SOFTI). Computational analyses predicted that SOFTI binds to the GDP-fucose-binding pocket of SPY and competitively inhibits GDP-fucose binding. In vitro assays confirmed that SOFTI interacts with SPY and inhibits its O-fucosyltransferase activity. Docking analysis identified additional SOFTI analogs that showed stronger inhibitory activities. SOFTI treatment of Arabidopsis seedlings decreased protein O-fucosylation and elicited phenotypes similar to the spy mutants, including early seed germination, increased root hair density, and defective sugar-dependent growth. In contrast, SOFTI did not visibly affect the spy mutant. Similarly, SOFTI inhibited the sugar-dependent growth of tomato (Solanum lycopersicum) seedlings. These results demonstrate that SOFTI is a specific SPY O-fucosyltransferase inhibitor that can be used as a chemical tool for functional studies of O-fucosylation and potentially for agricultural management.

Structure and dynamics of the Arabidopsis O-fucosyltransferase SPINDLY

SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that SPY is distinct from ER-localized POFUTs and contains N-terminal tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain resembling the O-linked-N-acetylglucosamine (GlcNAc) transferases (OGTs). However, the structural feature that determines the distinct enzymatic selectivity of SPY remains unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of SPY and its complex with GDP-fucose, revealing distinct active-site features enabling GDP-fucose instead of UDP-GlcNAc binding. SPY forms an antiparallel dimer instead of the X-shaped dimer in human OGT, and its catalytic domain interconverts among multiple conformations. Analysis of mass spectrometry, co-IP, fucosylation activity, and cryo-EM data further demonstrates that the N-terminal disordered peptide in SPY contains trans auto-fucosylation sites and inhibits the POFUT activity, whereas TPRs 1–5 dynamically regulate SPY activity by interfering with protein substrate binding.

SPINDLY mediates O-fucosylation of hundreds of proteins and sugar-dependent growth in Arabidopsis.

The recent discovery of SPINDLY (SPY)-catalyzed protein O-fucosylation revealed a novel mechanism for regulating nucleocytoplasmic protein functions in plants. Genetic evidence indicates the important roles of SPY in diverse developmental and physiological processes. However, the upstream signal controlling SPY activity and the downstream substrate proteins O-fucosylated by SPY remain largely unknown. Here, we demonstrated that SPY mediates sugar-dependent growth in Arabidopsis (Arabidopsis thaliana). We further identified hundreds of O-fucosylated proteins using lectin affinity chromatography followed by mass spectrometry. All the O-fucosylation events quantified in our proteomic analyses were undetectable or dramatically decreased in the spy mutants, and thus likely catalyzed by SPY. The O-fucosylome includes mostly nuclear and cytosolic proteins. Many O-fucosylated proteins function in essential cellular processes, phytohormone signaling, and developmental programs, consistent with the genetic functions of SPY. The O-fucosylome also includes many proteins modified by O-linked N-acetylglucosamine (O-GlcNAc) and by phosphorylation downstream of the target of rapamycin (TOR) kinase, revealing the convergence of these nutrient signaling pathways on key regulatory functions such as post-transcriptional/translational regulation and phytohormone responses. Our study identified numerous targets of SPY/O-fucosylation and potential nodes of crosstalk among sugar/nutrient signaling pathways, enabling future dissection of the signaling network that mediates sugar regulation of plant growth and development.

SPINDLY O-fucosylates nuclear and cytoplasmic proteins involved in diverse cellular processes in plants.

SPINDLY (SPY) is a novel nucleocytoplasmic protein O-fucosyltransferase that regulates target protein activity or stability via O-fucosylation of specific Ser/Thr residues. Previous genetic studies indicate that AtSPY regulates plant development during vegetative and reproductive growth by modulating gibberellin and cytokinin responses. AtSPY also regulates the circadian clock and plant responses to biotic and abiotic stresses. The pleiotropic phenotypes of spy mutants point to the likely role of AtSPY in regulating key proteins functioning in diverse cellular pathways. However, very few AtSPY targets are known. Here, we identified 88 SPY targets from Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana via the purification of O-fucosylated peptides using Aleuria aurantia lectin followed by electron transfer dissociation-MS/MS analysis. Most AtSPY targets were nuclear proteins that function in DNA repair, transcription, RNA splicing, and nucleocytoplasmic transport. Cytoplasmic AtSPY targets were involved in microtubule-mediated cell division/growth and protein folding. A comparison with the published O-linked-N-acetylglucosamine (O-GlcNAc) proteome revealed that 30% of AtSPY targets were also O-GlcNAcylated, indicating that these distinct glycosylations could co-regulate many protein functions. This study unveiled the roles of O-fucosylation in modulating many key nuclear and cytoplasmic proteins and provided a valuable resource for elucidating the regulatory mechanisms involved.

Structural insights into mechanism and specificity of the plant protein O-fucosyltransferase SPINDLY

Arabidopsis glycosyltransferase family 41 (GT41) protein SPINDLY (SPY) plays pleiotropic roles in plant development. Despite the amino acid sequence is similar to human O-GlcNAc transferase, Arabidopsis SPY has been identified as a novel nucleocytoplasmic protein O-fucosyltransferase. SPY-like proteins extensively exist in diverse organisms, indicating that O-fucosylation by SPY is a common way to regulate intracellular protein functions. However, the details of how SPY recognizes and glycosylates substrates are unknown. Here, we present a crystal structure of Arabidopsis SPY/GDP complex at 2.85 Å resolution. SPY adopts a head-to-tail dimer. Strikingly, the conformation of a ‘catalytic SPY’/GDP/‘substrate SPY’ complex formed by two symmetry-related SPY dimers is captured in the crystal lattice. The structure together with mutagenesis and enzymatic data demonstrate SPY can fucosylate itself and SPY’s self-fucosylation region negatively regulates its enzyme activity, reveal SPY’s substrate recognition and enzyme mechanism, and provide insights into the glycan donor substrate selection in GT41 proteins.

SPINDLY interacts with EIN2 to facilitate ethylene signalling-mediated fruit ripening in tomato.

The post-translational modification of proteins enables cells to respond promptly to dynamic stimuli by controlling protein functions. In higher plants, SPINDLY (SPY) and SECRET AGENT (SEC) are two prominent O-glycosylation enzymes that have both unique and overlapping roles; however, the effects of their O-glycosylation on fruit ripening and the underlying mechanisms remain largely unknown. Here we report that SlSPY affects tomato fruit ripening. Using slspy mutants and two SlSPY-OE lines, we provide biological evidence for the positive role of SlSPY in fruit ripening. We demonstrate that SlSPY regulates fruit ripening by changing the ethylene response in tomato. To further investigate the underlying mechanism, we identify a central regulator of ethylene signalling ETHYLENE INSENSITIVE 2 (EIN2) as a SlSPY interacting protein. SlSPY promotes the stability and nuclear accumulation of SlEIN2. Mass spectrometry analysis further identified that SlEIN2 has two potential sites Ser771 and Thr821 of O-glycans modifications. Further study shows that SlEIN2 is essential for SlSPY in regulating fruit ripening in tomatoes. Collectively, our findings reveal a novel regulatory function of SlSPY in fruit and provide novel insights into the role of the SlSPY-SlEIN2 module in tomato fruit ripening.

A cell wall-associated gene network shapes leaf boundary domains.

Boundary domains delimit and organize organ growth throughout plant development almost relentlessly, building plant architecture and morphogenesis. Boundary domains display reduced growth and orchestrate development of adjacent tissues in a non-cell-autonomous manner. How these two functions are achieved remains elusive despite the identification of several boundary-specific genes. Here, we show using morphometrics at the organ and cellular levels that leaf boundary domain development requires SPINDLY (SPY), an O-fucosyltransferase, to act as cell growth repressor. Furthermore, we show that SPY acts redundantly with the CUP-SHAPED COTYLEDON transcription factors (CUC2 and CUC3), which are major determinants of boundaries development. Accordingly, at the molecular level CUC2 and SPY repress a common set of genes involved in cell wall loosening, providing a molecular framework for the growth repression associated with boundary domains. Atomic force microscopy confirmed that young leaf boundary domain cells have stiffer cell walls than marginal outgrowth. This differential cell wall stiffness was reduced in spy mutant plants. Taken together, our data reveal a concealed CUC2 cell wall-associated gene network linking tissue patterning with cell growth and mechanics.

Selection of GDP‐fucose by active site residues in Arabidopsis thaliana SPY

O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) transferase (OGT) transfers O‐GlcNAc from the nucleotide‐sugar UDP‐GlcNAc to thousands of nuclear and cytoplasmic proteins. OGT is an essential enzyme, and misregulation of O‐GlcNAcylation has been linked to cancers or neurodegeneration. Most eukaryotic organisms possess a single structural homolog of OGT. However, Arabidopsis thaliana has two: Spindly (SPY) and Secret Agent (SEC). Whilst being structurally similar, SPY and SEC select different nucleotide sugars, GDP‐fucose and UDP‐GlcNAc, respectively. To determine which of SPY's active site amino acids govern GDP‐fucose selection, residues equivalent to those necessary for OGT's function were mutated and expressed in E. coli. Using the in vitro malachite green glycosyltransferase assay, the mutants’ activity was analyzed and compared to wildtype. One mutant displayed wild‐type levels of glycosylation, while another exhibited partial activity. The remaining three mutants lost glycosylation activity, suggesting essentiality for GDP‐fucose selection.

Identification and Characterization of Key Genes Responsible for Weedy and Cultivar Growth Types in Soybean.

In cultivated plants, shoot morphology is an important factor that influences crop economic value. However, the effects of gene expression patterns on shoot morphology are not clearly understood. In this study, the molecular mechanism behind shoot morphology (including leaf, stem, and node) was analyzed using RNA sequencing to compare weedy (creeper) and cultivar (stand) growth types obtained in F7 derived from a cross of wild and cultivated soybeans. A total of 12,513 (in leaves), 14,255 (in stems), and 11,850 (in nodes) differentially expressed genes were identified among weedy and cultivar soybeans. Comparative transcriptome and expression analyses revealed 22 phytohormone-responsive genes. We found that GIBBERELLIN 2-OXIDASE 8 (GA2ox), SPINDLY (SPY), FERONIA (FER), AUXIN RESPONSE FACTOR 8 (ARF8), CYTOKININ DEHYDROGENASE-1 (CKX1), and ARABIDOPSIS HISTIDINE KINASE-3 (AHK3), which are crucial phytohormone response genes, were mainly regulated in the shoot of weedy and cultivar types. These results indicate that interactions between phytohormone signaling genes regulate shoot morphology in weedy and cultivar growth type plants. Our study provides insights that are useful for breeding and improving crops to generate high-yield soybean varieties.

O-fucosylation of CPN20 by SPINDLY Derepresses Abscisic Acid Signaling During Seed Germination and Seedling Development.

SPINDLY is involved in some aspects of plant development. However, the nature of this protein as an O-fucosyltransferase was recently discovered. In this study, we show that SPINDLY (SPY) interacts with CPN20 in yeast two-hybrid and split-luc assays, and the interaction is promoted by ABA. CPN20 is a chloroplast-localized co-chaperonin that negatively regulates ABAR-mediated ABA signaling. By using Electron Transfer Dissociation-MS/MS analysis, two O-fucosylation sites, e.g., 116th and 119th threonines, were detected in ectopically expressed CPN20 in mammalian cells and in Arabidopsis. The O-fucosylation at both threonine residues was confirmed by in vitro peptide O-fucosylation assay. We further show that CPN20 accumulates in the chloroplast of spy mutants, suggesting that SPY negatively regulates CPN20 localization in the chloroplast. In vivo protein degradation assay along with CPN20 localization behavior suggest that import of CPN20 into the chloroplast is negatively regulated by SPY. Genetic analysis shows that ABA insensitive phenotypes of spy-3 in terms of seed germination and early seedling development are partially suppressed by the cpn20 mutation, suggesting that CPN20 acts downstream of SPY in this ABA signaling pathway and that there may exist other pathways in parallel with CPN20. Collectively, the above data support the notion that the O-fucosylation of CPN20 by SPY fine-tunes ABA signaling in Arabidopsis.

Ectopic expression of the Arabidopsis florigen gene FLOWERING LOCUS T in seeds enhances seed dormancy via the GA and DOG1 pathways.

Ectopic expression of specific genes in seeds could be a tool for molecular design of crops to alter seed dormancy and germination, thereby improving production. Here, a seed-specific vector, 12S-pLEELA, was applied to study the roles of genes in Arabidopsis seeds. Transgenic lines containing FLOWERING LOCUS T (FT) driven by the 12S promoter exhibited significantly increased seed dormancy and earlier flowering. Mutated FT(Y85H) and TERMINAL FLOWER1 (TFL1) transgenic lines also showed increased seed dormancy but without altered flowering time. FT(Y85H) and TFL1 caused weaker seed dormancy enhancement compared to FT. The FT and TFL1 transgenic lines showed hypersensitivity to paclobutrazol, but not to abscisic acid in seed germination. The levels of bioactive gibberellin 3 (GA3 ) and GA4 were significantly reduced, consistent with decreased expression of COPALYL DIPHOSPHATE SYNTHASE (CPS), KAURENE OXIDASE (KO), GIBBERELLIN 3-OXIDASE2 (GA3ox2), and GA20ox1 in p12S::FT lines. Exogenous GA4+7 could recover the germination ability of FT transgenic lines. These results revealed that FT regulates GA biosynthesis. A genetic analysis indicated that the GA signaling regulator SPINDLY (SPY) is epistatic to FT in GA-mediated seed germination. Furthermore, DELAY OF GERMINATION1 (DOG1) showed significantly higher transcript levels in p12S::FT lines. Seed dormancy analysis of dog1-2 spy-3 p12S::FT-2 indicated that the combination of SPY and DOG1 is epistatic to FT in the regulation of dormancy. Overall, we showed that ectopic expression of FT and TFL1 in seeds enhances dormancy through affecting GA and DOG1 pathways.

Balancing O-GlcNAc and O-fucose in plants.

O-linked modification of nuclear and cytosolic proteins with monosaccharides is essential in all eukaryotes. While many aspects of this post-translational modification are highly conserved, there are striking differences between plants and the animal kingdom. In animals, dynamic cycling of O-GlcNAc is established by two essential single copy enzymes, the O-GlcNAc transferase OGT and O-GlcNAc hydrolase OGA. In contrast, plants balance O-GlcNAc with O-fucose modifications, catalyzed by the OGT SECRET AGENT (SEC) and the protein O-fucosyltransferase (POFUT) SPINDLY (SPY). However, specific glycoside hydrolases for either of the two modifications have not yet been identified. Nucleocytoplasmic O-glycosylation is still not very well understood in plants, even though a high number of proteins were found to be affected. One important open question is how specificity is established in a system where only two enzymes modify hundreds of proteins. Here, we discuss the possibility that O-GlcNAc- and O-fucose-binding proteins could introduce an additional flexible layer of regulation in O-glycosylation-mediated signaling pathways, with the potential of integrating internal or external signals.

Arabidopsis ACINUS is O-glycosylated and regulates transcription and alternative splicing of regulators of reproductive transitions

O-GlcNAc modification plays important roles in metabolic regulation of cellular status. Two homologs of O-GlcNAc transferase, SECRET AGENT (SEC) and SPINDLY (SPY), which have O-GlcNAc and O-fucosyl transferase activities, respectively, are essential in Arabidopsis but have largely unknown cellular targets. Here we show that AtACINUS is O-GlcNAcylated and O-fucosylated and mediates regulation of transcription, alternative splicing (AS), and developmental transitions. Knocking-out both AtACINUS and its distant paralog AtPININ causes severe growth defects including dwarfism, delayed seed germination and flowering, and abscisic acid (ABA) hypersensitivity. Transcriptomic and protein-DNA/RNA interaction analyses demonstrate that AtACINUS represses transcription of the flowering repressor FLC and mediates AS of ABH1 and HAB1, two negative regulators of ABA signaling. Proteomic analyses show AtACINUS’s O-GlcNAcylation, O-fucosylation, and association with splicing factors, chromatin remodelers, and transcriptional regulators. Some AtACINUS/AtPININ-dependent AS events are altered in the sec and spy mutants, demonstrating a function of O-glycosylation in regulating alternative RNA splicing.

The Arabidopsis O-fucosyltransferase SPINDLY regulates root hair patterning independently of gibberellin signaling.

ABSTRACTRoot hairs are able to sense soil composition and play an important role in water and nutrient uptake. In Arabidopsis thaliana, root hairs are distributed in the epidermis in a specific pattern, regularly alternating with non-root hair cells in continuous cell files. This patterning is regulated by internal factors such as a number of hormones, as well as by external factors like nutrient availability. Thus, root hair patterning is an excellent model for studying the plasticity of cell fate determination in response to environmental changes. Here, we report that loss-of-function mutants for the Protein O-fucosyltransferase SPINDLY (SPY) show defects in root hair patterning. Using transcriptional reporters, we show that patterning in spy-22 is affected upstream of GLABRA2 (GL2) and WEREWOLF (WER). O-fucosylation of nuclear and cytosolic proteins is an important post-translational modification that is still not very well understood. So far, SPY is best characterized for its role in gibberellin signaling via fucosylation of the growth-repressing DELLA protein REPRESSOR OF ga1-3 (RGA). Our data suggest that the epidermal patterning defects in spy-22 are independent of RGA and gibberellin signaling.

Nuclear Localized O-Fucosyltransferase SPY Facilitates PRR5 Proteolysis to Fine-Tune the Pace of Arabidopsis Circadian Clock

Post-translational modifications play essential roles in finely modulating eukaryotic circadian clock systems. In plants, the effects of O-glycosylation on the circadian clock and the underlying mechanisms remain largely unknown. The O-fucosyltransferase SPINDLY (SPY) and the O-GlcNAc transferase SECRET AGENT (SEC) are two prominent O-glycosylation enzymes in higher plants, with both overlapped and unique functions in plant growth and development. Unlike the critical role of O-GlcNAc in regulating the animal circadian clock, here we report that nuclear-localized SPY, but not SEC, specifically modulates the pace of the Arabidopsis circadian clock. By identifying the interactome of SPY, we identified PSEUDO-RESPONSE REGULATOR 5 (PRR5), one of the core circadian clock components, as a new SPY-interacting protein. PRR5 can be O-fucosylated by SPY in planta, while point mutation in the catalytic domain of SPY abolishes the O-fucosylation of PRR5. The protein abundance of PRR5 is strongly increased in spy mutants, while the degradation rate of PRR5 is much reduced, suggesting that PRR5 proteolysis is promoted by SPY-mediated O-fucosylation. Moreover, multiple lines of genetic evidence indicate that PRR5 is a major downstream target of SPY to specifically mediate its modulation of the circadian clock. Collectively, our findings provide novel insights into the specific role of the O-fucosyltransferase activity of SPY in modulating the circadian clock and implicate that O-glycosylation might play an evolutionarily conserved role in modulating the circadian clock system, via O-GlcNAcylation in mammals, but via O-fucosylation in higher plants.

A New Insight into Flowering Regulation: Molecular Basis of Flowering Initiation in Magnolia × soulangeana 'Changchun'.

Magnolia × soulangeana ‘Changchun’ are trees that bloom in spring and summer respectively after flower bud differentiation. Here, we use phenological and morphological observation and RNA-seq technology to study the molecular basis of flowering initiation in ‘Changchun’. During the process of flowering initiation in spring and summer, the growth of expanded flower buds increased significantly, and their shape was obviously enlarged, which indicated that flowering was initiated. A total of 168,120 expressed genes were identified in spring and summer dormant and expanded flower buds, of which 11,687 genes showed significantly differential expression between spring and summer dormant and expanded flower buds. These differentially expressed genes (DEGs) were mainly involved in plant hormone signal transduction, metabolic processes, cellular components, binding, and catalytic activity. Analysis of differential gene expression patterns revealed that gibberellin signaling, and some transcription factors were closely involved in the regulation of spring and summer flowering initiation in ‘Changchun’. A qRT-PCR (quantitative Real Time Polymerase Chain Reaction) analysis showed that BGISEQ-500 sequencing platform could truly reflect gene expression patterns. It also verified that GID1B (GIBBERELLIN INSENSITIVE DWARF1 B), GID1C, SPL8 (SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 8), and GASA (GIBBERELLIC ACID-STIMULATED ARABIDOPSIS) family genes were expressed at high levels, while the expression of SPY (SPINDLY) was low during spring and summer flowering initiation. Meanwhile, the up- and down-regulated expression of, respectively, AGL6 (AGAMOUS-LIKE 6) and DREB3 (DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 3), AG15, and CDF1 (CYCLIC DOF FACTOR 1) might also be involved in the specific regulation of spring and summer flowering initiation. Obviously, flowering initiation is an important stage of the flowering process in woody plants, involving the specific regulation of relevant genes and transcription factors. This study provides a new perspective for the regulation of the flowering process in perennial woody plants.

SPINDLY is involved in ABA signaling bypassing the PYR/PYLs/RCARs-mediated pathway and partly through functional ABAR

SPINDLY (SPY) is a putative O-linked β-N-acetyl glucosamine transferase and an O-fucosyltransferase, involved in various signaling pathways. We report here that SPY is involved in ABA signaling. spy mutants have insensitive phenotypes in response to ABA in seed germination and early seedling development. SPY has a complex relationship with ABI5, ABI3 and ABI4, which are key transcription factors in ABA-mediated seed germination and early seedling development. This relationship is dependent of developmental stage. Quadruple mutants ♀spy-3♂pyl1-1 pyl4-1 pyr1-1 and ♀pyl1-1 pyl4-1 pyr1-1♂spy-3 display enhanced insensitivity to ABA compared with either pyl1-1/pyl4-1/pyr1-1 or spy-3 in terms of seed germination and cotyledon greening. In RNA-seq assay, the general transcription level of germinating seeds of spy-3 under ABA is up-regulated compared with that of Col-0. The transcription level of ABAR，another ABA receptor spanning the chloroplast envelope, is up-regulated in spy-3 whereas another two types of putative ABA receptors ABAR and GTG1/GTG2 remain largely unaltered. As uncovered by genetic analyses, insensitivity to ABA of spy-3 is hindered by cch, a mutant of ABAR, in terms of seed germination and cotyledon greening, and vice versa. Genetic analyses also reveal that SPY regulates chloroplast function and leaf development via ABAR. Altogether, we conclude that SPY is involved in ABA signaling bypassing PYR-mediated pathway but likely playing a role in ABA signaling partly via the ABAR-mediated pathway.

A rapid virus-induced gene silencing (VIGS) method for assessing resistance and susceptibility to cassava mosaic disease

BackgroundCassava mosaic disease (CMD) is a major constraint to cassava production in sub-Saharan Africa. Under field conditions, evaluation for resistance to CMD takes 12–18 months, often conducted across multiple years and locations under pressure from whitefly-mediated transmission. Under greenhouse or laboratory settings, evaluation for resistance or susceptibility to CMD involves transmission of the causal viruses from an infected source to healthy plants through grafting, or by using Agrobacterium-mediated or biolistic delivery of infectious clones. Following inoculation, visual assessment for CMD symptom development and recovery requires 12–22 weeks. Here we report a rapid screening system for determining resistance and susceptibility to CMD based on virus-induced gene silencing (VIGS) of an endogenous cassava gene.ResultsA VIGS vector was developed based on an infectious clone of the virulent strain of East African cassava mosaic virus (EACMV-K201). A sequence from the cassava (Manihot esculenta) ortholog of Arabidopsis SPINDLY (SPY) was cloned into the CP position of the DNA-A genomic component and used to inoculate cassava plants by Helios® Gene Gun microparticle bombardment. Silencing of Manihot esculenta SPY (MeSPY) using MeSPY1-VIGS resulted in shoot-tip necrosis followed by death of the whole plant in CMD susceptible cassava plants within 2–4 weeks. CMD resistant cultivars were not affected and remained healthy after challenge with MeSPY1-VIGS. Significantly higher virus titers were detected in CMD-susceptible cassava lines compared to resistant controls and were correlated with a concomitant reduction in MeSPY expression in susceptible plants.ConclusionsA rapid VIGS-based screening system was developed for assessing resistance and susceptibility to CMD. The method is space and resource efficient, reducing the time required to perform CMD screening to as little as 2–4 weeks. It can be employed as a high throughput rapid screening system to assess new cassava cultivars and for screening transgenic, gene edited and breeding lines under controlled growth conditions.