Reprogramming of sorghum proteome in response to sugarcane aphid infestation

Sugarcane aphid (SCA; Melanaphis sacchari Zehntner) is a key piercing-sucking pest of sorghum (Sorghum bicolor) that cause significant yield losses. While feeding on host plants, complex signaling networks are invoked from recognition of insect attack to induction of plant defenses. Consequently, these signaling networks lead to the production of insecticidal compounds or limited access of nutrients to insects. Previously, several studies were published on the transcriptomics analysis of sorghum in response to SCA infestation, but no information is available on the physiological changes of sorghum at the proteome level. We used the SCA resistant sorghum genotype SC265 for the global proteomics analysis after 1 and 7 days of SCA infestation using the TMT-plex technique. Peptides matching a total of 4211 proteins were identified and 158 proteins were differentially expressed at day 1 and 7. Overall, proteome profiling of SC265 after SCA infestation at days 1 and 7 revealed the suppression of plant defense-related proteins and upregulation of plant defense and signaling-related proteins, respectively. The plant defense responses based on proteome data were validated using electrical penetration graph (EPG) technique to observe changes in aphid feeding. Feeding behavior analyses revealed that SCA spent significantly longer time in phloem phase on SCA infested plants for day 1 and lesser time in day 7 SCA infested sorghum plants, compared to their respective control plants. Overall, our study provides insights into underlying mechanisms that contribute to sorghum resistance to SCA

Functional requirements for a Samd14-capping protein complex in stress erythropoiesis

Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes. Paracrine signals—involving cooperation between stem cell factor (SCF)/Kit signaling and other signaling inputs—are required for the increased erythroid precursor activity in anemia. Our prior work revealed that the sterile alpha motif (SAM) domain 14 (Samd14) gene increases the regenerative capacity of the erythroid system in a mouse genetic model and promotes stress-dependent Kit signaling. However, the mechanism underlying Samd14’s role in stress erythropoiesis is unknown. We identified a protein-protein interaction between Samd14 and the α- and β-heterodimers of the F-actin capping protein (CP) complex. Knockdown of the CP β subunit increased erythroid maturation in murine ex vivo cultures and decreased colony forming potential of stress erythroid precursors. In a genetic complementation assay for Samd14 activity, our results revealed that the Samd14-CP interaction is a determinant of erythroid precursor cell levels and function. Samd14-CP promotes SCF/Kit signaling in CD71med spleen erythroid precursors. Given the roles of Kit signaling in hematopoiesis and Samd14 in Kit pathway activation, this mechanism may have pathological implications in acute/chronic anemia

LncRNA Meg3 protects endothelial function by regulating the DNA damage response

The role of long non-coding RNAs (lncRNAs) in regulating endothelial function through the DNA damage response (DDR) remains poorly understood. In this study, we demonstrate that lncRNA maternally expressed gene 3 (Meg3) interacts with the RNA binding protein polypyrimidine tract binding protein 3 (PTBP3) to regulate gene expression and endothelial function through p53 signaling a major coordinator of apoptosis and cell proliferation triggered by the DDR. Meg3 expression is induced in endothelial cells (ECs) upon p53 activation. Meg3 silencing induces DNA damage, activates p53 signaling, increases the expression of p53 target genes, promotes EC apoptosis, and inhibits EC proliferation. Mechanistically, Meg3 silencing reduces the interaction of p53 with Mdm2, induces p53 expression, and promotes the association of p53 with the promoters of a subset of p53 target genes. PTBP3 silencing recapitulates the effects of Meg3 deficiency on the expression of p53 target genes, EC apoptosis and proliferation. The Meg3-dependent association of PTBP3 with the promoters of p53 target genes suggests that Meg3 and PTBP3 restrain p53 activation. Our studies reveal a novel role of Meg3 and PTBP3 in regulating p53 signaling and endothelial function, which may serve as novel targets for therapies to restore endothelial homeostasis

Variation in morpho‑physiological and metabolic responses to low nitrogen stress across the sorghum association panel

Background: Access to biologically available nitrogen is a key constraint on plant growth in both natural and agricultural settings. Variation in tolerance to nitrogen deficit stress and productivity in nitrogen limited conditions exists both within and between plant species. However, our understanding of changes in different phenotypes under long term low nitrogen stress and their impact on important agronomic traits, such as yield, is still limited. Results: Here we quantified variation in the metabolic, physiological, and morphological responses of a sorghum association panel assembled to represent global genetic diversity to long term, nitrogen deficit stress and the relationship of these responses to grain yield under both conditions. Grain yield exhibits substantial genotype by environment interaction while many other morphological and physiological traits exhibited consistent responses to nitrogen stress across the population. Large scale nontargeted metabolic profiling for a subset of lines in both conditions identified a range of metabolic responses to long term nitrogen deficit stress. Several metabolites were associated with yield under high and low nitrogen conditions. Conclusion: Our results highlight that grain yield in sorghum, unlike many morpho-physiological traits, exhibits substantial variability of genotype specific responses to long term low severity nitrogen deficit stress. Metabolic response to long term nitrogen stress shown higher proportion of variability explained by genotype specific responses than did morpho-pysiological traits and several metabolites were correlated with yield. This suggest, that it might be possible to build predictive models using metabolite abundance to estimate which sorghum genotypes will exhibit greater or lesser decreases in yield in response to nitrogen deficit, however further research needs to be done to evaluate such model

\u3ci\u3eFusarium graminearum\u3c/i\u3e Effector FgNls1 Targets Plant Nuclei to Induce Wheat Head Blight

Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most devastating diseases of wheat and barley worldwide. Effectors suppress host immunity and promote disease development. The genome of F. graminearum contains hundreds of effectors with unknown function. Therefore, investigations of the functions of these effectors will facilitate developing novel strategies to enhance wheat resistance to FHB. We characterized a F. graminearum effector, FgNls1, containing a signal peptide and multiple eukaryotic nuclear localization signals. A fusion protein of green fluorescent protein and FgNls1 accumulated in plant cell nucleiwhen transiently expressed in Nicotiana benthamiana. FgNls1 suppressed Bax-induced cell death when co-expressed in N. benthamiana.We revealed that the expression of FgNLS1 was induced in wheat spikes infected with F. graminearum. The Fgnls1 mutants significantly reduced initial infection andFHBspread within a spike.The function of FgNLS1 was restored in the Fgnls1-complemented strains.Wheat histone 2B was identified as an interacting protein by FgNls1-affinity chromatography. Furthermore, transgenic wheat plants that silence FgNLS1 expression had significantly lower FHB severity than control plants. This study demonstrates a critical role of FgNls1 in F. graminearum pathogenesis and indicates that host-induced gene silencing targeting F. graminearum effectors is a promising approach to enhance FHB resistance

Lipid Droplet Membrane Proteome Remodeling Parallels Ethanol-Induced Hepatic Steatosis and its Resolution

Lipid droplets (LDs) are composed of neutral lipids enclosed in a phospholipid monolayer, which harbors membrane-associated proteins that regulate LD functions. Despite the crucial role of LDs in lipid metabolism, remodeling of LD protein composition in disease contexts, such as steatosis, remains poorly understood. We hypothesized that chronic ethanol consumption, subsequent abstinence from ethanol, or fasting differentially affects the LD membrane proteome content and that these changes influence how LDs interact with other intracellular organelles. Here, male Wistar rats were pair-fed liquid control or ethanol diets for 6 weeks, and then, randomly chosen animals from both groups were either refed a control diet for 7 days or fasted for 48 h before euthanizing. From all groups, LD membrane proteins from purified liver LDs were analyzed immunochemically and by MS proteomics. Liver LD numbers and sizes were greater in ethanol-fed rats than in pair-fed control, 7-day refed, or fasted rats. Compared with control rats, ethanol feeding markedly altered the LD membrane proteome, enriching LD structural perilipins and proteins involved in lipid biosynthesis, while lowering LD lipase levels. Ethanol feeding also lowered LD-associated mitochondrial and lysosomal proteins. In 7-day refed (i.e., ethanol-abstained) or fasted-ethanol-fed rats, we detected distinct remodeling of the LD proteome, as judged by lower levels of lipid biosynthetic proteins, and enhanced LD interaction with mitochondria and lysosomes. Our study reveals evidence of significant remodeling of the LD membrane proteome that regulates ethanol-induced steatosis, its resolution after withdrawal and abstinence, and changes in LD interactions with other intracellular organelles

Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry

Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling pathways

Lipid droplet membrane proteome remodeling parallels ethanol-induced hepatic steatosis and its resolution

Abstract Lipid droplets (LDs) are composed of neutral lipids enclosed in a phospholipid monolayer, which harbors membrane-associated proteins that regulate LD functions. Despite the crucial role of LDs in lipid metabolism, remodeling of LD protein composition in disease contexts, such as steatosis, remains poorly understood. We hypothesized that chronic ethanol consumption, subsequent abstinence from ethanol, or fasting differentially affects the LD membrane proteome content and that these changes influence how LDs interact with other intracellular organelles. Here, male Wistar rats were pair-fed liquid control or ethanol diets for 6 weeks, and then, randomly chosen animals from both groups were either refed a control diet for 7 days or fasted for 48 h before euthanizing. From all groups, LD membrane proteins from purified liver LDs were analyzed immunochemically and by MS proteomics. Liver LD numbers and sizes were greater in ethanolfed rats than in pair-fed control, 7-day refed, or fasted rats. Compared with control rats, ethanol feeding markedly altered the LD membrane proteome, enriching LD structural perilipins and proteins involved in lipid biosynthesis, while lowering LD lipase levels. Ethanol feeding also lowered LDassociated mitochondrial and lysosomal proteins. In 7-day refed (i.e., ethanol-abstained) or fasted-ethanolfed rats, we detected distinct remodeling of the LD proteome, as judged by lower levels of lipid biosynthetic proteins, and enhanced LD interaction with mitochondria and lysosomes. Our study reveals evidence of significant remodeling of the LD membrane proteome that regulates ethanol-induced steatosis, its resolution after withdrawal and abstinence, and changes in LD interactions with other intracellular organelles

Delineating the role of FANCA in glucose-stimulated insulin secretion in β cells through its protein interactome

Hyperinsulinemia affects 72% of Fanconi anemia (FA) patients and an additional 25% experience lowered glucose tolerance or frank diabetes. The underlying molecular mechanisms contributing to the dysfunction of FA pancreas β cells is unknown. Therefore, we sought to evaluate the functional role of FANCA, the most commonly mutated gene in FA, in glucosestimulated insulin secretion (GSIS). This study reveals that FANCA or FANCB knockdown impairs GSIS in human pancreas β cell line EndoC-βH3. To identify potential pathways by which FANCA might regulate GSIS, we employed a proteomics approach to identify FANCA protein interactions in EndoC-βH3 differentially regulated in response to elevated glucose levels. Glucose-dependent changes in the FANCA interaction network were observed, including increased association with other FA family proteins, suggesting an activation of the DNA damage response in response to elevated glucose levels. Reactive oxygen species increase in response to glucose stimulation and are necessary for GSIS in EndoC-βH3 cells. Glucose-induced activation of the DNA damage response was also observed as an increase in the DNA damage foci marker γ-H2AX and dependent upon the presence of reactive oxygen species. These results illuminate the role of FANCA in GSIS and its protein interactions regulated by glucose stimulation that may explain the prevalence of β cell-specific endocrinopathies in FA patients