A stress inducible SUMO conjugating enzyme gene (SaSce9) from a grass halophyte Spartina alterniflora enhances salinity and drought stress tolerance in Arabidopsis

BACKGROUND: SUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulatory process found in all eukaryotes, mediated by SUMO activating enzyme, SUMO conjugating enzyme, and SUMO ligase for the attachment of SUMO to its target protein. Although the mechanism for regulation of SUMO conjugation pathway genes under abiotic stress has been studied to certain extent, the role of SUMO conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. Here, we have characterized a SUMO conjugating enzyme gene ‘SaSce9’ from a halophytic grass Spartina alterniflora and investigated its role in imparting abiotic stress tolerance. RESULTS: SaSce9 gene encodes for a polypeptide of 162 amino acids with a molecular weight of ~18 kD and isoelectric point 8.43. Amino acid sequence comparisons of SaSce9 with its orthologs from other plant species showed high degree (~85-93%) of structural conservation among each other. Complementation analysis using yeast SCE mutant, Ubc9, revealed functional conservation of SaSce9 between yeast and S. alterniflora. SaSce9 transcript was inducible by salinity, drought, cold, and exogenously supplied ABA both in leaves and roots of S. alterniflora. Constitutive overexpression of SaSce9 in Arabidopsis through Agrobacterium mediated transformation improved salinity and drought tolerance of Arabidopsis. SaSce9 overexpressing Arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. SaSce9 transgenic plants accumulated lower levels of reactive oxygen under salinity stress. Expression analysis of stress responsive genes in SaSce9 Arabidopsis plants revealed the increased expression of antioxidant genes, AtSOD and AtCAT, ion antiporter genes, AtNHX1 and AtSOS1, a gene involved in proline biosynthesis, AtP5CS, and a gene involved in ABA dependent signaling pathway, AtRD22. CONCLUSIONS: These results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. The study provides evidence that the overexpression of SaSce9 in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ROS, accumulation of an osmolyte, proline, and expression of stress responsive genes. In addition, this study demonstrates the potential of the halophyte grass S. alterniflora as a reservoir of abiotic stress related genes for crop improvement

Evidence for Directed Evolution of Larger Size Motif in Arabidopsis thaliana Genome

Transcription control of gene expression depends on a variety of interactions mediated by the core promoter region, sequence specific DNA-binding proteins, and their cognate promoter elements. The prominent group of cis acting elements in plants contains an ACGT core. The cis element with this core has been shown to be involved in abscisic acid, salicylic acid, and light response. In this study, genome-wide comparison of the frequency of occurrence of two ACGT elements without any spacers as well as those separated by spacers of different length was carried out. In the first step, the frequency of occurrence of the cis element sequences across the whole genome was determined by using BLAST tool. In another approach the spacer sequence was randomized before making the query. As expected, the sequence ACGTACGT had maximum occurrence in Arabidopsis thaliana genome. As we increased the spacer length, one nucleotide at a time, the probability of its occurrence in genome decreased. This trend continued until an unexpectedly sharp rise in frequency of (ACGT)N25(ACGT). The observation of higher probability of bigger size motif suggests its directed evolution in Arabidopsis thaliana genome

CRISPR/Cas9-mediated multi-allelic gene targeting in sugarcane confers herbicide tolerance

Sugarcane is the source of 80% of the sugar and 26% of the bioethanol produced globally. However, its complex, highly polyploid genome (2n = 100 – 120) impedes crop improvement. Here, we report efficient and reproducible gene targeting (GT) in sugarcane, enabling precise co-editing of multiple alleles via template-mediated and homology-directed repair (HDR) of DNA double strand breaks induced by the programmable nuclease CRISPR/Cas9. The evaluation of 146 independently transformed plants from five independent experiments revealed a targeted nucleotide replacement that resulted in both targeted amino acid substitutions W574L and S653I in the acetolactate synthase (ALS) in 11 lines in addition to single, targeted amino acid substitutions W574L or S653I in 25 or 18 lines, respectively. Co-editing of up to three ALS copies/alleles that confer herbicide tolerance was confirmed by Sanger sequencing of cloned long polymerase chain reaction (PCR) amplicons. This work will enable crop improvement by conversion of inferior alleles to superior alleles through targeted nucleotide substitutions

Molecular Detection Method Developed to Track the Koinobiont Larval Parasitoid Apanteles opuntiarum (Hymenoptera: Braconidae) Imported from Argentina to Control Cactoblastis cactorum (Lepidoptera: Pyralidae)

Apanteles opuntiarum Martínez & Berta (Hymenoptera: Braconidae) is a native natural enemy of the cactus moth, Cactoblastis cactorum Berg (Lepidoptera: Pyralidae) in Argentina, where the 2 species are believed to have co-evolved. Cactoblastis cactorum is an established invasive pest in the US that is rapidly spreading throughout the southeast. Apanteles opuntiarum was imported from Argentina, and reared at the Division of Plant Industry containment facility in Gainesville, Florida, for study as a possible biocontrol agent for release in the US to control C. cactorum. A DNA barcode was developed to enable the identification of the reared parasitoid population. The mitochondrial cytochrome oxidase I (COI) gene of the A. opuntiarum reared in Florida containment was found to be identical to its Argentine founders, but distinctly different from the COI sequences of all other reported Apanteles species in the NCBI (National Center for Biotechnology Information) GenBank. Additionally, the AoF1 and AoR1 primer pair developed in this study specifically amplified the COI gene of A. opuntiarum, but did not amplify the COI gene of the host C. cactorum. Therefore, the COI gene fragment identified in this study has the potential to be used as a DNA barcode specific to A. opuntiarum that can aid in tracking and identifying this parasitoid inside hosts.Apanteles opuntiarum Martínez & Berta (Hymenoptera: Braconidae) es un enemigo natural de la polilla de la tuna Cactoblastis cactorum Berg (Lepidoptera: Pyralidae) en Argentina, su rango nativo, donde han co-evolucionado. Cactoblastis cactorum es una especie invasora establecida en Estados Unidos, que se está dispersando rápidamente hacia el sudeste de este país. Apanteles opuntiarum fue importado desde Argentina y es criado en la cuarentena de Gainesville, Florida (Division of Plant Industry), donde está siendo evaluado como posible agente de control de C. cactorum, para ser liberado en Estados Unidos. Se desarrolló un código de barras de ADN para permitir la identificación de la población de parasitoides criada. Se encontró que el gen de la citocromo oxidasa mitocondrial I (COI) de los A. opuntiarum criados en Florida fue idéntico al de sus fundadores argentinos, y claramente diferente de las secuencias de COI de todas las demás especies de Apanteles reportados en el GenBank del NCBI (Centro Nacional de información sobre biotecnología). Además, el par “primer” AoF1 y AoR1 desarrollado en este estudio amplificó específicamente el gen COI de A. opuntiarum, y no amplificó el gen de la COI del hospedador C. cactorum. Por lo tanto, el fragmento del gen COI identificado en este estudio tiene el potencial para ser utilizados como un código de barras de ADN específico para A. opuntiarum que puede ayudar en el seguimiento y la identificación de este parasitoide dentro de los hospedadoresFil: Srivastava, Mrittunjai. Florida Department of Agriculture and Consumer Services; Estados UnidosFil: Srivastava, Pratibha. Florida Department of Agriculture and Consumer Services; Estados UnidosFil: Karan, Ratna. Florida Department of Agriculture and Consumer Services; Estados UnidosFil: Jeyaprakash, Ayyamperumal. Florida Department of Agriculture and Consumer Services; Estados UnidosFil: Whilby, Leroy. Florida Department of Agriculture and Consumer Services; Estados UnidosFil: Rohrig, Eric. Florida Department of Agriculture and Consumer Services; Estados UnidosFil: Howe, Amy C.. Florida Department of Agriculture and Consumer Services; Estados UnidosFil: Hight, Stephen D.. United States Department of Agriculture. Agriculture Research Service; Estados UnidosFil: Varone, Laura. Fundación para el Estudio de Especies Invasivas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Author Correction: Cancer Testis Antigen Promotes Triple Negative Breast Cancer Metastasis and is Traceable in the Circulating Extracellular Vesicles (Scientific Reports, (2019), 9, 1, (11632), 10.1038/s41598-019-48064-w)

Triple negative breast cancer (TNBC) has poor survival, exhibits rapid metastases, lacks targeted therapies and reliable prognostic markers. Here, we examined metastasis promoting role of cancer testis antigen SPANXB1 in TNBC and its utility as a therapeutic target and prognostic biomarker. Expression pattern of SPANXB1 was determined using matched primary cancer, lymph node metastatic tissues and circulating small extracellular vesicles (sEVs). cDNA microarray analysis of TNBC cells stably integrated with a metastasis suppressor SH3GL2 identified SPANXB1 as a potential target gene. TNBC cells overexpressing SH3GL2 exhibited decreased levels of both SPANXB1 mRNA and protein. Silencing of SPANXB1 reduced migration, invasion and reactive oxygen species production of TNBC cells. SPANXB1 depletion augmented SH3GL2 expression and decreased RAC-1, FAK, A-Actinin and Vinculin expression. Phenotypic and molecular changes were reversed upon SPANXB1 re-expression. SPANXB1 overexpressing breast cancer cells with an enhanced SPANXB1:SH3GL2 ratio achieved pulmonary metastasis within 5 weeks, whereas controls cells failed to do so. Altered expression of SPANXB1 was detected in the sEVs of SPANXB1 transduced cells. Exclusive expression of SPANXB1 was traceable in circulating sEVs, which was associated with TNBC progression. SPANXB1 represents a novel and ideal therapeutic target for blocking TNBC metastases due to its unique expression pattern and may function as an EV based prognostic marker to improve TNBC survival. Uniquely restricted expression of SPANXB1 in TNBCs, makes it an ideal candidate for targeted therapeutics and prognostication

Salt Stress Induced Variation in DNA Methylation Pattern and Its Influence on Gene Expression in Contrasting Rice Genotypes

BACKGROUND: Salinity is a major environmental factor limiting productivity of crop plants including rice in which wide range of natural variability exists. Although recent evidences implicate epigenetic mechanisms for modulating the gene expression in plants under environmental stresses, epigenetic changes and their functional consequences under salinity stress in rice are underexplored. DNA methylation is one of the epigenetic mechanisms regulating gene expression in plant's responses to environmental stresses. Better understanding of epigenetic regulation of plant growth and response to environmental stresses may create novel heritable variation for crop improvement. METHODOLOGY/PRINCIPAL FINDINGS: Methylation sensitive amplification polymorphism (MSAP) technique was used to assess the effect of salt stress on extent and patterns of DNA methylation in four genotypes of rice differing in the degree of salinity tolerance. Overall, the amount of DNA methylation was more in shoot compared to root and the contribution of fully methylated loci was always more than hemi-methylated loci. Sequencing of ten randomly selected MSAP fragments indicated gene-body specific DNA methylation of retrotransposons, stress responsive genes, and chromatin modification genes, distributed on different rice chromosomes. Bisulphite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied with genotypes and tissue types irrespective of the level of salinity tolerance of rice genotypes. CONCLUSIONS/SIGNIFICANCE: The gene body methylation may have an important role in regulating gene expression in organ and genotype specific manner under salinity stress. Association between salt tolerance and methylation changes observed in some cases suggested that many methylation changes are not "directed". The natural genetic variation for salt tolerance observed in rice germplasm may be independent of the extent and pattern of DNA methylation which may have been induced by abiotic stress followed by accumulation through the natural selection process

Post-intervention Status in Patients With Refractory Myasthenia Gravis Treated With Eculizumab During REGAIN and Its Open-Label Extension

OBJECTIVE: To evaluate whether eculizumab helps patients with anti-acetylcholine receptor-positive (AChR+) refractory generalized myasthenia gravis (gMG) achieve the Myasthenia Gravis Foundation of America (MGFA) post-intervention status of minimal manifestations (MM), we assessed patients' status throughout REGAIN (Safety and Efficacy of Eculizumab in AChR+ Refractory Generalized Myasthenia Gravis) and its open-label extension. METHODS: Patients who completed the REGAIN randomized controlled trial and continued into the open-label extension were included in this tertiary endpoint analysis. Patients were assessed for the MGFA post-intervention status of improved, unchanged, worse, MM, and pharmacologic remission at defined time points during REGAIN and through week 130 of the open-label study. RESULTS: A total of 117 patients completed REGAIN and continued into the open-label study (eculizumab/eculizumab: 56; placebo/eculizumab: 61). At week 26 of REGAIN, more eculizumab-treated patients than placebo-treated patients achieved a status of improved (60.7% vs 41.7%) or MM (25.0% vs 13.3%; common OR: 2.3; 95% CI: 1.1-4.5). After 130 weeks of eculizumab treatment, 88.0% of patients achieved improved status and 57.3% of patients achieved MM status. The safety profile of eculizumab was consistent with its known profile and no new safety signals were detected. CONCLUSION: Eculizumab led to rapid and sustained achievement of MM in patients with AChR+ refractory gMG. These findings support the use of eculizumab in this previously difficult-to-treat patient population. CLINICALTRIALSGOV IDENTIFIER: REGAIN, NCT01997229; REGAIN open-label extension, NCT02301624. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that, after 26 weeks of eculizumab treatment, 25.0% of adults with AChR+ refractory gMG achieved MM, compared with 13.3% who received placebo

Minimal Symptom Expression' in Patients With Acetylcholine Receptor Antibody-Positive Refractory Generalized Myasthenia Gravis Treated With Eculizumab

The efficacy and tolerability of eculizumab were assessed in REGAIN, a 26-week, phase 3, randomized, double-blind, placebo-controlled study in anti-acetylcholine receptor antibody-positive (AChR+) refractory generalized myasthenia gravis (gMG), and its open-label extension

A stress inducible SUMO conjugating enzyme gene (<it>SaSce9</it>) from a grass halophyte <it>Spartina alterniflora</it> enhances salinity and drought stress tolerance in <it>Arabidopsis</it>

Abstract Background SUMO (Small Ubiquitin related Modifier) conjugation is a post translational regulatory process found in all eukaryotes, mediated by SUMO activating enzyme, SUMO conjugating enzyme, and SUMO ligase for the attachment of SUMO to its target protein. Although the mechanism for regulation of SUMO conjugation pathway genes under abiotic stress has been studied to certain extent, the role of SUMO conjugating enzyme in improving abiotic stress tolerance to plant is largely unexplored. Here, we have characterized a SUMO conjugating enzyme gene ‘SaSce9’ from a halophytic grass Spartina alterniflora and investigated its role in imparting abiotic stress tolerance. Results SaSce9 gene encodes for a polypeptide of 162 amino acids with a molecular weight of ~18 kD and isoelectric point 8.43. Amino acid sequence comparisons of SaSce9 with its orthologs from other plant species showed high degree (~85-93%) of structural conservation among each other. Complementation analysis using yeast SCE mutant, Ubc9, revealed functional conservation of SaSce9 between yeast and S. alterniflora. SaSce9 transcript was inducible by salinity, drought, cold, and exogenously supplied ABA both in leaves and roots of S. alterniflora. Constitutive overexpression of SaSce9 in Arabidopsis through Agrobacterium mediated transformation improved salinity and drought tolerance of Arabidopsis. SaSce9 overexpressing Arabidopsis plants retained more chlorophyll and proline both under salinity and drought stress. SaSce9 transgenic plants accumulated lower levels of reactive oxygen under salinity stress. Expression analysis of stress responsive genes in SaSce9 Arabidopsis plants revealed the increased expression of antioxidant genes, AtSOD and AtCAT, ion antiporter genes, AtNHX1 and AtSOS1, a gene involved in proline biosynthesis, AtP5CS, and a gene involved in ABA dependent signaling pathway, AtRD22. Conclusions These results highlight the prospect of improving abiotic stress tolerance in plants through genetic engineering of the sumoylation pathway. The study provides evidence that the overexpression of SaSce9 in plant can improve salinity and drought stress tolerance by protecting the plant through scavenging of ROS, accumulation of an osmolyte, proline, and expression of stress responsive genes. In addition, this study demonstrates the potential of the halophyte grass S. alterniflora as a reservoir of abiotic stress related genes for crop improvement.</p