Vitamin E biofortification: Maximizing oilseed tocotrienol and total vitamin E tocochromanol production by use of metabolic bypass combinations.

Vitamin E tocochromanols are generated in plants by prenylation of homogentisate using geranylgeranyl diphosphate (GGDP) for tocotrienol biosynthesis and phytyl diphosphate (PDP) for tocopherol biosynthesis. Homogentisate geranylgeranyl transferase (HGGT), which uses GGDP for prenylation, is a proven target for oilseed tocochromanol biofortification that effectively bypasses the chlorophyll-linked pathway that limits PDP for vitamin E biosynthesis. In this report, we explored the feasibility of maximizing tocochromanol production in the oilseed crop camelina (Camelina sativa) by combining seed-specific HGGT expression with increased biosynthesis and/or reduced homogentisate catabolism. Plastid-targeted Escherichia coli TyrA-encoded chorismate mutase/prephenate dehydrogenase and Arabidopsis hydroxyphenylpyruvate dioxygenase (HPPD) cDNA were co-expressed in seeds to bypass feedback-regulated steps and increase flux into homogentisate biosynthesis. Homogentisate catabolism was also suppressed by seed-specific RNAi of the gene for homogentisate oxygenase (HGO), which initiates homogentisate degradation. In the absence of HGGT expression, tocochromanols were increased by ∼2.5-fold with HPPD/TyrA co-expression, and ∼1.4-fold with HGO suppression compared to levels in non-transformed seeds. No further increase in tocochromanols was observed in HPPD/TyrA lines with the addition of HGO RNAi. HGGT expression alone increased tocochromanol concentrations in seeds by âˆ¼four-fold to ≤1400 µg/g seed weight. When combined with HPPD/TyrA co-expression, we obtained an additional three-fold increase in tocochromanol concentrations indicating that homogentisate concentrations limit HGGT's capacity for maximal tocochromanol production. The addition of HGO RNAi further increased tocochromanol concentrations to 5000 µg/g seed weight, an unprecedented tocochromanol concentration in an engineered oilseed. Metabolomic data obtained from engineered seeds provide insights into phenotypic changes associated with "extreme" tocochromanol production.

Mechanisms of Primed Defense: Plant Immunity Induced by Endophytic Colonization of a Mycovirus-Induced Hypovirulent Fungal Pathogen.

How mycovirus-induced hypovirulence in fungi activates plant defense is still poorly understood. The changes in plant fitness and gene expression caused by the inoculation of the fungus Sclerotinia sclerotiorum harboring and made hypovirulent by the mycovirus soybean leaf-associated gemygorvirus-1 (SlaGemV-1) of the species Gemycircularvirus soybe1 were examined in this study. As the hypovirulent fungus (DK3V) colonized soybean Glycine max, plant transcriptomic analysis indicated changes in defense responses and photosynthetic activity, supported by an upregulation of individual genes and overrepresentation of photosystem gene ontology groups. The upregulated genes include genes relating to both pathogen-associated molecular pattern-triggered immunity and effector-triggered immunity as well as various genes relating to the induction of systemic acquired resistance and the biosynthesis of jasmonic acid. Plants colonized with DK3V showed a resistant phenotype to virulent S. sclerotiorum infection. Plant height and leaf area were also determined to be larger in plants grown with the virus-infected fungus. Here, we hypothesize that inoculation of soybean with DK3V can result in the triggering of a wide range of defense mechanisms to prime against later infection. The knowledge gained from this study about plant transcriptomics and phenotype will help prime plant immunity with mycovirus-infected hypovirulent fungal strains more effectively. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Unpublished clinical trials of common rheumatic diseases.

OBJECTIVES: Randomized controlled trials (RCTs) provide high-quality evidence for treatment efficacy, but many RCTs remain unpublished. The objective of this study was to describe the proportion of unpublished RCTs in five rheumatic diseases and to identify factors associated with publication. METHODS: Registered RCTs for five rheumatic diseases (SLE, vasculitis, spondyloarthritis, SS and PsA) with over 30 months since study completion were identified using ClinicalTrials.gov. Index publications were identified by NCT ID numbers and structured text searches of publication databases. The results of unpublished studies were identified in abstracts and press releases; reasons for non-publication were assessed by surveying corresponding authors. RESULTS: Out of 203 studies that met eligibility criteria, 17.2% remained unpublished, representing data from 4281 trial participants. Higher proportions of published trials were phase 3 RCTs (57.1% vs 28.6% unpublished, P < 0.05) or had a positive primary outcome measure (64.9% vs 25.7% unpublished, P < 0.001). In a multivariable Cox proportional hazards model, a positive outcome was independently associated with publication (hazard ratio 1.55; 95% CI: 1.09, 2.22). Corresponding authors of 10 unpublished trials cited ongoing preparation of the manuscript (50.0%), sponsor/funder issues (40.0%) and unimportant/negative result (20.0%) as reasons for lack of publication. CONCLUSIONS: Nearly one in five RCTs in rheumatology remain unpublished 2 years after trial completion, and publication is associated with positive primary outcome measures. Efforts to encourage universal publication of rheumatology RCTs and reanalysis of previously unpublished trials should be undertaken.

Genome of Paspalum vaginatum and the role of trehalose mediated autophagy in increasing maize biomass.

A number of crop wild relatives can tolerate extreme stress to a degree outside the range observed in their domesticated relatives. However, it is unclear whether or how the molecular mechanisms employed by these species can be translated to domesticated crops. Paspalum (Paspalum vaginatum) is a self-incompatible and multiply stress-tolerant wild relative of maize and sorghum. Here, we describe the sequencing and pseudomolecule level assembly of a vegetatively propagated accession of P. vaginatum. Phylogenetic analysis based on 6,151 single-copy syntenic orthologues conserved in 6 related grass species places paspalum as an outgroup of the maize-sorghum clade. In parallel metabolic experiments, paspalum, but neither maize nor sorghum, exhibits a significant increase in trehalose when grown under nutrient-deficit conditions. Inducing trehalose accumulation in maize, imitating the metabolic phenotype of paspalum, results in autophagy dependent increases in biomass accumulation.

Characterization of Transcriptional Responses to Genomovirus Infection of the White Mold Fungus, Sclerotinia sclerotiorum.

Soybean leaf-associated gemygorvirus-1 (SlaGemV-1) is a CRESS-DNA virus classified in the family Genomoviridae, which causes hypovirulence and abolishes sclerotia formation in infected fungal pathogens under the family Sclerotiniaceae. To investigate the mechanisms involved in the induction of hypovirulence, RNA-Seq was compared between virus-free and SlaGemV-1-infected Sclerotinia sclerotiorum strain DK3. Overall, 4639 genes were differentially expressed, with 50.5% up regulated and 49.5% down regulated genes. GO enrichments suggest changes in integral membrane components and transmission electron microscopy images reveal virus-like particles localized near the inner cell membrane. Differential gene expression analysis focused on genes responsible for cell cycle and DNA replication and repair pathways, ubiquitin proteolysis, gene silencing, methylation, pathogenesis-related, sclerotial development, carbohydrate metabolism, and oxalic acid biosynthesis. Carbohydrate metabolism showed the most changes, with two glycoside hydrolase genes being the most down regulated by -2396.1- and -648.6-fold. Genes relating to pathogenesis showed consistent down regulation with the greatest being SsNep1, SsSSVP1, and Endo2 showing, -4555-, -14.7-, and -12.3-fold changes. The cell cycle and DNA replication/repair pathways were almost entirely up regulated including a putative cyclin and separase being up regulated 8.3- and 5.2-fold. The oxalate decarboxylase genes necessary for oxalic acid catabolism and oxalic acid precursor biosynthesis genes and its metabolism show down regulations of -17.2- and -12.1-fold changes. Sclerotial formation genes also appear differentially regulated including a melanin biosynthesis gene Pks1 and a sclerotia formation gene Sl2 with fold changes of 3.8 and -2.9.

Comparative Efficacy Randomized Controlled Trials in Rheumatology Guidelines.

BACKGROUND: Comparative efficacy randomized controlled trials (RCTs) compare two active interventions in a head-to-head design. They are useful for informing clinical practice guidelines, but the degree to which such trials inform clinical practice guidelines in rheumatology is unknown. METHODS: The American College of Rheumatology (ACR) and European Alliance of Associations for Rheumatology (EULAR) websites were searched from January 1, 2017, to June 12, 2021, for clinical practice guidelines. RCTs referenced by each guideline were identified, and information regarding design and outcomes were extracted. Clinical practice recommendations from each guideline were also analyzed. RESULTS: Fifteen ACR- and nine EULAR-endorsed guidelines were included, which cited 609 RCTs and provided 481 recommendations. Referenced RCTs enrolled an average of 418 patients (SD 985), most commonly evaluated biologic/targeted synthetic disease-modifying antirheumatic drugs (70.1%), and infrequently used a head-to-head design (28%). A minority of recommendations received a high level of evidence (LOE) by the Grades of Recommendation, Assessment, Development, and Evaluation (GRADE) methodology (2.9%) or an "A" grade by the Oxford Centre for Evidence based Medicine Standards (OCEBM) methodology (28.9%). LOE was higher for recommendations informed by RCTs (P < 0.001) or head-to-head RCTs (P = 0.008). Many recommendations received a strong recommendation despite low (8 [2.6%]) or very low (25 [8.3%]) LOE. CONCLUSION: Less than one in six rheumatology guideline recommendations are informed by head-to-head RCTs. Recommendations that were informed by head-to-head RCTs were more likely to have a high LOE by both GRADE and OCEBM. Efforts to introduce more comparative efficacy RCTs should be undertaken.

Fusarium Graminearum Virus-1 Strain FgV1-SD4 Infection Eliminates Mycotoxin Deoxynivalenol Synthesis by Fusarium graminearum in FHB.

Deoxynivalenol (DON) toxin production during the infection of F. graminearum in small grain crops is one of the most harmful virulence factors associated with economic losses. Metatranscriptome sequencing and RT-qPCR traced back that the only mycovirus infecting an F. graminearum isolate, designated as Fg-4-2, was a novel strain of Fusarium graminearum virus 1 (FgV1), designated as FgV1-SD4. The isolate Fg-4-2 showed significantly reduced virulence against wheat compared to the virus-free culture, designated as isolate Fg-4-1, which was obtained by deep freezing and single conidial germination. Notably, no DON accumulation was detected in the harvested wheat seeds infected by Fg-4-2, whereas ~18 ppm DON was detected in seeds infected by Fg-4-1. Comparison of the genome sequence of FgV1-SD4 with other identified strains of FgV1, i.e., FgV1-DK21 and FgV1-ch, indicates mutations on ORF-2 and the 3'-UTR in the genome that might be associated with hypovirulence. This mycovirus strain alone and specific genetic components of FgV1-SD4 can be further optimized to be developed as a biocontrol agent to reduce Fusarium head blight and to lower the DON accumulation levels in small grain crops due to this fungal disease.

Direct Metatranscriptomic Survey of the Sunflower Microbiome and Virome.

Sunflowers (Helianthus annuus L.) are susceptible to multiple diseases in field production. In this study, we collected diseased sunflower leaves in fields located in South Dakota, USA, for virome investigation. The leaves showed visible symptoms on the foliage, indicating phomopsis and rust infections. To identify the viruses potentially associated with the disease diagnosed, symptomatic leaves were obtained from diseased plants. Total RNA was extracted corresponding to each disease diagnosed to generate libraries for paired-end high throughput sequencing. Short sequencing reads were assembled de novo and the contigs with similarities to viruses were identified by aligning against a custom protein database. We report the discovery of two novel mitoviruses, four novel partitiviruses, one novel victorivirus, and nine novel totiviruses based on similarities to RNA-dependent RNA polymerases and capsid proteins. Contigs similar to bean yellow mosaic virus and Sclerotinia sclerotiorum hypovirulence-associated DNA virus were also detected. To the best of our knowledge, this is the first report of direct metatranscriptomics discovery of viruses associated with fungal infections of sunflowers bypassing culturing. These newly discovered viruses represent a natural genetic resource from which we can further develop potential biopesticide to control sunflower diseases.

Identification of the Viral Determinant of Hypovirulence and Host Range in Sclerotiniaceae of a Genomovirus Reconstructed from the Plant Metagenome.

Uncharacterized viral genomes that encode circular replication-associated proteins of single-stranded DNA viruses have been discovered by metagenomics/metatranscriptomics approaches. Some of these novel viruses are classified in the newly formed family Genomoviridae. Here, we determined the host range of a novel genomovirus, SlaGemV-1, through the transfection of Sclerotinia sclerotiorum with infectious clones. Inoculating with the rescued virions, we further transfected Botrytis cinerea and Monilinia fructicola, two economically important members of the family Sclerotiniaceae, and Fusarium oxysporum. SlaGemV-1 causes hypovirulence in S. sclerotiorum, B. cinerea, and M. fructicola. SlaGemV-1 also replicates in Spodoptera frugiperda insect cells but not in Caenorhabditis elegans or plants. By expressing viral genes separately through site-specific integration, the replication protein alone was sufficient to cause debilitation. Our study is the first to demonstrate the reconstruction of a metagenomically discovered genomovirus without known hosts with the potential of inducing hypovirulence, and the infectious clone allows for studying mechanisms of genomovirus-host interactions that are conserved across genera. IMPORTANCE Little is known about the exact host range of widespread genomoviruses. The genome of soybean leaf-associated gemygorvirus-1 (SlaGemV-1) was originally assembled from a metagenomic/metatranscriptomic study without known hosts. Here, we rescued SlaGemV-1 and found that it could infect three important plant-pathogenic fungi and fall armyworm (S. frugiperda Sf9) insect cells but not a model nematode, C. elegans, or model plant species. Most importantly, SlaGemV-1 shows promise for inducing hypovirulence of the tested fungal species in the family Sclerotiniaceae, including Sclerotinia sclerotiorum, Botrytis cinerea, and Monilinia fructicola. The viral determinant of hypovirulence was further identified as replication initiation protein. As a proof of concept, we demonstrate that viromes discovered in plant metagenomes can be a valuable genetic resource when novel viruses are rescued and characterized for their host range.