Identification of phytotoxins produced by fusarium virguliforme associated with foliar symptoms of sudden death syndrome and genome-wide association studies for soybean disease resistance

Plant pathology is a multidisciplinary subject and the scientific progress in plant pathology follows the advent of sciences. Beginning in the 1800s, the father of plant pathology, Dr. Anton de Bary, initiated studies on the identification of oomycetes and fungal plant pathogens. This was the first integration for the subjects of "microbiology" and "botany", which initiated the science of plant pathology. Dr. Flor proposed the gene-for-gene model for plant-microbe interaction at 1940s, which introduced Mendelian "genetics" to the subject that eventually became the foundation for resistance breeding. The development of "molecular biology" and "biotechnology" in the 1950s to 2000s enabled molecular studies using forward and reverse genetics to understand the biological and physiological mechanisms of microbes, plants, and their interactions. Although studies at this period were limited in functional analyses for a single gene or few target genes, the accumulation of knowledge over decades inspired the zigzag plant immunity model including the concepts of pathogen-associated molecular pattern (PAMP)-trigger immunity (PTI) and effector-trigger immunity (ETI), which has become the dogma for plant pathology. With the development of the microarray or biochip technology in early 2000s and the maturity of next-generation sequencing around 2010s, genomic or transcriptomic level of biology studies have become regular experiments when I started my academia career in plant pathology. When "informatics" joined plant pathology; I was provided with an opportunity to "dig" biological information from the big data, with specific interests on soybean pathology. In my first project, I applied the RNA-Seq technology to identify additional phytotoxins produced by the fungus Fusarium virguliforme, which causes soybean sudden death syndrome (SDS). A robust and comprehensive bioinformatics-searching pipeline was established and I successfully identified three secondary metabolites and 11 phytotoxic effectors. One of the effectors, FvNIS1, induced identical foliar symptoms to field-observed SDS through an overexpression system via Soybean mosaic virus. Results of phytotoxicity assay on eighty plant introductions (PIs), genome-wide association study (GWAS), and phytotoxicity assay for FvNIS1 gene knockout mutants supported that FvNIS1 is one of the phytotoxins responsible for SDS foliar symptoms. My second project focused on annotation of carbohydrate-active enzymes and plant cell wall degrading enzymes (PCWDEs) in the genome of F. virguliforme. I focused on the polymorphisms of GH28 polygalacturonase and GH11 xylanase because several Fusarium species have amino acid substitutions on these enzymes that allow them to escape PCWDEs-inhibiting proteins released by plants as a counteract defense mechanism. The results indicated F. virguliforme has conserved xylanases and development of transgenic soybean with wheat xylanase-inhibitor protein might enhance soybean resistance to F. virguliforme. In my third project, I incorporated soybean sensitivity to Tobacco ringspot virus (TRSV) to the single nucleotide polymorphism (SNP) markers from SoySNP50K, and performed a GWAS to identify SNP that associate with TRSV sensitivity. I further applied genomic selection to predict TRSV sensitivity for the unscreened soybean PIs in the USDA Soybean Germplasm Collection. In this project, I identified a single locus and two candidate genes that may involve in TRSV sensitivity, and showed genomic prediction has higher performance than single SNP-based marker-assisted selection. My interests in GWAS extended to my fourth project, for which I adopted phenotypes of 13 soybean diseases deposited in the United States Department of Agriculture of Agriculture Research Service (USDA-ARS) Germplasm Resources Information Network (GRIN) database and performed GWAS for each disease. In the study, I discovered SNPs locate in previously reported loci, I found novel SNPs for diseases such as Diaporthe stem canker, and I presented the power and challenges of GWAS in searching soybean resistance sources. In summary, my dissertation contains demonstrations on the impact of informatics in soybean pathology regarding finding genes involved in soybean-microbes interactions

Intravitreal Aflibercept for the treatment of diabetic macular edema in routine clinical practice: results from the 24-Month AURIGA observational study

Introduction AURIGA is the largest real-world study to date to evaluate intravitreal aflibercept (IVT-AFL) in the treatment of diabetic macular edema (DME) or macular edema secondary to retinal vein occlusion in routine clinical practice. The 24-month outcomes in the DME cohort from across 11 participating countries are reported here.MethodsAURIGA (NCT03161912) was a prospective observational study. The study enrolled eligible patients with DME for whom the decision to treat with IVT-AFL had previously been made by the attending physician. Patients were treated with IVT-AFL for up to 24 months at physician discretion according to local practice. The primary endpoint was mean change in visual acuity (VA; Early Treatment Diabetic Retinopathy Study [ETDRS] letters) from baseline to month 12 (M12). All statistical analyses were descriptive.ResultsIn 1478 treatment-naive and 384 previously treated patients with DME, the mean (95% confidence interval) change in VA from baseline was +6.7 (5.7, 7.6) and +7.4 (5.5, 9.4) letters by M12 and +5.9 (4.9, 6.9) and +8.1 (6.1, 10.1) letters by M24 (baseline [mean +/- standard deviation]: 56.0 +/- 19.8 and 50.8 +/- 19.5 letters), respectively; 25.9% of treatment-naive and 32.8% of previously treated patients achieved >= 15-letter gains by M24. The mean change in central retinal thickness from baseline to M24 was -110 (-119, -102) mu m in treatment-naive patients and -169 (-188, -151) mu m in previously treated patients. By M6, M12, and M24, treatment-naive patients had received 3.8 +/- 1.7, 4.9 +/- 2.8, and 5.7 +/- 3.9 injections, respectively, and previously treated patients had received 3.9 +/- 1.5, 4.9 +/- 2.4, and 6.2 +/- 3.6 injections, respectively. The safety profile of IVT-AFL was consistent with previous studies.ConclusionIn AURIGA, treatment-naive and previously treated patients with DME achieved clinically relevant functional and anatomic improvements following IVT-AFL treatment for up to 24 months in routine clinical practice. Even with the decreasing injection frequency observed, these gains were largely maintained throughout the study, suggesting long-term durability of the positive effects of IVT-AFL treatment. Infographic available for this article.Trial RegistrationClinicalTrials.gov Identifier: NCT03161912 (May 19, 2017)

In vitro anti-Helicobacter pylori activity and the underlining mechanism of an empirical herbal formula – Hezi Qingyou

BackgroundHelicobacter pylori (H. pylori) is thought to primarily colonize the human stomach and lead to various gastrointestinal disorders, such as gastritis and gastric cancer. Currently, main eradication treatment is triple or quadruple therapy centered on antibiotics. Due to antibiotic resistance, the eradication rate of H. pylori is decreasing gradually. Therefore, searching for anti-H. pylori drugs from herbal sources has become a strategy for the treatment. Our team proposed a Hezi Qingyou Formula (HZQYF), composed of Chebulae Fructus, Ficus hirta Vahl and Cloves, and studied its anti-H. pylori activity and mechanism.MethodsChemical components of HZQYF were studied using UHPLC–MS/MS and HPLC. Broth microdilution method and agar dilution method were used to evaluate HZQYF’s antibacterial activity. The effects of HZQYF on expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF), and flagellar genes (flaA, flaB) were explored using Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) technology. Effects on morphology and permeability of the extracellular membrane were studied using scanning electron microscopy (SEM) and N-phenylnaphthalen-1-amine (NPN) uptake. Effect on urease activity was studied using a urease kinetics analysis in vitro. Immunofluorescence staining method was used to examine the effect on adhesion. Western blot was used to examine the effect on cagA protein.ResultsMinimum inhibitory concentration (MIC) values of the formula against H. pylori clinical strains and standard strains were 80–160 μg/mL, and minimum bactericidal concentration (MBC) values were 160–320 μg/mL. The formula could down-regulate the expression of adhesion genes (alpA, alpB, babA), urease genes (ureE, ureF) and flagellar genes (flaA, flaB), change the morphology of H. pylori, increase its extracellular membrane permeability, and decrease its urease activity.ConclusionPresent studies confirmed that HZQYF had promising in vitro anti-H. pylori activities and demonstrated its possible mechanism of action by down-regulating the bacterial adhesion, urease, and flagellar gene expression, which provided scientific bases for further clinical investigations

Characterization of Soybean STAY-GREEN Genes in Susceptibility to Foliar Chlorosis of Sudden Death Syndrome

Fusarium virguliforme causes sudden death syndrome (SDS) of soybean (Glycine max) in the United States. This fungal pathogen inhabits soil and produces multiple phytotoxins, which are translocated from infected roots to leaves, causing SDS foliar chlorosis and necrosis (Hartman et al., 2015). Because SDS foliar symptoms are solely induced by phytotoxins, it represents a unique pathosystem to study plant-phytotoxin interactions (Chang et al., 2016). SDS foliar symptoms typically appear near flowering through late reproductive growth stages, with chlorotic spots that gradually develop into interveinal chlorosis and necrosis (Fig. 1A). The sudden appearance of SDS foliar symptoms not only explains the origin of the disease name, but also reflects the difficulty of early detection in managing this disease. Yield reductions caused by SDS have been documented at 5% to15%, and the economic loss was estimated up to $669 million U.S. dollars in a single year (Navi and Yang, 2016). Seed treatments have been used to manage SDS, but performance differs by year and location. Alternatively, partially resistant soybean cultivars provide a sustainable option for SDS management, but the genetic architecture of SDS resistance is quantitative and complicated. Among more than 80 quantitative trait loci reported for SDS, only a few quantitative trait loci are reproducible due to the complexity of SDS etiology and environmental interactions (Chang et al., 2018)