ABSTRACT
Coumarins can fight pathogens and are thus promising for crop protection. Their biosynthesis, however, has not yet been engineered in crops. We tailored the constitutive accumulation of coumarins in transgenic Nicotiana benthamiana, Glycine max and Arabidopsis thaliana plants, as well as in Nicotiana tabacum BY-2 suspension cells. We did so by overexpressing A. thaliana feruloyl-CoA 6-hydroxylase 1 (AtF6'H1), encoding the key enzyme of scopoletin biosynthesis. Besides scopoletin and its glucoside scopolin, esculin at low level was the only other coumarin detected in transgenic cells. Mechanical damage of scopolin-accumulating tissue led to a swift release of scopoletin, presumably from the scopolin pool. High scopolin levels in A. thaliana roots coincided with reduced susceptibility to the root-parasitic nematode Heterodera schachtii. In addition, transgenic soybean plants were more tolerant to the soil-borne pathogenic fungus Fusarium virguliforme. Because mycotoxin-induced accumulation of reactive oxygen species and cell death were reduced in the AtF6'H1-overexpressors, the weaker sensitivity to F. virguliforme may be caused by attenuated oxidative damage of coumarin-hyperaccumulating cells. Together, engineered coumarin accumulation is promising for enhanced disease resilience of crops.
Subject(s)
Arabidopsis , Mycotoxins , Arabidopsis/metabolism , Scopoletin/metabolism , Mycotoxins/metabolism , Disease Susceptibility/metabolism , Coumarins/metabolism , Oxidative Stress , Plant Roots/genetics , Plant Roots/metabolismABSTRACT
BACKGROUND: Owing to organ shortage, transplantation of organs from HCV (hepatitis C virus) viremic donors into HCV negative individuals is getting more and more accepted. However, transmission of HCV to the host is nearly universal. Until now it is unknown if preservation solutions (PS) might alter infectivity and stability of HCV in the transplant setting. Therefore, seven different preservation solutions (PS) with variable composition were tested in vitro for their direct anti- and proviral effects on HCV. METHODS: In vitro grown HCV based on the JFH-1 isolate was used to characterize the effect of seven different PS on the HCV replication cycle including HCV attachment, entry, replication, and assembly. In addition, HCV stability in PS was tested. RESULTS: Overall, 6/7 PS enhanced HCV infectivity: IGL-1 increased HCV attachment and entry, UW Belzer and Perfadex boosted HCV entry. Production of novel viral particles was enhanced in HTK, UW Belzer, and IGL-1. In contrast, viral replication was significantly reduced in HTK solution while all other PS had no effect on HCV RNA replication. HCV was significantly more stable in HTK solution. Euro Collins was the only PS that did not support HCV infectivity in cell culture. None of the used PS showed cytotoxic effects. CONCLUSION: Our data indicate that HCV infectivity and stability is maintained by several PS.
Subject(s)
Hepacivirus/drug effects , Organ Preservation Solutions/pharmacology , Virus Replication/drug effects , Cell Line , Hepacivirus/physiology , Humans , Virus Attachment/drug effects , Virus Internalization/drug effectsABSTRACT
Background: Although organ shortage is a rising problem, organs from hepatitis C virus (HCV) ribonucleic acid (RNA)-positive donors are not routinely transplanted in HCV-negative individuals. Because HCV only infects hepatocytes, other organs such as kidneys are merely contaminated with HCV via the blood. In this study, we established a protocol to reduce HCV virions during the cold ischemic time. Methods: Standard virological assays were used to investigate the effect of antivirals, including methylene blue (MB), in different preservation solutions. Kidneys from mini pigs were contaminated with Jc1 or HCV RNA-positive human serum. Afterwards, organs were flushed with MB. Hypothermic machine perfusion was used to optimize reduction of HCV. Results: Three different antivirals were investigated for their ability to inactivate HCV in vitro. Only MB completely inactivated HCV in the presence of all perfusion solutions. Hepatitis C virus-contaminated kidneys from mini pigs were treated with MB and hypothermic machine perfusion without any negative effect on the graft. Human liver-uPA-SCID mice did not establish HCV infection after inoculation with flow through from these kidneys. Conclusions: This proof-of-concept study is a first step to reduce transmission of infectious HCV particles in the transplant setting and might serve as a model for other relevant pathogens.
