ABSTRACT
L-amino acid oxidase (ThLAAO) secreted by Trichoderma harzianum ETS323 is a ï¬avoenzyme with antimicrobial characteristics. In this study, we transformed the ThLAAO gene into tobacco to elucidate whether ThLAAO can activate defense mechanisms and confer resistance against phytopathogens. Transgenic tobacco overexpressing ThLAAO showed enhanced resistance against Sclerotinia sclerotiorum and Botrytis cinerea and activated the expression of defense-related genes and the genes involved in salicylic acid, jasmonic acid, and ethylene biosynthesis accompanied by substantial accumulation of H2O2 in chloroplasts, cytosol around chloroplasts, and cell membranes of transgenic tobacco. Scavenge of H2O2 with ascorbic acid abolished disease resistance against B. cinerea infection and decreased the expression of defense-related genes. ThLAAO-FITC application on tobacco protoplast or overexpression of ThLAAO-GFP in tobacco revealed the localization of ThLAAO in chloroplasts. Chlorophyll a/b binding protein (CAB) was isolated through ThLAAO-ConA affinity chromatography. The pull down assay results confirmed ThLAAO-CAB binding. Application of ThLAAO-Cy5.5 on cabbage roots promptly translocated to the leaves. Treatment of ThLAAO on cabbage roots induces systemic resistance against B. cinerea. Overall, these results demonstrate that ThLAAO may target chloroplast and activate defense mechanisms via H2O2 signaling to confer resistance against S. sclerotiorum and B. cinerea.
Subject(s)
Ascomycota , Botrytis , Disease Resistance/genetics , Fungal Proteins/genetics , Hypocreales/genetics , L-Amino Acid Oxidase/genetics , Nicotiana/immunology , Plant Diseases/immunology , Fungal Proteins/physiology , Hydrogen Peroxide/metabolism , Hypocreales/enzymology , L-Amino Acid Oxidase/physiology , Plant Diseases/microbiology , Plants, Genetically Modified , Real-Time Polymerase Chain Reaction , Nicotiana/genetics , Nicotiana/microbiologyABSTRACT
In the mouse, L-amino acid oxidase (LAO) produces hydrogen peroxide by utilizing free amino acids and is a proven antibacterial factor in mammary glands. Mastitis, a bacterial infection of the mammary gland, is the most frequent disease in dairy cattle. Here, we investigate whether LAO is expressed in the mammary gland of dairy cattle and is antibacterial. In dairy cattle, the expression level of LAO mRNA in the mammary gland was considerably lower than that in mice, and LAO activity was not observed in cattle milk that produced hydrogen peroxide. The expression of LAO mRNA was also low in Japanese Black cattle, the same as in Holstein cattle. A higher LAO mRNA expression was observed in the mastitis glands than in the lactating glands. Furthermore, spleen and lymph nodes expressed high levels of LAO mRNA in dairy cattle. We conclude that mammary glands in dairy cattle have lower ability to express the LAO gene compared to that in mice, which may result in a high incidence of mastitis.
Subject(s)
Gene Expression , L-Amino Acid Oxidase/genetics , Mammary Glands, Animal/enzymology , Mastitis, Bovine/enzymology , Mastitis, Bovine/genetics , Amino Acids/metabolism , Animals , Anti-Bacterial Agents , Cattle , Female , Hydrogen Peroxide/metabolism , L-Amino Acid Oxidase/physiology , Lactation/genetics , Lactation/metabolism , Mice , Mice, Inbred ICR , RNA, Messenger/metabolismABSTRACT
Traditionally, small molecules (<1kDa) have dominated the study of the chemistry and chemical ecology of marine natural products. However, as reported in a recent publication, Yang and co-workers have isolated a 60-kDa antibacterial protein from the defensive secretions of the sea hare Aplysia californica. This protein, escapin, has been characterized as an l-amino acid oxidase with bacteriostatic and bacteriocidal activities. Their work highlights the largely untapped biomedical potential of marine organism-derived proteins and addresses the problems of supply associated with invertebrate natural products. It also leads to intriguing hypotheses about the ecological function(s) of the new protein.
