Abstract
Curvularia protuberata, an endophytic fungus in the Ascomycota, provides plants with thermotolerance only when it carries a mycovirus known as Curvularia thermotolerance virus (CThTV), and forms a three-way symbiotic relationship among these organisms. Under heat stress, several genes are expressed differently between virus-free C. protuberata (VF) and C. protuberata carrying CThTV (AN). We developed an expression vector, pM2Z-fun, carrying a zeocin resistance gene driven by the ToxA promoter, to study gene functions in C. protuberata to better understand this three-way symbiosis. Using this new 3.7-kb vector, five genes that are differentially expressed in C. protuberata—including genes involved in the trehalose, melanin, and catalase biosynthesis pathways—were successfully overexpressed or downregulated in VF or AN C. protuberata strains, respectively. The VF overexpression lines showed higher metabolite and enzyme activity than in the control VF strain. Furthermore, downregulation of expression of the same genes in the AN strain resulted in lower metabolite and enzyme activity than in the control AN strain. The newly generated expression vector, pM2Z-fun, has been successfully used to express target genes in C. protuberata and will be useful in further functional expression studies in other Ascomycota fungi.
Highlights
The endophytic fungus Curvularia protuberata carrying the mycovirus Curvularia thermotolerance virus (CThTV) can participate in a three-way symbiosis with plants that leads to extreme thermotolerance [1]
Because several C. protuberata genes are differently expressed between AN and virus-free fungus (VF) strains under heat stress, we hypothesize that these genes are involved in the thermotolerance mechanism that results from this three-way symbiosis [2]
We demonstrate the differences in metabolite and enzyme activity between overexpressed and downregulated targeted genes in C. protuberata VF and AN stains, respectively, using the newly developed expression vector pM2Z-fun, to further the understanding of the molecular mechanisms that drive this plant, fungus, and virus three-way symbiotic relationship
Summary
The endophytic fungus Curvularia protuberata carrying the mycovirus Curvularia thermotolerance virus (CThTV) can participate in a three-way symbiosis with plants that leads to extreme thermotolerance [1]. Because several C. protuberata genes are differently expressed between AN and VF strains under heat stress, we hypothesize that these genes are involved in the thermotolerance mechanism that results from this three-way symbiosis [2]. Five of these genes were chosen to test the new expression vector. We demonstrate the differences in metabolite and enzyme activity between overexpressed and downregulated targeted genes in C. protuberata VF and AN stains, respectively, using the newly developed expression vector pM2Z-fun, to further the understanding of the molecular mechanisms that drive this plant, fungus, and virus three-way symbiotic relationship
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