Development of an Expression Vector to Overexpress or Downregulate Genes in Curvularia protuberata

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.

Infection of Nigrospora nonsegmented RNA Virus 1 Has Important Biological Impacts on a Fungal Host

“…Here, we reported the interesting phenomenon that NoNRV1 infection promoted pigmentation in host hyphae on solid media (Figure 5B). A similar impact has been reported previously in C. protuberata with infection by CThTV [32]. CThTV infection markedly increases melanin content in host cells, leading to darkened mycelia.…”

“…A previous work reported that CThTV upregulates the expression of host scytalone dehydratase-a key enzyme in the melanin biosynthetic pathway [21]-suggesting that CThTV infection might promote melanin accumulation in fungal host cells. This suggestion is supported by recently published work on the same virus [32].…”

“…The 3 UTR sequence was folded into two hairpins with perfect base paring in their stems (Figure 2D). Such hairpin structures are common in the UTRs of other dsRNA mycoviruses [32,[43][44][45]66,67] and are presumed to play an important role in viral replication and assembly [32,46,47]. The IGR sequence between ORF1 and ORF2 is 58 nt long and forms two hairpins that are separated by a 9 nt stretch (Figure 2D).…”

Dark Septate Endophytes Isolated From Wild Licorice Roots Grown in the Desert Regions of Northwest China Enhance the Growth of Host Plants Under Water Deficit Stress

“…The effect of osmotic stress on the melanin content of DSE depended on the DSE species. Melanin, an antioxidant agent that reduces oxidative damage, protects organisms from environmental stress ( Zhan et al, 2011 ; Liu et al, 2018 ; Hosseini et al, 2019 ). Eisenman et al (2020) also reported that melanin protects fungi from a range of stresses in the environment.…”