title: Conditional gene expression and promoter replacement in Zymoseptoria tritici using fungal nitrate reductase promoters.
abstract: Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a β-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets.
authors.affiliation: UMR 1290 BIOGER-CPP, INRA, AgroParisTech, Campus AgroParisTech, Ave Louis Bretignières, F75850 Thiverval-Grignon, France.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Marchegiani
authors.affiliation: Biosciences, College of Life and Environmental Sciences, Geoffrey Pope, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Yaadwinder
authors.initials: Y
authors.lastname: Sidhu
authors.affiliation: Biosciences, College of Life and Environmental Sciences, Geoffrey Pope, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.
authors.forename: Ken
authors.initials: K
authors.lastname: Haynes
authors.affiliation: UMR 1290 BIOGER-CPP, INRA, AgroParisTech, Campus AgroParisTech, Ave Louis Bretignières, F75850 Thiverval-Grignon, France.
authors.forename: Marc-Henri
authors.initials: MH
authors.lastname: Lebrun
mesh_terms: D019677:Artificial Gene Fusion; D001203:Ascomycota / Q000235:genetics* / Q000254:growth & development; D015870:Gene Expression; D020869:Gene Expression Profiling; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D018390:Gene Targeting; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D050901:Nitrate Reductase / Q000235:genetics*; D009566:Nitrates / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic
chemical_list: D009566:Nitrates; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D050901:Nitrate Reductase
keywords: Conditional promoter; Essential gene; Mycosphaerella; Nitrate reductase; Promoter exchange; Zymoseptor