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United States Department of Agriculture

Agricultural Research Service

Research Project: CHARACTERIZATION OF HOST-PATHOGEN INTERACTIONS IN BARLEY AND WHEAT Title: RNA-mediated Gene Silencing in the Cereal Fungal Pathogen Cochliobolus sativus

Authors
item Leng, Yueqiang -
item Wu, Chengxiang -
item Liu, Zhaohui -
item Friesen, Timothy
item Rasmussen, Jack -
item Zhong, Shaobin -

Submitted to: Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 1, 2010
Publication Date: April 21, 2011
Citation: Leng, Y., Wu, C., Liu, Z., Friesen, T.L., Rasmussen, J.B., Zhong, S. 2011. RNA-mediated Gene Silencing in the Cereal Fungal Pathogen Cochliobolus sativus. Molecular Plant Pathology. 12(3):289-298.

Interpretive Summary: Little is known about the mechanisms of disease of the fungal pathogen Cochliobolus sativus (anamorph: Bipolaris sorokiniana) which causes spot blotch, common root rot and black point in barley and wheat. In this study, we developed a high-throughput RNA-mediated gene silencing system for the evaluation and characterization of genes of this fungal pathogen. The green fluorescent protein (GFP) gene and the proteinous host selective toxin gene(ToxA) were first introduced into an isolate of C. sativus. A gene silencing vector was used to construct RNA interference (RNAi) vectors for GFP and ToxA. Silencing of the GFP or ToxA genes in the GFP or ToxA transformants were demonstrated by introduction of an RNAi construct of each target gene. The polyketide synthase gene (CsPKS1), involved in melanin biosynthesis pathways in C. sativus, was also used to construct an RNAi vector and introduction of this construct into C. sativus by transformation resulted in albino phenotypes and reduction of melanization, suggesting the effective knock down of the endogenous CsPKS1 in this plant pathogen. This high-throughput gene silencing system will be an alternate tool for functional genomics studies in C. sativus and other filamentous fungi.

Technical Abstract: Cochliobolus sativus (anamorph: Bipolaris sorokiniana) is the causal agent of spot blotch, common root rot and black point in barley and wheat. However, little is known about the mechanisms underlying the pathogenicity and virulence of the pathogen. In this study, we developed a high-throughput RNA-mediated gene silencing system for functional genomics of the fungus. The green fluorescent protein (GFP) gene and the proteinous host-selective toxin gene (ToxA) were first introduced into an isolate of C. sativus via the polyethylene glycol (PEG)-mediated transformation method. Transformants with a high level expression of the GFP or ToxA were generated. A silencing vector (pSGate1) based on the Gateway Cloning system was used to construct RNA interference (RNAi) vectors for GFP and ToxA. Silencing of the GFP or ToxA genes in the GFP or ToxA transformants were demonstrated by introduction of an RNAi construct that expressed the hairpin RNA of each target gene. The polyketide synthase gene (CsPKS1), involved in melanin biosynthesis pathways in C. sativus, was also used to construct an RNAi vector (pSGate1-CsPKS1) encoding hairpin RNA of the CsPKS1 gene. Introduction of pSGate1-CsPKS1 into C. sativus by transformation resulted in albino phenotypes and reduction of melanization, suggesting the effective knock down of the endogenous CsPKS1 in this plant pathogen. The high-throughput gene silencing system established will be an alternate tool for functional genomics studies in C. sativus and other filamentous fungi.

Last Modified: 11/21/2014
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