Transient expression and insertional mutagenesis of Puccinia triticina using biolistics

Craig A. Webb, Les J. Szabo, Guus Bakkeren, Clarke Garry, Richard C. Staples, Merle Eversmeyer, John P. Fellers

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

The fungal genus Puccinia contains more than 4,000 species. Puccinia triticina, causal agent of wheat leaf rust, is an economically significant, biotrophic basidiomycete. Little is known about the molecular biology of this group, and tools for understanding gene function have not yet been established. A set of parameters was established for the transient transformation of urediniospores. The expression of three heterologous promoters (actin, elongation factor 1-α, and Hss1, Heat Shock 70 protein), derived from Puccinia graminis, was evaluated along with the potential for insertional mutagenesis. The UidA (GUS) gene was used as a marker for transient expression. When transferred into P. triticina urediniospores, transient expression was observed across four helium pressures using one size of gold and three sizes of tungsten microprojectiles. Each of the three promoters displayed strong transient expression in germinated urediniospores; however, higher numbers of GUS-positive urediniospores were observed when either the actin or Hss 1 promoters were used. Possible concomitant insertional mutagenesis of several avirulence genes was selected in wheat cultivars harboring the cognate resistance genes. Using a linearized cloning plasmid, stable integration into the genome was achieved as demonstrated by PCR and sequencing analysis.

Original languageEnglish (US)
Pages (from-to)250-260
Number of pages11
JournalFunctional and Integrative Genomics
Volume6
Issue number3
DOIs
StatePublished - Jul 2006

Bibliographical note

Funding Information:
Acknowledgements The authors acknowledge C. Elburn Parker, Barb Bremenkamp, and Marie Herbel for their technical assistance in establishing and maintaining rust cultures; Stephanie Dahl for technical assistance in constructing expression vectors; Jacolyn Morrison for preparation of the line drawing of the vector constructs; Tim Todd for statistical assistance; and Dr. Barbara Valent for the use of the Zeiss microscope and imaging system housed in her laboratory. We also thank Dr. Kristi Hill-Ambroz, Dr. Barbara Valent, and Dr. Frank White for critical review of the manuscript. Partial funding for this research was provided by the Lieberman– Okinow Endowed Chair for Disease Resistance Breeding in Cereal Crops, University of Minnesota. This work was also supported by USDA-ARS CRIS project #5430-21000-005-00D. This article is contribution no. 04-345-J from the Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS.

Keywords

  • Biolistics
  • Leaf rust
  • Stable integration

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