Abstract
Biotrophic fungi (Puccinia spp.) cause devastating diseases of wheat and other cereal species globally. The function of large repertories of genes from Puccinia spp. still needs to be discovered to understand the infection process of these obligate parasites, eventually to protect plants from rust diseases. Functional analysis of targeted genes is challenging due to the inherent difficulties with culturing the fungus and transforming the host. RNA interference (RNAi) is a conserved gene regulation process in eukaryotes and known to be a powerful genetic tool in plant biotechnology. More recently, host-induced gene silencing (HIGS) has been developed to assess pathogen gene function in plants. HIGS is an RNAi-based process where double stranded RNA (dsRNA) homologous to a pathogen gene can be expressed in a plant to induce targeted silencing of the pathogen gene. Here we described a detailed HIGS protocol for functional analysis of rust genes from Puccinia species in cereals. As an example we describe an experiment silencing the tryptophan 2-monooxygenase gene (Pgt-IaaM) from Puccinia graminis f. sp. tritici (Pgt) that is involved in virulence to wheat.
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References
Baulcombe DC (2004) RNA silencing in plants. Nature 431:356–363
Cakir C, Gillespie ME, Scofield SR (2010) Rapid determination of gene function by virus-induced gene silencing in wheat and barley. Crop Sci 50:S77–S84
Scofield SR, Nelson RS (2009) Resources for virus-induced gene silencing in the grasses. Plant Physiol 149:152–157
Nowara D et al (2010) HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22(9):3130–3141
Tinoco MLP et al (2010) In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA. BMC Biol 31:27
Yin C, Jurgenson JE, Hulbert SH (2011) Development of a host-induced RNAi system in the wheat stripe rust fungus Puccinia striiformis f. sp. tritici. Mol Plant-Microbe Interact 24:554–561
Yin C et al (2015) Identification of promising host-induced silencing targets among genes preferentially transcribed in haustoria of Puccinia. BMC Genomics 16(1):579
Yin C, Park JJ, Gang DR, Hulbert SH (2014) Characterization of a tryptophan 2-monooxygenase gene from Puccinia graminis f. sp. tritici involved in auxin biosynthesis and rust pathogenicity. Mol Plant-Microbe Interact 27:227–235
Panwar V, McCallum B, Bakkeren G (2013) Endogenous silencing of Puccinia triticina pathogenicity genes through in planta-expressed sequences leads to the suppression of rust diseases on wheat. The Plant J 73:521–532
Andrade CM et al (2015) Host-induced gene silencing in the necrotrophic fungal pathogen Sclerotinia Sclerotiorum. Plant Pathol 65(4):626–632
Koch A et al (2013) Host-induced gene silencing of cytochrome P450 lanosterol C14 ademethylase-encoding genes confers strong resistance to Fusarium species. Proc Natl Acad Sci U S A 110:19324–19329
Hu ZL et al (2015) Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance. Front Chem 3:1
Govindarajulu M, Epstein L, Wroblewski T, Michelmore RW (2015) Host-induced gene silencing inhibits the biotrophic pathogen causing downy mildew of lettuce. Plant Biotechnol J 13:875–883
Koch A, Kogel KH (2014) New wind in the sails: improving the agronomic value of crop plants through RNAi-mediated gene silencing. Plant Biotech J 12:821–831
Scofield SR et al (2005) Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol 138:2165–2173
Brueggeman R et al (2008) The stem rust resistance gene Rpg5 encodes a protein with nucleotide-binding-site, leucine-rich, and protein kinase domains. Proc Natl Acad Sci U S A 105:14970–14975
Stakman EC, Stewart DM, Loegering WQ (1962) Identification of physiologic races of Puccinia graminis var. tritici. Agric Res Serv E 617. U. S. Dep. Agric, Washington, DC
Line RF, Qayoum A (1992) Virulence, aggressiveness, evolution, and distribution of races of Puccinia striiformis (the cause of stripe rust of wheat) in North America, 1968-87. In: Tech. Bull. No. 1788. United States Department of Agriculture, Agricultural Research Service, Washington, DC
Acknowledgments
This work was funded by National Institute of Food and Agriculture to S.H. (Grant No. 2010-65108-20568).
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Yin, C., Hulbert, S.H. (2018). Host-Induced Gene Silencing (HIGS) for Elucidating Puccinia Gene Function in Wheat. In: Ma, W., Wolpert, T. (eds) Plant Pathogenic Fungi and Oomycetes. Methods in Molecular Biology, vol 1848. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8724-5_12
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DOI: https://doi.org/10.1007/978-1-4939-8724-5_12
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