HIGS: Host-Induced Gene Silencing in the Obligate Biotrophic Fungal Pathogen Blumeria graminis[W][OA]

This work examines the effects of RNA interference constructs expressed in host cells on target RNAs in Blumeria graminis, an obligate biotrophic fungal pathogen of barley, and finds that RNAs in the host can affect pathogen transcript levels and pathogen development, thereby providing both a useful research tool and a potentially important means for engineering plant disease resistance. Powdery mildew fungi are obligate biotrophic pathogens that only grow on living hosts and cause damage in thousands of plant species. Despite their agronomical importance, little direct functional evidence for genes of pathogenicity and virulence is currently available because mutagenesis and transformation protocols are lacking. Here, we show that the accumulation in barley (Hordeum vulgare) and wheat (Triticum aestivum) of double-stranded or antisense RNA targeting fungal transcripts affects the development of the powdery mildew fungus Blumeria graminis. Proof of concept for host-induced gene silencing was obtained by silencing the effector gene Avra10, which resulted in reduced fungal development in the absence, but not in the presence, of the matching resistance gene Mla10. The fungus could be rescued from the silencing of Avra10 by the transient expression of a synthetic gene that was resistant to RNA interference (RNAi) due to silent point mutations. The results suggest traffic of RNA molecules from host plants into B. graminis and may lead to an RNAi-based crop protection strategy against fungal pathogens.

[1]  D. Douchkov,et al.  A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells. , 2005, Molecular plant-microbe interactions : MPMI.

[2]  J. Lötvall,et al.  Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells , 2007, Nature Cell Biology.

[3]  U Seiffert,et al.  A pattern recognition tool for quantitative analysis of in planta hyphal growth of powdery mildew fungi. , 2005, Molecular plant-microbe interactions : MPMI.

[4]  Jonathan D. G. Jones,et al.  Multiple Avirulence Paralogues in Cereal Powdery Mildew Fungi May Contribute to Parasite Fitness and Defeat of Plant Resistance , 2006, The Plant Cell Online.

[5]  P. Schulze-Lefert,et al.  Extracellular transport and integration of plant secretory proteins into pathogen-induced cell wall compartments. , 2009, The Plant journal : for cell and molecular biology.

[6]  R. Hückelhoven,et al.  Multivesicular compartments proliferate in susceptible and resistant MLA12-barley leaves in response to infection by the biotrophic powdery mildew fungus. , 2006, The New phytologist.

[7]  I. Somssich,et al.  Nuclear Activity of MLA Immune Receptors Links Isolate-Specific and Basal Disease-Resistance Responses , 2007, Science.

[8]  James K. M. Brown,et al.  Coevolution between a Family of Parasite Virulence Effectors and a Class of LINE-1 Retrotransposons , 2009, PloS one.

[9]  R. Panstruga,et al.  Terrific Protein Traffic: The Mystery of Effector Protein Delivery by Filamentous Plant Pathogens , 2009, Science.

[10]  J. Kumlehn,et al.  Efficient generation of transgenic barley: the way forward to modulate plant-microbe interactions. , 2008, Journal of plant physiology.

[11]  Rodrigo Lopez,et al.  Clustal W and Clustal X version 2.0 , 2007, Bioinform..

[12]  Qinxi Li,et al.  Axin determines cell fate by controlling the p53 activation threshold after DNA damage , 2009, Nature Cell Biology.

[13]  R. Hussey,et al.  Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene , 2006, Proceedings of the National Academy of Sciences.

[14]  P. Schweizer,et al.  Transcriptome analysis of mlo-mediated resistance in the epidermis of barley. , 2005, Molecular plant pathology.

[15]  Uwe Scholz,et al.  CR-EST: a resource for crop ESTs , 2004, Nucleic Acids Res..

[16]  S. Hippe Ultrastructure of haustoria oferysiphe graminis f. sp.hordei preserved by freeze-substitution , 1985, Protoplasma.

[17]  A. Beauvais,et al.  Deletion of GEL2 encoding for a β(1–3)glucanosyltransferase affects morphogenesis and virulence in Aspergillus fumigatus , 2005, Molecular microbiology.

[18]  S. Holzberg,et al.  Barley stripe mosaic virus-induced gene silencing in a monocot plant. , 2002, The Plant journal : for cell and molecular biology.

[19]  M. Bruun-Rasmussen,et al.  Stability of Barley stripe mosaic virus-induced gene silencing in barley. , 2007, Molecular plant-microbe interactions : MPMI.

[20]  Pari Skamnioti,et al.  Of genes and genomes, needles and haystacks: Blumeria graminis and functionality. , 2005, Molecular plant pathology.

[21]  A. Di Pietro,et al.  Fusarium oxysporum gas1 encodes a putative beta-1,3-glucanosyltransferase required for virulence on tomato plants. , 2005, Molecular plant-microbe interactions : MPMI.

[22]  P. Schulze-Lefert,et al.  Cell-autonomous complementation of mlo resistance using a biolistic transient expression system , 1999 .

[23]  P. Schweizer,et al.  Protein Polyubiquitination Plays a Role in Basal Host Resistance of Barley[W][OA] , 2006, The Plant Cell Online.

[24]  P. Schulze-Lefert,et al.  Double-stranded RNA interferes with gene function at the single-cell level in cereals , 2000, The Plant Journal.

[25]  R. Panstruga Establishing compatibility between plants and obligate biotrophic pathogens. , 2003, Current opinion in plant biology.

[26]  Jia-Wei Wang,et al.  Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol , 2007, Nature Biotechnology.

[27]  G. Dimopoulos,et al.  Transcript profiles of Blumeria graminis development during infection reveal a cluster of genes that are potential virulence determinants. , 2005, Molecular plant-microbe interactions : MPMI.

[28]  R. Hückelhoven,et al.  Mutations in Ror1 and Ror2 genes cause modification of hydrogen peroxide accumulation in mlo-barley under attack from the powdery mildew fungus. , 2000, Molecular plant pathology.

[29]  B. Gill,et al.  Development of a Virus-Induced Gene-Silencing System for Hexaploid Wheat and Its Use in Functional Analysis of the Lr21-Mediated Leaf Rust Resistance Pathway1 , 2005, Plant Physiology.

[30]  P. Waterhouse,et al.  Viruses Face a Double Defense by Plant Small RNAs , 2006, Science.

[31]  P. Schulze-Lefert,et al.  Diversity at the Mla powdery mildew resistance locus from cultivated barley reveals sites of positive selection. , 2010, Molecular plant-microbe interactions : MPMI.

[32]  Geert Plaetinck,et al.  Control of coleopteran insect pests through RNA interference , 2007, Nature Biotechnology.

[33]  J. Westwood,et al.  RNA translocation between parasitic plants and their hosts. , 2009, Pest management science.

[34]  P. Schulze-Lefert,et al.  Closing the ranks to attack by powdery mildew. , 2000, Trends in plant science.

[35]  D. Douchkov,et al.  A Set of Modular Binary Vectors for Transformation of Cereals1[W][OA] , 2007, Plant Physiology.

[36]  M. Sudarshana,et al.  Methods for engineering resistance to plant viruses. , 2007, Methods in molecular biology.

[37]  Jeppe Emmersen,et al.  Powdery mildew fungal effector candidates share N-terminal Y/F/WxC-motif , 2010, BMC Genomics.

[38]  Thierry Fontaine,et al.  The Gas family of proteins of Saccharomyces cerevisiae: characterization and evolutionary analysis , 2007, Yeast.

[39]  P. Schweizer,et al.  Heat-induced resistance in barley to the powdery mildew fungus Erysiphe graminis f.sp. hordei , 1995 .

[40]  Catherine Rabouille,et al.  Mechanisms of regulated unconventional protein secretion , 2009, Nature Reviews Molecular Cell Biology.

[41]  Olivier Voinnet,et al.  Non‐cell autonomous RNA silencing , 2005, FEBS letters.