Transgenic Research

, Volume 22, Issue 6, pp 1119–1131 | Cite as

Antisense expression of peach mildew resistance locus O (PpMlo1) gene confers cross-species resistance to powdery mildew in Fragaria x ananassa

  • Derick Jiwan
  • Eric H. Roalson
  • Dorrie Main
  • Amit Dhingra
Original Paper


Powdery mildew (PM) is one of the major plant pathogens. The conventional method of PM control includes frequent use of sulfur-based fungicides adding to production costs and potential harm to the environment. PM remains a major scourge for Rosaceae crops where breeding approaches mainly resort to gene-for-gene resistance. We have tested an alternate source of PM resistance in Rosaceae. Mildew resistance locus O (MLO) has been well studied in barley due to its role in imparting broad spectrum resistance to PM. We identified PpMlo1 (Prunus persica Mlo) in peach and characterized it further to test if a similar mechanism of resistance is conserved in Rosaceae. Due to its recalcitrance in tissue culture, reverse genetic studies involving PpMloI were not feasible in peach. Therefore, Fragaria x ananassa LF9 line, a taxonomic surrogate, was used for functional analysis of PpMlo1. Agrobacterium-mediated transformation yielded transgenic strawberry plants expressing PpMlo1 in sense and antisense orientation. Antisense expression of PpMlo1 in transgenic strawberry plants conferred resistance to Fragaria-specific powdery mildew, Podosphaera macularis. Phylogenetic analysis of 208 putative Mlo gene copies from 35 plant species suggests a large number of duplications of this gene family prior to the divergence of monocots and eudicots, early in eudicot diversification. Our results indicate that the Mlo-based resistance mechanism is functional in Rosaceae, and that Fragaria can be used as a host to test mechanistic function of genes derived from related tree species. To the best of our knowledge, this work is one of the first attempts at testing the potential of using a Mlo-based resistance strategy to combat powdery mildew in Rosaceae.


Rosaceae Powdery mildew Peach Fragaria Mildew locus O 



This work was supported by Washington State University start-up funds to DM and AD. The authors would like to thank Albert G. Abbott, Donna Lalli, Tatyana Zhebentyayeva and Chris Saski at Clemson University for useful discussions and BAC sequencing; Kevin Folta at University of Florida for the Fragaria x ananassa LF9 line; Washington State University colleagues Patrick Moore for providing Fragaria-virulent powdery mildew Podosphaera macularis, Frank Dugan for discussions on pathogen challenge experiments and Pat Okubara for her advice on quantifying pathogen infection. Authors would like to acknowledge the assistance of Nathan Tarlyn in the AD laboratory for maintenance of plants used in this study.

Supplementary material

11248_2013_9715_MOESM1_ESM.docx (444 kb)
Supplementary material 1 (DOCX 443 kb)


  1. Attanayake RN, Glawe DA, McPhee KE, Dugan FM, Chen W (2010) Erysiphe trifolii—a newly recognized powdery mildew pathogen of pea. Plant Pathol 59(4):712–720. doi: 10.1111/j.1365-3059.2010.02306.x CrossRefGoogle Scholar
  2. Burge C, Karlin S (1997) Prediction of complete gene structures in human genomic DNA. J Mol Biol 268(1):78–94PubMedCrossRefGoogle Scholar
  3. Buschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, VanDaelen R, VanderLee T, Diergaarde P, Groenendijk J, Topsch S, Vos P, Salamini F, Schulze-Lefert P (1997) The barley mlo gene: a novel control element of plant pathogen resistance. Cell 88(5):695–705PubMedCrossRefGoogle Scholar
  4. Chen ZY, Noir S, Kwaaitaal M, Hartmann HA, Wu MJ, Mudgil Y, Sukumar P, Muday G, Panstruga R, Jones AM (2009) Two seven-transmembrane domain mildew resistance locus O proteins cofunction in Arabidopsis root thigmomorphogenesis. Plant Cell 21(7):1972–1991PubMedCrossRefGoogle Scholar
  5. Collins TJ (2007) ImageJ for microscopy. Biotechniques 43(1 Suppl):25–30PubMedCrossRefGoogle Scholar
  6. Consonni C, Humphry ME, Hartmann HA, Livaja M, Durner J, Westphal L, Vogel J, Lipka V, Kemmerling B, Schulze-Lefert P, Somerville SC, Panstruga R (2006) Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat Genet 38(6):716–720PubMedCrossRefGoogle Scholar
  7. Devoto A, Piffanelli P, Nilsson I, Wallin E, Panstruga R, von Heijne G, Schulze-Lefert P (1999) Topology, subcellular localization, and sequence diversity of the Mlo family in plants. J Biol Chem 274(49):34993–35004PubMedCrossRefGoogle Scholar
  8. Devoto A, Hartmann HA, Piffanelli P, Elliott C, Simmons C, Taramino G, Goh CS, Cohen FE, Emerson BC, Schulze-Lefert P, Panstruga R (2003) Molecular phylogeny and evolution of the plant-specific seven-transmembrane MLO family. J Mol Evol 56(1):77–88PubMedCrossRefGoogle Scholar
  9. Edgar RC (2004) Muscle: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32(5):1792–1797PubMedCrossRefGoogle Scholar
  10. Ewing B, Hillier L, Wendl MC, Green P (1998) Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8(3):175–185PubMedCrossRefGoogle Scholar
  11. Feechan A, Jermakow AM, Torregrosa L, Panstruga R, Dry IB (2008) Identification of grapevine MLO gene candidates involved in susceptibility to powdery mildew. Funct Plant Biol 35(12):1255–1266CrossRefGoogle Scholar
  12. Flor HH (1971) Current status of the gene-for-gene concept. Annu Rev Phytopathol 9:275–276CrossRefGoogle Scholar
  13. Folta KM, Dhingra A, Howard L, Stewart PJ, Chandler CK (2006) Characterization of LF9, an octoploid strawberry genotype selected for rapid regeneration and transformation. Planta 224(5):1058–1067PubMedCrossRefGoogle Scholar
  14. Galtier N (2001) Maximum-likelihood phylogenetic analysis under a covarion-like model. Mol Biol Evol 18(5):866–873PubMedCrossRefGoogle Scholar
  15. Georgi LL, Wang Y, Yvergniaux D, Ormsbee T, Inigo M, Reighard G, Abbott AG (2002) Construction of a BAC library and its application to the identification of simple sequence repeats in peach (Prunus persica (L.) Batsch). Theor App Genetics 105(8):1151–1158CrossRefGoogle Scholar
  16. Glawe DA (2008) The powdery mildews: a review of the world’s most familiar (yet poorly known) plant pathogens. Annu Rev Phytopathol 46:27–51. doi: 10.1146/annurev.phyto.46.081407.104740 PubMedCrossRefGoogle Scholar
  17. Gordon D, Abajian C, Green P (1998) Consed: a graphical tool for sequence finishing. Genome Res 8(3):195–202PubMedCrossRefGoogle Scholar
  18. Huckelhoven R, Fodor J, Preis C, Kogel KH (1999) Hypersensitive cell death and papilla formation in barley attacked by the powdery mildew fungus are associated with hydrogen peroxide but not with salicylic acid accumulation. Plant Physiol 119(4):1251–1260PubMedCrossRefGoogle Scholar
  19. Huelsenbeck JP, Ronquist F (2001) MRBAYES: bayesian inference of phylogenetic trees. Bioinformatics 17(8):754–755PubMedCrossRefGoogle Scholar
  20. Huelsenbeck JP, Larget B, Miller RE, Ronquist F (2002) Potential applications and pitfalls of Bayesian inference of phylogeny. Syst Biol 51(5):673–688. doi: 10.1080/10635150290102366 PubMedCrossRefGoogle Scholar
  21. Humphry M, Reinstadler A, Ivanov S, Bisseling T, Panstruga R (2011) Durable broad-spectrum powdery mildew resistance in pea er1 plants is conferred by natural loss-of-function mutations in PsMLO1. Mol Plant Pathol. doi: 10.1111/j.1364-3703.2011.00718.x PubMedGoogle Scholar
  22. Jorgensen JH (1992) Discovery, characterization and exploitation of Mlo powdery mildew resistance in barley. Euphytica 63(1–2):141–152CrossRefGoogle Scholar
  23. Kim MC, Panstruga R, Elliott C, Muller J, Devoto A, Yoon HW, Park HC, Cho MJ, Schulze-Lefert P (2002) Calmodulin interacts with MLO protein to regulate defence against mildew in barley. Nature 416(6879):447–450PubMedCrossRefGoogle Scholar
  24. Korber B (2000) HIV signature and sequence variation analysis. Kluwer Academic Publishers, Computational Analysis of HIV Molecular SequencesGoogle Scholar
  25. Lalli DA, Decroocq V, Blenda AV, Schurdi-Levraud V, Garay L, Le Gall O, Damsteegt V, Reighard GL, Abbott AG (2005) Identification and mapping of resistance gene analogs (RGAs) in Prunus: a resistance map for Prunus. Theor Appl Genetics 111(8):1504–1513CrossRefGoogle Scholar
  26. Lewis PO (2001) Phylogenetic systematics turns over a new leaf. Trends Ecol Evol 16(1):30–37PubMedCrossRefGoogle Scholar
  27. Lukashin AV, Borodovsky M (1998) GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res 26(4):1107–1115PubMedCrossRefGoogle Scholar
  28. Lyngkjaer MF, Carver TL (2000) Conditioning of cellular defence responses to powdery mildew in cereal leaves by prior attack. Mol Plant Pathol 1(1):41–49. doi: 10.1046/j.1364-3703.2000.00006.x PubMedCrossRefGoogle Scholar
  29. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  30. Panstruga R (2005) Discovery of novel conserved peptide domains by ortholog comparison within plant multi-protein families. Plant Mol Biol 59(3):485–500PubMedCrossRefGoogle Scholar
  31. Pavan S, Jacobsen E, Visser RGF, Bai Y (2010) Loss of susceptibility as a novel breeding strategy for durable and broad-spectrum resistance. Mol Breed 25(1):1–12. doi: 10.1007/s11032-009-9323-6 PubMedCrossRefGoogle Scholar
  32. Peries OS (1962) Studies on strawberry mildew, caused by Spareotheca macularis (Wallr. Ex. Fries) Jaczewski: I. biology of the fungus. Ann Appl Biol 50:211–224CrossRefGoogle Scholar
  33. Quevillon E, Silventoinen V, Pillai S, Harte N, Mulder N, Apweiler R, Lopez R (2005) InterProScan: protein domains identifier. Nucleic Acids Res 33:W116–W120PubMedCrossRefGoogle Scholar
  34. Rusterucci C, Aviv DH, Holt BF, Dangl JL, Parker JE (2001) The disease resistance signalling components EDS1 and PAD4 are essential regulators of the cell death pathway controlled by LSD1 in arabidopsis. Plant Cell 13(10):2211–2224PubMedGoogle Scholar
  35. Salamov AA, Solovyev VV (2000) Ab initio gene finding in Drosophila genomic DNA. Genome Res 10(4):516–522PubMedCrossRefGoogle Scholar
  36. Schultheiss H, Dechert C, Kogel KH, Huckelhoven R (2002) A small GTP-binding host protein is required for entry of powdery mildew fungus into epidermal cells of barley. Plant Physiol 128(4):1447–1454PubMedCrossRefGoogle Scholar
  37. Schultz J, Milpetz F, Bork P, Ponting CP (1998) SMART, a simple modular architecture research tool: identification of signalling domains. Proc Natl Acad Sci USA 95(11):5857–5864PubMedCrossRefGoogle Scholar
  38. Shulaev V, Korban SS, Sosinski B, Abbott AG, Aldwinckle HS, Folta KM, Iezzoni A, Main D, Arus P, Dandekar AM, Lewers K, Brown SK, Davis TM, Gardiner SE, Potter D, Veilleux RE (2008) Multiple models for Rosaceae genomics. Plant Physiol 147(3):985–1003PubMedCrossRefGoogle Scholar
  39. Shulaev V, Sargent DJ, Crowhurst RN, Mockler TC, Folkerts O, Delcher AL, Jaiswal P, Mockaitis K, Liston A, Mane SP, Burns P, Davis TM, Slovin JP, Bassil N, Hellens RP, Evans C, Harkins T, Kodira C, Desany B, Crasta OR, Jensen RV, Allan AC, Michael TP, Setubal JC, Celton JM, Rees DJ, Williams KP, Holt SH, Rojas JJ, Chatterjee M, Liu B, Silva H, Meisel L, Adato A, Filichkin SA, Troggio M, Viola R, Ashman TL, Wang H, Dharmawardhana P, Elser J, Raja R, Priest HD, Bryant DW Jr, Fox SE, Givan SA, Wilhelm LJ, Naithani S, Christoffels A, Salama DY, Carter J, Girona EL, Zdepski A, Wang W, Kerstetter RA, Schwab W, Korban SS, Davik J, Monfort A, Denoyes-Rothan B, Arus P, Mittler R, Flinn B, Aharoni A, Bennetzen JL, Salzberg SL, Dickerman AW, Velasco R, Borodovsky M, Veilleux RE, Folta KM (2011) The genome of woodland strawberry (Fragaria vesca). Nat Genet 43(2):109–116. doi: 10.1038/ng.740 PubMedCrossRefGoogle Scholar
  40. Staskawicz BJ, Ausubel FM, Baker BJ, Ellis JG, Jones JDG (1995) Molecular-genetics of plant-disease resistance. Science 268(5211):661–667PubMedCrossRefGoogle Scholar
  41. Takamatsu S, Niinomi S, Harada M, Havrylenko M (2010) Molecular phylogenetic analyses reveal a close evolutionary relationship between Podosphaera (Erysiphales: Erysiphaceae) and its rosaceous hosts. Persoonia 24:38–48PubMedCrossRefGoogle Scholar
  42. Thompson JD, Higgins DG, Gibson TJ (1994) Clustal-W—improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680PubMedCrossRefGoogle Scholar
  43. ThordalChristensen H, Zhang ZG, Wei YD, Collinge DB (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11(6):1187–1194CrossRefGoogle Scholar
  44. Tor M, Lotze MT, Holton N (2009) Receptor-mediated signalling in plants: molecular patterns and programmes. J Exp Bot 60(13):3645–3654PubMedCrossRefGoogle Scholar
  45. Tuffley C, Steel M (1998) Modeling the covarion hypothesis of nucleotide substitution. Math Biosci 147(1):63–91PubMedCrossRefGoogle Scholar
  46. Vogel J, Somerville S (2000) Isolation and characterization of powdery mildew-resistant Arabidopsis mutants. Proc Natl Acad Sci USA 97(4):1897–1902PubMedCrossRefGoogle Scholar
  47. von Arnim AG, Deng XW, Stacey MG (1998) Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants. Gene 221(1):35–43CrossRefGoogle Scholar
  48. Yin ZC, Chen J, Zeng LR, Goh ML, Leung H, Khush GS, Wang GL (2000) Characterizing rice lesion mimic mutants and identifying a mutant with broad-spectrum resistance to rice blast and bacterial blight. Mol Plant Microbe Interact 13(8):869–876PubMedCrossRefGoogle Scholar
  49. Zhou JM, Loh YT, Bressan RA, Martin GB (1995) The tomato gene Pti1 encodes a serine/threonine kinase that is phosphorylated by Pto and is involved in the hypersensitive response. Cell 83(6):925–935PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Derick Jiwan
    • 1
  • Eric H. Roalson
    • 2
  • Dorrie Main
    • 1
  • Amit Dhingra
    • 1
  1. 1.Department of HorticultureWashington State UniversityPullmanUSA
  2. 2.School of Biological SciencesWashington State UniversityPullmanUSA

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