Advertisement

Effectoromics-Based Identification of Cell Surface Receptors in Potato

  • Emmanouil Domazakis
  • Xiao Lin
  • Carolina Aguilera-Galvez
  • Doret Wouters
  • Gerard Bijsterbosch
  • Pieter J. Wolters
  • Vivianne G. A. A. VleeshouwersEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1578)

Abstract

In modern resistance breeding, effectors have emerged as tools for accelerating and improving the identification of immune receptors. Effector-assisted breeding was pioneered for identifying resistance genes (R genes) against Phytophthora infestans in potato (Solanum tuberosum). Here we show that effectoromics approaches are also well suitable for identifying pathogen recognition receptors (PRRs) that recognize apoplastic effectors. To detect genotypes that recognize apoplastic proteins of P. infestans, routine agroinfiltration and potato virus X (PVX) agroinfection methods can be applied. In addition, protein infiltrations are feasible for assessing responses to apoplastic effectors and aid in confirming results obtained from the aforementioned methods. Protocols for the effectoromics pipeline are provided, starting from phenotyping for effector responses, up to genotyping and PRR gene identification.

Key words

Pattern recognition receptors (PRRs) Apoplastic effector Genetic mapping Agroinfiltration PVX agroinfection Protein infiltration Effectoromics Yeast protein production Solanum 

Notes

Acknowledgments

This work was supported by a NWO-VIDI grant 12378 (ED, XL, DW, VGAAV), the China Scholarship Council Program for Graduate Students (XL), Colciendas (CAG), Veenhuizen Tulp Fonds (CAG), J.R. Simplot Company (PJW), and COST FA1208 (XL).

References

  1. 1.
    Vleeshouwers VGAA, Raffaele S, Vossen JH et al (2011) Understanding and exploiting late blight resistance in the age of effectors. Annu Rev Phytopathol 49:507–531. doi: 10.1146/annurev-phyto-072910-095326 CrossRefPubMedGoogle Scholar
  2. 2.
    Vleeshouwers VGAA, Rietman H, Krenek P et al (2008) Effector genomics accelerates discovery and functional profiling of potato disease resistance and Phytophthora infestans avirulence genes. PLoS One 3(8):e2875. doi: 10.1371/journal.pone.0002875 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Takken FL, Luderer R, Gabriels SH et al (2000) A functional cloning strategy, based on a binary PVX-expression vector, to isolate HR-inducing cDNAs of plant pathogens. Plant J 24(2):275–283. doi: 10.1046/j.1365-313x.2000.00866.x CrossRefPubMedGoogle Scholar
  4. 4.
    Vleeshouwers VGAA, Driesprong JD, Kamphuis LG et al (2006) Agroinfection-based high-throughput screening reveals specific recognition of INF elicitins in Solanum. Mol Plant Pathol 7(6):499–510. doi: 10.1111/j.1364-3703.2006.00355.x CrossRefPubMedGoogle Scholar
  5. 5.
    Kapila J, De Rycke R, Van Montagu M et al (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122(1):101–108. doi: 10.1016/s0168-9452(96)04541-4 CrossRefGoogle Scholar
  6. 6.
    Kanneganti T-D, Huitema E, Kamoun S (2007) In planta expression of oomycete and fungal Genes. In: Plant-pathogen interactions, vol 354. Methods Mol Biol, pp 35–43. doi: 10.1385/1-59259-966-4:35
  7. 7.
    Du J, Vleeshouwers VGAA (2014) The do’s and don’ts of effectoromics. In: Plant-pathogen interactions, vol 1127. Methods Mol Biol, pp 257–268. doi: 10.1007/978-1-62703-986-4_19
  8. 8.
    Du J, Verzaux E, Chaparro-Garcia A et al (2015) Elicitin recognition confers enhanced resistance to Phytophthora infestans in potato. Nat Plants 1(4):15034. doi: 10.1038/nplants.2015.34 CrossRefPubMedGoogle Scholar
  9. 9.
    Oliver RP, Friesen TL, Faris JD et al (2012) Stagonospora nodorum: from pathology to genomics and host resistance. Annu Rev Phytopathol 50:23–43. doi: 10.1146/annurev-phyto-081211-173019 CrossRefPubMedGoogle Scholar
  10. 10.
    Vleeshouwers VGAA, Oliver RP (2014) Effectors as tools in disease resistance breeding against biotrophic, hemibiotrophic, and necrotrophic plant pathogens. Mol Plant Microbe Interact 27(3):196–206. doi: 10.1094/MPMI-10-13-0313-IA CrossRefPubMedGoogle Scholar
  11. 11.
    Huang S, van der Vossen EA, Kuang H et al (2005) Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato. Plant J 42(2):251–261. doi: 10.1111/j.1365-313X.2005.02365.x CrossRefPubMedGoogle Scholar
  12. 12.
    Armstrong MR, Whisson SC, Pritchard L et al (2005) An ancestral oomycete locus contains late blight avirulence gene Avr3a, encoding a protein that is recognized in the host cytoplasm. Proc Natl Acad Sci U S A 102(21):7766–7771. doi: 10.1073/pnas.0500113102 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Torto TA, Li S, Styer A et al (2003) EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora. Genome Res 13(7):1675–1685. doi: 10.1101/gr.910003 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Du J, Rietman H, Vleeshouwers VGAA (2014) Agroinfiltration and PVX agroinfection in potato and Nicotiana benthamiana. J Vis Exp 83:e50971. doi: 10.3791/50971 Google Scholar
  15. 15.
    Tan KC, Oliver RP, Solomon PS et al (2010) Proteinaceous necrotrophic effectors in fungal virulence. Funct Plant Biol 37(10):907–912. doi: 10.1071/FP10067 CrossRefGoogle Scholar
  16. 16.
    Liu Z, Zhang Z, Faris JD et al (2012) The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1. PLoS Pathog 8(1):e1002467. doi: 10.1371/journal.ppat.1002467 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Saunders DGO, Win J, Cano LM et al. (2012) Using hierarchical clustering of Secreted protein families to classify and rank candidate effectors of rust fungi. PLoS One 7(1):e29847. doi: 10.1371/journal.pone.0029847
  18. 18.
    Mirzadi Gohari A, Ware SB, Wittenberg AH et al (2015) Effector discovery in the fungal wheat pathogen Zymoseptoria tritici. Mol Plant Pathol 16(9):931–945. doi: 10.1111/mpp.12251 CrossRefPubMedGoogle Scholar
  19. 19.
    Petersen TN, Brunak S, von Heijne G et al (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8(10):785–786CrossRefPubMedGoogle Scholar
  20. 20.
    Haas BJ, Kamoun S, Zody MC et al (2009) Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans. Nature 461(7262):393–398. doi: 10.1038/nature08358 CrossRefPubMedGoogle Scholar
  21. 21.
    Ma LJ, van der Does HC, Borkovich KA et al (2010) Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature 464(7287):367–373. doi: 10.1038/nature08850 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Schmidt SM, Houterman PM, Schreiver I et al (2013) MITEs in the promoters of effector genes allow prediction of novel virulence genes in Fusarium oxysporum. BMC Genomics 14(1):119. doi: 10.1186/1471-2164-14-119 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Bolton MD, van Esse HP, Vossen JH et al (2008) The novel Cladosporium fulvum lysin motif effector Ecp6 is a virulence factor with orthologues in other fungal species. Mol Microbiol 69(1):119–136. doi: 10.1111/j.1365-2958.2008.06270.x CrossRefPubMedGoogle Scholar
  24. 24.
    Manning VA, Hamilton SM, Karplus PA et al (2008) The Arg-Gly-Asp-containing, solvent-exposed loop of Ptr ToxA is required for internalization. Mol Plant Microbe Interact 21(3):315–325. doi: 10.1094/Mpmi-21-3-0315 CrossRefPubMedGoogle Scholar
  25. 25.
    Whisson SC, Boevink PC, Moleleki L et al (2007) A translocation signal for delivery of oomycete effector proteins into host plant cells. Nature 450(7166):115–118. doi: 10.1038/nature06203 CrossRefPubMedGoogle Scholar
  26. 26.
    Ahmad M, Hirz M, Pichler H et al (2014) Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Appl Microbiol Biotechnol 98(12):5301–5317. doi: 10.1007/s00253-014-5732-5 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Brondyk WH, Richard RB, Murray PD (2009) Selecting an appropriate method for expressing a recombinant protein. In: Methods Enzymol, vol 463. Academic Press, New York, pp 131–147. doi: 10.1016/S0076-6879(09)63011-1
  28. 28.
    Jupe F, Witek K, Verweij W et al (2013) Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations. Plant J 76(3):530–544. doi: 10.1111/tpj.12307 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9(10):963–967CrossRefPubMedGoogle Scholar
  30. 30.
    Koncz C, Schell J (1986) The promoter of Tl-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204(3):383–396. doi: 10.1007/Bf00331014 CrossRefGoogle Scholar
  31. 31.
    Van der Hoorn RA, Laurent F, Roth R et al (2000) Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis. Mol Plant Microbe Interact 13(4):439–446. doi: 10.1094/MPMI.2000.13.4.439 CrossRefPubMedGoogle Scholar
  32. 32.
    Karimi M, Inze D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7(5):193–195CrossRefPubMedGoogle Scholar
  33. 33.
    Lu R, Malcuit I, Moffett P et al (2003) High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance. EMBO J 22(21):5690–5699. doi: 10.1093/emboj/cdg546 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    PichiaPink™ Expression System manual (2014). Life Technol, MAN0000717Google Scholar
  35. 35.
    Milbourne D, Meyer RC, Collins AJ et al (1998) Isolation, characterisation and mapping of simple sequence repeat loci in potato. Mol Gen Genet 259(3):233–245CrossRefPubMedGoogle Scholar
  36. 36.
    Hardigan MA, Crisovan E, Hamilton JP et al (2016) Genome reduction uncovers a large dispensable genome and adaptive role for copy number variation in asexually propagated Solanum tuberosum. Plant Cell 28(2):388–405. doi: 10.1105/tpc.15.00538 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Potato Genome Sequencing Consortium, Xu X, Pan S et al (2011) Genome sequence and analysis of the tuber crop potato. Nature 475(7355):189–195Google Scholar
  38. 38.
    Jones JD, Dangl JL (2006) The plant immune system. Nature 444(7117):323–329. doi: 10.1038/nature05286 CrossRefPubMedGoogle Scholar
  39. 39.
    Katagiri F, Tsuda K (2010) Understanding the plant immune system. Mol Plant Microbe Interact 23(12):1531–1536. doi: 10.1094/MPMI-04-10-0099 CrossRefPubMedGoogle Scholar
  40. 40.
    Cheng C, Gao X, Feng B et al (2013) Plant immune response to pathogens differs with changing temperatures. Nat Commun 4:2530. doi: 10.1038/ncomms3530 PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Emmanouil Domazakis
    • 1
  • Xiao Lin
    • 1
  • Carolina Aguilera-Galvez
    • 1
  • Doret Wouters
    • 1
  • Gerard Bijsterbosch
    • 1
  • Pieter J. Wolters
    • 1
  • Vivianne G. A. A. Vleeshouwers
    • 1
    Email author
  1. 1.Plant BreedingWageningen University & ResearchWageningenThe Netherlands

Personalised recommendations