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The role of cuscutain-propeptide inhibitor in haustoria parasitism and enhanced resistance to dodder in transgenic alfalfa expressing this propeptide

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Cuscutain is a cysteine protease produced by dodder (the most important weeds of alfalfa) that is essential for the development and penetration of the haustoria in host. The propeptide subunit of cuscutain has a specific inhibitory function and inhibits the enzymatic activity of the cuscutain. In this study, we introduced the gene encoding the propeptide segment of the cuscutain (signal peptide-less inhibitor) into alfalfa and investigated its roles in parasitism and the alfalfa resistance to C. reflexa. Results demonstrated that cuscutain is mainly expressed in haustoria and the expression of propeptide in transgenic alfalfa plants effectively inhibited cuscutain enzyme activity and consequently interrupted haustoria development at the pathogenic stage. Digitate cells of haustoria could not differentiate into the xylem and phloem hyphae in dodder grown on transgenic alfalfa. Dodder development on transgenic alfalfa lines showed an overall reduction in fecundity and vigor due to imperfect attachment of haustoria. Morphology, nodule development and biomass of transgenic plants indicate that the inhibitory transgene exhibits exquisite specificity for cuscutain enzyme and by expression of the inhibitor in transgenic plants, there was no obvious adverse effect on them. The increased development and growth of dodder-challenged alfalfa transgenic plants compared to controls, showed the efficacy of propeptide in dodder control.

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  1. Albert M, Kaiser B, Van der Krol S, Kaldenhoff R (2010) Calcium signaling during the plant-plant interaction of parasitic Cuscuta reflexa with its hosts. Plant Signal Behav 59:1144–1146

  2. Amini M, Saify Nabiabad H, Deljou A (2017) Host-synthesized cysteine protease-specific inhibitor disrupts Cuscuta campestris parasitism in tomato. Plant Biotechnol Rep 2(5):1–10

  3. Ashigh J, Marquez E (2010) Dodder (Cuscuta spp.) biology and management. NM State University, Guide A-615

  4. Birschwilks M, Sauer N, Scheel D, Neumann S (2007) Arabidopsis thaliana is a susceptible host plant for the holoparasite Cuscuta spec. Planta 226:1231–1241

  5. Bleischwitz M, Albert M, Fuchsbauer H, Kaldenhoff R (2010) Significance of cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions. BMC Plant Biol 10:227

  6. Furuhashi T, Furuhashi K, Weckwerth W (2011) The parasitic mechanism of the holostemparasitic plant Cuscuta. Journal of Plant Interaction 6:207–219

  7. Hong L, Shen H, Chen H, Li L (2011) The morphology and anatomy of the haustoria of the holoparasitic angiosperm Cuscuta campestris. Pak J Bot 43(4):1853–1859

  8. Hosseinkhani S, Mortazavi M, Emamzadeh A (2008) Expression and purification of the luciferase enzyme and in Vivo ATP Assay. J Physiol Pharmacol 12(2):109–114

  9. Kim SH, Hamada T (2005) Rapid and reliable method of extracting DNA and RNA from sweet potato, Ipomoea batatas (L.). Lam Biotechnology Letters 27:1841–1845

  10. Lee KB (2007) Structure and development of the upper haustorium in the parasitic flowering plant Cuscuta japonica (Convolvulaceae). Am J Bot 94(5):737–745

  11. Martinez M, Cambra I, Gonzalez-Melendi P, Santamaria ME, Diaz I (2012) C1A cysteine-proteases and their inhibitors in plants. Physiol Plant 145(1):85–94

  12. Nga BTT, Takeshita Y, Yamamoto M (2014) Studies of inhibitory mechanisms of propeptide-like cysteine protease inhibitors. Enzyme Res 2014:1–10

  13. Okamoto T, Shimada T, Hara-Nishimura I, Nishimura M, Minamikawa T (2003) C-terminal KDEL sequence of a KDEL-tailed cysteine-proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle in efficient vacuolar transport of sulfhydryl-endopeptidase. Plant Physiol 132:1892–1900

  14. Rzychon M, Chmiel D, Stec-Niemczyk J (2004) Modes of inhibition of cysteine proteases. Acta Biochim Pol 51:861–873

  15. Saify Nabiabad H, Yaghoobi M, Jalali-Javaran M, Hosseinkhani S (2011) Expression analysis and purification of human recombinant tissue type plasminogen activator (rtPA) from transgenic tobacco plants. Preparative Biochem Biotechnol 41:175–186

  16. Santamaria M, Arnaiz A, Mendoza M, Martinez M, Diaz I (2015) Inhibitory properties of cysteine protease pro-peptides from barley confer resistance to spider mite feeding. PLOS One 10(6):e0128323

  17. Saric-Krsmanovic M, Bozic D, Malidza G, Radivojevic L et al (2015) Chemical control of field dodder in alfalfa. Pesticides Phytomedicine 30:107–114

  18. Sheokand S, Dahiya P, Vincent JL, Brewin N (2005) Modified expression of cysteine protease affects seed germination, vegetative growth and nodule development in transgenic lines of Medicago truncatula. Plant Sci 169:966–975

  19. Tohidfar M, Zare N, Salehi- Jouzani GH, Eftekhari SM (2013) Agrobacterium-mediated transformation of alfalfa (Medicago sativa) using a synthetic cry3a gene to enhance resistance against alfalfa weevil. Plant Cell Tissue Organ Cult113:227–235

  20. Weigel D, Glazebrook J (2006) Transformation of Agrobacterium using the freeze-thaw method. Cold Spring Harbor Protocols

  21. Wenting L, Liang Z, Shan C, Marsolais F, Tian L (2013) Genetic transformation and full recovery of alfalfa plants via secondary somatic embryogenesis. In Vitro Cell Dev Biol Plant 49(1):17–23

  22. Yoder JI, Scholes JD (2010) Host plant resistance to parasitic weeds; recent progress and bottlenecks. Curr Opin Plant Biol 13:478–484

  23. Yousefi N, Chehregani A, Malayeri B, Lorestani M. (Chenopodiaceae (2011) Effect of the heavy metals on the developmental stages of ovule and seed proteins in Chenopodium botrys L. Biol Trace Elem Res 144(1):1142–1149

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The authors are grateful to the University of Bu Ali Sina for the material support [grant no. 23.7.92].

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All authors contributed equally to this study. Amini M. performed transformation, Saify H. performed protein purification and enzy assay, and data was analysed done by A. Deljou.

Correspondence to Haidar Saify Nabiabad.

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The authors declare that they have no conflict of interest.

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Amini, M., Saify Nabiabad, H. & Deljou, A. The role of cuscutain-propeptide inhibitor in haustoria parasitism and enhanced resistance to dodder in transgenic alfalfa expressing this propeptide. Plant Biotechnol Rep 12, 165–173 (2018). https://doi.org/10.1007/s11816-018-0481-z

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  • Cuscuta reflexa
  • Cuscutain, Haustoria
  • Propeptide
  • Medicago sativa
  • Resistance