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Prediction of Leymus arenarius (L.) antimicrobial peptides based on de novo transcriptome assembly

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Abstract

Leymus arenarius is a unique wild growing Poaceae plant exhibiting extreme tolerance to environmental conditions. In this study we for the first time performed whole-transcriptome sequencing of lymegrass seedlings using Illumina platform followed by de novo transcriptome assembly and functional annotation. Our goal was to identify transcripts encoding antimicrobial peptides (AMPs), one of the key components of plant innate immunity. Using the custom software developed for this study that predicted AMPs and classified them into families, we revealed more than 160 putative AMPs in lymegrass seedlings. We classified them into 7 families based on their cysteine motifs and sequence similarity. The families included defensins, thionins, hevein-like peptides, snakins, cyclotide, alfa-hairpinins and LTPs. This is the first communication about the presence of almost all known AMP families in trascriptomic data of a single plant species. Additionally, cysteine-rich peptides that potentially represent novel families of AMPs were revealed. We have confirmed by RT-PCR validation the presence of 30 transcripts encoding selected AMPs in lymegrass seedlings. In summary, the presented method of pAMP prediction developed by us can be applied for relatively fast and simple screening of novel components of plant immunity system and is well suited for whole-transcriptome or genome analysis of uncharacterized plants.

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References

  • Andreev YA, Korostyleva TV, Slavokhotova AA, Rogozhin EA, Utkina LL, Vassilevski AA, Grishin EV, Egorov TA, Odintsova TI (2012) Genes encoding hevein-like defense peptides in wheat: distribution, evolution, and role in stress response. Biochimie 94:1009–1016

    Article  CAS  PubMed  Google Scholar 

  • Berrocal-Lobo M, Segura A, Moreno M, Lopez G, Garcia-Olmedo F, Molina A (2002) Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection. Plant Physiol 128:951–961

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Bureau TE, Ronald PC, Wessler SR (1996) A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild-type rice genes. Proc Natl Acad Sci USA 93:8524–8529

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chen KC, Lin CY, Kuan CC, Sung HY, Chen CS (2002) A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid. J Agric Food Chem 50:7258–7263

    Article  CAS  PubMed  Google Scholar 

  • Chen S, Huang X, Yan X, Liang Y, Wang Y, Li X, Peng X, Ma X, Zhang L, Cai Y, Ma T, Cheng L, Qi D, Zheng H, Yang X, Li X, Liu G (2013) Transcriptome analysis in sheepgrass (Leymus chinensis): a dominant perennial grass of the Eurasian Steppe. PLoS ONE 8:e67974

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Chen S, Cai Y, Zhang L, Yan X, Cheng L, Qi D, Zhou Q, Li X, Liu G (2014) Transcriptome analysis reveals common and distinct mechanisms for sheepgrass (Leymus chinensis) responses to defoliation compared to mechanical wounding. PLoS ONE 9:e89495

    Article  PubMed Central  PubMed  Google Scholar 

  • Choi JY, Roh JY, Wang Y, Zhen Z, Tao XY, Lee JH, Liu Q, Kim JS, Shin SW, Je YH (2012) Analysis of genes expression of Spodoptera exigua larvae upon AcMNPV infection. PLoS ONE 7:e42462

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676

    Article  CAS  PubMed  Google Scholar 

  • Conners R, Konarev AV, Forsyth J, Lovegrove A, Marsh J, Joseph-Horne T, Shewry P, Brady RL (2007) An unusual helix-turn-helix protease inhibitory motif in a novel trypsin inhibitor from seeds of Veronica (Veronica hederifolia L.). J Biol Chem 282:27760–27768

    Article  CAS  PubMed  Google Scholar 

  • Egorov Ts A, Odintsova TI (2012) Defense peptides of plant immune system. Bioorg Khim 38:7–17

    CAS  PubMed  Google Scholar 

  • Egorov TA, Odintsova TI, Pukhalsky VA, Grishin EV (2005) Diversity of wheat anti-microbial peptides. Peptides 26:2064–2073

    Article  CAS  PubMed  Google Scholar 

  • Fan X, Sha LN, Yang RW, Zhang HQ, Kang HY, Ding CB, Zhang L, Zheng YL, Zhou YH (2009) Phylogeny and evolutionary history of Leymus (Triticeae; Poaceae) based on a single-copy nuclear gene encoding plastid acetyl-CoA carboxylase. BMC Evolut Biol 9:247

    Article  Google Scholar 

  • Florack DE, Stiekema WJ (1994) Thionins: properties, possible biological roles and mechanisms of action. Plant Mol Biol 26:25–37

    Article  CAS  PubMed  Google Scholar 

  • Greipsson S, Davy AJ (1994) Leymus arenarius. Characteristics and uses of a dune-building grass. Icel Agr Sci 8:41–50

    Google Scholar 

  • Guimaraes PM, Brasileiro AC, Morgante CV, Martins AC, Pappas G, Silva OB Jr, Togawa R, Leal-Bertioli SC, Araujo AC, Moretzsohn MC, Bertioli DJ (2012) Global transcriptome analysis of two wild relatives of peanut under drought and fungi infection. BMC Genom 13:387

    Article  CAS  Google Scholar 

  • Jagadish K, Camarero JA (2010) Cyclotides, a promising molecular scaffold for peptide-based therapeutics. Biopolymers 94:611–616

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Jaworski DC, Zou Z, Bowen CJ, Wasala NB, Madden R, Wang Y, Kocan KM, Jiang H, Dillwith JW (2010) Pyrosequencing and characterization of immune response genes from the American dog tick, Dermacentor variabilis (L.). Insect Mol Biol 19:617–630

    Article  CAS  PubMed  Google Scholar 

  • Kader JC (1996) Lipid-transfer proteins in plants. Annu Rev Plant Physiol Plant Mol Biol 47:627–654

    Article  CAS  PubMed  Google Scholar 

  • Koike M, Okamoto T, Tsuda S, Imai R (2002) A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation. Biochem Biophys Res Commun 298:46–53

    Article  CAS  PubMed  Google Scholar 

  • Li F, Yang XX, Xia HC, Zeng R, Hu WG, Li Z, Zhang ZC (2003) Purification and characterization of Luffin P1, a ribosome-inactivating peptide from the seeds of Luffa cylindrica. Peptides 24:799–805

    Article  CAS  PubMed  Google Scholar 

  • Liu YJ, Cheng CS, Lai SM, Hsu MP, Chen CS, Lyu PC (2006) Solution structure of the plant defensin VrD1 from mung bean and its possible role in insecticidal activity against bruchids. Proteins 63:777–786

    Article  CAS  PubMed  Google Scholar 

  • Luna-Ramirez K, Quintero-Hernandez V, Vargas-Jaimes L, Batista CV, Winkel KD, Possani LD (2013) Characterization of the venom from the Australian scorpion Urodacus yaschenkoi: molecular mass analysis of components, cDNA sequences and peptides with antimicrobial activity. Toxicon 63:44–54

    Article  CAS  PubMed  Google Scholar 

  • Ma Y, Liu C, Liu X, Wu J, Yang H, Wang Y, Li J, Yu H, Lai R (2010) Peptidomics and genomics analysis of novel antimicrobial peptides from the frog, Rana nigrovittata. Genomics 95:66–71

    Article  CAS  PubMed  Google Scholar 

  • Marcus JP, Green JL, Goulter KC, Manners JM (1999) A family of antimicrobial peptides is produced by processing of a 7S globulin protein in Macadamia integrifolia kernels. Plant J 19:699–710

    Article  CAS  PubMed  Google Scholar 

  • Melo FR, Rigden DJ, Franco OL, Mello LV, Ary MB, Grossi de Sa MF, Bloch C Jr (2002) Inhibition of trypsin by cowpea thionin: characterization, molecular modeling, and docking. Proteins 48:311–319

    Article  CAS  PubMed  Google Scholar 

  • Mendez E, Moreno A, Colilla F, Pelaez F, Limas GG, Mendez R, Soriano F, Salinas M, de Haro C (1990) Primary structure and inhibition of protein synthesis in eukaryotic cell-free system of a novel thionin, gamma-hordothionin, from barley endosperm. Eur J Biochem 194:533–539

    Article  CAS  PubMed  Google Scholar 

  • Moreira R, Balseiro P, Planas JV, Fuste B, Beltran S, Novoa B, Figueras A (2012) Transcriptomics of in vitro immune-stimulated hemocytes from the Manila clam Ruditapes philippinarum using high-throughput sequencing. PLoS ONE 7:e35009

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Nguyen GK, Lim WH, Nguyen PQ, Tam JP (2012) Novel cyclotides and uncyclotides with highly shortened precursors from Chassalia chartacea and effects of methionine oxidation on bioactivities. J Biol Chem 287:17598–17607

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Nguyen GK, Lian Y, Pang EW, Nguyen PQ, Tran TD, Tam JP (2013) Discovery of linear cyclotides in monocot plant Panicum laxum of Poaceae family provides new insights into evolution and distribution of cyclotides in plants. J Biol Chem 288:3370–3380

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Nolde SB, Vassilevski AA, Rogozhin EA, Barinov NA, Balashova TA, Samsonova OV, Baranov YV, Feofanov AV, Egorov TA, Arseniev AS, Grishin EV (2011) Disulfide-stabilized helical hairpin structure and activity of a novel antifungal peptide EcAMP1 from seeds of barnyard grass (Echinochloa crus-galli). J Biol Chem 286:25145–25153

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Odintsova TI, Rogozhin EA, Baranov Y, Musolyamov A, Yalpani N, Egorov TA, Grishin EV (2008) Seed defensins of barnyard grass Echinochloa crusgalli (L.) Beauv. Biochimie 90:1667–1673

    Article  CAS  PubMed  Google Scholar 

  • Odintsova TI, Vassilevski AA, Slavokhotova AA, Musolyamov AK, Finkina EI, Khadeeva NV, Rogozhin EA, Korostyleva TV, Pukhalsky VA, Grishin EV, Egorov TA (2009) A novel antifungal hevein-type peptide from Triticum kiharae seeds with a unique 10-cysteine motif. FEBS J 276:4266–4275

    Article  CAS  PubMed  Google Scholar 

  • Oparin PB, Mineev KS, Dunaevsky YE, Arseniev AS, Belozersky MA, Grishin EV, Egorov TA, Vassilevski AA (2012) Buckwheat trypsin inhibitor with helical hairpin structure belongs to a new family of plant defence peptides. Biochem J 446:69–77

    Article  CAS  PubMed  Google Scholar 

  • Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786

    Article  CAS  PubMed  Google Scholar 

  • Porto WF, Pires AS, Franco OL (2012) CS-AMPPred: an updated SVM model for antimicrobial activity prediction in cysteine-stabilized peptides. PLoS ONE 7:e51444

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Prasath D, Karthika R, Habeeba NT, Suraby EJ, Rosana OB, Shaji A, Eapen SJ, Deshpande U, Anandaraj M (2014) Comparison of the transcriptomes of ginger (Zingiber officinale Rosc.) and mango ginger (Curcuma amada Roxb.) in response to the bacterial wilt infection. PLoS ONE 9:e99731

    Article  PubMed Central  PubMed  Google Scholar 

  • Pushpanathan M, Gunasekaran P, Rajendhran J (2013) Antimicrobial peptides: versatile biological properties. Int J Pept 2013:675391

    Article  PubMed Central  PubMed  Google Scholar 

  • Segura A, Moreno M, Molina A, Garcia-Olmedo F (1998) Novel defensin subfamily from spinach (Spinacia oleracea). FEBS Lett 435:159–162

    Article  CAS  PubMed  Google Scholar 

  • Segura A, Moreno M, Madueno F, Molina A, Garcia-Olmedo F (1999) Snakin-1, a peptide from potato that is active against plant pathogens. Mol Plant Microbe Interact 12:16–23

    Article  CAS  PubMed  Google Scholar 

  • Silverstein KA, Graham MA, Paape TD, VandenBosch KA (2005) Genome organization of more than 300 defensin-like genes in Arabidopsis. Plant Physiol 138:600–610

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Silverstein KA, Moskal WA Jr, Wu HC, Underwood BA, Graham MA, Town CD, VandenBosch KA (2007) Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants. Plant J 51:262–280

    Article  CAS  PubMed  Google Scholar 

  • Slavokhotova AA, Odintsova TI, Rogozhin EA, Musolyamov AK, Andreev YA, Grishin EV, Egorov TA (2011) Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds. Biochimie 93:450–456

    Article  CAS  PubMed  Google Scholar 

  • Slavokhotova AA, Naumann TA, Price NP, Rogozhin EA, Andreev YA, Vassilevski AA, Odintsova TI (2014a) Novel mode of action of plant defense peptides—hevein-like antimicrobial peptides from wheat inhibit fungal metalloproteases. FEBS J 281:4754–4764

    Article  CAS  PubMed  Google Scholar 

  • Slavokhotova AA, Rogozhin EA, Musolyamov AK, Andreev YA, Oparin PB, Berkut AA, Vassilevski AA, Egorov TA, Grishin EV, Odintsova TI (2014b) Novel antifungal alpha-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and evolution. Plant Mol Biol 84:189–202

    Article  CAS  PubMed  Google Scholar 

  • Soltis DE, Soltis PS, Tate JA (2004) Advances in the study of polyploidy since plant speciation. New Phytol 161:173–191

    Article  CAS  Google Scholar 

  • Stotz HU, Waller F, Wang K (2013) Innate immunity in plants: the role of antimicrobial peptides. Antimicrobial peptides and innate immunity. Progress in Inflammation Research pp 29–51

  • Sun Q, Jiang H, Zhu X, Wang W, He X, Shi Y, Yuan Y, Du X, Cai Y (2013a) Analysis of sea-island cotton and upland cotton in response to Verticillium dahliae infection by RNA sequencing. BMC Genom 14:852

    Article  Google Scholar 

  • Sun Y, Wang F, Wang N, Dong Y, Liu Q, Zhao L, Chen H, Liu W, Yin H, Zhang X, Yuan Y, Li H (2013b) Transcriptome exploration in Leymus chinensis under saline-alkaline treatment using 454 pyrosequencing. PLoS ONE 8:e53632

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Terras FR, Schoofs HM, De Bolle MF, Van Leuven F, Rees SB, Vanderleyden J, Cammue BP, Broekaert WF (1992) Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds. J Biol Chem 267:15301–15309

    CAS  PubMed  Google Scholar 

  • Terras F, Schoofs H, Thevissen K, Osborn RW, Vanderleyden J, Cammue B, Broekaert WF (1993) Synergistic enhancement of the antifungal activity of wheat and barley thionins by radish and oilseed rape 2S albumins and by barley trypsin inhibitors. Plant Physiol 103:1311–1319

    PubMed Central  CAS  PubMed  Google Scholar 

  • Utkina LL, Andreev YA, Rogozhin EA, Korostyleva TV, Slavokhotova AA, Oparin PB, Vassilevski AA, Grishin EV, Egorov TA, Odintsova TI (2013) Genes encoding 4-Cys antimicrobial peptides in wheat Triticum kiharae Dorof. et Migush.: multimodular structural organization, instraspecific variability, distribution and role in defence. FEBS J 280:3594–3608

    Article  CAS  PubMed  Google Scholar 

  • Van Parijs J, Broekaert WF, Goldstein IJ, Peumans WJ (1991) Hevein: an antifungal protein from rubber-tree (Hevea brasiliensis) latex. Planta 183:258–264

    Article  PubMed  Google Scholar 

  • Vilcinskas A, Mukherjee K, Vogel H (2013) Expansion of the antimicrobial peptide repertoire in the invasive ladybird Harmonia axyridis. Proc Biol Sci 280:20122113

    Article  PubMed Central  PubMed  Google Scholar 

  • Wang X, Thoma RS, Carroll JA, Duffin KL (2002) Temporal generation of multiple antifungal proteins in primed seeds. Biochem Biophys Res Commun 292:236–242

    Article  CAS  PubMed  Google Scholar 

  • Zhao Z, Tan L, Dang C, Zhang H, Wu Q, An L (2012) Deep-sequencing transcriptome analysis of chilling tolerance mechanisms of a subnival alpine plant, Chorispora bungeana. BMC Plant Biol 12:222

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Zhou P, Silverstein KA, Gao L, Walton JD, Nallu S, Guhlin J, Young ND (2013) Detecting small plant peptides using SPADA (small peptide alignment discovery application). BMC Bioinformatics 14:335

    Article  PubMed Central  PubMed  Google Scholar 

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Acknowledgments

This work was supported in part by the Biodiversity Program of the Russian Academy of Sciences and in part by Grants Nos. 15-04-04680/15 and 15-29-02480/ofi_m from the Russian Foundation for Basic Research. AAS is a recipient of the fellowship of the President of Russian Federation (MK-5568.2015.4). A part of this work was performed using the equipment of EIMB RAS “Genome” center (http://www.eimb.ru/RUSSIAN_NEW/INSTITUTE/ccu_genome_c.php). We thank Dr. A.A. Shijan for lymegrass plants’ collection and Dr. I.F. Wonderplush for useful suggestions.

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    1. Vavilov Institute of General Genetics, Russian Academy of Sciences, 3 Gubkina Str., Moscow, Russia, 119991

      Anna A. Slavokhotova, Andrey A. Shelenkov & Tatyana I. Odintsova

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    1. Anna A. Slavokhotova
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    2. Andrey A. Shelenkov
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    3. Tatyana I. Odintsova
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    Correspondence to Anna A. Slavokhotova.

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    Anna A. Slavokhotova and Andrey A. Shelenkov have contributed equally to this work.

    The Illumina sequencing data supporting the results of this study are available at NCBI Short Read Archive under accession number SRR1812885.

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    Slavokhotova, A.A., Shelenkov, A.A. & Odintsova, T.I. Prediction of Leymus arenarius (L.) antimicrobial peptides based on de novo transcriptome assembly. Plant Mol Biol 89, 203–214 (2015). https://doi.org/10.1007/s11103-015-0346-6

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