Variation in the LRR region of Pi54 protein alters its interaction with the AvrPi54 protein revealed by in silico analysis

Autoři: Chiranjib Sarkar aff001;  Banita Kumari Saklani aff003;  Pankaj Kumar Singh aff003;  Ravi Kumar Asthana aff004;  Tilak Raj Sharma aff003
Působiště autorů: ICAR-Indian Agricultural Research Institute, New Delhi, India aff001;  ICAR-Indian Agricultural Statistics Research Institute, New Delhi, India aff002;  ICAR-National Research Centre on Plant Biotechnology, New Delhi, India aff003;  Banaras Hindu University, Varanasi, Uttar Pradesh, India aff004;  National Agri-Food Biotechnology Institute, Mohali, Punjab, India aff005
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224088


Rice blast, caused by the ascomycete fungus Magnaporthe oryzae is a destructive disease of rice and responsible for causing extensive damage to the crop. Pi54, a dominant blast resistance gene cloned from rice line Tetep, imparts a broad spectrum resistance against various M. oryzae isolates. Many of its alleles have been explored from wild Oryza species and landraces whose sequences are available in the public domain. Its cognate effector gene AvrPi54 has also been cloned from M. oryzae. Complying with the Flor’s gene-for-gene system, Pi54 protein interacts with AvrPi54 protein following fungal invasion leading to the resistance responses in rice cell that prevents the disease development. In the present study Pi54 alleles from 72 rice lines were used to understand the interaction of Pi54 (R) proteins with AvrPi54 (Avr) protein. The physiochemical properties of these proteins varied due to the nucleotide level polymorphism. The ab initio tertiary structures of these R- and Avr- proteins were generated and subjected to the in silico interaction. In this interaction, the residues in the LRR region of R- proteins were shown to interact with the Avr protein. These R proteins were found to have variable strengths of binding due to the differential spatial arrangements of their amino acid residues. Additionally, molecular dynamic simulations were performed for the protein pairs that showed stronger interaction than Pi54tetep (original Pi54 from Tetep) protein. We found these proteins were forming h-bond during simulation which indicated an effective binding. The root mean square deviation values and potential energy values were stable during simulation which validated the docking results. From the interaction studies and the molecular dynamics simulations, we concluded that the AvrPi54 protein interacts directly with the resistant Pi54 proteins through the LRR region of Pi54 proteins. Some of the Pi54 proteins from the landraces namely Casebatta, Tadukan, Varun dhan, Govind, Acharmita, HPR-2083, Budda, Jatto, MTU-4870, Dobeja-1, CN-1789, Indira sona, Kulanji pille and Motebangarkaddi cultivars show stronger binding with the AvrPi54 protein, thus these alleles can be effectively used for the rice blast resistance breeding program in future.

Klíčová slova:

Biochemical simulations – Protein interactions – Protein structure – Protein structure comparison – Protein structure prediction – Rice – Sequence alignment


1. Wilson RA, Talbot NJ (2009) Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Nature Revi Microbiol 7: 185.

2. Fisher MC, Henk DA, Briggs CJ, Brownstein JS, Madoff LC, McCraw SL et al. (2012) Emerging fungal threats to animal, plant and ecosystem health. Nature 484: 186. doi: 10.1038/nature10947 22498624

3. Sharma TR, Rai AK, Gupta SK, Vijayan J, Devanna BN, Ray S (2012) Rice blast management through host-plant resistance: retrospect and prospects. Agricult Res 1: 37–52.

4. Fukuoka S, Yamamoto SI, Mizobuchi R, Yamanouchi U, Ono K, Kitazawa N, et al. (2014) Multiple functional polymorphisms in a single disease resistance gene in rice enhance durable resistance to blast. Scientific Rep 4: 4550.

5. Ma J, Lei C, Xu X, Hao K, Wang J, Cheng Z et al. (2015) Pi64, encoding a novel CC-NBS-LRR protein, confers resistance to leaf and neck blast in rice. Mol Plant-Microbe Interact 28: 558–568. doi: 10.1094/MPMI-11-14-0367-R 25650828

6. Sirisathaworn T, Srirat T, Longya A, Jantasuriyarat C (2017) Evaluation of mating type distribution and genetic diversity of three Magnaporthe oryzae avirulence genes, PWL-2, AVR-Pii and Avr-Piz-t, in Thailand rice blast isolates. Agricult Nat Resour 51: 7–14.

7. Flor HH (1971) Current status of the gene-for-gene concept. Annu Rev Phytopathol 9: 275–296.

8. Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411: 826. doi: 10.1038/35081161 11459065

9. Zipfel C, Rathjen JP (2008) Plant immunity: AvrPto targets the frontline Curr Biol 18: 218–220.

10. Khush GS, Jena KK (2009) Current status and future prospects for research on blast resistance in rice (Oryza sativa L) In Advances in genetics, genomics and control of rice blast disease. 1–10 Springer, Dordrecht.

11. Sharma TR, Madhav MS, Singh BK, Shanker P, Jana TK, Dalal V, et al. (2005) High-resolution mapping, cloning and molecular characterization of the Pi-k h gene of rice, which confers resistance to Magnaporthe griseae. Mol Genet Genomics 274: 569–578. doi: 10.1007/s00438-005-0035-2 16228246

12. Sharma TR, Rai AK, Gupta SK, Singh NK (2010) Broad-spectrum blast resistance gene Pi-kh cloned from rice line Tetep designated as Pi54. J Plant Biochem Biotechnol 19: 87–89.

13. Rai AK, Kumar SP, Gupta SK, Gautam N, Singh NK, Sharma TR (2011) Functional complementation of rice blast resistance gene Pi-k h (Pi54) conferring resistance to diverse strains of Magnaporthe oryzae. J Plant Biochem Biotechnol 20: 55–65.

14. Gupta SK, Rai AK, Kanwar SS, Chand D, Singh NK, Sharma TR (2011) The single functional blast resistance gene Pi54 activates a complex defence mechanism in rice. J Exp Bot 63: 757–772. doi: 10.1093/jxb/err297 22058403

15. Gupta SK, Rai AK, Kanwar SS, Sharma TR (2012) Comparative analysis of zinc finger proteins involved in plant disease resistance. PLOS One 7: Se42578.

16. Takken FLW, Tameling WIL (2009) To nibble at plant resistance proteins. Science 324: 744–746. doi: 10.1126/science.1171666 19423813

17. Thakur S, Singh PK, Rathour R, Variar M, Prashanthi SK, Singh AK et al. (2013) Positive selection pressure on rice blast resistance allele Piz-t makes it divergent in Indian land races. J Plant Interact 8: 34–44.

18. Das A, Soubam D, Singh PK, Thakur S, Singh NK, Sharma TR (2012) A novel blast resistance gene, Pi54rh cloned from wild species of rice, Oryza rhizomatis confers broad spectrum resistance to Magnaporthe oryzae. Funct Integr Genomics 12: 215–228. doi: 10.1007/s10142-012-0284-1 22592658

19. Devanna NB, Vijayan J, Sharma TR (2014) The blast resistance gene Pi54of cloned from Oryza officinalis interacts with Avr-Pi54 through its novel non-LRR domains. PLOS One 9: e104840. doi: 10.1371/journal.pone.0104840 25111047

20. Ray S, Singh PK, Gupta DK, Mahato AK, Sarkar C, Rathour R, et al, (2016) Analysis of Magnaporthe oryzae genome reveals a fungal effector, which is able to induce resistance response in transgenic rice line containing resistance gene, Pi54. Front Plant Sci 7, 1140. doi: 10.3389/fpls.2016.01140 27551285

21. Yavuz C, Öztürk ZN (2017) Working with Proteins in silico: A Review of Online Available Tools for Basic Identification of Proteins Turk J Agri -Food Sci Technol 5: 65–70.

22. Meng XY, Zhang HX, Mezei M, Cui M (2011) Molecular docking: a powerful approach for structure-based drug discovery. Current Comput-aided Drug Des 7: 146–157.

23. Ehrenfeld N, Gonzalez A, Canon P, Medina C, Perez-Acle T, Arce-Johnson P (2008) Structure–function relationship between the tobamovirus TMV-Cg coat protein and the HR-like response. J Gen Virol 89: 809–817. doi: 10.1099/vir.0.83355-0 18272773

24. Rozas J and Rozas R (1995) DnaSP, DNA sequence polymorphism: an interactive program for estimating population genetics parameters from DNA sequence data. Bioinformatics 11: 621–625

25. Waterhouse AM, Procter JB, Martin DM, Clamp M and Barton GJ, 2009 Jalview Version 2—a multiple sequence alignment editor and analysis workbench Bioinformatics 25: 189–1191.

26. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H et al. (2000) The protein data bank. Nucleic Acids Res 28: 235–242. doi: 10.1093/nar/28.1.235 10592235

27. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410. doi: 10.1016/S0022-2836(05)80360-2 2231712

28. Sayle RA, Milner-White EJ (1995) RASMOL: biomolecular graphics for all Trends. Biochem Sci 20: 374–376.

29. Ramachandran GT, Sasisekharan V (1968) Conformation of polypeptides and proteins. Adv Protein Chem 23: 283–437. 4882249

30. Brooks BR (1983) A program for macromolecular energy, minimization, and dynamics calculations. J Comput Chem 4: 187–217.

31. Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, et al. (2015) GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1: 19–25.

32. Schmid N, Eichenberger AP, Choutko A, Riniker S, Winger M, Mark AE et al. (2011) Definition and testing of the GROMOS force-field versions 54A7 and 54B7. Eur Biophys J 40: 843. doi: 10.1007/s00249-011-0700-9 21533652

33. Ikai A (1980) Thermostability and aliphatic index of globular proteins. J Biochem 88: 1895–1898. 7462208

34. Singh PK, Thakur S, Rathour R, Variar M, Prashanthi SK, Singh AK, et al. (2014) Transposon-based high sequence diversity in Avr-Pita alleles increases the potential for pathogenicity of Magnaporthe oryzae populations. Funct Integr Genomics 14: 419–429. doi: 10.1007/s10142-014-0369-0 24633351

35. Singh PK, Nag A, Arya P, Kapoor R, Singh A, Jaswal R et al. (2018) Prospects of Understanding the Molecular Biology of Disease Resistance in Rice. Int J Mol Sci 19: 1141.

36. Thakur S, Gupta YK, Singh PK, Rathour R, Varia M, Prashanthi SK et al. (2013) Molecular diversity in rice blast resistance gene Pi-ta makes it highly effective against dynamic population of Magnaporthe oryzae. Funct Integr Genomics 13: 309–322. doi: 10.1007/s10142-013-0325-4 23818197

37. Yang S, Gu T, Pan C, Feng Z, Ding J, Hang Y, et al. (2008) Genetic variation of NBS-LRR class resistance genes in rice lines. Theor Appl Genet 1:165–177.

38. Yamamoto T, Nagasaki H, Yonemaru JI, Ebana K, Nakajima M, Shibaya T et al. (2010) Fine definition of the pedigree haplotypes of closely related rice cultivars by means of genome-wide discovery of single-nucleotide polymorphisms. BMC genomics 11: 267. doi: 10.1186/1471-2164-11-267 20423466

39. Thakur S., Singh P.K., Das A., Rathour R., Variar M., Prashanthi S.K., Singh A.K., Singh U.D., Chand D., Singh N.K. and Sharma T.R., 2015. Extensive sequence variation in rice blast resistance gene Pi54 makes it broad spectrum in nature. Frontiers in plant science, 6: 345. doi: 10.3389/fpls.2015.00345 26052332

40. Singh PK, Ray S, Thakur S, Rathour R, Sharma V Sharma TR (2018) Co-evolutionary interactions between host resistance and pathogen avirulence genes in rice-Magnaporthe oryzae pathosystem. Fungal Genet Biol 115, 9–19 doi: 10.1016/j.fgb.2018.04.005 29630984

41. Jiang L, Lai L (2002) CH··· O hydrogen bonds at protein-protein interfaces. J Biol Chem 277: 37732–37740. doi: 10.1074/jbc.M204514200 12119293

42. Eildal JN, Hultqvist G, Balle T, Stuhr-Hansen N, Padrah S, Gianni S et al. (2013) Probing the role of backbone hydrogen bonds in protein–peptide interactions by amide-to-ester mutations. J Am Chem Soc 135: 12998–13007. doi: 10.1021/ja402875h 23705582

43. Kobe B, Deisenhofer J (1994) The leucine-rich repeat: a versatile binding motif. Trends Biochem Sci 19: 415–421. doi: 10.1016/0968-0004(94)90090-6 7817399

44. Wulff BB, Thomas CM, Smoker M, Grant M, Jones JD (2001) Domain swapping and gene shuffling identify sequences required for induction of an Avr-dependent hypersensitive response by the tomato Cf-4 and Cf-9 proteins. Plant Cell 13: 255–272. doi: 10.1105/tpc.13.2.255 11226184

45. Leckie F, Mattei B, Capodicasa C, Hemmings A, Nuss L, Aracri B, et al. (1999) The specificity of polygalacturonase‐inhibiting protein (PGIP): a single amino acid substitution in the solvent‐exposed β‐strand/β‐turn region of the leucine‐rich repeats (LRRs) confers a new recognition capability. EMBO J 18: 2352–2363. doi: 10.1093/emboj/18.9.2352 10228150

46. Warren RF, Henk A, Mowery P, Holub E, Innes RW (1998) A mutation within the leucine-rich repeat domain of the Arabidopsis disease resistance gene RPS5 partially suppresses multiple bacterial and downy mildew resistance genes. Plant Cell 10: 1439–1452. doi: 10.1105/tpc.10.9.1439 9724691

47. Zhou B, Qu S, Liu G, Dolan M, Sakai H, Lu G, et al. 2006. The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity to Magnaporthe grisea. Mol Plant-Microbe Interact 19: 1216–28. doi: 10.1094/MPMI-19-1216 17073304

48. Dill KA, MacCallum JL (2012) The protein-folding problem, 50 years on Science. 338: 1042–1046. doi: 10.1126/science.1219021 23180855

49. Kanzaki H, Yoshida K, Saitoh H, Fujisaki K, Hirabuchi A, Alaux L, et al. (2012) Arms race co‐evolution of Magnaporthe oryzae AVR‐Pik and rice Pik genes driven by their physical interactions. Plant J 72: 894–907. doi: 10.1111/j.1365-313X.2012.05110.x 22805093

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2019 Číslo 11