Journal of Plant Biochemistry and Biotechnology

, Volume 28, Issue 4, pp 447–459 | Cite as

Functional annotation and characterization of hypothetical protein involved in blister blight tolerance in tea (Camellia sinensis (L) O. Kuntze)

  • Gagandeep Singh
  • Gopal Singh
  • Romit Seth
  • Rajni Parmar
  • Pradeep Singh
  • Vikram Singh
  • Sanjay Kumar
  • Ram Kumar SharmaEmail author
Original Article


Tea [Camellia sinensis (L) O. Kuntze], worldwide known source of popular non-alcoholic beverage, is severely affected by various biotic and abiotic stresses. Among these, blister blight (BB) disease caused by the obligate biotrophic fungus Exobasidium vexan is responsible for significant depletion of its yield and quality. Our comparative NGS transcriptomic analysis although elucidated global gene expression pattern of BB defense transitions, yet left with 12,022 transcripts categorized as ‘hypothetical proteins (HPs)’. In this study, efforts were made for assigning functions to HPs derived from RNA-Seq data and successfully identified novel putative candidates involved in BB defense in tea. Domain and family-based characterization identified 9390 HPs representing 2867 protein families and 953 super families. Of these, 213 HPs were assigned with novel putative defense related functional categories (LRR, WRKY, NAC, chitinases and peroxidases). Further, sub-cellular localization (cytosolic,133 HPs; transmembranic, 80 HPs) with abundance of HPs exhibiting of acidic (133) and basic (80) nature suggests their wider functional range. 36 HPs upregulated in tolerant genotype having significant interactions with defense responsive candidates in Protein–Protein Interaction Network analysis, possibly suggests their key regulatory role in BB defense. Interestingly, 12 stereo-dynamically stable structures [LRR (5), NAC (4), WRKY, Chitinase & Peroxidase (1 each)] of HP’s were successfully modelled based on their conserved signature sequences and empirically validated using qRT-PCR analysis, therefore, can be potential novel candidates possibly involved in signal transduction and pathogen recognition during BB defense in tea. Futuristically, novel genes identified in this study can be potentially utilized to expedite genetic improvement efforts in tea. The approach successfully employed in tea can also be adopted for assigning molecular function to the HPs in other plant species.


Blister blight Hypothetical proteins In-silico Structural modelling Tea Transcriptome 



Differential gene expression


Gene ontology


Hypothetical proteins


Kyoto encyclopedia of genes and genomes


Next generation sequencing


Protein–protein interaction network


Quantitative real-time PCR


Transcription factor



The financial support provided by Council of Scientific and Industrial Research (CSIR), New Delhi Research Grants (BSC 0301, MLP-0146), and DST grant in the form of Indo-Sri Lanka joint Research project is acknowledged. This is CSIR-IHBT Publication No. 4190.

Author contributions

GDS, RKS: Conceived and designed the experiments; GDS, GS, RS, RP: Performed the experiments; GDS, PS: Analyzed the data; GDS, GS, RS: wrote the paper; VS, SK: Helped in manuscript editing; RKS: Overall editing and approval of final version of manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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  1. Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC et al (2015) GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1:19–25Google Scholar
  2. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z et al (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedPubMedCentralGoogle Scholar
  3. Axtell MJ, Staskawicz BJ (2003) Initiation of RPS2-specified disease resistance in Arabidopsis is coupled to the AvrRpt2-directed elimination of RIN4. Cell 112:369–377PubMedGoogle Scholar
  4. Baby UI, Balasubramanian S, Ajay D, Premkumar R (2004) Effect of ergosterol biosynthesis inhibitors on blister blight disease, the tea plant and quality of made tea. Crop Prot 23:795–800Google Scholar
  5. Barth M, Holstein SEH (2004) Identification and functional characterization of Arabidopsis AP180, a binding partner of plant αC-adaptin. J Cell Sci 117:2051–2062PubMedGoogle Scholar
  6. Bhandawat A, Singh G, Seth R, Singh P, Sharma RK (2017) Genome-wide transcriptional profiling to elucidate key candidates involved in bud burst and rattling growth in a subtropical bamboo (Dendrocalamus hamiltonii). Front Plant Sci 7:2038PubMedPubMedCentralGoogle Scholar
  7. Calhoun LN, Kwon YM (2011) Structure, function and regulation of the DNA-binding protein Dps and its role in acid and oxidative stress resistance in Escherichia coli: a review. J Appl Microbiol 110:375–386PubMedGoogle Scholar
  8. Campa A (1991) Biological roles of plant peroxidases: known and potential function. In: Everse J, Everse KE, Grisham MB (eds) Peroxidases in chemistry & biology. CRC Press, Boca Raton, pp 25–50Google Scholar
  9. Caño-Delgado A, Yin Y, Yu C, Vafeados D, Mora-García S et al (2004) BRL1 and BRL3 are novel brassinosteroid receptors that function in vascular differentiation in Arabidopsis. Development 131:5341–5351PubMedGoogle Scholar
  10. da Fonsêca MM, Zaha A, Caffarena ER, Vasconcelos ATR (2012) Structure-based functional inference of hypothetical proteins from Mycoplasma hyopneumoniae. J Mol Model 18:1917–1925PubMedGoogle Scholar
  11. DeLano WL (2002) The PyMOL molecular graphics system.
  12. Dhanyalakshmi KH, Naika MBN, Sajeevan RS, Mathew OK, Shafi KM et al (2016) An approach to function annotation for proteins of unknown function (PUFs) in the transcriptome of Indian mulberry. PLoS ONE 11:e0151323PubMedPubMedCentralGoogle Scholar
  13. Di Matteo A, Federici L, Mattei B, Salvi G, Johnson KA et al (2003) The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense. Proc Natl Acad Sci 100:10124–10128PubMedGoogle Scholar
  14. Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res 38:W64–W70PubMedPubMedCentralGoogle Scholar
  15. Duval M, Hsieh T-F, Kim SY, Thomas TL (2002) Molecular characterization of AtNAM: a member of the Arabidopsis NAC domain superfamily. Plant Mol Biol 50:237–248PubMedGoogle Scholar
  16. Emanuelsson O, Brunak S, Von Heijne G, Nielsen H (2007) Locating proteins in the cell using TargetP, SignalP and related tools. Nat Protoc 2:953–972PubMedGoogle Scholar
  17. Fang Y, Liao K, Du H, Xu Y, Song H et al (2015) A stress-responsive NAC transcription factor SNAC3 confers heat and drought tolerance through modulation of reactive oxygen species in rice. J Exp Bot 66:6803–6817PubMedPubMedCentralGoogle Scholar
  18. Finn RD, Bateman A, Clements J, Coggill P, Eberhardt RY et al (2013) Pfam: the protein families database. Nucleic Acids Res 42:D222–D230PubMedPubMedCentralGoogle Scholar
  19. Galperin MY, Koonin EV (2004) ‘Conserved hypothetical’ proteins: prioritization of targets for experimental study. Nucleic Acids Res 32:5452–5463PubMedPubMedCentralGoogle Scholar
  20. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR et al (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The Proteomics Protocols Handbook. Humana Press, New York, pp 571–607Google Scholar
  21. Goda H, Sawa S, Asami T, Fujioka S, Shimada Y et al (2004) Comprehensive comparison of auxin-regulated and brassinosteroid-regulated genes in Arabidopsis. Plant Physiol 134:1555–1573PubMedPubMedCentralGoogle Scholar
  22. Gollery M, Harper J, Cushman J, Mittler T, Mittler R (2007) POFs: what we don’t know can hurt us. Trends Plant Sci 12:492–496PubMedGoogle Scholar
  23. Gou X, He K, Yang H, Yuan T, Lin H et al (2010) Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana. BMC Genom 11:19Google Scholar
  24. Gupta R, Lee SE, Agrawal GK, Rakwal R, Park S, Wang Y, Kim ST (2015) Understanding the plant–pathogen interactions in the context of proteomics-generated apoplastic proteins inventory. Front Plant Sci 6:352PubMedPubMedCentralGoogle Scholar
  25. Guruprasad K, Reddy BVB, Pandit MW (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng Des Sel 4:155–161Google Scholar
  26. Hsu F-C, Chou M-Y, Chou S-J, Li Y-R, Peng H-P et al (2013) Submergence confers immunity mediated by the WRKY22 transcription factor in Arabidopsis. Plant Cell 25:2699–2713PubMedPubMedCentralGoogle Scholar
  27. Ijaq J, Chandrasekharan M, Poddar R, Bethi N, Sundararajan VS (2015) Annotation and curation of uncharacterized proteins-challenges. Front Genet 6:119PubMedPubMedCentralGoogle Scholar
  28. Jayaswall K, Mahajan P, Singh G, Parmar R, Seth R et al (2016) Transcriptome analysis reveals candidate genes involved in blister blight defense in tea (Camellia sinensis (L) Kuntze). Sci Rep 6:30412PubMedPubMedCentralGoogle Scholar
  29. Jeyaramraja PR, Pius PK, Manian S, Meenakshi SN (2005) Certain factors associated with blister blight resistance in Camellia sinensis (L.) O. Kuntze. Physiol Mol Plant Pathol 67:291–295Google Scholar
  30. Jones P, Binns D, Chang H-Y, Fraser M, Li W et al (2014) InterProScan 5: genome-scale protein function classification. Bioinformatics 30:1236–1240PubMedPubMedCentralGoogle Scholar
  31. Kanehisa M, Goto S, Sato Y, Kawashima M, Furumichi M et al (2013) Data, information, knowledge and principle: back to metabolism in KEGG. Nucleic Acids Res 42:D199–D205PubMedPubMedCentralGoogle Scholar
  32. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858PubMedPubMedCentralGoogle Scholar
  33. Krogh A, Larsson B, Von Heijne G, Sonnhammer ELL (2001) Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580PubMedGoogle Scholar
  34. Kumar S, Paul A, Bhattacharya A, Sharma RK, Ahuja PS (2012a) Tea: present status and strategies to improve abiotic stress tolerance. Improv Crop Resist Abiotic Stress 1–2:1401–1424Google Scholar
  35. Kumar V, Singh G, Verma AK, Agrawal S (2012b) In Silico characterization of histidine acid phytase sequences. Enzyme Res 2012:845465PubMedPubMedCentralGoogle Scholar
  36. Kumar V, Singh G, Sangwan P, Verma AK, Agrawal S (2014) Cloning, sequencing, and in silico analysis of-propeller phytase Bacillus licheniformis strain PB-13. Biotechnol Res Int 2014:191–201Google Scholar
  37. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359PubMedPubMedCentralGoogle Scholar
  38. Li H, Greene LH (2010) Sequence and structural analysis of the chitinase insertion domain reveals two conserved motifs involved in chitin-binding. PLoS ONE 5:e8654PubMedPubMedCentralGoogle Scholar
  39. Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY et al (2012) CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res 41:D348–D352PubMedPubMedCentralGoogle Scholar
  40. Matunis MJ, Coutavas E, Blobel G (1996) A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol 135:1457–1470PubMedGoogle Scholar
  41. Matunis MJ, Wu J, Blobel G (1998) SUMO-1 modification and its role in targeting the Ran GTPase-activating protein, RanGAP1, to the nuclear pore complex. J Cell Biol 140:499–509PubMedPubMedCentralGoogle Scholar
  42. Meier S, Bastian R, Donaldson L, Murray S, Bajic V et al (2008) Co-expression and promoter content analyses assign a role in biotic and abiotic stress responses to plant natriuretic peptides. BMC Plant Biol 8:24PubMedPubMedCentralGoogle Scholar
  43. Mosher RA, Durrant WE, Wang D, Song J, Dong X (2006) A comprehensive structure–function analysis of Arabidopsis SNI1 defines essential regions and transcriptional repressor activity. Plant Cell 18:1750–1765PubMedPubMedCentralGoogle Scholar
  44. Pandey SP, Somssich IE (2009) The role of WRKY transcription factors in plant immunity. Plant Physiol 150:1648–1655PubMedPubMedCentralGoogle Scholar
  45. Passardi F, Longet D, Penel C, Dunand C (2004) The class III peroxidase multigenic family in rice and its evolution in land plants. Phytochemistry 65:1879–1893PubMedGoogle Scholar
  46. Pieterse CMJ, Leon-Reyes A, Van der Ent S, Van Wees SCM (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5:308–316Google Scholar
  47. Porto WF, Maria-Neto S, Nolasco DO, Franco OL (2014) Screening and functional prediction of conserved hypothetical proteins from Escherichia coli. J Proteomics Bioinform 7:203–213Google Scholar
  48. Prymula K, Roterman I (2009) Functional characteristics of small proteins (70 amino acid residues) forming protein-nucleic acid complexes. J Biomol Struct Dyn 26:663–677PubMedGoogle Scholar
  49. Rao VS, Srinivas K, Sujini GN, Kumar GN (2014) Protein–protein interaction detection: methods and analysis. Int J Proteomics 2014:35–46Google Scholar
  50. Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140PubMedGoogle Scholar
  51. Ross CA, Liu Y, Shen QJ (2007) The WRKY gene family in rice (Oryza sativa). J Integr Plant Biol 49:827–842Google Scholar
  52. Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcription factors. Trends Plant Sci 15:247–258PubMedGoogle Scholar
  53. Shanmugam V (2005) Role of extracytoplasmic leucine rich repeat proteins in plant defence mechanisms. Microbiol Res 160:83–94PubMedGoogle Scholar
  54. Sigrist CJA, De Castro E, Cerutti L, Cuche BA, Hulo N et al (2012) New and continuing developments at PROSITE. Nucleic Acids Res 41:D344–D347PubMedPubMedCentralGoogle Scholar
  55. Singh G, Singh V (2017) Functional elucidation of hypothetical proteins for their indispensable roles toward drug designing targets from Helicobacter pylori strain HPAG1. J Biomol Struct Dyn 36:1–13Google Scholar
  56. Singh G, Singh G, Singh P, Parmar R, Paul N et al (2017a) Molecular dissection of transcriptional reprogramming of steviol glycosides synthesis in leaf tissue during developmental phase transitions in Stevia rebaudiana Bert. Sci Rep 7:11835PubMedPubMedCentralGoogle Scholar
  57. Singh P, Singh G, Bhandawat A, Singh G, Parmar R et al (2017b) Spatial transcriptome analysis provides insights of key gene (s) involved in steroidal saponin biosynthesis in medicinally important herb Trillium govanianum. Sci Rep 7:45295PubMedPubMedCentralGoogle Scholar
  58. Skolnick J, Fetrow JS (2000) From genes to protein structure and function: novel applications of computational approaches in the genomic era. Trends Biotechnol 18:34–39PubMedGoogle Scholar
  59. Smoot ME, Ono K, Ruscheinski J, Wang P-L, Ideker T (2010) Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics 27:431–432PubMedPubMedCentralGoogle Scholar
  60. Song Y, Jing S, Yu D (2009) Overexpression of the stress-induced OsWRKY08 improves osmotic stress tolerance in Arabidopsis. Chin Sci Bull 54:4671–4678Google Scholar
  61. Songsiriritthigul C, Pantoom S, Aguda AH, Robinson RC, Suginta W (2008) Crystal structures of Vibrio harveyi chitinase A complexed with chitooligosaccharides: implications for the catalytic mechanism. J Struct Biol 162:491–499PubMedGoogle Scholar
  62. Tusnady GE, Simon I (2001) The HMMTOP transmembrane topology prediction server. Bioinformatics 17:849–850PubMedGoogle Scholar
  63. Unamba CI, Nag A, Sharma RK (2015) Next generation sequencing technologies: the doorway to the unexplored genomics of non-model plants. Front Plant Sci 6:1074PubMedPubMedCentralGoogle Scholar
  64. Vernon DM, Forsthoefel NR (2002) Leucine-rich repeat proteins in plants: diverse roles in signaling and development. Res Signpost Recent Res Dev Plant Biol 2120021:202–214Google Scholar
  65. Wan J, Zhang X-C, Neece D, Ramonell KM, Clough S et al (2008) A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis. Plant Cell 20:471–481PubMedPubMedCentralGoogle Scholar
  66. Wang Z, Gerstein M, Snyder M (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet 10:57–63PubMedPubMedCentralGoogle Scholar
  67. Wang Y-N, Tang L, Hou Y, Wang P, Yang H et al (2016) Differential transcriptome analysis of leaves of tea plant (Camellia sinensis) provides comprehensive insights into the defense responses to Ectropis oblique attack using RNA-Seq. Funct Integr Genom 16:383–398Google Scholar
  68. Wilson D, Madera M, Vogel C, Chothia C, Gough J (2006) The SUPERFAMILY database in 2007: families and functions. Nucleic Acids Res 35:D308–D313PubMedPubMedCentralGoogle Scholar
  69. Yang S-D, Seo PJ, Yoon H-K, Park C-M (2011) The Arabidopsis NAC transcription factor VNI2 integrates abscisic acid signals into leaf senescence via the COR/RD genes. Plant Cell 23:2155–2168PubMedPubMedCentralGoogle Scholar
  70. Yao D, Zhang X, Zhao X, Liu C, Wang C et al (2011) Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of cotton (Gossypium hirsutum L.). Genomics 98:47–55PubMedGoogle Scholar
  71. Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11:1–12Google Scholar
  72. Yu C, Chen Y, Lu C, Hwang J (2006) Prediction of protein subcellular localization. Proteins Struct Funct Bioinform 64:643–651Google Scholar
  73. Zhang S, Klessig DF (2001) MAPK cascades in plant defense signaling. Trends Plant Sci 6:520–527PubMedGoogle Scholar
  74. Zhang LJ, Wang XE, Peng X, Wei YJ, Cao R et al (2006) Proteomic analysis of low-abundant integral plasma membrane proteins based on gels. Cell Mol Life Sci C 63:1790–1804Google Scholar
  75. Zheng XH, Lu F, Wang Z-Y, Zhong F, Hoover J et al (2004) Using shared genomic synteny and shared protein functions to enhance the identification of orthologous gene pairs. Bioinformatics 21:703–710PubMedGoogle Scholar
  76. Zhu L, Zhang Y, Guo W, Wang Q (2014) Gleditsia sinensis: transcriptome sequencing, construction, and application of its protein–protein interaction network. Biomed Res Int 2014:404578PubMedPubMedCentralGoogle Scholar
  77. Zorzatto C, Machado JPB, Lopes KVG, Nascimento KJT, Pereira WA et al (2015) NIK1-mediated translation suppression functions as a plant antiviral immunity mechanism. Nature 520:679–682PubMedPubMedCentralGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2019

Authors and Affiliations

  1. 1.Biotechnology DepartmentCouncil of Scientific & Industrial Research-Institute of Himalayan Bioresource TechnologyPalampurIndia
  2. 2.Centre for Computational Biology and Bioinformatics, Central University of Himachal PradeshDharamshalaIndia

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