Integrating long noncoding RNAs and mRNAs expression profiles of response to Plasmodiophora brassicae infection in Pakchoi (Brassica campestris ssp. chinensis Makino)

Autoři: Hongfang Zhu aff001;  Xiaofeng Li aff001;  Dandan Xi aff001;  Wen Zhai aff003;  Zhaohui Zhang aff001;  Yuying Zhu aff001
Působiště autorů: Horticulture Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, China aff001;  Shanghai Key Lab of Protected Horticultural Technology, Shanghai, China aff002;  East China University of Technology, Nanchang, China aff003
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


The biotrophic protist Plasmodiophora brassicae causes serious damage to Brassicaceae crops grown worldwide. However, the molecular mechanism of the Brassica rapa response remains has not been determined. Long noncoding RNA and mRNA expression profiles in response to Plasmodiophora brassicae infection were investigated using RNA-seq on the Chinese cabbage inbred line C22 infected with P. brassicae. Approximately 5,193 mRNAs were significantly differentially expressed, among which 1,345 were upregulated and 3,848 were downregulated. The GO enrichment analysis shows that most of these mRNAs are related to the defense response. Meanwhile, 114 significantly differentially expressed lncRNAs were identified, including 31 upregulated and 83 downregulated. Furthermore, a total of 2,344 interaction relationships were detected between 1,725 mRNAs and 103 lncRNAs with a correlation coefficient greater than 0.8. We also found 15 P. brassicaerelated mRNAs and 16 lncRNA interactions within the correlation network. The functional annotation showed that 15 mRNAs belong to defense response proteins (66.67%), protein phosphorylation (13.33%), root hair cell differentiation (13.33%) and regulation of salicylic acid biosynthetic process (6.67%). KEGG annotation showed that the vast majority of these genes are involved in the biosynthesis of secondary metabolism pathways and plant-pathogen interactions. These results provide a new perspective on lncRNA-mRNA network function and help to elucidate the molecular mechanism of P. brassicae infection.

Klíčová slova:

Brassica – Cellular stress responses – Gene expression – Gene ontologies – Gene regulation – Long non-coding RNAs – Metabolic processes – RNA sequencing


1. Ludwig-Müller J, Prinsen E, Rolfe SA, Scholes JD. Metabolism and plant hormone action during clubroot disease. Journal of Plant Growth Regulation. 2009; 28(3): 229–244.

2. Jia H, Wei X, Yang Y, Yuan Y, Wei F, Zhao Y, et al. Root RNA-seq analysis reveals a distinct transcriptome landscape between clubroot-susceptible and clubroot-resistant Chinese cabbage lines after Plasmodiophora brassicae infection. Plant and Soil. 2017; 421(1–2): 93–105.

3. Kageyama K, Asano T. Life cycle of Plasmodiophora brassicae. Journal of Plant Growth Regulation. 2009; 28(3): 203.

4. Zhao Y, Bi K, Gao Z, Chen T, Liu H, Xie J, et al. Transcriptome analysis of Arabidopsis thaliana in response to Plasmodiophora brassicae during early infection. Frontiers in microbiology. 2017; 8: 673. doi: 10.3389/fmicb.2017.00673 28484434.

5. Ueno H, Matsumoto E, Aruga D, Kitagawa S, Matsumura H, Hayashida N. Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa. Plant molecular biology. 2012; 80(6): 621–629. doi: 10.1007/s11103-012-9971-5 23054353.

6. Hatakeyama K, Suwabe K, Tomita RN, Kato T, Nunome T, Fukuoka H, et al. Identification and characterization of Crr1a, a gene for resistance to clubroot disease (Plasmodiophora brassicae Woronin) in Brassica rapa L. PloS one. 2013; 8(1): e54745. doi: 10.1371/journal.pone.0054745 23382954.

7. Hatakeyama K, Niwa T, Kato T, Ohara T, Kakizaki T, Matsumoto S. The tandem repeated organization of NB-LRR genes in the clubroot-resistant CRb locus in Brassica rapa L. Molecular genetics and genomics. 2017; 292(2): 397–405. doi: 10.1007/s00438-016-1281-1 28013378.

8. Suwabe K, Tsukazaki H, Iketani H, Hatakeyama K, Fujimura M, Nunome T, et al. Identification of two loci for resistance to clubroot (Plasmodiophora brassicae Woronin) in Brassica rapa L. Theoretical and Applied Genetics. 2003; 107(6): 997–1002. doi: 10.1007/s00122-003-1309-x 12955203.

9. Hirai M, Harada T, Kubo N, Tsukada M, Suwabe K, Matsumoto S. A novel locus for clubroot resistance in Brassica rapa and its linkage markers. Theoretical and applied genetics. 2004; 108(4): 639–643. doi: 10.1007/s00122-003-1475-x 14551685.

10. Saito M, Kubo N, Matsumoto S, Suwabe K, Tsukada M, Hirai M. Fine mapping of the clubroot resistance gene, Crr3, in Brassica rapa. Theoretical and applied genetics. 2006; 114(1): 81. doi: 10.1007/s00122-006-0412-1 17039346.

11. Suwabe K, Tsukazaki H, Iketani H, Hatakeyama K, Kondo M, Fujimura M, et al. Simple sequence repeat-based comparative genomics between Brassica rapa and Arabidopsis thaliana: the genetic origin of clubroot resistance. Genetics. 2006; 173(1): 309–319. doi: 10.1534/genetics.104.038968 16723420.

12. Sakamoto K, Saito A, Hayashida N, Taguchi G, Matsumoto E. Mapping of isolate-specific QTLs for clubroot resistance in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Theoretical and applied genetics. 2008; 117(5): 759–767. doi: 10.1007/s00122-008-0817-0 18612625.

13. Chu M, Song T, Falk KC, Zhang X, Liu X, Chang A, et al. Fine mapping of Rcr1 and analyses of its effect on transcriptome patterns during infection by Plasmodiophora brassicae. BMC genomics. 2014; 15(1): 1166. doi: 10.1186/1471-2164-15-1166 25532522.

14. Yu F, Zhang X, Huang Z, Chu M, Song T, Falk KC, et al. Identification of genome-wide variants and discovery of variants associated with Brassica rapa clubroot resistance gene Rcr1 through bulked segregant RNA sequencing. PLoS One. 2016; 11(4): e0153218. doi: 10.1371/journal.pone.0153218 27078023.

15. Chen J, Jing J, Zhan Z, Zhang T, Zhang C, Piao Z. Identification of novel QTLs for isolate-specific partial resistance to Plasmodiophora brassicae in Brassica rapa. PloS one. 2013; 8(12): e85307. doi: 10.1371/journal.pone.0085307 24376876.

16. Pang WX, Liang S, Li XN, Li PP, Yu S, Lim YP, et al. Genetic detection of clubroot resistance loci in a new population of Brassica rapa. Horticulture, Environment, and Biotechnology. 2014; 55(6): 540–547.

17. Cho KH, Park SH, Kim KT, Kim S, Kim JS, Park BS, et al. Mapping quantitative trait loci (QTL) for clubroot resistance in Brassica rapa L. Journal of Horticultural Science & Biotechnology. 2012; 87(4): 325–333.

18. Yu F, Zhang X, Peng G, Falk KC, Strelkov SE, Gossen BD. Genotyping-by-sequencing reveals three QTL for clubroot resistance to six pathotypes of Plasmodiophora brassicae in Brassica rapa. Sci Rep. 2017; 7(1): 4516. doi: 10.1038/s41598-017-04903-2 28674416.

19. Dangl JL, Jones JDG. Plant pathogens and integrated defence responses to infection. Nature. 2001; 411(6839): 826–833. doi: 10.1038/35081161 11459065.

20. Bernoux M, Ellis JG, Dodds PN. New insights in plant immunity signaling activation. Current Opinion in Plant Biology. 2011; 14(5): 512–518. doi: 10.1016/j.pbi.2011.05.005 21723182

21. Piao ZY, Ramchiary N, Lim YP. Genetics of Clubroot Resistance in Brassica Species. J Plant Growth Regul. 2009; 28(3): 252–264.

22. Cao T, Srivastava S, Rahman MH, Kav NNV, Hotte N, Ddyholos MK, et al. Proteome-level changes in the roots of Brassica napus as a result of Plasmodiophora brassicae infection. Plant Science. 2008; 174(1): 97–115.

23. Chen JJ, Pang WX, Chen B, Zhang CY, Piao ZY. Transcriptome Analysis of Brassica rapa Near-Isogenic Lines Carrying Clubroot-Resistant and–Susceptible Alleles in Response to Plasmodiophora brassicae during Early Infection. Frontiers in Plant Science. 2016; 6. doi: 10.3389/fpls.2015.01183 26779217.

24. Zhang Y, Chen Y. Long noncoding RNAs: new regulators in plant development. Biochemical and biophysical research communications. 2013; 436(2): 111–114. doi: 10.1016/j.bbrc.2013.05.086 23726911.

25. Chen S, Liu T, Gao Y, Zhang C, Peng S, Bai M, et al. Discovery of clubroot-resistant genes in Brassica napus by transcriptome sequencing. Genet Mol Res. 2016; 15(3). 27525940.

26. Heo JB, Lee Y-S, Sung S. Epigenetic regulation by long noncoding RNAs in plants. Chromosome Research. 2013; 21(6–7): 685–693. doi: 10.1007/s10577-013-9392-6 24233054.

27. Qin T, Zhao H, Cui P, Albesher N, Xiong L. A nucleus-localized long non-coding RNA enhances drought and salt stress tolerance. Plant physiology. 2017; 175(3): 1321–1336. doi: 10.1104/pp.17.00574 28887353.

28. Zaynab M, Fatima M, Abbas S, Umair M, Sharif Y, Raza MA. Long non-coding RNAs as molecular players in plant defense against pathogens. Microbial pathogenesis. 2018. doi: 10.1016/j.micpath.2018.05.050 29859899.

29. Zhu QH, Stephen S, Taylor J, Helliwell CA, Wang MB. Long noncoding RNAs responsive to Fusarium oxysporum infection in Arabidopsis thaliana. New Phytologist. 2014; 201(2): 574–584. doi: 10.1111/nph.12537 24117540.

30. Seo JS, Sun H-X, Park BS, Huang C-H, Yeh S-D, Jung C, et al. ELF18-INDUCED LONG-NONCODING RNA associates with mediator to enhance expression of innate immune response genes in Arabidopsis. The Plant Cell. 2017; 29(5): 1024–1038. doi: 10.1105/tpc.16.00886 28400491.

31. Kwenda S, Birch PR, Moleleki LN. Genome-wide identification of potato long intergenic noncoding RNAs responsive to Pectobacterium carotovorum subspecies brasiliense infection. BMC genomics. 2016; 17(1): 614. doi: 10.1186/s12864-016-2967-9 27515663.

32. Cui J, Luan Y, Jiang N, Bao H, Meng J. Comparative transcriptome analysis between resistant and susceptible tomato allows the identification of lncRNA 16397 conferring resistance to Phytophthora infestans by co‐expressing glutaredoxin. The plant journal. 2017; 89(3): 577–589. doi: 10.1111/tpj.13408 27801966

33. Yang Y, Liu T, Shen D, Wang J, Ling X, Hu Z, et al. Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms. PLoS pathogens. 2019; 15(1): e1007534. doi: 10.1371/journal.ppat.1007534 30668603.

34. Jiang N, Cui J, Shi Y, Yang G, Zhou X, Hou X, et al. Tomato lncRNA23468 functions as a competing endogenous RNA to modulate NBS-LRR genes by decoying miR482b in the tomato-Phytophthora infestans interaction. Horticulture research. 2019; 6(1): 28. doi: 10.1038/s41438-018-0096-0 30729018.

35. Joshi RK, Megha S, Basu U, Rahman MH, Kav NN. Genome wide identification and functional prediction of long non-coding RNAs responsive to Sclerotinia sclerotiorum infection in Brassica napus. PLoS One. 2016; 11(7): e0158784. doi: 10.1371/journal.pone.0158784 27388760.

36. Song X, Liu G, Huang Z, Duan W, Tan H, Li Y, et al. Temperature expression patterns of genes and their coexpression with LncRNAs revealed by RNA-Seq in non-heading Chinese cabbage. BMC genomics. 2016; 17(1): 297. doi: 10.1186/s12864-016-2625-2 27103267.

37. Shen E, Zhu X, Hua S, Chen H, Ye C, Zhou L, et al. Genome-wide identification of oil biosynthesis-related long non-coding RNAs in allopolyploid Brassica napus. BMC genomics. 2018; 19(1): 745. doi: 10.1186/s12864-018-5117-8 30314449.

38. Williams PH. A system for the determination of races of Plasmodiophora brassicae that infect cabbage and rutabaga. Phytopathology. 1966; 56(6): 624–626.

39. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–2120. doi: 10.1093/bioinformatics/btu170 24695404.

40. Daehwan K, Ben L, Salzberg SL. HISAT: a fast spliced aligner with low memory requirements. Nature Methods. 2015; 12(4): 357–360. doi: 10.1038/nmeth.3317 25751142.

41. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map format and SAMtools. Bioinformatics. 2009; 25(16): 2078–2079. doi: 10.1093/bioinformatics/btp352 19505943.

42. Mihaela P, Pertea GM, Antonescu CM, Tsung-Cheng C, Mendell JT, Salzberg SL. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nature Biotechnology. 2015; 33(3): 290–295. doi: 10.1038/nbt.3122 25690850.

43. Ghosh S, Chan C-KK. Analysis of RNA-Seq data using TopHat and Cufflinks. Methods Mol Biol. 2016; 1374: 339–361. doi: 10.1007/978-1-4939-3167-5_18 26519415.

44. Lei K, Yong Z, Zhi-Qiang Y, Xiao-Qiao L, Shu-Qi Z, Liping W, et al. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Research. 2007; 35(Web Server issue): W345. doi: 10.1093/nar/gkm391 17631615.

45. Liang S, Haitao L, Dechao B, Guoguang Z, Kuntao Y, Changhai Z, et al. Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts. Nucleic Acids Research. 2013; 41(17): e166–e166. doi: 10.1093/nar/gkt646 23892401.

46. Li A, Zhang J, Zhou Z. PLEK: a tool for predicting long non-coding RNAs and messenger RNAs based on an improved k-mer scheme. Bmc Bioinformatics. 2014; 15(1): 311. doi: 10.1186/1471-2105-15-311 25239089.

47. Sonnhammer ELL, Eddy SR, Birney E, Bateman A, Durbin R. Pfam: multiple sequence alignments and HMM-profiles of protein domains. Nucleic Acids Research. 1998; 26(1): 320–322. doi: 10.1093/nar/26.1.320 9399864.

48. Roberts A, Pachter L. Streaming fragment assignment for real-time analysis of sequencing experiments. Nature Methods. 2013; 10(1): 71–U99. doi: 10.1038/nmeth.2251 23160280.

49. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature methods. 2012; 9(4): 357. doi: 10.1038/nmeth.1923 22388286.

50. Anders S, Huber W. Differential expression of RNA-Seq data at the gene level–the DESeq package. Heidelberg, Germany: European Molecular Biology Laboratory (EMBL). 2012. doi: 10.12688/f1000research.15398.1 PMID: 30356428.

51. Gutierrez L, Mauriat M, Guénin S, Pelloux J, Lefebvre JF, Louvet R, et al. The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription‐polymerase chain reaction (RT‐PCR) analysis in plants. Plant biotechnology journal. 2008; 6(6): 609–618. doi: 10.1111/j.1467-7652.2008.00346.x 18433420.

52. Yassour M, Kaplan T, Fraser HB, Levin JZ, Pfiffner J, Adiconis X, et al. Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing. Proceedings of the National Academy of Sciences. 2009; 106(9): 3264–3269. doi: 10.1073/pnas.0812841106 19208812.

53. Wang X, Wang H, Wang J, Sun R, Wu J, Liu S, et al. The genome of the mesopolyploid crop species Brassica rapa. Nature genetics. 2011; 43(10): 1035. doi: 10.1038/ng.919 21873998.

54. Roberts A, Trapnell C, Donaghey J, Rinn JL, Pachter L. Improving RNA-Seq expression estimates by correcting for fragment bias. Genome biology. 2011; 12(3): R22. doi: 10.1186/gb-2011-12-3-r22 21410973.

55. Szcześniak MW, Rosikiewicz W, Makałowska I. CANTATAdb: a collection of plant long non-coding RNAs. Plant and Cell Physiology. 2015; 57(1): e8–e8. doi: 10.1093/pcp/pcv201 26657895.

56. Ponting CP, Oliver PL, Reik W. Evolution and functions of long noncoding RNAs. Cell. 2009; 136(4): 629–641. doi: 10.1016/j.cell.2009.02.006 19239885.

57. Li L, Eichten SR, Shimizu R, Petsch K, Yeh C-T, Wu W, et al. Genome-wide discovery and characterization of maize long non-coding RNAs. Genome biology. 2014; 15(2): R40. doi: 10.1186/gb-2014-15-2-r40 24576388.

58. Zaratiegui M, Irvine DV, Martienssen RA. Noncoding RNAs and gene silencing. Cell. 2007; 128(4): 763–776. doi: 10.1016/j.cell.2007.02.016 17320512

59. Hainer SJ, Pruneski JA, Mitchell RD, Monteverde RM, Martens JA. Intergenic transcription causes repression by directing nucleosome assembly. Genes & development. 2011; 25(1): 29–40. doi: 10.1101/gad.1975011 21156811.

60. Wang KC, Yang YW, Liu B, Sanyal A, Corces-Zimmerman R, Chen Y, et al. A long noncoding RNA maintains active chromatin to coordinate homeotic gene expression. Nature. 2011; 472(7341): 120. doi: 10.1038/nature09819 21423168.

61. Paul P, Dhandapani V, Choi SR, Lim YP. Genome wide identification and functional prediction of long non-coding RNAs in Brassica rapa. Genes & Genomics. 2016; 38(6): 547–555.

62. Clarke JD, Volko SM, Ledford H, Ausubel FM, Dong X. Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in Arabidopsis. The Plant Cell. 2000; 12(11): 2175–2190. doi: 10.1105/tpc.12.11.2175 11090217.

63. Shiryaev A, Moens U. Mitogen-activated protein kinase p38 and MK2, MK3 and MK5: menage a trois or menage a quatre? Cellular signalling. 2010; 22(8): 1185–1192. doi: 10.1016/j.cellsig.2010.03.002 20227494.

64. Nelson DR, Zeldin DC, Hoffman SM, Maltais LJ, Wain HM, Nebert DW. Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes, including nomenclature recommendations for genes, pseudogenes and alternative-splice variants. Pharmacogenetics and Genomics. 2004; 14(1): 1–18. doi: 10.1097/00008571-200401000-00001 15128046.

65. Pang W, Fu P, Li X, Zhan Z, Yu S, Piao Z. Identification and mapping of the Clubroot resistance gene CRd in Chinese cabbage (Brassica rapa ssp. pekinensis). Frontiers in plant science. 2018; 9: 653. doi: 10.3389/fpls.2018.00653 29868100.

66. Laila R, Park J-I, Robin AHK, Natarajan S, Vijayakumar H, Shirasawa K, et al. Mapping of a novel clubroot resistance QTL using ddRAD-seq in Chinese cabbage (Brassica rapa L.). BMC plant biology. 2019; 19(1): 13. doi: 10.1186/s12870-018-1615-8 30621588.

67. Heo JB, Sung S. Vernalization-mediated epigenetic silencing by a long intronic noncoding RNA. Science. 2011; 331(6013): 76–79. doi: 10.1126/science.1197349 21127216

68. Shin J-H, Chekanova JA. Arabidopsis RRP6L1 and RRP6L2 function in FLOWERING LOCUS C silencing via regulation of antisense RNA synthesis. PLoS genetics. 2014; 10(9): e1004612. doi: 10.1371/journal.pgen.1004612 25211139

69. Zhu H, Zhai W, Li X, Zhu Y. Two QTLs controlling Clubroot resistance identified from Bulked Segregant Sequencing in Pakchoi (Brassica campestris ssp. chinensis Makino). Scientific reports. 2019; 9(1): 9228. doi: 10.1038/s41598-019-44724-z 31239512.

70. Dijkstra JM, Alexander DB. The “NF-ĸB interacting long noncoding RNA”(NKILA) transcript is antisense to cancer-associated gene PMEPA1. F1000Research. 2015; 4: 96. doi: 10.12688/f1000research.6400.1 26069731.

71. Isah T. Stress and defense responses in plant secondary metabolites production. Biological research. 2019; 52(1): 39. doi: 10.1186/s40659-019-0246-3 31358053.

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