Abstract
Aims
Clubroot, caused by the soil-borne protist Plasmodiophora brassicae, is one of the most destructive disease for Brassica oleracea worldwide. However, the molecular mechanism of clubroot resistance still remains poorly elucidated. Therefore, we aim at identifying key genes responsive to P. brassicae infection and deducing possible molecular mechanism regulating clubroot resistance in cabbage.
Methods
A clubroot-resistant line (XG) and a clubroot-susceptible line (JF) were employed to conduct histological observation and transcriptome analysis at 7 and 28 DAI (days after inoculation) following inoculation with P. brassicae. Differentially expressed genes (DEGs) obtained by comparing infected roots with mock-infected roots were assigned to Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for enrichment analysis.
Results
TEM observation showed obvious histological differences of root cells between JF and XG after inoculation with P. brassicae. At 7 DAI, the number of DEGs identified in JF was much higher than that of XG, and most of them were enriched in metabolic pathways, metabolites biosynthesis and starch, sucrose metabolism. More DEGs were identified at 28 DAI compared to 7 DAI in XG, and most of these DEGs involved in biosynthesis of secondary metabolites, plant-pathogen interaction and plant hormone transduction. Genes related to cell wall biosynthesis, pattern recognition receptors (PRRs), disease resistance proteins, SA signal transduction, calcium influx, respiratory burst oxidase homolog (RBOH), MAPK cascades, transcription factors and chitinase were mainly up-regulated in XG at 28 DAI, while most of them were repressed in JF.
Conclusions
Our research work suggest drastic and complex defense response to P. brassicae infection at 28 DAI (secondary infection stage) at transcriptional level. Results generated in the present study could provide comprehensive insights into the transcriptomic landscape for better understanding of molecular regulatory mechanism of clubroot resistance in cabbage.
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Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983. https://doi.org/10.1038/415977a
Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B 57:289–300. https://doi.org/10.2307/2346101
Chen JJ, Pang WX, Chen B, Zhang CY, Piao ZY (2016) Transcriptome analysis of Brassica rapa near-isogenic lines carrying clubroot-resistant and-susceptible alleles in response to Plasmodiophora brassicae during early infection. Front Plant Sci 6:1183. https://doi.org/10.3389/fpls.2015.01183
Cheng SH, Willmann MR, Chen HC, Sheen J (2002) Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol 129:469–485. https://doi.org/10.1104/pp.005645
Chu MG, Song T, Falk KC, Zhang XG, Liu XJ, Chang A, Lahlali R, Yu FQ, McGregor L, Gossen BD, Peng G (2014) Fine mapping of Rcr1 and analyses of its effect on transcriptome patterns during infection by Plasmodiophora brassicae. BMC Genomics 15:1471–2164. https://doi.org/10.1186/1471-2164-15-1166
Colcombet J, Hirt H (2008) Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem J 413:217–226. https://doi.org/10.1042/BJ20080625
Devos S, Laukens K, Deckers P, Van Der Straeten D, Beeckman T, Inzé D, Van Onckelen H, Witters E, Prinsen E (2006) A hormone and proteome approach to picturing the initial metabolic events during Plasmodiophora brassicae infection on Arabidopsis. Mol Plant-Microbe Interact 19:1431–1443. https://doi.org/10.1094/MPMI-19-1431
Dixon GR (2009) The occurrence and economic impact of Plasmodiophora brassicae and clubroot disease. J Plant Growth Regul 28:194–202. https://doi.org/10.1007/s00344-009-9090-y
Djavaheri M, Ma L, Klessig DF, Mithofer A, Gropp G, Borhan MH (2019) Mimicking the host regulation of SA: a virulence strategy by the clubroot pathogen Plasmodiophora brassicae. Mol Plant-Microbe Interact 32:296–305. https://doi.org/10.1094/MPMI-07-18-0192-R
Dodds PN, Rathjen JP (2010) Plant immunity: towards an integrated view of plant-pathogen interactions. Nat Rev Genet 11:539–548. https://doi.org/10.1038/nrg2812
Donald C, Porter IJ (2009) Integrated control of Clubroot. J Plant Growth Regul 28:289–303. https://doi.org/10.1007/s00344-009-9094-7
FAO (2017) Food and agriculture organization of the United Nations, statistics division. http://www.fao.org/faostat/en/#data
Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227. https://doi.org/10.1146/annurev.phyto.43.040204.135923
Gotz S, Garcia-Gomez JM, Terol J, Williams TD, Nagaraj SH, Nueda MJ, Robles M, Talon M, Dopazo J, Conesa A (2008) High-throughput functional annotation and data mining with the Blast2GO suite. Nucleic Acids Res 36:3420–3435. https://doi.org/10.1093/nar/gkn176
Group M, Ichimura K, Shinozaki K, Tena G, Sheen J, Henry Y, Champion A, Kreis M, Zhang S, Hirt H, Wilson C, Heberle-Bors E, Ellis BE, Morris PC, Innes RW, Ecker JR, Scheel D, Klessig DF, Machida Y, Mundy J, Ohashi Y, Walker JC (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7:301–308. https://doi.org/10.1016/S1360-1385(02)02302-6
Hatakeyama K, Suwabe K, Tomita NR, Kato T, Nunome T, Fukuoka H, Matsumoto S (2013) Identification and characterization of Crr1a, a gene for resistance to clubroot disease (Plasmodiophora brassicae Woronin) in Brassica rapa L. PLoS One 8:1–10. https://doi.org/10.1371/journal.pone.0054745
Hatakeyama K, Niwa T, Kato T, Ohara T, Kakizaki T, Matsumoto S (2017) The tandem repeated organization of NB-LRR genes in the clubroot-resistant CRb locus in Brassica rapa L. Mol Gen Genomics 292:397–405. https://doi.org/10.1007/s00438-016-1281-1
Hernandez-Blanco C, Feng DX, Hu J, Sanchez-Vallet A, Deslandes L, Llorente F, Berrocal-Lobo M, Keller H, Barlet X, Sanchez-Rodriguez C, Anderson LK, Somerville S, Marco Y, Molina A (2007) Impairment of cellulose synthases required for Arabidopsis secondary cell wall formation enhances disease resistance. Plant Cell 19:890–903. https://doi.org/10.1105/tpc.106.048058
Holland N, Holland D, Helentjaris T, Dhugga KS, Xoconostle-Cazares B, Delmer DP (2000) A comparative analysis of the plant cellulose synthase(CesA) gene family. Plant Physiol 123:1313–1324. https://doi.org/10.1104/pp.123.4.1313
Huang Z, Peng G, Liu X, Deora A, Falk KC, Gossen BD, McDonald MR, Yu F (2017) Fine mapping of a clubroot resistance gene in Chinese cabbage using SNP markers identified from bulked segregant RNA sequencing. Front Plant Sci 8:1448. https://doi.org/10.3389/fpls.2017.01448
Irani S, Trost B, Waldner M, Nayidu N, Tu J, Kusalik AJ, Todd CD, Wei Y, Bonham-Smith PC (2018) Transcriptome analysis of response to Plasmodiophora brassicae infection in the Arabidopsis shoot and root. BMC Genomics 19:23. https://doi.org/10.1186/s12864-017-4426-7
Jammes F, Hu HC, Villiers F, Bouten R, Kwak JM (2011) Calcium-permeable channels in plant cells. FEBS J 278:4262–4276. https://doi.org/10.1111/j.1742-4658.2011.08369.x
Ji R, Wang Y, Wang X, Liu Y, Shen X, Feng H (2018) Proteomic analysis of the interaction between Plasmodiophora brassicae and Chinese cabbage (Brassica rapa L. ssp. Pekinensis ) at the initial infection stage. Sci Hortic 233:386–393. https://doi.org/10.1016/j.scienta.2018.02.006
Jia H, Wei X, Yang Y, Yuan Y, Wei F, Zhao Y, Yang S, Yao Q, Wang Z, Tian B, Zhang X (2017a) Root RNA-seq analysis reveals a distinct transcriptome landscape between clubroot-susceptible and clubroot-resistant Chinese cabbage lines after Plasmodiophora brassicae infection. Plant Soil 421:93–105. https://doi.org/10.1007/s11104-017-3432-5
Jia PF, Li HJ, Yang WC (2017b) Analysis of peroxisome biogenesis in pollen by confocal microscopy and transmission electron microscopy. Plant Germline Development 1669:173–180. https://doi.org/10.1007/978-1-4939-7286-9_14
Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323–329. https://doi.org/10.1038/nature05286
Kageyama K, Asano T (2009) Life cycle of Plasmodiophora brassicae. J Plant Growth Regul 28:203–211. https://doi.org/10.1007/s00344-009-9101-z
Kanehisa M, Goto S (2000) KEGG: Kyoto Encyclopaedia of genes and genomes. Nucleic Acids Res 28:27–30. https://doi.org/10.1093/nar/28.1.27
Kant S, Bi YM, Zhu T, Rothstein SJ (2009) SAUR39, a small auxin-up RNA gene, acts as a negative regulator of auxin synthesis and transport in rice. Plant Physiol 151:691–701. https://doi.org/10.1104/pp.109.143875
Kazan K, Manners JM (2012) JAZ repressors and the orchestration of phytohormone crosstalk. Trends Plant Sci 17:22–31. https://doi.org/10.1016/j.tplants.2011.10.006
Kim SH, Kwon SI, Saha D, Anyanwu NC, Gassmann W (2009) Resistance to the Pseudomonas syringae effector HopA1 is governed by the TIR-NBS-LRR protein RPS6 and is enhanced by mutations in SRFR1. Plant Physiol 150:1723–1732. https://doi.org/10.1104/pp.109.139238
Kim KH, Kang YJ, Kim DH, Yoon MY, Moon JK, Kim MY, Van K, Lee SH (2011) RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and-susceptible alleles. DNA Res 18:483–497. https://doi.org/10.1093/dnares/dsr033
Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14:36. https://doi.org/10.1186/gb-2013-14-4-r36
Kurusu T, Kuchitsu K, Tada Y (2015) Plant signaling networks involving Ca2+ and Rboh/Nox-mediated ROS production under salinity stress. Front Plant Sci 6:427. https://doi.org/10.3389/fpls.2015.00427
Lacombe E, Hawkins S, Doorsselaere J, Piquemal J, Goffner D, Poeydomenge O, Boudet A, Grima-Pettenati J (1997) Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant J 11:429–441. https://doi.org/10.1046/j.1365-313X.1997.11030429.x
Lahlali R, Song T, Chu M, Yu F, Kumar S, Karunakaran C, Peng G (2017) Evaluating changes in cell-wall components associated with clubroot resistance using fourier transform infrared spectroscopy and RT-PCR. Int J Mol Sci 18:2058. https://doi.org/10.3390/ijms18102058
Lee J, Izzah NK, Choi BS, Joh HJ, Lee SC, Perumal S, Seo J, Ahn K, Jo EJ, Choi GJ, Nou IS, Yu Y, Yang TJ (2016) Genotyping-by-sequencing map permits identification of clubroot resistance QTLs and revision of the reference genome assembly in cabbage (Brassica oleracea L.). DNA Res 23:29–41. https://doi.org/10.1093/dnares/dsv034
Lemarie S, Robert-Seilaniantz A, Lariagon C, Lemoine J, Marnet N, Jubault M, Manzanares-Dauleux MJ, Gravot A (2015) Both the jasmonic acid and the salicylic acid pathways contribute to resistance to the biotrophic clubroot agent Plasmodiophora brassicae in Arabidopsis. Plant Cell Physiol 56:2158–2168. https://doi.org/10.1093/pcp/pcv127
Li CY, Deng GM, Yang J, Viljoen A, Jin Y, Kuang RB, Zuo CW, Lv ZC, Yang QS, Sheng O (2012) Transcriptome profiling of resistant and susceptible Cavendish banana roots following inoculation with Fusarium oxysporum f. sp. cubense tropical race 4. BMC Genomics 13:374. https://doi.org/10.1186/1471-2164-13-374
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lovelock DA, Donald CE, Conlan XA, Cahill DM (2013) Salicylic acid suppression of clubroot in broccoli (Brassicae oleracea var. italica) caused by the obligate biotroph Plasmodiophora brassicae. Australas Plant Pathol 42:141–153. https://doi.org/10.1007/s13313-012-0167-x
Ludwig-Müller J (2014) Auxin homeostasis, signaling, and interaction with other growth hormones during the clubroot disease of Brassicaceae. Plant Signal Behav 9:e28593. https://doi.org/10.4161/psb.28593
Ludwig-Müller J, Prinsen E, Rolfe SA, Scholes JD (2009) Metabolism and plant hormone action during clubroot disease. J Plant Growth Regul 28:229–244. https://doi.org/10.1007/s00344-009-9089-4
Luo HC, Chen GK, Liu CP, Huang Y, Xiao CG (2013) An improved culture solution technique for Plasmodiophora brassicae infection and the dynamic infection in the root hair. Austral Plant Pathol 43:53–60. https://doi.org/10.1007/s13313-013-0240-0
Luo Y, Dong D, Su Y, Wang X, Peng Y, Peng J, Zhou C (2018) Transcriptome analysis of Brassica juncea var. tumida Tsen responses to Plasmodiophora brassicae primed by the biocontrol strain Zhihengliuella aestuarii. Funct Integr Genomic 18:301–314. https://doi.org/10.1007/s10142-018-0593-0
Ma W (2011) Roles of Ca2+ and cyclic nucleotide gated channel in plant innate immunity. Plant Sci 181:342–346. https://doi.org/10.1016/j.plantsci.2011.06.002
Moxham SE, Buczacki ST (1983) Chemical composition of the resting spore wall of Plasmodiophora brassicae. Trans Br Mycol Soc 80:297–304. https://doi.org/10.1016/S0007-1536(83)80013-8
Nagaharu U (1935) Genome analysis in Brassica with special reference to the experimental formation of B. napus and peculiar mode of fertilization. Jpn J Bot 7:389–452
Ning Y, Wang Y, Fang Z, Zhuang M, Zhang Y, Lv H, Liu Y, Li Z, Yang L (2018) Identification and characterization of resistance for Plasmodiophora brassicae race 4 in cabbage (Brassica oleracea var. capitata). Australas Plant Pathol 47:531–541. https://doi.org/10.1007/s13313-018-0590-8
Pandey SP, Somssich IE (2009) The role of WRKY transcription factors in plant immunity. Plant Physiol 150:1648–1655. https://doi.org/10.1104/pp.109.138990
Piao Z, Ramchiary N, Lim YP (2009) Genetics of clubroot resistance in Brassica species. J Plant Growth Regul 28:252–264. https://doi.org/10.1007/s00344-009-9093-8
Rolfe SA, Strelkov SE, Links MG, Clarke WE, Robinson SJ, Djavaheri M, Malinowski R, Haddadi P, Kagale S, Parkin IAP, Taheri A, Borhan MH (2016) The compact genome of the plant pathogen Plasmodiophora brassicaeis adapted to intracellular interactions with host Brassica spp. BMC Genomics 17:272. https://doi.org/10.1186/s12864-016-2597-2
Scheller HV, Ulvskov P (2010) Hemicelluloses. Annu Rev Plant Biol 61:263–289. https://doi.org/10.1146/annurev-arplant-042809-112315
Schwelm A, Fogelqvist J, Knaust A, Jülke S, Lilja T, Bonilla-Rosso G, Karlsson M, Shevchenko A, Dhandapani V, Choi SR, Kim HG, Park JY, Lim YP, Ludwig-Müller J, Dixelius C (2015) The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases. Sci Rep 5:11153–11165. https://doi.org/10.1038/srep11153
Shinshi H, Mohnen D, Meins F (1987) Regulation of a plant pathogenesis-related enzyme: inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proc Natl Acad Sci 84:89–93. https://doi.org/10.1073/pnas.84.1.89
Sitbon F, Hennion S, Little CHA, Sundberg B (1999) Enhanced ethylene production and peroxidase activity in IAA-overproducing transgenic tobacco plants is associated with increased lignin content and altered lignin composition. Plant Sci 141:165–173. https://doi.org/10.1016/S0168-9452(98)00236-2
Sterling JD, Quigley HF, Orellana A, Mohnen D (2001) The catalytic site of the pectin biosynthetic enzyme α-1,4-galacturonosyltransferase is located in the lumen of the golgi. Plant Physiol 127:360–371. https://doi.org/10.1104/pp.127.1.360
Strelkov SE, Dixon GR (2014) Clubroot (Plasmodiophora brassicae) on canola and other Brassica species-disease development, epidemiology and management. Can J Plant Pathol 36:1–4. https://doi.org/10.1080/07060661.2013.875338
Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L (2010) Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:511–515. https://doi.org/10.1038/nbt.1621
Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and cufflinks. Nat Protoc 7:562–578. https://doi.org/10.1038/nprot.2012.016
Ueno H, Matsumoto E, Aruga D, Kitagawa S, Matsumura H, Hayashida N (2012) Molecular characterization of the CRa gene conferring clubroot resistance in Brassica rapa. Plant Mol Biol 80:621–629. https://doi.org/10.1007/s11103-012-9971-5
Williams PH (1966) A system for the determination of races of Plasmodiophora brassicae that infect cabbage and rutabaga. Phytopathology 56:624–626
Xing P, Zhang X, Bao Y, Wang Y, Wang H, Li X (2017) Comparative transcriptome analyses of resistant and susceptible near-isogenic wheat lines following inoculation with Blumeria graminis f. sp. tritici. Int J. Genomics 2017:7305684. https://doi.org/10.1155/2017/7305684
Yahaya N, Petriacq P, Burrell M, Walker H, Malinowski R, Rolfe S (2017) Changes of metabolites status in plant pathogen interaction. Adv Sci Lett 23:4623–4626. https://doi.org/10.1166/asl.2017.8947
Ye ZH, Zhong R, Morrison Iii WH, Himmelsbach DS (2001) Caffeoyl coenzyme a O-methyltransferase and lignin biosynthesis. Phytochemistry 57:1177–1185. https://doi.org/10.1016/S0031-9422(01)00051-6
Yu D, Chen C, Chen Z (2001) Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13:1527–1540. https://doi.org/10.1105/TPC.010115
Zhang X, Liu Y, Fang Z, Li Z, Yang L, Zhuang M, Zhang Y, Lv H (2016) Comparative transcriptome analysis between broccoli (Brassica oleracea var. italica) and wild cabbage (Brassica macrocarpa Guss.) in response to Plasmodiophora brassicae during different infection stages. Front. Plant Sci 7:1929. https://doi.org/10.3389/fpls.2016.01929
Zhao Y, Bi K, Gao Z, Chen T, Liu H, Xie J, Cheng J, Fu Y, Jiang D (2017) Transcriptome analysis of Arabidopsis thaliana in response to Plasmodiophora brassicae during early infection. Front Microbiol 8:673. https://doi.org/10.3389/fmicb.2017.00673
Acknowledgements
This work was supported by the National Key Research and Development Program (2017YFD0101804), the National Natural Science Foundation of China (31801876), the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2013-IVFCAAS), the National High Technology Research and Development Program of China (863 Program, 2012AA100101), the Key Projects in the National Science and Technology Pillar Program during the Twelfth Five-Year Plan Period (2012BAD02B01), the Modern Agro-Industry Technology Research System (CARS-25-B-01), and the Project of Science and Technology Commission of Beijing Municipality (Z141105002314020-1).
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YN designed and performed the experiments, analyzed the data, wrote and revised the manuscript; ZF guided the experimental design. ZL and YL planted and managed the seedlings. YW and HL performed histological observation of infection process. MZ, YZ and LY critically reviewed the manuscript. All authors approved the final manuscript.
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Fig. S1
Phenotypes of roots infected by P. brassicae. (a) JF at 7DAI, (b) XG at 7 DAI, (c) JF at 28 DAI and (d) XG at 28DAI. (PNG 2859 kb)
Fig. S2
Identification of significant DEGs and their enriched pathways in JF(a) and XG (b) at 7DAI and in JF (c) and XG (d) at 28 DAI. The left section of the x-axis indicates the pathways that are common between genotypes at the same time point. The y-axis shows the number of DEGs. Columns in red mean up-regulated genes and in green mean down-regulated genes. (PNG 208 kb)
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Table S1
Description and primer sequences of genes used for qRT-PCR verification (XLSX 156 kb)
Table S2
Statistics for the RNA-seq data of 24 libraries from JF and XG at 7 and 28 DAI. (XLSX 13 kb)
Table S3
Significantly enriched GO terms of DEGs obtained by comparing infected library with control in both genotypes at different time points (XLSX 25 kb)
Table S4
Significantly enriched KEGG pathways of DEGs in both JF and XG at 7 and 28 DAI (XLSX 14 kb)
Table S5
Expression pattern of DEGs involving in clubroot resistance in two genotypes at different infection stages of P. brassicae (XLSX 19 kb)
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Ning, Y., Wang, Y., Fang, Z. et al. Comparative transcriptome analysis of cabbage (Brassica oleracea var. capitata) infected by Plasmodiophora brassicae reveals drastic defense response at secondary infection stage. Plant Soil 443, 167–183 (2019). https://doi.org/10.1007/s11104-019-04196-6
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DOI: https://doi.org/10.1007/s11104-019-04196-6