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Comparison of transcriptome profiles by Fusarium oxysporum inoculation between Fusarium yellows resistant and susceptible lines in Brassica rapa L.

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Abstract

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Resistant and susceptible lines in Brassica rapa have different immune responses against Fusarium oxysporum inoculation.

Abstract

Fusarium yellows caused by Fusarium oxysporum f. sp. conglutinans (Foc) is an important disease of Brassicaceae; however, the mechanism of how host plants respond to Foc is still unknown. By comparing with and without Foc inoculation in both resistant and susceptible lines of Chinese cabbage (Brassica rapa var. pekinensis), we identified differentially expressed genes (DEGs) between the bulked inoculated (6, 12, 24, and 72 h after inoculation (HAI)) and non-inoculated samples. Most of the DEGs were up-regulated by Foc inoculation. Quantitative real-time RT-PCR showed that most up-regulated genes increased their expression levels from 24 HAI. An independent transcriptome analysis at 24 and 72 HAI was performed in resistant and susceptible lines. GO analysis using up-regulated genes at 24 HAI indicated that Foc inoculation activated systemic acquired resistance (SAR) in resistant lines and tryptophan biosynthetic process and responses to chitin and ethylene in susceptible lines. By contrast, GO analysis using up-regulated genes at 72 HAI showed the overrepresentation of some categories for the defense response in susceptible lines but not in the resistant lines. We also compared DEGs between B. rapa and Arabidopsis thaliana after F. oxysporum inoculation at the same time point, and identified genes related to defense response that were up-regulated in the resistant lines of Chinese cabbage and A. thaliana. Particular genes that changed expression levels overlapped between the two species, suggesting that they are candidates for genes involved in the resistance mechanisms against F. oxysporum.

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Acknowledgements

We thank Dr. Zijing Pu for excellent technical assistance. We also thank Dr. Qian-Hao Zhu and Dr. Ming-Bo Wang for providing us the data of DEGs in A. thaliana. This work was supported in part by a Grant-in-Aid for Young Scientists (B) (2478002) (JSPS) to R. Fujimoto, Research Fellowships of JSPS for Young Scientists (12J05450) to M. Shimizu, and by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (26022A) and by the Matching Planner Program from Japan Science and Technology Agency, JST (MP28116808421) to K. Okazaki.

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  1. Naomi Miyaji and Motoki Shimizu have contributed equally to this work.

Authors and Affiliations

  1. Graduate School of Agricultural Science, Kobe University, Rokkodai, Nada-ku, Kobe, 657-8501, Japan

    Naomi Miyaji, Satoko Takada & Ryo Fujimoto

  2. Iwate Biotechnology Research Center, Narita, Kitakami, Iwate, 024-0003, Japan

    Motoki Shimizu

  3. Department of Animal, Plant and Soil Sciences, Centre for AgriBioscience, La Trobe University, Bundoora, VIC, 3086, Australia

    Junji Miyazaki

  4. Plant Epigenetics Unit, Okinawa Institute of Science and Technology Graduate University, Onna-Son, Okinawa, 904-0495, Japan

    Kenji Osabe

  5. Graduate School of Science and Technology, Niigata University, Ikarashi-ninocho, Niigata, 950-2181, Japan

    Maho Sato, Yusuke Ebe & Keiichi Okazaki

  6. Watanabe seed Co., Ltd, Machiyashiki, Misato-Cho, Miyagi, 987-8607, Japan

    Makoto Kaji

  7. CSIRO Agriculture, Canberra, ACT, 2601, Australia

    Elizabeth S. Dennis

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  1. Naomi Miyaji
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Correspondence to Ryo Fujimoto.

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Communicated by Dr. Kinya Toriyama.

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299_2017_2198_MOESM1_ESM.pptx

Figure S1. Gene expression levels of up-regulated genes after Foc inoculation measured by quantitative real-time RT-PCR. Six genes, BG3 (Group-2), Bra037877 (Group-1), GULLO2 (Group-2), JRG21 (Group-3), PDR12 (Group-2), and PRB1 (Group-2), whose expression was up-regulated in R-24 h-inf and/or S-24 h-inf, were examined. Expression levels of these six genes were measured by real-time RT-PCR at 6, 12, 24, and 72 h after Foc inoculation (HAI). Values are mean ± SE (three biological and technical replicates) of relative expression levels compared with Bractin were shown. NI, non-inoculated samples (PPTX 92 kb)

299_2017_2198_MOESM2_ESM.pptx

Figure S2. Validation of six genes at 24 HAI by quantitative real-time RT-PCR. The expression levels quantified by real-time RT-PCR and RNA-seq data at 24 HAI were compared in six genes, whose expression was up-regulated in Rb-inf and/or Sb-inf. Values are mean ± SE (three biological and technical replicates) of relative expression levels compared with Bractin are shown. The values of fragments per kilo-base per million (FPKM) were calculated by data from RNA-seq analysis. R-24 h-mock, mock-inoculated samples of the resistant line (RJKB-T23); S-24 h-mock; mock-inoculated samples of the susceptible line (RJKB-T24). R-24 h-inf, inoculated samples at 24 HAI of the resistant line; S-24 h-inf, inoculated samples at 24 HAI of the susceptible line; HAI, hours after inoculation. NI, non-inoculated samples (PPTX 105 kb)

299_2017_2198_MOESM3_ESM.pptx

Figure S3. Validation of six genes at 24 HAI by quantitative real-time RT-PCR. The expression levels quantified by real-time RT-PCR and RNA-seq data at 24 HAI were compared in six genes, whose expression was up-regulated in R-24 h-inf and/or S-24 h-inf. Values are mean ± SE (three biological and technical replicates) of relative expression levels compared with Bractin are shown. The values of fragments per kilo-base per million (FPKM) were calculated by data from RNA-seq analysis. R-24 h-mock, mock-inoculated samples of the resistant line (RJKB-T23); S-24 h-mock; mock-inoculated samples of the susceptible line (RJKB-T24). R-24 h-inf, inoculated samples at 24 HAI of the resistant line; S-24 h-inf, inoculated samples at 24 HAI of the susceptible line; HAI, hours after inoculation. NI, non-inoculated samples (PPTX 107 kb)

299_2017_2198_MOESM4_ESM.pptx

Figure S4. Validation of six genes at 72 HAI by quantitative real-time RT-PCR. The expression levels quantified by real-time RT-PCR and RNA-seq data at 72 HAI were compared in six genes, whose expression was up-regulated in Rb-inf and/or Sb-inf. Values are mean ± SE (three biological and technical replicates) of relative expression levels compared with Bractin are shown. The values of fragments per kilo-base per million (FPKM) were calculated by data from RNA-seq analysis. R-72 h-mock, mock-inoculated samples of the resistant line (RJKB-T23); S-72 h-mock; mock-inoculated samples of the susceptible line (RJKB-T24). R-72 h-inf, inoculated samples at 72 HAI of the resistant line; S-72 h-inf, inoculated samples at 72 HAI of the susceptible line; HAI, hours after inoculation. NI, non-inoculated samples (PPTX 100 kb)

299_2017_2198_MOESM5_ESM.pptx

Figure S5. Validation of six genes at 72 HAI by quantitative real-time RT-PCR. The expression levels quantified by real-time RT-PCR and RNA-seq data at 72 HAI were compared in six genes, whose expression was up-regulated in R-24 h-inf and/or S-24 h-inf. Values are mean ± SE (three biological and technical replicates) of relative expression levels compared with Bractin are shown. The values of fragments per kilo-base per million (FPKM) were calculated by data from RNA-seq analysis. R-72 h-mock, mock-inoculated samples of the resistant line (RJKB-T23); S-72 h-mock; mock-inoculated samples of the susceptible line (RJKB-T24). R-72 h-inf, inoculated samples at 72 HAI of the resistant line; S-72 h-inf, inoculated samples at 72 HAI of the susceptible line; HAI, hours after inoculation. NI, non-inoculated samples (PPTX 103 kb)

299_2017_2198_MOESM6_ESM.pptx

Figure S6. Venn diagram of up- or down-regulated genes in inoculated samples of B. rapa (24 HAI) compared with A. thaliana (1 DPI). R-24 h-inf UP/DOWN, up-/down-regulated genes in the inoculated whole plants compared with genes in the mock-inoculated whole plants in resistant line (RJKB-T23) at 24 HAI (hours after inoculation). S-24 h-inf UP/DOWN, up-/down-regulated genes in the inoculated whole plants compared with genes in the mock-inoculated whole plants in the susceptible line (RJKB-T24) at 24 HAI. At UP/DOWN, up-/down-regulated genes in inoculated samples at 1 DPI (day-post-inoculation) compared with genes in mock-inoculated samples in A. thaliana (PPTX 59 kb)

299_2017_2198_MOESM7_ESM.pptx

Figure S7. Validation of four genes at 24 HAI by quantitative real-time RT-PCR. The expression levels quantified by real-time RT-PCR and RNA-seq data at 24 HAI were compared in four genes, whose expression was up-regulated at 24 HAI in resistant line of B. rapa and 1 DPI (day-post-inoculation) in A. thaliana. Values are mean ± SE (three biological and technical replicates) of relative expression levels compared with Bractin are shown. The values of fragments per kilo-base per million (FPKM) were calculated by data from RNA-seq analysis. R-24 h-mock, mock-inoculated samples of the resistant line (RJKB-T23); S-24 h-mock; mock-inoculated samples of the susceptible line (RJKB-T24). R-24 h-inf, inoculated samples at 24 HAI of the resistant line; S-24 h-inf, inoculated samples at 24 HAI of the susceptible line; HAI, hours after inoculation. NI, non-inoculated samples (PPTX 84 kb)

299_2017_2198_MOESM8_ESM.pptx

Figure S8. Validation of four genes at 72 HAI by quantitative real-time RT-PCR. The expression levels quantified by real-time RT-PCR and RNA-seq data at 72 HAI were compared in four genes, whose expression was up-regulated at 24 HAI in resistant line of B. rapa (RJKB-T23) and 1 DPI (day-post-inoculation) in A. thaliana. Values are mean ± SE (three biological and technical replicates) of relative expression levels compared with Bractin are shown. The values of fragments per kilo-base per million (FPKM) were calculated from the RNA-seq data analysis. R-72 h-mock, mock-inoculated samples of the resistant line (RJKB-T23); S-72 h-mock; mock-inoculated samples of the susceptible line (RJKB-T24). R-72 h-inf, inoculated samples at 72 HAI of the resistant line (RJKB-T23); S-72 h-inf, inoculated samples at 72 HAI of the susceptible line (RJKB-T24); HAI, hours after inoculation. NI, non-inoculated samples (PPTX 84 kb)

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Miyaji, N., Shimizu, M., Miyazaki, J. et al. Comparison of transcriptome profiles by Fusarium oxysporum inoculation between Fusarium yellows resistant and susceptible lines in Brassica rapa L.. Plant Cell Rep 36, 1841–1854 (2017). https://doi.org/10.1007/s00299-017-2198-9

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