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Functional Characterization of a Putative Bacterial Wilt Resistance Gene (RE-bw) in Eggplant

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An Erratum to this article was published on 11 January 2015

Abstract

In this study, a putative resistant gene RE-bw was isolated from bacterial wilt-resistant eggplant inbred line (E-31) by cDNA amplified fragment length polymorphism (cDNA-AFLP) and rapid amplification of cDNA end (RACE). RE-bw has a length of 1,210 bp with 1,116 bp open reading frame (ORF) which encodes 371 amio acids. Bioinformation analysis showed that RE-bw include an NB-ACR and WRKY domain and involved in protein binding and defense response progress. Overexpression and RNAi experiments indicated that the RE-bw was an important gene for bacterial wilt resistance. Further research result showed that RE-bw is involved in enhanced SA content, ROS-scavenging enzymatic activities, cell wall, and lignin to limit the colonization of Ralstonia solanacearum. Bimolecular fluorescence complementation assay and yeast two-hybrid assay confirmed that RE-bw interacted with EDS1, PAD4, NPR1, SGT1, WRKY70, and avirulence effector Popp2 of R. solanacearum. The results provide an important new clue on understanding the mechanism of bacterial wilt disease resistance in eggplant.

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Acknowledgments

This work was supported by the State Natural Science Fundation of China [Grant No. 3097200], the Natural Science Fund of Guangdong Province [Grant No. S2011030001410], and the Key Laboratory of Innovation and Utilization for Germplasm Resources in Horticultural Crops in Southern China of Guangdong Higher Education Institutes, South China Agricultural University [Grant No. KBL11008].

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  1. South China Agriculture University, Room 722, College of horticulture, No.483 Wushan Road, Tianhe District, Guangzhou, People’s Republic of China, 510642

    Xiao Xi’ou, Cao Bihao, Li Guannan, Lei Jianjun, Chen Qinghua, Jiang Jin & Cheng Yujing

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  1. Xiao Xi’ou
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  2. Cao Bihao
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Correspondence to Cao Bihao.

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Fig. S1

cDNA-AFLP analysis of primers combination T3/A8 in resistant and susceptible eggplant. 1: E-31(R); 2: E-32 × E-31(R);3: E-32(S);4 ~ 8,13 ~ 16,18,19: resistant plants in F2 of E-32 × E-31 ;9 ~ 12,17,20,21: susceptible plants in F2 of E-32 × E-31; the arrows show difference special band. (GIF 35 kb)

High resolution image (TIFF 3115 kb)

Fig. S2

The expression level of RE-bw. A: the expression of RE-bw in the different part of resistant plant (E-31) under normal condition. Lanes 1 and 2 showing the leaf of E-31. Lanes 3 and 4 showing the stem of E-31. Lanes 5 and 6 showing the root of E-31. B: the expression of RE-bw in stem of the resistant plants and susceptible plants under normal condition. Lanes 1 showing the stem of resistant plant E-31 (R). Lane 2 showing the stem of resistant plant E-32x E-31(F1 , R). Lane 3 showing the stem of susceptible plant E-32 (S). Lanes 4 and 6 showing the stem of resistant plant of E-32x E-31(F2) (R). Lanes 5 and 7 showing the the stem of susceptible plant of E-32 x E-31(F2) (S). C: the expression of RE-bw in stem of the resistant plants (E-31) after inoculation pathogen (R. solanacearum), Lanes 1 showing control. Lane 2 showing Expression of RE-bw after inoculation for 6 h. Lane 3 Expression of RE-bw after inoculation for 12 h. Lanes 4 Expression of RE-bw after inoculation for 24 h. Lanes 5 Expression of RE-bw after inoculation for 36 h. D: Expression of RE-bw in resistant plants (E-31) after treatment of 20 mg/L SA for different times. Lane1 showing 0 h; lane 2 showing treatment for 2 h; lane 3 showing treatment for 4 h; lane 4 showing treatment for 6 h; lane 5 showing treatment for 8 h. E: Expression of RE-bw in resistant plants (E-31) after treatment of 30 mg/L JA for different times. Lane1 showing 0 h; lane 2 showing treatment for 2 h; lane 3 showing treatment for 4 h; lane 4 showing treatment for 6 h; lane 5 showing treatment for 8 h. (GIF 10 kb)

High resolution image (TIFF 3559 kb)

Fig. S3

The Southern blotting analysis of RE-bw in resistant plant (E-31) genomic. ‘E-31′ genomic DNA was degested by BamHI, EcoRI and Hind III, XbaI and SacI, souther blotting revealed that two bands were detected in those DNA samples digested with BamH, EcoR I,Hind III, five hybridization signal bands were showed in those simple DNA digested with XbaIand Sac I (GIF 37 kb)

High resolution image (TIFF 7617 kb)

Fig. S4

Analysis of co-segregation of RE-bw in F2 of E-31× E-32. Lane 1: showing E-31(R), lane 2 showing E-32(S), lane 3 showing F1 ;lanes 4–136 showing the F2 segregation individuals of E-31× E-32. DNA was extracted by CTAB and digested with Hind III, Southern blotting was carried out in F2 segregation individuals, and RE-bw was used as probe. (GIF 343 kb)

High resolution image (TIFF 105263 kb)

Fig. S5

Detection of Southern blotting and expression of RE-bw in transgenic plant, RNAi pant and VIGS eggplant plant. A: Detection of southern blotting of RE-bw in transgenic tomato plant. Lane 1 showing non-transgenic plant, lane2 showing positive CK; lanes 3-7showing transgenic plants. B: Detection of expression of RE-bw in transgenic tomato. C: Detection of southern blotting of eggplant RE-bw RNAi plant. Lane 1 showing positive CK, lane 2 showing non-transgenic plant, lanes 3–6 showing transgenic RNAi plant. D: Detection of expression of RE-bw in RNAi eggplant. E: Detection of expression of RE-bw in VIGS eggplant. (GIF 60 kb)

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Fig. S6

Analysis of SA level, PPO, POD and PAL activity. A:SA content after inoculation R.solanacearum. B:PPO activity after inoculation R.solanacearum. C:POD activity after inoculation R.solanacearum. D:PAL activity after inoculation R.solanacearum. Each sample time point for each treatment was comprised of three independent replicates. Least significant differences were calculated to compare the significance at the 5 % level using SPSS software (version 16.0). (GIF 99 kb)

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Fig. S7

The expression qPCR analysis of nine signal genes in resistant eggplant (E-31) and RE- bw RNAi-4 eggplant plants after inoculation pathogen for different time. EDS1, PAD4, NPR1, SGT1, WRYK70 were increased with the addition of induction time, and their levels of expression were higher in resistant plants (E-31) than that of in RE-bw-RNAi-4 plant (S), but the levels of expression of JAR1, NDR1, EIN2, RAR1 were not significant different after both resistance and susceptibility plants were induced by inoculation pathogen. (GIF 111 kb)

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11105_2014_814_MOESM8_ESM.doc

S1 Isolation of RE-bw sequence. a TDF sequence of RE-bw, b 5′ end sequence of RE-bw gene , c 3′ end sequence of RE-bw gene (DOC 22 kb)

Table S1

Primer for qPCR. (DOC 36 kb)

Table S2

The primers used in BiFC assay. (DOC 34 kb)

Table S3

Evaluation of BW-resistance in eggplant (DOC 45 kb)

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Xi’ou, X., Bihao, C., Guannan, L. et al. Functional Characterization of a Putative Bacterial Wilt Resistance Gene (RE-bw) in Eggplant. Plant Mol Biol Rep 33, 1058–1073 (2015). https://doi.org/10.1007/s11105-014-0814-1

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