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Inducible and constitutive expression of an elicitor gene Hrip1 from Alternaria tenuissima enhances stress tolerance in Arabidopsis

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Abstract

Hrip1 is a novel hypersensitive response-inducing protein secreted by Alternaria tenuissima that activates defense responses and systemic acquired resistance in tobacco. This study investigates the role that Hrip1 plays in responses to abiotic and biotic stress using transgenic Arabidopsis thaliana expressing the Hrip1 gene under the control of the stress-inducible rd29A promoter or constitutive cauliflower mosaic virus 35S promoter. Bioassays showed that inducible Hrip1 expression in rd29A∷Hrip1 transgenic lines had a significantly higher effect on plant height, silique length, plant dry weight, seed germination and root length under salt and drought stress compared to expression in 35S∷Hrip1 lines and wild type plants. The level of enhancement of resistance to Botrytis cinerea by the 35S∷Hrip1 lines was higher than in the rd29A∷Hrip1 lines. Moreover, stress-related gene expression in the transgenic Arabidopsis lines was significantly increased by 200 mM NaCl and 200 mM mannitol treatments, and defense genes in the jasmonic acid and ethylene signaling pathway were significantly up-regulated after Botrytis inoculation in the Hrip1 transgenic plants. Furthermore, the activity of some antioxidant enzymes, such as peroxidase and catalase increased after salt and drought stress and Botrytis infection. These results suggested that the Hrip1 protein contributes to abiotic and biotic resistance in transgenic Arabidopsis and may be used as a useful gene for resistance breeding in crops. Although the constitutive expression of Hrip1 is suitable for biotic resistance, inducible Hrip1 expression is more responsive for abiotic resistance.

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Abbreviations

CaMV35S:

Cauliflower mosaic virus 35S promoter

rd29A:

Promoter of desiccation-responsive rd29A gene in Arabidopsis thaliana

POD:

Peroxidase

CAT:

Catalase (Kong et al. 2011)

dpi:

Days post inoculate

hpi:

Hours post inoculate

SA:

Salicylic acid

JA/ET:

Jasmonates/ethylene

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Acknowledgments

We thank Dr. Jianping Yang (Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China) for assistance with Arabidopsis planting. This work was supported by the National technology research and development program (863 program) project (Grant Nos. 2011AA10A205 and 2012AA101504), The State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant protection, Chinese Academy of Agricultural science, No. 12 Zhong-guan-cun South Street, Beijing, China.

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Authors and Affiliations

  1. The State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Science, No. 12 Zhong-guan-cun South Street, Beijing, 100081, China

    Xue-Cong Peng, De-Wen Qiu, Hong-Mei Zeng, Li-Hua Guo, Xiu-Fen Yang & Zheng Liu

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  1. Xue-Cong Peng
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  2. De-Wen Qiu
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  5. Xiu-Fen Yang
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Corresponding authors

Correspondence to Xiu-Fen Yang or Zheng Liu.

Electronic supplementary material

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11248_2014_9824_MOESM1_ESM.tif

Supplemental Fig. 1 Expression of Hrip1 in transgenic Arabidopsis a The transcript and expression levels of Hrip1 in the 35S∷Hrip1 and rd29A∷Hrip1 expression lines, as analyzed by PCR, RT-PCR, and western blotting. Total RNA and protein were extracted from WT plants and Hrip1-expressing plants grown under normal or salt conditions for 8 h. Different expression lines were analyzed by western blotting. All lines were analyzed using an unquantified concentration of protein extract. The protein is 17 kDa. b Representative leaves photographed at 8 dpi for GUS detection of rd29A∷Hrip1∷GUS and 35S∷Hrip1∷GUS transgenic plants and WT under control and stress conditions. The plants were grown on MS agar containing 25 µg mL−1 kanamycin and then exposed to salt for 8 h; GUS activity is shown in rosette plants (TIFF 2767 kb)

11248_2014_9824_MOESM2_ESM.tif

Supplemental Fig. 2 Germination of transgenic Arabidopsis on MS medium and MS supplemented with 75 mM NaCl and 50 mM mannitol. a Germination of seeds on medium at three days after stratification. b Germination rate of transgenic Arabidopsis under salt and drought stresses. The results are presented as the means and standard errors from three independent experiments; three lines of each construct were used. A total of 60 seeds of each line were used for each experiment (TIFF 4447 kb)

11248_2014_9824_MOESM3_ESM.tif

Supplemental Fig. 3 Relative root length of transgenic Arabidopsis. Phenotypes on MS medium supplemented with different concentrations of NaCl or mannitol. The relative root lengths were measured on day 7 after stratification (n = 90 for each condition), and photographs were taken. The results are presented as the means and standard errors from three independent experiments. * and **and *** indicate significant differences at P < 0.05 (TIFF 4276 kb)

11248_2014_9824_MOESM4_ESM.tif

Supplemental Fig. 4 Expression of stress tolerance-related genes in WT and Hrip1-expressing plants, as analyzed by qRT-PCR. Total RNA was extracted from 21-d-old plants grown under normal or stress treatment for 0, 1.5 h, and 10.5 h. Actin was used as an internal control. Each column represents an average of three replicates, and the error bars indicate the standard deviation. Different * indicate significant differences in comparison with WT at P < 0.05. a Expression of DREB2A and P5CS2 with 200 mM NaCl. b Expression of LOS6 and NCED3 with 200 mM mannitol. c Expression of PR1 and BGL2 with Botrytis. d Expression of PR4 and Thi2.1 with Botrytis (TIFF 9421 kb)

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Supplementary material 7 (DOC 26 kb)

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Peng, XC., Qiu, DW., Zeng, HM. et al. Inducible and constitutive expression of an elicitor gene Hrip1 from Alternaria tenuissima enhances stress tolerance in Arabidopsis . Transgenic Res 24, 135–145 (2015). https://doi.org/10.1007/s11248-014-9824-x

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  • DOI: https://doi.org/10.1007/s11248-014-9824-x

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