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Nicotiana benthamiana NbbZIP28, a possible regulator of unfolded protein response, plays a negative role in viral infection

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Abstract

The unfolded protein response (UPR), a highly conserved cellular defense pathway, is activated by endoplasmic reticulum (ER) stress. In Arabidopsis, the transcription factor AtbZIP28 mediates ER stress responses induced by tunicamycin. Here, we identified NbbZIP28 in Nicotiana benthamiana. Sequence analyses showed that the NbbZIP28 encodes a protein of 812 amino acids with a putative transmembrane domain (TMD) near its C terminus next to a basic leucine zipper domain. In localization analyses, NbbZIP28 was located in the ER, and NbbZIP28∆C, a truncated form of NbbZIP28 that lacked the region from the TMD to the carboxy terminus, localized in the nucleus. The transcript levels of NbbZIP28 were significantly up-regulated at 48 h post inoculation with Cucumber mosaic virus and Tobacco mosaic virus. NbbZIP28-silenced N. benthamiana plants showed no significant phenotypic change, but viruses were able to accumulate to higher levels in the silenced plants than in control plants. The NbbZIP28-silenced plants showed lower transcript levels of UPR genes including BiP and NF-YC2 at the early stage of virus infection, although the transcript levels recovered or even increased at a later stage, indicating that NbbZIP28 may be a key regulator, among others, of UPR,. In summary, the ER-localized transcription factor NbbZIP28 may play a role in the defense response against viruses by promoting the UPR pathway activation in N. benthamiana.

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Acknowledgements

This study was funded by the Agricultural Science and Technology Innovation Program (ASTIP-TRIC04, HYHY2016YL02, 1610232016004).

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Author notes

  1. Lili Shen and Fangfang Li contributed equally to this work.

Authors and Affiliations

  1. Shenyang Agricultural University, Shenyang, 110161, China

    Lili Shen & Yuanhua Wu

  2. Tobacco Research Institute, Chinese Academy of Agricultural Science, Qingdao, 266101, China

    Lili Shen, Fangfang Li, Wenfeng Dong, Wei Liu, Yumei Qian, Jinguang Yang & Fenglong Wang

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  1. Lili Shen
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  2. Fangfang Li
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Correspondence to Fenglong Wang or Yuanhua Wu.

Electronic supplementary material

ESM 1

Virus-induced gene silencing (VIGS) of NbbZIP28 in Nicotiana benthamiana. (a) Appearance of control and NbbZIP28-silenced N. benthamiana plants. (b) qRT-PCR analysis of NbbZIP28 transcript levels in NbbZIP28-silenced plants at 15 days post agro-infiltration. Actin was used as the internal reference gene. (c) qRT-PCR analysis of TRV transcript levels in NbbZIP28-silenced plants and TRV:00 plants. The values represent the endogenous control (PP2A or L23) and calibrator (TRV:00) subtracted from the target sample value to provide the 2 − ∆∆CT with three independent replications. Significant differences (P < 0.05) are denoted by different lowercase letters. (JPEG 469 kb)

High Resolution Image (TIFF 5497 kb)

ESM 2

qRT-PCR analysis of unfolded protein response (UPR) gene BiP and NF-YC2 transcript levels in NbbZIP28-silenced or TRV:00 plants infected with Cucumber mosaic virus (CMV), compared with 0 h plants respectively (a), and the relative expression of BiP and NF-YC2 in NbbZIP28-silenced plants compared to the TRV:00 control (b). PP2A was used as the internal reference gene. “0 h” indicates plants inoculated with phosphate buffered saline (PBS). (JPEG 419 kb)

High Resolution Image (TIFF 2240 kb)

ESM 3

qRT-PCR analysis of unfolded protein response gene transcript levels in NbbZIP28-silenced plants compared to the TRV:00 control in response to infection by Cucumber mosaic virus (a) or Tobacco mosaic virus (b). The values represent the endogenous control (PP2A) and calibrator (a constant quantity of TRV:00 at different time points) subtracted from the target sample value to provide the 2 − ∆∆CT with three independent replications. Significant differences (P < 0.05) are denoted by different lowercase letters. (JPEG 567 kb)

High Resolution Image (TIFF 3882 kb)

ESM 4

qRT-PCR analysis of BiP transcript levels in NbbZIP28-silenced plants compared to the TRV:00 control in response to infection by Tobacco mosaic virus. L23 was used as the internal reference gene. Significant differences (P < 0.05) are denoted by different lowercase letters. (JPEG 116 kb)

High Resolution Image (TIFF 780 kb)

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Shen, L., Li, F., Dong, W. et al. Nicotiana benthamiana NbbZIP28, a possible regulator of unfolded protein response, plays a negative role in viral infection. Eur J Plant Pathol 149, 831–843 (2017). https://doi.org/10.1007/s10658-017-1231-8

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