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An efficient transient mesophyll protoplast system for investigation of the innate immunity responses in the rubber tree (Hevea brasiliensis)

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Abstract

The transient mesophyll protoplast expression system has become a powerful tool for rapid gene functional analysis, and it has been used successfully in several plant species, including model plants Arabidopsis and rice, but not in rubber tree. In this study, we describe an improved method to isolate high quality rubber tree mesophyll protoplasts. The transient expression of exogenous DNA constructs in this mesophyll protoplast system was observed by using green fluorescent protein, and was detected by immunoblot. Furthermore, we used two typical pathogen-associated molecular patterns flg22 and chitin from bacteria and fungi, respectively, as inducers of plant innate immunity responses to detect the immunity response of rubber tree. Our data showed that flg22 and chitin induced activation of mitogen-activated protein kinases, generation and accumulation of reactive oxygen species, and transcription of defense genes. Our study demonstrated that the rubber tree mesophyll protoplast system can be used for functional analysis of immune responses of rubber tree and the dissection of cell signaling pathways. This transient mesophyll protoplast system may be applied to analyse gene function of rubber tree, a perennial plant for which the applicable transformation system was limited.

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Acknowledgments

This work was supported by the National Natural Science Foundation (Project No. 31160151), Hainan Science and Technology Project (Project No. ZDZX2013023).

Authors’ contribution

HLL and CZH designed the experiments. XDZ, HLL and CZH wrote the paper. XDZ and LJW performed the experiments. XDZ and HLL analyzed the data.

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Authors and Affiliations

  1. Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources, Hainan University, 58 Renming Road, Haikou, 570228, Hainan, China

    Xiaodong Zhang, Liangjie Wang, Chaozu He & Hongli Luo

  2. College of Agriculture, Hainan University, 58 Renming Road, Haikou, 570228, Hainan, China

    Xiaodong Zhang, Liangjie Wang, Chaozu He & Hongli Luo

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  1. Xiaodong Zhang
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  2. Liangjie Wang
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  3. Chaozu He
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Correspondence to Chaozu He or Hongli Luo.

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Figure S1

Sequence comparison between HbMAPK3 and MAPK3 proteins from other plants. The black shaded residues are identical in all the proteins compared, and gray shading indicates residues identical to the HbMAPK3 protein. Dashes denote gaps introduced to maximize alignment. The red box showed the conserved TRW motif. Sequences used in the alignments are HbMAPK3 (Hevea brasiliensis, unpublished), CsMAPK3 (Citrus sinensis, ABM67698.1), VvMAPK3 (Vitis vinifera, XP_002284807.1), GmMAPK3 (Glycine max, XP_003539740.1) and AtMAPK3 (Arabidopsis thaliana, XP_002875737.1) (TIFF 869 kb)

Figure S2

Sequence comparison between HbMAPK6 and MAPK6 proteins from other plants. The black shaded residues are identical in all the proteins compared, and gray shading indicates residues identical to the HbMAPK6 protein. Dashes denote gaps introduced to maximize alignment. The red box showed the conserved TRW motif. Sequences used in the alignments are HbMAPK6 (Hevea brasiliensis, unpublished), AtMAPK6 (Arabidopsis thaliana, NP_181907.1), PcMAPK6 (Petroselinum crispum, AAN65179.1), OsMAPK6 (Oryza sativa, ACD76439.1) and PtMAPK6 (Pinus taeda, ACT63866.1) (TIFF 948 kb)

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Zhang, X., Wang, L., He, C. et al. An efficient transient mesophyll protoplast system for investigation of the innate immunity responses in the rubber tree (Hevea brasiliensis). Plant Cell Tiss Organ Cult 126, 281–290 (2016). https://doi.org/10.1007/s11240-016-0997-2

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