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NtRING1, putative RING-finger E3 ligase protein, is a positive regulator of the early stages of elicitin-induced HR in tobacco

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Abstract

Key message

NtRING1 is a RING-finger protein with a putative E3 ligase activity. NtRING1 regulates HR establishment against different pathogens. Loss-/gain-of-function of NtRING1 altered early stages of HR phenotype establishment.

Abstract

Plant defence responses against pathogens often involve the restriction of pathogens by inducing a hypersensitive response (HR). cDNA clones DD11-39, DD38-11 and DD34-26 were previously obtained from a differential screen aimed at characterising tobacco genes with an elicitin-induced HR-specific pattern of expression. Our precedent observations suggested that DD11-39, DD38-11 and DD34-26 might play roles in the HR establishment. Only for DD11-39 a full-length cDNA sequence was obtained and the corresponding protein encoded for a type-HC RING-finger/putative E3 ligase protein which we termed NtRING1. The expression of NtRING1 was upregulated upon HR induction by elicitin, Ralstonia solanacearum, or tobacco mosaic virus (TMV) in tobacco. Silencing of NtRING1 remarkably delayed the establishment of elicitin-induced HR in tobacco as well as the expression of different early induction genes in tissues undergoing HR. Accordingly, transient overexpression of NtRING1 accelerated the HR launching upon elicitin treatment. Taking together, our data suggests that NtRING1 plays a functional role in the early establishment of HR.

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Acknowledgments

Authors wish to thank Michael Metzlaff (Bayer Crop Science) for providing SVISS system. A. Ghannam was thankfully supported by a PhD fellowship from Atomic Energy Commission of Syria, Department of Molecular Biology and Biotechnology. A. Jacques was supported by a PhD fellowship from the French ministry of research.

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  1. Ahmed Ghannam

    Present address: Laboratory Functional Genomics for Plant Immunomodulation, Plant Pathology Division, Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria, P.O. Box 6091, Damascus, Syria

  2. Alban Jacques

    Present address: Ecole d’ingénieurs de Purpan, Laboratoire d’Agro-Physiologie, 75 voie du TOEC, 31076, Toulouse Cedex 3, France

Authors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes du CNRS, Université Louis Pasteur, 12 rue du Général Zimmer, 67084, Strasbourg, France

    Ahmed Ghannam, Alban Jacques, Patrice de Ruffray & Serge Kauffmann

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299_2015_1893_MOESM1_ESM.jpg

Supplementary material 1 (JPEG 682 kb) Figure S1. NtRING1 sequence Analyses. (A) cDNA sequence and deduced primary structure of NtRING1. Amino acids involved in the different structural domains are shown as follows: in the black box those of RING finger domain; in the grey box those of membrane anchor domain. (B) Hydrophobicity profile of NtRING1. The bar indicates a putative membrane anchoring domain. (C) Amino acid sequence alignment of NtRING1 and AtRMA1 (Arabidopsis thaliana RING finger protein with membrane anchor 1, AAO50721). The RING-finger domain is highlighted in black and the anchoring domain is in grey. Conserved residues are shown on the bottom line: identical amino acids are indicated by asterisks and similar amino acids by dots.

299_2015_1893_MOESM2_ESM.jpg

Supplementary material 2 (JPEG 20 kb). Figure S2. Three dimensional structure of NtRING1 RING-finger domain. (A) Amino acid alignment of NtRING1RING finger domain and the template of 4ktp.1.B of the protein TRIM5alpha E3 ligase. The RING finger domain residues are highlighted in orange. (B) Homology model showing the overall fold of RING-finger domain of NtRING1 and TRIM5alpha E3 ligase. The two Zinc ions are shown as rose small circles. Structure representation was done using SWISS MODEL online software (Bordoli et al. 2009).

299_2015_1893_MOESM3_ESM.jpg

Supplementary material 3 (JPEG 43 kb) Figure S3. Monitoring of electrolyte leakage during HR induced by infiltration of β-megaspermin in NtRING1 Silenced tobacco leaf tissues. NtRING1 (triangles) and controls (squares) were silenced in tobacco leaf tissue using SVISS technology (S-NtRING1 = Silenced NtRING1). Fifteen days after silencing, treated tissues were infiltrated with 10 nM β-megaspermin (closed symbols) or water (open symbols) as control. Electrolyte leakage was then measured from leaf discs punched out at different times after treatments (each hour from 0 to 12 h). Electrolyte leakage is expressed as the conductivity of tissues. Student t test was applied and asterisks indicate a statistical difference at P < 0.05 on each sample mean. The error bars correspond to the 95 % confidence interval calculated from the Student t test.

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Ghannam, A., Jacques, A., de Ruffray, P. et al. NtRING1, putative RING-finger E3 ligase protein, is a positive regulator of the early stages of elicitin-induced HR in tobacco. Plant Cell Rep 35, 415–428 (2016). https://doi.org/10.1007/s00299-015-1893-7

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