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Identification and characterization of genes associated with thermo-tolerance using virus induced gene silencing in Nicotiana benthamiana

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Abstract

Crop acclimatization to elevated temperature conditions often demands transcriptional reprogramming of an array of genes to evoke protection from heat stress. Previously, we addressed such question in potato and identified GLP1 (Germin-like protein 1), nsLTP (Non specific lipid transfer protein), PI-PLC (phosphoinositide-specific phospholipase-c), CHP (Conserved hypothetical protein) and RPL4 (60 S Ribosomal L4/L1 protein) as candidate thermo-tolerant genes. Herein, Nicotiana benthamiana knockdown lines were developed to study their functional relevance in high-temperature stress using virus induced gene silencing (VIGS). Among all the VIGS lines tested, silencing of GLP1 and CHP showed significant reduction (more than 75 %) in their endogenous transcript levels, whereas the expression of nsLTP, PI-PLC and RPL4 was reduced by 50 %. Thus suggesting the silencing of endogenous target gene was successful using VIGS. Among those genes tested, VIGS knockdown lines generated against GLP1 and nsLTP produced a very strong hypersensitive phenotype under gradual heat stress and sudden heat shock conditions. Knockdown of GLP1 and nsLTP also showed higher chlorophyll degradation and less cell viability upon stress compared with mock plants. Interestingly, down-regulation of PI-PLC plants showed thermo-sensitive to sudden heat shock, whereas CHP and RPL4 silenced plants showed relatively less sensitive phenotypes. Most importantly, silencing of GLP1 and nsLTP genes compromised acclimation induced H2O2 accumulation, which subsequently failed to activate reactive oxygen species (ROS) scavenging signaling pathways that controlling transcription of antioxidant and other heat stress related genes. These data suggest that GLP1 or nsLTP dependent H2O2 production is required for acquiring thermo-tolerance mediated via activation of ROS-related genes. These analyses will aid in an effort to engineer broad spectrum thermo-tolerance in economically important crop.

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Acknowledgments

This work was supported by the next-Generation Bio Green 21 Program (No. PJ008182), Rural Development Administration, Republic of Korea.

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Author notes

  1. Baniekal Hiremath Gangadhar

    Present address: Department of Biotechnology and Crop Improvement, University of Horticultural Sciences, Bagalkot, India

Authors and Affiliations

  1. Department of Molecular Biotechnology, Konkuk University, Seoul, 143701, South Korea

    Baniekal Hiremath Gangadhar, Jelli Venkatesh, Venkidasamy Baskar, So Hyun Moon & Jae Woong Yu

  2. School of Applied Biosciences, Kyungpook National University, Daegu, 702701, South Korea

    Kappachery Sajeesh

  3. Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada

    Kumar Abhinandan

  4. Department of Biotechnology, Andhra University, Visakhapatnam, Andhra Pradesh, 530003, India

    Tura Safwo Jarso

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  1. Baniekal Hiremath Gangadhar
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Correspondence to Baniekal Hiremath Gangadhar or Jae Woong Yu.

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Fig. 6S

(a): APX (b): CAT (c): SOD and (d): GR enzyme activities (nmol/ min/ mg of protein) in GLP1 and nsLTP silenced lines and mock plants under non-stress, sudden heat shock and gradual heat stress. Means of three independent samples and standard errors are presented. The same letter above the column indicates no significant difference at P<0.05. (TIFF 532 kb)

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Gangadhar, B.H., Sajeesh, K., Venkatesh, J. et al. Identification and characterization of genes associated with thermo-tolerance using virus induced gene silencing in Nicotiana benthamiana . Plant Growth Regul 80, 355–366 (2016). https://doi.org/10.1007/s10725-016-0175-x

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