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Suppressive Subtractive Hybridization Approach Revealed Differential Expression of Hypersensitive Response and Reactive Oxygen Species Production Genes in Tea (Camellia sinensis (L.) O. Kuntze) Leaves during Pestalotiopsis thea Infection

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Abstract

Tea (Camellia sinensis (L.) O. Kuntze) is an economically important plant cultivated for its leaves. Infection of Pestalotiopsis theae in leaves causes gray blight disease and enormous loss to the tea industry. We used suppressive subtractive hybridization (SSH) technique to unravel the differential gene expression pattern during gray blight disease development in tea. Complementary DNA from P. theae-infected and uninfected leaves of disease tolerant cultivar UPASI-10 was used as tester and driver populations respectively. Subtraction efficiency was confirmed by comparing abundance of β-actin gene. A total of 377 and 720 clones with insert size >250 bp from forward and reverse library respectively were sequenced and analyzed. Basic Local Alignment Search Tool analysis revealed 17 sequences in forward SSH library have high degree of similarity with disease and hypersensitive response related genes and 20 sequences with hypothetical proteins while in reverse SSH library, 23 sequences have high degree of similarity with disease and stress response-related genes and 15 sequences with hypothetical proteins. Functional analysis indicated unknown (61 and 59 %) or hypothetical functions (23 and 18 %) for most of the differentially regulated genes in forward and reverse SSH library, respectively, while others have important role in different cellular activities. Majority of the upregulated genes are related to hypersensitive response and reactive oxygen species production. Based on these expressed sequence tag data, putative role of differentially expressed genes were discussed in relation to disease. We also demonstrated the efficiency of SSH as a tool in enriching gray blight disease related up- and downregulated genes in tea. The present study revealed that many genes related to disease resistance were suppressed during P. theae infection and enhancing these genes by the application of inducers may impart better disease tolerance to the plants.

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Acknowledgement

The authors are thankful to Dr. P. Mohankumar, Director, UPASI TRI for his constant support and encouragement. The authors are also thankful to Tea Board, Govt. of India for their financial support under XI-five-year plan.

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Authors and Affiliations

  1. Plant Physiology and Biotechnology Division, UPASI Tea Research Institute, Valparai, Tamil Nadu, India, 642127

    Palanisamy Senthilkumar & Krishnaraj Thirugnanasambantham

  2. School of Biosciences and Technology, VIT University, Vellore, Tamil Nadu, India, 632014

    Abul Kalam Azad Mandal

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  1. Palanisamy Senthilkumar
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  2. Krishnaraj Thirugnanasambantham
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Correspondence to Abul Kalam Azad Mandal.

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Table S1

Putative identities of cDNA clones from forward SSH library (DOC 176 kb)

Table S2

Putative identities of cDNA clones from reverse SSH library (DOC 164 kb)

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Senthilkumar, P., Thirugnanasambantham, K. & Mandal, A.K.A. Suppressive Subtractive Hybridization Approach Revealed Differential Expression of Hypersensitive Response and Reactive Oxygen Species Production Genes in Tea (Camellia sinensis (L.) O. Kuntze) Leaves during Pestalotiopsis thea Infection. Appl Biochem Biotechnol 168, 1917–1927 (2012). https://doi.org/10.1007/s12010-012-9907-1

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