Quantitative RT-PCR analysis of differentially expressed genes in Quercus suber in response to Phytophthora cinnamomi infection.

Ghazal Ebadzad,Alfredo Cravador

doi:10.1186/2193-1801-3-613

Ghazal Ebadzad, Alfredo Cravador

Open Access

https://doi.org/10.1186/2193-1801-3-613

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Journal: SpringerPlus	Publication Date: Oct 17, 2014
Citations: 72	License type: CC BY 4.0

Affiliation: University of Algarve

Abstract

cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-β-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-β-glucanase (QsGlu).Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), β-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work.Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-613) contains supplementary material, which is available to authorized users.

Highlights

Cork oak (Quercus suber L.) forests are part of Mediterranean ecosystems of Portugal, Spain, France, Italy and Morocco that, besides their high socio-economic value resulting from the exploitation of cork, play a significant ecological role
We report the results of cDNA-AFLP analysis to identify defence related transcripts in micropropagated clonal Q. suber during infection
Isolation, cloning and sequencing of cDNA fragments Those gene fragments present only in the mRNA profile of roots infected with P. cinnamomi and those that showed an increase or decrease in their expression were eluted from the gel

Summary

Introduction

Cork oak (Quercus suber L.) forests are part of Mediterranean ecosystems of Portugal, Spain, France, Italy and Morocco that, besides their high socio-economic value resulting from the exploitation of cork, play a significant ecological role. Understanding the host specific interaction between Q. suber and the pathogen involves studying the genes expressed as a cellular response to the infection, and their role in the plant disease.

Results

Conclusion