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

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.

Characterization of the cork formation and production transcriptome in Quercus cerris × suber hybrids

“…Note that due to the complexity of representing the expression values of those genes, the RGB color scale goes from the most down-regulated in red to the most up-regulated in green (color figure online) pathways. The thaumatin-like protein is related with the defense by displaying antimicrobial (Ebadzad and Cravador 2014) and antifugical activity, inhibiting the growth of pathogens (Wang and Ng 2002). The enzyme chitinase was also identified in the synthesis of suberin, a relevant metabolic pathway involved in the formation of cork cells (Pereira-Leal et al 2014), which is in accordance with the cork-producing phenotype displayed by this group of trees.…”

Differential physiological and biochemical responses of Quercus infectoria and Q. libani to drought and charcoal disease

“…Furthermore, both pathogen attack and water shortage often result in the establishment of oxidative stress, because of an overproduction of reactive oxygen species (ROS) and/or an inhibition of the plant antioxidant system (Soares et al ). Thus, in an attempt to maintain the cellular homeostasis and avoid cellular oxidative damage (Zarafshar et al ), trees also employ several morphological, physiological and biochemical strategies, which include the osmotic adjustment and the activation of enzymatic and non‐enzymatic antioxidant systems (Soares et al 2019), but also the expression and the synthesis of pathogen‐related genes and proteins, respectively (Ebadzad and Cravador ).…”

Reference gene selection for normalization of RT-qPCR gene expression data from Actinidia deliciosa leaves infected with Pseudomonas syringae pv. actinidiae

“…Four of these genes were used in previous studies as RGs for kiwifruit: actin ( ACT ) 31 32 , eukaryotic elongation factor 1α ( eEF-1a ) 33 , protein phosphatase 2A ( PP2A ) 33 and ubiquitin conjugating enzyme 9 ( UBC9 ) 34 . Other genes were described as RGs, either to pathosystems or biotic stress: glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) 35 , SAND family protein ( SAND ) 13 16 36 , β-tubulin ( TUB ) 14 36 , 7s-globulin ( GLO7A ) 37 and cyclophilin ( CYP ) 38 39 .…”