Identification of genes differentially expressed in a resistant reaction to Mycosphaerella pinodes in pea using microarray technology

Abstract

BackgroundAscochyta blight, caused by Mycosphaerella pinodes is one of the most important pea pathogens. However, little is known about the genes and mechanisms of resistance acting against M. pinodes in pea. Resistance identified so far to this pathogen is incomplete, polygenic and scarce in pea, being most common in Pisum relatives. The identification of the genes underlying resistance would increase our knowledge about M. pinodes-pea interaction and would facilitate the introgression of resistance into pea varieties. In the present study differentially expressed genes in the resistant P. sativum ssp. syriacum accession P665 comparing to the susceptible pea cv. Messire after inoculation with M. pinodes have been identified using a M. truncatula microarray.ResultsOf the 16,470 sequences analysed, 346 were differentially regulated. Differentially regulated genes belonged to almost all functional categories and included genes involved in defense such as genes involved in cell wall reinforcement, phenylpropanoid and phytoalexins metabolism, pathogenesis- related (PR) proteins and detoxification processes. Genes associated with jasmonic acid (JA) and ethylene signal transduction pathways were induced suggesting that the response to M. pinodes in pea is regulated via JA and ET pathways. Expression levels of ten differentially regulated genes were validated in inoculated and control plants using qRT-PCR showing that the P665 accession shows constitutively an increased expression of the defense related genes as peroxidases, disease resistance response protein 39 (DRR230-b), glutathione S-transferase (GST) and 6a-hydroxymaackiain methyltransferase.ConclusionsThrough this study a global view of genes expressed during resistance to M. pinodes has been obtained, giving relevant information about the mechanisms and pathways conferring resistance to this important disease. In addition, the M. truncatula microarray represents an efficient tool to identify candidate genes controlling resistance to M. pinodes in pea.

Pea (Pisum sativum L.) in the Genomic Era

“…Notably, the effect of mutations in genes involved in primary metabolism or hormone deficiency on the seed transcriptome has been assessed [122][123][124][125]. Transcriptome variation for reaction to biotic stress was also analyzed using this microarray [126]. More recently, next generation sequencing (NGS) technology has provided the opportunity to generate transcriptome repertoires for non-model species, such as pea, without a sequenced genome [127].…”

Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (<i>Pisum sativum</i> L.) Subjected to Abiotic and Biotic Stress

“…However, numerous technical challenges associated with this method, including the requirement for large quantities of RNA and standardization of blotting, probe labeling, hybridization and blot washing conditions limits its application for gene expression analysis in terms of both sensitivity and sample size. Fondevilla et al (2011) [9] used microarrays to examine differential gene expression in pea in response to infection by Mycosphaerella pinodes. Unlike Northern Blot analysis, which allows for at best the examination of only a few transcripts at a time, microarray analysis allows for the simultaneous examination of thousands of genes in a single reaction.…”

Transcriptomic Studies in Non-Model Plants: Case of Pisum sativum L. and Medicago lupulina L.

“…Despite the fact that pea genome is not sequenced yet, several studies of pea gene expression have been carried out by microarray technology. Analysis of gene expression during Mycosphaerella pinodes infection was carried out using a microarray [45] containing 16,470 diferent 70-mer oligonucleotides from M. truncatula and only 25 did not show a detectable signal [46]. In another study, microarray transcriptome proiling based on known pea Expressed Sequence Tags (ESTs) revealed altered expression of genes associated with programmed cell death, oxidative stress and protein ubiquitylation during seed aging [47].…”