Evaluation of suitable reference genes for normalization of quantitative real-time PCR analysis in rice plants under Xanthomonas\xa0oryzae\xa0pv. oryzae--infection and melatonin supplementation

Xian Chen,Fengquan Liu,Yan Dong,Pedro Laborda

doi:10.1186/s43014-020-00035-9

Abstract

Exogenous melatonin (MT) was found to be an interesting tool for enhancing the resistance of rice to Xanthomonasoryzaepv. oryzae (Xoo)-caused bacterial blight (BB). However, the accurate comparison of the expression levels across samples was a challenging task. In this work, the stability of 10 common used housekeeping genes under Xoo-infection and MT supplementation in rice was analyzed using quantitative real-time PCR (qRT-PCR), and algorithms geNorm, NormFinder and BestKeeper. Our results indicated that most reference genes remained stable in Xoo-infected rice plants, while a number of reference genes were affected by MT supplementation. Among all studied genes, the transcript levels of 18S(18S ribosomal RNA) and UBC (Ubiquitin-conjugating enzyme E2) remained unaltered by Xoo infection, while UBC and UBQ5(Ubiquitin 5) were the most stable genes when examining simultaneous Xoo-infection and MT supplementation, demonstrating that UBC is a suitable reference gene for qRT-PCR data normalization in rice under Xoo-infection and MT supplementation.

Highlights

Rice is one of the most important crops worldwide, providing essential food supply to most of the world's population
We have investigated for the first time the expression stability of 10 candidate reference genes, including 18S ribosomal RNA (18S), 25S ribosomal RNA (25S), Ubiquitin 5 (UBQ5), Ubiquitin 10 (UBQ10), Actin (ACT), β-Tubulin (β-TUB), Eukaryotic elongation factor 1-alpha, Eukaryotic initiation factor 4-alpha, Ubiquitin-conjugating enzyme E2 (UBC) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under Xanthomonasoryzaepv. oryzae (Xoo)-infection and MT supplementation (Jain et al 2006; Kim et al 2003; Li et al 2010)
Four parallel treatment conditions were studied: 1. rice leaves were treated with water, while the plant roots were cultivated in water in the absence of MT; 2. rice leaves were inoculated with Xoo, and the plant roots were submerged in water in the absence of MT (Xoo group); 3. rice leaves were inoculated with water, and the plant roots were submerged in an aqueous solution containing 20 μg/mL melatonin (MT-water group); and 4. rice leaves were inoculated with Xoo, and the plant roots were submerged in an aqueous solution containing 20 μg/mL melatonin (MTXoo group)

Summary

Introduction

Rice is one of the most important crops worldwide, providing essential food supply to most of the world's population. Oryzae (Xoo), the causal agent of bacterial blight (BB), is a member of γ-proteobacteria, and is able to invade plant through wounds and hydathodes to colonize the xylem (White and Yang 2009). This pathogen can rapidly spread through the vascular system, leading to systemic infection (Yuan et al 2010). Chen et al Food Production, Processing and Nutrition (2020) 2:21 et al 2015) Those DEGs were associated to defense responses, protein phosphorylation, hormone-mediated signaling pathways and metabolic processes. The validation of the expression stabilities of reference genes is necessary for the accurate acquisition of qRT-PCR data. The final outcome of this feasible study will benefit further quantifications of gene expression by qRT-PCR and RNA-seqbased transcriptomic studies in rice

Materials and methods

UBQ-10

Results and discussion

Conclusions