Validation of Reliable Reference Genes for RT-qPCR Studies of Target Gene Expression in Colletotrichum camelliae During Spore Germination and Mycelial Growth and Interaction With Host Plants.

Shengnan He,Runa A,Shouan Liu,Tai An

doi:10.3389/fmicb.2019.02055

Abstract

The tea plant [Camellia sinensis (L.) O. Kuntze] is one of the most important leaf crops, and it is widely used for the production of non-alcoholic beverages worldwide. Tea also has a long history of medicinal use. Colletotrichum camelliae Massee is one of the dominant fungal pathogens that infects tea leaves and causes severe tea anthracnose disease. To analyze the molecular biology of C. camelliae, the quantification of pathogen gene expression by the RT-qPCR method is necessary. Reliable RT-qPCR results require the use of stable reference genes for data normalization. However, suitable reference genes have not been reported in C. camelliae thus far. In this study, 12 candidate genes (i.e., CcSPAC6B12.04c, CcWDR83, Cchp11, Ccnew1, CcHplo, CcRNF5, CcHpcob, CcfaeB-2, CcYER010C, CcRNM1, CcUP18, and CcACT) were isolated from C. camelliae and assessed as potential reference genes. The expression stability of these genes in C. camelliae during spore germination and mycelial growth and interaction with host plants was first evaluated using several statistical algorithms, such as geNorm, NormFinder, and Bestkeeper. A web-based analysis program, Refinder, was then used to find the most suitable reference genes. Our results indicated that Cenew1, CcHplo, and CcSPAC6B12.04c were the most stable reference genes in C. camelliae under all conditions. Our work provided the most suitable reference genes for future studies performed to quantify the target gene expression levels of C. camelliae.

Highlights

Colletotrichum includes a wide range of fungal pathogens that cause serious diseases in various plants in tropical, subtropical, and temperate regions (Kubo, 2012; Yan et al, 2018)
The transcripts of these genes were slightly changed during C. camelliae spore germination and during its interaction with host plants, thereby we chose them for further analysis
Protein-protein BLAST analysis indicated that two genes had the highest homology (89.5 and 98.9%) with C. fructicola Nara gc5, while others had the highest homology with C. gloeosporioides Cg-14 (Alkan et al, 2013; Gan et al, 2013)

Summary

Introduction

Colletotrichum includes a wide range of fungal pathogens that cause serious diseases in various plants in tropical, subtropical, and temperate regions (Kubo, 2012; Yan et al, 2018). Their economic impacts have led to extensive studies on diverse aspects of fungal biology, including fungi–plant interactions, genomics and genetics, the cell biology of pathogen infection and colonization, and fungal virulence factors (De Silva et al, 2017; Villa-Rivera et al, 2017; Yan et al, 2018). The suitable reference genes in C. camelliae should be constantly expressed among the samples, and their expression is assumed to be unaffected by different experimental conditions (Bustin, 2002; Galli et al, 2015)

Methods

Results

Conclusion