Identification and validation of reference genes for qRT-PCR studies of the obligate aphid pathogenic fungus Pandora neoaphidis during different developmental stages.

Shutao Zhang,Tingna Xie,Chun Chen,Sudan Ye,Raffaella Balestrini

doi:10.1371/journal.pone.0179930

Shutao Zhang, Tingna Xie + Show 3 more

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https://doi.org/10.1371/journal.pone.0179930

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Abstract

The selection of stable reference genes is a critical step for the accurate quantification of gene expression. To identify and validate the reference genes in Pandora neoaphidis–an obligate aphid pathogenic fungus—the expression of 13classical candidate reference genes were evaluated by quantitative real-time reverse transcriptase polymerase chain reaction(qPCR) at four developmental stages (conidia, conidia with germ tubes, short hyphae and elongated hyphae). Four statistical algorithms, including geNorm, NormFinder, BestKeeper and Delta Ct method were used to rank putative reference genes according to their expression stability and indicate the best reference gene or combination of reference genes for accurate normalization. The analysis of comprehensive ranking revealed that ACT1and 18Swas the most stably expressed genes throughout the developmental stages. To further validate the suitability of the reference genes identified in this study, the expression of cell division control protein 25 (CDC25) and Chitinase 1(CHI1) genes were used to further confirm the validated candidate reference genes. Our study presented the first systematic study of reference gene(s) selection for P. neoaphidis study and provided guidelines to obtain more accurate qPCR results for future developmental efforts.

Highlights

Pandora neoaphidis (Entomophthoromycotina, Entomophthorales) is an obligate aphid pathogenic fungus and has great potential for use in biological control applications[1,2,3]
For demonstrating the efficacy of the selected reference genes, we investigated the expression of cell division control protein 25 (CDC25), a key protein controlling entry into and progression through various phases of the cell cycle[23], and Chitinase 1 (CHI1), responsible for the carbohydrate active enzymes[24]
Identification and validation of reference genes for Pandora neoaphidis curve of each Quantitative real-time reverse transcription polymerase chain reaction (qPCR) reaction, but there were no signals detected within the controls, indicating that primer dimers or other non-specific amplification products had not appeared(S2A Fig, Appendix)

Summary

Introduction

Pandora neoaphidis (Entomophthoromycotina, Entomophthorales) is an obligate aphid pathogenic fungus and has great potential for use in biological control applications[1,2,3]. Elucidation of the infection processes and pathogenicity by modern genomic tools is desirable because it provides clues for understanding the main elements of host-pathogen evolution and the function of virulence genes in pathogenic fungi[4,5]. A good understanding of the GOIs requires good knowledge of gene expression under a broad set of conditions. The study of P. neoaphidis gene expression in different conditions may provide useful information regarding the molecular mechanisms highlighting the epizootic ability enabling for the development of genetic engineering approaches and field application. One constant challenge is the selection of suitable reference genes because it requires a species-specific solving process[13,14]. An ideal reference gene set should have constant expression across all the samples to be investigated, regardless of the developmental stage or any other biological or technical variability

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