mRNA extraction of Xanthomonas fragariae in strawberry and validation of reference genes for the RT-qPCR for study of bacterial gene expression

Abstract

This is the first study which describes a unique procedure of isolating of high-quality, intact RNA from strawberry leaves of Xanthomonas fragariae, three most suitable reference genes, crucial for the normalization of RT-qPCR data for this pathogen and accurate expression analysis of target genes. In our study, various mathematic algorithms: NormFinder geNorm, Best-Keeper, the delta CT method, RefFinder were adopted for validation of most stable reference genes from nine candidate genes (ffh, glyA, gyrA, gyrB, proC, pykA, recA, rpoB, rpoD). The analyses allowing to select three most suitable pioneer reference genes, gyrB, ffh, and pykA, that we recommend for the normalization of RT-qPCR data and for the study of the expression of target genes in Xf. Moreover, their combination as references allowed for an accurate expression analysis and computation of the fold change of the flhF and iroN2 genes in Xf. These two genes are important for the success of the colonization of plant tissue and pathogenicity and sequences of primers designed to study these genes, are presented.

Selection and validation of reference genes for gene expression studies in Xanthomonas arboricola pv. juglandis subjected to abiotic stress

“…AU10 (García‐Laviña et al, 2019), while other studies have indicated that this gene is not as reliable as internal control and is the least stably expressed in Aeromonas salmonicida subsp. salmonicida (Rivera et al, 2015) and Xanthomonas fragariae (Kałużna et al, 2019). Furthermore, rpoD gene expression has also been used as an indicator of bacterial pathogens such as P. syringae pv.…”

“…campestris (Yan et al, 2019), Pectobacterium atrosepticum (Takle et al, 2007), Erwinia amylovora (Kałużna et al, 2017), Ralstonia pseudosolanacearum (Yang et al, 2018), Clavibacter michiganensis (Jiang et al, 2019), Xanthomonas citri subsp. citri (Jacob et al, 2011), and Xanthomonas fragariae (Kałużna et al, 2019).…”

Xanthomonas rydalmerensis sp. nov., a non-pathogenic member of Group 1 Xanthomonas

“…Inoculation of the strawberry cv. elsanta plantlets with DAR 34855 T was adapted from the protocol by Kałużna et al [68] in their second trial. Plantlets used in the study were derived from the stolons of 3 month-old strawberry plants.…”

Transcriptome analysis of Xanthomonas fragariae in strawberry leaves

“…For RNA isolation, young leaves of strawberry cv. Elsanta grown in a quarantine greenhouse (25-30 °C; relative humidity level of 70-90%, natural light conditions) were inoculated with a water bacterial suspension of 10 6 cfu mL −1 grown for 20 h in liquid Wilbrink's medium 46 by high-pressure spraying of the abaxial and adaxial surfaces of leaves as previously described 47 .…”

“…Fifteen days after inoculation, samples were processed, and total RNA was isolated using a Total RNA Purification Kit (Norgen Biotek) after the Genomic Mini AX SOIL Spin kit (A&A Biotechnology, Gdynia, Poland) as described by Kałużna et al 47 . At this time point, RNA was isolated separately from three samples of 1.5 to 2.5 g of water-soaked parts of strawberry leaves.…”