Abstract
Phakopsora pachyrhizi is a devastating pathogen on soybean, endangering soybean production worldwide. Use of Host Induced Gene Silencing (HIGS) and the study of effector proteins could provide novel strategies for pathogen control. For both approaches quantification of transcript abundance by RT-qPCR is essential. Suitable stable reference genes for normalization are indispensable to obtain accurate RT-qPCR results. According to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and using algorithms geNorm and NormFinder we tested candidate reference genes from P. pachyrhizi and Glycine max for their suitability in normalization of transcript levels throughout the infection process. For P. pachyrhizi we recommend a combination of CytB and PDK or GAPDH for in planta experiments. Gene expression during in vitro stages and over the whole infection process was found to be highly unstable. Here, RPS14 and UbcE2 are ranked best by geNorm and NormFinder. Alternatively CytB that has the smallest Cq range (Cq: quantification cycle) could be used. We recommend specification of gene expression relative to the germ tube stage rather than to the resting urediospore stage. For studies omitting the resting spore and the appressorium stages a combination of Elf3 and RPS9, or PKD and GAPDH should be used. For normalization of soybean genes during rust infection Ukn2 and cons7 are recommended.
Highlights
Phakopsora pachyrhizi, the causative agent of Asian soybean rust, is one of the most devastating pathogens of soybean
In vitro structures of P. pachyrhizi normally tested in gene expression studies are resting urediospores (s), germ tubes, and appressoria
In order to ensure that our reference genes are not affected by virus infection, we included samples of plants infected with Bean Pod Mottle Virus (BPMV)
Summary
Phakopsora pachyrhizi, the causative agent of Asian soybean rust, is one of the most devastating pathogens of soybean. To elucidate the biotrophic interaction in more detail, genes responsible for nutrient uptake and involved in primary metabolism have been investigated Results from these experiments support the idea that a major switch in the fungal metabolism occurs after the plant is penetrated [8,9]. One way to influence gene expression of plant pathogens is Host Induced Gene Silencing (HIGS). It is common to use housekeeping genes—genes involved in primary metabolic processes—for normalization, assuming that these important genes need to be expressed unchangingly during every stage of development or in every tissue. We determined P. pachyrhizi reference genes suitable for normalization in transcript profiling during the infection process including in vitro stages and in measuring transcript levels in HbV experiments. We determined Glycine max reference genes for normalizing transcript levels of soybean genes during infection with P. pachyrhizi
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