Testing reference genes for transcript profiling in Uromyces appendiculatus during urediospore infection of common...

Area of Science:

• Plant Pathology
• Molecular Biology
• Mycology

Background:

• Uromyces appendiculatus is a significant pathogen affecting common bean production.
• Rust fungi, including U. appendiculatus, utilize effector proteins to manipulate host plants during infection.
• Accurate quantification of effector gene expression requires stably expressed reference genes for normalization in RT-qPCR studies.

Purpose of the Study:

• To identify and validate the most suitable reference genes for RT-qPCR normalization in Uromyces appendiculatus during the urediospore-based infection process.
• To evaluate the expression stability of 14 candidate reference genes across seven developmental stages of infection.

Main Methods:

• Quantitative real-time PCR (RT-qPCR) was used to measure the expression levels of 14 candidate reference genes.
• Gene expression data were analyzed using geNorm and NormFinder algorithms to assess stability.
• Candidate genes were evaluated based on their coefficient of variation (Cq) values and algorithmic rankings.

Main Results:

• UbcE2, Elf3, and Tub consistently ranked among the top reference genes according to both geNorm and NormFinder analyses.
• RPS14 exhibited the narrowest Cq range, indicating stable expression.
• While UbcE2, Elf3, and Tub are recommended, most tested genes, except RPS9, SDH, Ubc, and PDK, showed acceptable stability.

Conclusions:

• A combination of UbcE2 and Tub, or UbcE2, Tub, and Elf3, is recommended for reliable normalization in U. appendiculatus gene expression studies.
• The identified reference genes will facilitate accurate analysis of effector gene expression and host-pathogen interactions.
• This study provides essential tools for advancing research on Uromyces appendiculatus pathogenesis and common bean resistance.