@article{fdi:010053872,
  title = {{V}alidation of {RT}-q{PCR} reference genes for in planta expression studies in {H}emileia vastatrix, the causal agent of coffee leaf rust},
  author = {{V}ieira, {A}. and {T}alhinhas, {P}. and {L}oureiro, {A}. and {D}uplessis, {S}. and {F}ernandez, {D}iana and {S}ilva, {M}. {D}. and {P}aulo, {O}. {S}. and {A}zinheira, {H}. {G}.},
  editor = {},
  language = {{ENG}},
  abstract = {{H}emileia vastatrix is a biotrophic fungus, causing coffee leaf rust in all coffee growing countries, leading to serious social and economic problems. {G}ene expression studies may have a key role unravelling the transcriptomics of this pathogen during interaction with the plant host. {R}everse transcription quantitative real-time polymerase chain reaction ({RT}-q{PCR}) is currently the golden standard for gene expression analysis, although an accurate normalisation is essential for adequate conclusions. {R}eference genes are often used for this purpose, but the stability of their expression levels requires validation under experimental conditions. {M}oreover, pathogenic fungi undergo important biomass variations along their infection process in planta, which raises the need for an adequate method to further normalise the proportion of fungal c{DNA} in the total plant and fungus c{DNA} pool. {I}n this work, the expression profiles of seven reference genes [glyceraldehyde-3-phosphate dehydrogenase ({GADPH}), elongation factor ({EF}-1), {B}eta tubulin (beta-tubulin), cytochrome c oxidase subunit {III} ({C}yt {III}), cytochrome b ({C}yt b), {H}v00099, and 40{S} ribosomal protein (40{S}_{R}ib)] were analysed across 28 samples, obtained in vitro (germinated uredospores and appressoria) and in planta (post-penetration fungal growth phases). {G}ene stability was assessed using the statistical algorithms incorporated in ge{N}orm and {N}orm{F}inder tools. {C}yt b, 40{S}_{R}ib, and {H}v00099 were the most stable genes for the in vitro dataset, while 40{S}_{R}ib, {GADPH}, and {C}yt {III} were the most stable in planta. {F}or the combined datasets (in vitro and in planta), 40{S}_{R}ib, {GADPH}, and {H}v00099 were selected as the most stable. {S}ubsequent expression analysis for a gene encoding an alpha subunit of a heterotrimeric {G}-protein showed that the reference genes selected for the combined dataset do not differ significantly from those selected specifically for the in vitro and in planta datasets. {O}ur study provides tools for correct validation of reference genes in obligate biotrophic plant pathogens, as well as the basis for {RT}-q{PCR} studies in {H}. vastatrix.},
  keywords = {{B}asidiomycete plant pathogen ; {C}offea arabica ; {C}offee leaf rust ; {H}ousekeeping gene ; {N}ormalisation factor},
  booktitle = {},
  journal = {{F}ungal {B}iology},
  volume = {115},
  numero = {9},
  pages = {891--901},
  ISSN = {1878-6146},
  year = {2011},
  DOI = {10.1016/j.funbio.2011.07.002},
  URL = {https://www.documentation.ird.fr/hor/fdi:010053872},
}