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Expression of target mRNAs during Popsipviroid pathogenesis in the "leaf factory" system
SELINGER, Martin
The aim of this work was to identify potential mRNA targets of PTGS triggered by viroid-derived small RNAs (vsRNAs) in PSTVd-infected tomato plants (S. lycopersicum L.). We selected 47 possible gene targets using data provided by Prof. Dr. Steger (Heinrich Heine Universität, Düsseldorf, Germany) - the list of 1633 possible target mRNAs from tomato based on vsRNA:mRNA duplex prediction. The vsRNA sequences were obtained by Illumina sequencing of small RNA libraries from healthy and PSTVd-infected tomato plants. By qRT-PCR analysis we identified 6 genes with significantly altered levels of mRNA in PSTVd-infected tomato plants: CUL1 (protein ubiquitination), ERF4 (transcription factor of abiotic stress signalling pathway), H/ACA1 (rRNA pseudouridylation), NPH3 (transcription factor of fototropic signalling pathway), Sl-MYB (transcription factor regulating leaf development) and TCP3 (transcription factor regulating leaf development). The binary vector pLV07 with inserted expression cassette containing coding sequence of Sl-MYB was prepared for experiments in ?leaf factory? system in N. benthamiana plants. Expression analyses in ?leaf factory? system after 1,5 DPI using qRT-PCR and RNA blots revealed strong inhibition of expression of Sl-MYB in leaf sectors infiltrated with severe PSTVd AS1 strain, while mild PSTVd QFA strain showed minimal change in expression comparing to control sectors. Moreover, the overexpression of Sl-MYB in leaf sectors resulted in development of necroses after 2,5-3 DPI, in presence of silencing suppressor p19 after 2 DPI. The development of necroses was largely inhibited in PSTVd AS1-infiltrated leaf sectors in comparison with PSTVd QFA- and control-infiltrated sectors.
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