Regulation of potato late blight resistance genes by different promoters and translation regulators

Abstract Late blight, caused by Phytophthora infestans , is one of the most devastative global plant threats. Pyramiding multiple resistance genes ( R genes) was initially considered as a promising strategy for breeding broad-spectrum late blight-resistance potato varieties. However, stacking multiple R genes may induce detrimental effects on plant health. Fine-tuning the expression of R genes is critical for their effective stacking in potato breeding. This study confirms that low-level expression of individual potato late blight resistance genes like RB , R8 , and Vnt1.1 , controlled by the CaMV 35S promoter, does not trigger cell death (CD). However, co-expression of these genes in Nicotiana benthamiana leaves induces CD. Plant upstream open reading frames (uORFs) attenuate the translation of the downstream main ORF. We demonstrates that N. benthamiana open reading frame (NbuORF) and Solanum tuberosum L. open reading frame (StuORF) completely suppress green fluorescent protein (GFP) translation, whereas Arabidopsis thaliana open reading frames (AtuORFs) only partially inhibit GFP translation in N. benthamiana leaves. Simultaneous expression of AtuORFs or StuORF and RB , under the control of either pathogen-inducible potato vetispiradiene synthase gene 3 (PVS3) promoter or the 35S promoter, leads to inhibition of CD. Transgenic potatoes with 35S::RB exhibit superior late blight resistance compared to PVS3::RB transgenic lines. Plants expressing 35S::StuORF-RB show increased resistance, although slightly lower than that of 35S::RB transgenic plants. However, 35S::AtuORFs-RB and PVS3::AtuORFs-RB transgenic potatoes are susceptible to P. infestans , suggesting that AtuORFs and StuORF suppress RB translation in these plants. These findings highlight the potential of different promoter and uORF combinations for finely tuning R gene expression, which may optimize the balance between plant resistance and normal growth.