Cloning and functional analysis of downy mildew resistance gene RPF1 in spinach (Spinacia oleracea L.)

Downy mildew, caused by the obligate pathogen Peronospora effusa, is a devastating disease in spinach (Spinacia oleracea L.). Resistance to P. effusa is largely controlled by a dominant resistance locus RPF1, for which two candidate genes have been previously proposed but lacked functional validation. In this study, we employed virus-induced gene silencing (VIGS) to demonstrate that silencing of Spo12903 significantly compromised resistance to P. effusa, thereby confirming Spo12903 as the functional gene underlying RPF1. Sequence characterization and comparative analyses revealed that RPF1 possesses conserved features of plant NLR immune receptors, including a conserved NB-ARC domain and LRR regions. Subcellular localization analysis indicated that RPF1 is predominantly located in the nucleus. Transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) revealed that genes associated with RPF1-mediated resistance were enriched in defense-related pathways, including Ras-related and MAPK signaling pathways. Furthermore, co-expression and orthology-based interaction analyses identified candidate genes potentially associated with RPF1, including receptor-like proteins and receptor-like kinases involved in plant immunity. Population genetic analysis of 154 spinach accessions revealed that the RPF1 resistance locus in cultivated spinach was likely derived from introgression from Spinacia tetrandra. Collectively, our findings establish Spo12903 as the functional gene of RPF1, provide new insights into the molecular basis of spinach downy mildew resistance, and offer valuable resources for resistance breeding.