**Background:** Peronospora species are obligate biotrophic oomycetes that cause downy mildew diseases in economically important crops including basil (P. belbahrii), spinach (P. effusa), and tobacco (P. tabacina). These pathogens are completely dependent on living host tissues for nutrition and cannot be cultured on artificial media. Previous genomic analyses revealed that Peronospora species have reduced numbers of transporter genes compared to Phytophthora species, suggesting limitations in the host substrates they can transport. However, the expression patterns of transporter genes during host infection remained poorly characterized. This study aimed to identify all transporter genes in three Peronospora species and determine which are highly expressed during host infection.
**Methods:** Putative transporter genes were identified from the P. effusa genome using InterPro annotations and BLAST searches against P. belbahrii and P. tabacina genomes. Proteins were screened for transporter motifs using the Conserved Domain Database and hmmscan (threshold E-value 10⁻⁵). RNA sequencing data were analyzed from infected leaf tissue at two time points: P. tabacina-infected tobacco at 2 and 5 days post-inoculation (DPI), P. effusa-infected spinach at 2 and 7 DPI, and P. belbahrii-infected basil at 3 and 6 DPI. Gene expression was quantified as transcripts per million (TPM) and normalized using trimmed mean of M-values. Protein modeling was performed using I-TASSER, and phylogenetic analyses were conducted with MEGA version 10.
**Key Results:** Approximately 170–190 nonredundant transporter genes were identified per species: 192 in P. effusa, 174 in P. belbahrii, and 173 in P. tabacina. The largest groups belonged to the Major Facilitator Superfamily (MFS; 40–45 genes), Mitochondrial Carriers (MC; 33–35 genes), and Drug/Metabolite Transporter group (30–36 genes). At early time points (2–3 DPI), transporter gene expression was generally low, with the exception of the ADP/ATP translocase gene RMX67221, which showed high expression (23,647 TPM in P. effusa at 2 DPI; 163,026 TPM in P. belbahrii at 3 DPI; 98,033 TPM in P. tabacina at 2 DPI). At later time points (5–7 DPI), 16 transporter genes were among the top 45 most highly expressed in all three species. Seven of these were mitochondrial carriers, including ADP/ATP translocase (RMX67221) and mitochondrial phosphate carrier (RMX65655). Four MFS transporters were also commonly expressed, including three putative glucose transporters. The ADP/ATP translocase showed the highest expression levels, with TPM values of 51,876 (P. effusa, 7 DPI), 156,778 (P. belbahrii, 6 DPI), and 69,417 (P. tabacina, 5 DPI). Protein modeling revealed high structural conservation between Peronospora transporters and mammalian orthologs, with TM-scores of 0.69 (ADP/ATP translocase), 0.77 (phosphate carrier), and 0.83 (glucose transporter).
**Clinical Implications:** This study identifies 16 transporter genes that are consistently highly expressed across three Peronospora species during host infection, suggesting they are critical for pathogenesis. The predominance of mitochondrial carriers (especially ADP/ATP translocase and phosphate carrier) highlights the importance of energy metabolism for these obligate biotrophs. These transporters represent potential targets for novel crop protection strategies, such as RNA interference-based approaches targeting essential transporter genes. The identification of species-specific transporter expression patterns (11–18 unique genes per species among the top 45) may reflect differences in host metabolite availability or pathogen metabolism, which could inform the development of species-specific control measures. Given that downy mildew pathogens can develop resistance to current fungicides, these findings provide a foundation for developing new, targeted control strategies.