Table 1.
Characteristics of genes encoding key enzymes of the P. capsici RNAi pathway.
Fig 1.
Phylogenetic reconstruction of Dcl, Rdr, and Exportin-5 in Phytophthora species.
(A) Phylogenetic tree of Dicer-like proteins from P. capsici and other species. A. thaliana Dcl proteins 1-4, and Dicer and Drosha from H. sapiens and M. musculus were used as outgroups. (B) Phylogenetic tree of RNA-dependent RNA polymerase of P. capsici with Rdr from other oomycetes, using A. thaliana Rdr6 protein as an outgroup. (C) Phylogenetic tree of Exportin-5 proteins in Phytophthora species, with Exportin-5 from M. musculus and H. sapiens used as outgroups. The conserved domains of the proteins are highlighted in color on the right side of each panel. The scale bar indicates the size of the protein in amino acids. Colored rectangles with rounded corners indicate the protein domains.
Fig 2.
Phylogenetic reconstruction of Ago in Phytophthora species.
Phylogenetic reconstruction of Argonaute proteins from Phytophthora capsici and other oomycetes. The scale bar indicates the size of the protein in amino acids. The conserved domains of the proteins are shown in color on the right side of the phylogeny.
Fig 3.
Genomic neighborhood and conserved domains of Exportin-5.
(A) Genomic neighborhood of P. capsici EXPORTIN-5 gene shown in red stripe, located in gene-dense region and highly conserved regions between P. infestans, P. sojae, and P. ramorum genomes, conserved region cyan: UTR, blue: exons, red: introns. (B) Sequence alignment of exportin-5 proteins divided by the conserved domains IBN_N, Xpo1, and Exportin-5 in various species of Phytophthora, H. sapiens, and M. musculus, linked to a weblogo plot of abundance of amino acids at a determined position. * Amino acid conservation among all ranked organisms. - Amino acid misalignment.
Fig 4.
Synteny and protein-protein interaction networks of key genes in the RNAi pathway.
(A) Synteny of key genes in the RNAi pathway in four oomycete species. Gray lines in the background indicate collinear blocks within the P. capsici and other oomycete genomes, whereas the colored lines highlight syntenic genes. (B) Protein-protein interaction networks of Dcl1, Dcl2, Exp5, and Rdr from P. capsici. Red nodes: interaction network of proteins related to siRNAs in P. capsici; black nodes: first shell of interactions; white nodes: second shell of interactions; shell number: proteome ID from the P. capsici v11 JGI database. A thicker dark line indicates a higher score for protein-protein interaction. IDs: 504092 (GTPase Ran/TC4/GSP1 nuclear protein transport), 506453 (trafic intracellular, nuclear pore complex, Nup214/CAN component), 538731 (Ran GTPase-activating protein, RNA processing and modification), 525817 (ubiquitin-protein ligase), 545320 (karyopherin importin beta 1, intracellular trafficking, secretion and vesicular transport), 555389 (nuclear export signal-RNA export factor), 533480 (nuclear porin, structural constituent of nuclear pore), and 103847 (DNA excision repair protein XPA/XPAC/RAD14).
Fig 5.
Infection caused by P. capsici.
(A) Infection caused by P. capsici D3 in chili and broccoli leaves at 24, 48, and 72 hours post-inoculation (hpi). The bars indicate the standard error of the mean (SEM) of the infected area in mm2 from five independent experiments. Groups A, B, C, and D indicate ANOVA significant differences (p < 0.05) in the development of infection within each plant species. (B) Phenotype of P. capsici infection in chili and broccoli. Left leaf of each host: visualization in natural light; the dark regions show the infected area. Right leaf: visualization with UV light (340 nm); the red regions indicate healthy tissue, the dark regions show the infected area in necrosis, and the orange regions show infection development in the biotrophic phase. Infection zoom: before and after infection (72 hpi) at 10X and 40X.
Fig 6.
Relative expression of key genes in the RNAi pathway in P. capsici.
(A) DCL1. (B) DCL2. (C) EXP5. (D) AGO1. (E) AGO2. (F) AGO4. (G) AGO5. (H) AGO6 and (I) RDR by 2∆Ct (Log2 fold change) from the mycelium of P. capsici D3 (control), Pc-chili and Pc-broccoli, Pc-ch-ch and Pc-br-ch, corresponding to two biological replicates in triplicate. The bars represent the standard error. Groups A, B, C, D, and E indicate significant ANOVA differences (p < 0.05) between treatments for the same gene; EF-1α was used as an endogenous gene.
Fig 7.
Hypothetical model of biogenesis, transport, and processing of small interference RNAs in P. capsici.
1. Biogenesis of small interference RNAs by Dcl1 and Dcl2, 2. Transport of small interference RNAs by Exp5, 3. Processing of mature siRNAs, 4. Regulation of target genes by Agos, 5. Synthesis of dsRNAs by Rdr to amplify the gene silencing signal.