Figure 1.
The Phytophthora capsici cysts germinating on different surfaces.
The cysts were germinated on a cellophane membrane that was placed on the top of a Nicotiana benthamiana leaf (A), or directly on N. benthamiana leaves (B-C), or directly in water (D). Photos were taken at 70 (A), 60 (B-C) and 90 (D) min post-inoculation. To make samples for observation in (B), free water around the inoculation sites was absorbed away by filter paper and germinating cysts with some leaf tissue were peeled off using the sticky tape method [79]. The cysts germinating directly on N. benthamiana leaves were also observed using Cryoscanning electron microscopy (Hitachi S-4800 SEM) according to the instruction manual (C). The germinating cysts (A, B and D) were observed under an Olympus microscope. Scale bar = 10 µm.
Table 1.
Description of three Phytophthora capsici RNA-Seq libraries.
Table 2.
Summary of Illumina reads mapping to Phytophthora capsici reference genome by BLAT analysis.
Figure 2.
The distribution of gene expression levels.
Total reads from each of three libraries that match to the 19,805 P. capsici gene models were plotted as integrated log2 values. The distribution is based on the number of genes falling in each log2 gene expression category.
Figure 3.
Changes in gene expression profile among the different developmental stages.
The number of up-regulated and down-regulated genes between library pairs is summarized.
Figure 4.
Differentially expressed genes in different stages.
The ‘x’ axis represents fold-change of differentially expressed unique reads in different library pairs. The ‘y’ axis represents the number of unique reads (log10). Differentially accumulating unique reads within 5-fold difference between pairs are shown in the red region. The blue and green regions represent unique reads that are up- and down-regulated more than 5 fold in the stages, respectively. Library pairs: (A), GC vs. MY; (B) ZO vs. MY; (C) GC vs. ZO.
Table 3.
The effector genes differentially expressed during the pre-infection stages of Phytophthora capsici.
Table 4.
List of first ten pathways for up- and down-regulated DEGs between library pairs.
Figure 5.
Expression patterns of 19 Phytophthora capsici genes during pre-infection and infection stages.
Lanes: MY, plate-grown mycelia; SP, sporangia; ZO, zoospores; GC, germinating cysts; Mock, mock-inoculated plants; 1.5 – 72 hpi, Nicotiana benthamiana roots at 1.5, 3, 6, 12, 24, 36, 72 h post-inoculation (hpi); W, sterile distilled water. Gene names under different categories are listed on the left and sizes are listed on the right. All sizes were as expected, and the experiment was performed with at least three biological replicates.
Figure 6.
Alignment of four protein sequences from Phytophthora capsici against different Phytophthora species and Pythium ultimum.
Full-length P. capsici homologous sequences were translated using BioEdit. (A) Pc22053. Red and blue boxes denote putative RXLR and dEER motifs, respectively. (B) Pc506611. Red and blue boxes denote putative LXLFLAK and HVLVVVP motifs, respectively. (C) Pc508761; (D) Pc118548. Red arrows indicate conserved cysteines and key functional residues previously described [24]. The putative GHRHDWE motif was denoted by red box. All motifs mentioned above were also marked by black asterisks for visualization.
Table 5.
Summary and comparison of putative RXLR, CRN, elicitin and NLP protein sequences from Phytophthora capsici with Phytophthora infestans.
Figure 7.
Transient assays of Phytophthora capsici RXLR, CRN, NLP and elicitin effectors in Nicotiana benthamiana.
(A) Pc118548 (NLP) and Pc508761 (elicitin) trigger cell death in N. benthamiana. The plant leaves were infiltrated with A. tumefaciens (strain GV3101) cells to express Pc22053 (RXLR), Pc506611 (CRN), Ps118548, Pc508761, P. infestans INF1 (positive control) or GFP (negative control). Photographs were taken 5 d after infiltration. (B) – (I), Pc22053 and Pc506611 suppress cell death caused by different cell death inducers in N. benthamiana. Agro-infiltration sites in each N. benthamiana leaf expressing Pc22053 (top left), Pc506611 (top right), or GFP (middle right) were challenged with A. tumefaciens expressing Bax (B), INF1 (C), Pc508761 (D), PsojNIP (E), Pc118548 (F), PsAvh241 (G), PsCRN63 (H) and Avr3a/R3a (I). The other three sites in each leaf expressing GFP only (middle left), inducer itself (bottom right) or infiltrated by 10 mM MgCl2 buffer only (bottom left) served as controls. Photographs were taken 5 d after cell death inducer infiltration.