Fig 1.
(A) mating initiated using parallel strips of A1 and A2 inoculum. The zone excised for isolating RNA or protein is denoted by the dashed box. For obtaining RNA or protein, a polycarbonate membrane was placed on top of the agar prior to inoculation, in order to facilitate tissue recovery. An orange disk of paper was placed under the plate to increase contrast in the photograph. (B) MTT assay showing red-stained viable (top) and inviable black-stained (bottom) oospores (bottom). Oospores are typically about 32 μm in diameter. (C) oospore production in pairings of strains 8811 or 88069 (A1) with strains 618 or E13 (A2). Graphed are the density of oospores and the fraction of oospores that were determined to be viable (red) or inviable (black). The image in panel A is taken from a prior publication [1].
Fig 2.
RNA-seq analysis of transcriptional changes in P. infestans during mating.
(A) number of genes induced (right) or repressed (left) during mating compared to non-mating A1 and A2 controls. The bar labeled 4, for example, denotes the number of genes with a ≥4-fold increase. Color-coding indicates the number of genes changing in one (orange), two (yellow), or all three (red) crosses. (B) Venn diagram comparing genes showing >10-fold mating induction (FDR<0.05) in the three crosses. (C) Heatmap of genes showing >10-fold induction in at least one cross. Labeled as "mating experiment" are the 10-day nonmating and mating samples, which were used for the analyses in panel A. The samples labeled "nonmating timecourse" are from 2 to 7.5-day old cultures of an A1 isolate. (D) Classification of genes showing >10-fold induction in all three crosses, which are listed along with their predicted functions in S5 Table.
Fig 3.
Evolution of two mating-induced gene families.
(A) Maximum likelihood phylogenetic tree of M96 proteins from eleven species of Phytophthora. The species are color-coded and denoted by gene prefixes, which are P. capsici (PCAP), P. cinnamomi (PCIN), P. infestans (PINF), P. kernoviae (PKER), P. mirabilis (PMIR), P. multivora (PMUL), P. parasitica (PPAR), P. phaseoli (PPHA), P. pluvalis (PPLU), P. ramorum (PRAM), and P. sojae (PSOJ). (B) Copy number estimates of M96 genes based on hits in Illumina libraries of genomic DNA (bars). Arrowheads denote the number of loci annotated as genes in each species’ respective genome projects that have similarity to M96. (C) Prolyl hydroxylase (2OG) family in nine species of Phytophthora (labeled as in panels a and b) plus Phytopythium vexans (PYVE) and Py. ultimum (PYUL). (D) Number of prolyl hydroxylase gene copies calculated as described in panel B.
Fig 4.
Analysis of self-fertile strain 6.11 of P. infestans.
(A) Production of oospores and asexual spores by 6.11 compared to normal A1 and A2 strains (8811, E13) and a 8811 × E13 mating. (B) mRNA levels of the 455 mating-induced genes. The x-axis shows the CPM ratio of the genes in 6.11 compared to the nonmating controls, and the y-axis shows their ratio in the self-fertile strain compared to the nonmating controls. Orange symbols portray genes that are consistently elevated in expression in the oospore-forming cultures, based on 4- and 2-fold induction ratio thresholds in normal matings and in 6.11, respectively. Pearson's correlation coefficient (r) between the datasets was 0.74.
Fig 5.
Processing of P. infestans mating zones for proteomics analysis.
Indicated from left to right are the initial homogenate obtained by disrupting the sample with 50 μm beads, the sample after the final passage through nylon mesh, the sample after washing and differential centrifugation, and unclarified lysate obtained by shaking with 6 mm ceramic and 0.2 mm glass beads.
Fig 6.
Analysis of proteins in oospores and vegetative hyphae.
(A) Ratio of protein levels in oospores compared to hyphae based on iTraq analysis. Orange spots correspond to those that show >2-fold changes (log2 of 1) at P<0.05. (B) Enrichment of proteins in different functional classes. Values were determined by summing the median-normalized pseudospectral counts of all proteins in each functional class, and dividing the value for oospores by the sum from vegetative hyphae. Stars represent differences significant at p<0.05 based on Bayesian estimation, with error bars reflecting the biological replicates. Proteins in each category are shown in S6 Table.
Fig 7.
Expression stability of P. infestans genes based on 20 tissues analyzed by RNA-seq.
Indicated by arrows are genes examined in this study (PITG_01604, 02745, 09862, 21219, 11766) and those used as housekeeping controls in prior studies of Phytophthora (ActA, ActB, EF1α, TubA, TubB, RS3A).
Fig 8.
RT-qPCR of primer sets for five genes against 21 tissues.
(A) Cq values. The dashed line represents the average for all samples, which are described in more detail in Materials and Methods. Each condition was represented by two biological replicates, each analyzed with three technical replicates. Error bars represent standard deviations. (B) ranking of performance of each primer set based on the Normfinder, geNorm, delta Ct, and BestKeeper algorithms, plus a comprehensive ranking based on the RefFinder program.
Fig 9.
Validation of mating-induced genes by RT-qPCR.
Grey bars illustrate results using primers against genes PITG_12027, 03562, 00260, 03995, and 10837 in strains 8811 (A1), E13 (A2) and a 8811 × E13 mating (MAT) using gene PITG_09862 as a housekeeping control. Black bars are the results from RNA-seq. The data are adjusted to show the mRNA levels in mating cultures as 1.0.
Fig 10.
Polyadenylated RNA fraction of P. infestans tissues.
Values are based on two biological replicates, using an oligo-dT binding assay.