Fig 1.
The RB transgene confers resistance against P. infestans in potato foliage and tubers.
(A) Foliage phenotypes. Photo was taken three weeks after spray inoculation with P. infestans under moist greenhouse conditions (see methods for details) (B) Tuber phenotypes. Six week old (post-harvest) potato tuber was inoculated with P. infestans, photo was taken two weeks after inoculation (see reference [8] for details on inoculation procedures). In both cases (tuber or foliage) WT indicates nontransformed ‘Russet Burbank’, +RB indicates transgenic line SP2211 (‘Russet Burbank’ +RB), which carries 15 copies of the RB transgene [7].
Fig 2.
Contrasting foliage and tuber RB transcript levels.
(A) RB transcript level differences between foliage and tuber averaged across select time points (0, 6 and 24 hpi for foliage; 0, 24 and 48 hpi for tubers). (B) Effects of P. infestans infection on RB transcript levels in foliage and tubers. Cross time point dynamics of RB transcript levels are presented. Time points 1, 2, and 3 indicate 0, 24, and 48 hpi for tubers and 0, 6, and 24 hpi for foliage, respectively. Y axis shows normalized RB transcript levels. Infection with P. infestans results in an increase in RB transcript levels in foliage but not in tubers.
Fig 3.
PCA analysis of foliage and tuber transcriptomes.
(A) Potato foliage and tubers have distinct transcriptomes. Analysis of log2 transformed FPKM values of 31,239 genes from potato foliage and tubers. PC1 explains 25.8% of observed variation, clearly separating foliar and tuber transcriptomes. (B) WT and +RB foliage responds similarly to P. infestans infection. Foliage transcriptomes from water- (black circles) and P. infestans- (red triangles) inoculated samples. P. infestans-inoculated tissues were collected at 6 and 24 hpi. Sample names starting with “R” are from the nontransformed ‘Russet Burbank’ (WT) line, sample names starting with “H” are from high copy transgenic line SP2211 (+RB). PC2 roughly separates water- and P. infestans-inoculated samples. Neither PC1 nor PC2 separates WT from +RB samples.
Fig 4.
Hierarchical clustering of foliage DE genes (between treatment comparisons).
Foliage of nontransformed ‘Russet Burbank’ (WT) and transgenic SP2211 (+RB) was inoculated with P. infestans or water. Foliage samples collected 0, 6, and 24 hpi were subjected to RNA-seq, revealing a total of 475 DE genes between water- and P. infestans-inoculated comparisons within the same genotype and the same time point. Log2(FPKM_p.inf/FPKM_mock) values were used to cluster these 475 DE genes (FDR<0.01) in Cluster 3.0 [20] using uncentered correlation and the complete linkage method. Results were visualized using Treeview [20]. Red indicates genes that are up-regulated, green indicates genes that are down-regulated. (A) Global visualization of the 475 DE genes; (B) a small gene cluster differentially regulated in +RB and WT at 24 hpi; (C) a small gene cluster generally up-regulated in +RB and WT. These clusters highlight the role of cysteine protease inhibitors and other pathogenesis related proteins (e.g., PR1) in foliar defense response to P. infestans.
Fig 5.
Hierarchical clustering of shared (foliage and tuber) DE genes.
Foliage and tubers of nontransformed ‘Russet Burbank’ (WT) and transgenic SP2211 (+RB) were inoculated with P. infestans or water. Tuber samples collected at 0, 24, 48 hpi and foliage samples collected 0, 6, and 24 hpi were subjected to RNA-seq, revealing a total of 1,102 (for tubers) [8] and 475 (for foliage) DE genes between water- and P. infestans-inoculated comparisons within the same genotype and the same time point. A total of 127 DE genes are shared between both foliage (FDR<0.01) and tubers (FDR< = 0.001). Log2(FPKM_p.inf/FPKM_mock) values were used to cluster these 127 DE genes in Cluster 3.0 [20] as described in detail previously [8]. Results were visualized using Treeview [20]. Red indicates genes that are up-regulated, green indicates genes that are down-regulated. (A) Global visualization of the 127 DE genes; (B) a small cluster of photosynthesis genes down-regulated in all tissues; (C) A small gene cluster harboring carbonic anhydrase (CA); (D) a small cluster representing genes that are generally up-regulated under all conditions. (E-F) Small clusters of DE genes showing higher induction in +RB in both foliage (at 24 hpi) and tubers (at 48 hpi).
Fig 6.
Foliage of the +RB line has a higher frequency of DE genes 24 hpi with Phytophthora infestans.
Foliage samples collected 0, 6, and 24 hpi were subjected to RNA-seq, revealing a total of 475 DE genes between water- and P. infestans-inoculated comparisons within the same genotype and the same time point. (A) All 475 DE genes were analyzed using the “Venn count” function in the limma package of R [22] and results were summarized as a Venn diagram. Red: WT 6 hpi; orange: +RB 6 hpi; blue: WT 24 hpi; green: +RB 24 hpi. The results show that the +RB line is the main contributor of DE genes during water- vs. P. infestans-inoculated comparisons. (B) All 475 DE genes were also assigned to a MapMan ontology based on the Mercator mapping file (see methods), and subjected to Fisher’s exact test. As described previously [8], bins in red were significantly up-regulated; bins in blue were significantly down-regulated; transcription of bins in white did not change significantly. The results indicate that ontology bins encompassing ET metabolism and stress are enriched for DE genes in +RB but not in WT at 24 hpi.
Fig 7.
Faster and stronger activation of defense related genes or gene groups correlates with successful foliage and tubers resistance against P. infestans.
Foliage and tubers of ‘Russet Burbank’ (WT) and SP2211 (+RB) were inoculated with P. infestans and water. We compared the RNA-seq FPKM counts for WT and +RB using all 39,031 gene models included in the Potato Genome Sequencing Consortium (PGSC) v3 dataset. Genes were grouped into ontology bins using a MapMan mapping file. (A) Each column represents a tuber comparison between the two genotypes at a defined time point post inoculation, as indicated. (B) Each column represents a foliar comparison between the two genotypes at a defined time point post inoculation, as indicated. As described previously [8], bins in blue are transcribed at higher levels in WT than in +RB; bins in red are transcribed at higher levels in +RB than in WT; bins in white did not significantly differ in transcript levels between WT and +RB. Results indicate that faster and stronger activation of defense bins, most notably biotic stress response and receptor kinase bins, occurred in tubers and foliage of the tuber late blight resistant +RB line.