Comparative Transcriptome Analysis Reveals Novel Candidate Resistance Genes Involved in Defence against Phytophthora cactorum in Strawberry

Abstract

Crown rot, caused by Phytophthora cactorum, is a devastating disease of strawberry. While most commercial octoploid strawberry cultivars (Fragaria × ananassa Duch) are generally susceptible, the diploid species Fragaria vesca is a potential source of resistance genes to P. cactorum. We previously reported several F. vesca genotypes with varying degrees of resistance to P. cactorum. To gain insights into the strawberry defence mechanisms, comparative transcriptome profiles of two resistant genotypes (NCGR1603 and Bukammen) and a susceptible genotype (NCGR1218) of F. vesca were analysed by RNA-Seq after wounding and subsequent inoculation with P. cactorum. Differential gene expression analysis identified several defence-related genes that are highly expressed in the resistant genotypes relative to the susceptible genotype in response to P. cactorum after wounding. These included putative disease resistance (R) genes encoding receptor-like proteins, receptor-like kinases, nucleotide-binding sites, leucine-rich repeat proteins, RPW8-type disease resistance proteins, and 'pathogenesis-related protein 1'. Seven of these R-genes were expressed only in the resistant genotypes and not in the susceptible genotype, and these appeared to be present only in the genomes of the resistant genotypes, as confirmed by PCR analysis. We previously reported a single major gene locus RPc-1 (Resistance to Phytophthora cactorum 1) in F. vesca that contributed resistance to P. cactorum. Here, we report that 4-5% of the genes (35-38 of ca 800 genes) in the RPc-1 locus are differentially expressed in the resistant genotypes compared to the susceptible genotype after inoculation with P. cactorum. In particular, we identified three defence-related genes encoding wall-associated receptor-like kinase 3, receptor-like protein 12, and non-specific lipid-transfer protein 1-like that were highly expressed in the resistant genotypes compared to the susceptible one. The present study reports several novel candidate disease resistance genes that warrant further investigation for their role in plant defence against P. cactorum.

Keywords: Fragaria vesca; crown rot; disease resistance (R-genes); oomycete.

Figure 1

Fragaria vesca genotypes NCGR1218, NCGR1603, and Bukammen after inoculation with Phytophthora cactorum. (a). Phenotyping of crown rot resistance in the three F. vesca genotypes. The image of the susceptible genotype NCGR1218 was taken two weeks after inoculation with P. cactorum, while the images of the resistant genotypes NCGR1603 and Bukammen were taken four weeks after inoculation. Rectangular boxes with dashed white lines represent the strawberry rhizomes used for scoring disease development. (b). The disease scores of three Fragaria vesca genotypes NCGR1218, NCGR1603, and Bukammen after inoculation with Phytophthora cactorum. The scores are based on visual symptom observations, where scores 8, 7, 6, and 5 represent plants that showed wilting and collapsing of the whole plant during the first, second, third, and fourth weeks after inoculation, respectively, whereas scores <5 represent different degrees of necrosis visually observed in the rhizome for plants that survived four weeks after inoculation: 4 = clear necrosis covering at least 50% of the rhizome area; 3 = small patches of necrosis; 2 = minor brown/dark speckles; 1 = no symptoms as previously described [19,48]. Untreated and mock-inoculated (wounded and inoculated with water) plants were used as controls. The data represent the mean disease score of sixteen biological replicates for each genotype. The asterisk indicates a significant difference in the mean disease score between the genotype NCGR1218 and the two genotypes NCGR1603 and Bukammen after inoculation with P. cactorum (p-value < 0.01).

Figure 2

Multidimensional scaling plot of differentially expressed genes in the three Fragaria vesca genotypes NCGR1218, NCGR1603, and Bukammen in response to Phytophthora cactorum. The samples were harvested from rhizome tissue inoculated with P. cactorum after wounding and harvested 48 h later (infected); mock control (wounded); and untreated control (control). Each symbol in the plot represents a biological replicate.

Figure 3

GO enrichment analysis of the differentially expressed genes (DEGs) in the resistant F. vesca genotypes (a) NCGR1603 and (b) Bukammen, relative to the inoculated susceptible genotype NCGR1218, 48 h after inoculation with Phytophthora cactorum (p-value < 0.05, Pearson chi-square test).

Figure 4

Expression levels of the 31 putative disease resistance genes in three Fragaria vesca genotypes 48 h after inoculation with Phytophthora cactorum. Genes that were uniquely expressed, or two-fold or more upregulated (p < 0.05), in the resistant genotypes (NCGR1603 and Bukammen) relative to the susceptible genotype (NCGR1218) are shown. The susceptible genotype NCGR1218 is indicated by S, and the two resistant genotypes NCGR1603 and Bukammen are indicated by R. A blue to grey colour gradient indicates transcript abundance in terms of transcripts per million (TPM), while white indicates no expression. The TPM values are the mean of the four biological replicates, each consisting of four individual plants for each genotype and treatment (in total, 16 plants per genotype and treatment).

Figure 5

Number of shared differentially expressed genes (DEGs) in response to Phytophthora cactorum in the RPc-1 locus of three Fragaria vesca genotypes. (a) The susceptible genotype NCGR1218, as well as the two resistant genotypes NCGR1603 and Bukammen, 48 h after wounding and inoculation with P. cactorum (their expression is relative to their untreated controls). The shared DEGs are listed in Supplementary Material S3. (b) Shared DEGs in the resistant genotypes NCGR1603 and Bukammen 48 h after wounding and inoculation with P. cactorum (the expression is relative to the susceptible genotype NCGR1218). DEGs were defined as fold change ≥2 or ≤−2 (p < 0.05). The DEGs shared by the resistant genotypes are listed in Supplementary Material S2.

Figure 6

The number of upregulated and downregulated transcription factor genes in different TF families in the resistant Fragaria vesca genotypes NCGR1603 and Bukammen, 48 h after inoculation with Phytophthora cactorum, relative to the inoculated susceptible genotype NCGR1218. Genes with a fold change ≥2 or ≤−2 (p < 0.05) were defined as upregulated and downregulated, respectively.

Figure 7

Fragaria vesca genes expressed in the two resistant genotypes NCGR1603 and Bukammen after wounding and inoculation with Phytophthora cactorum, while absent in the susceptible genotype NCGR1218. Agarose gel electrophoresis of PCR amplified transcripts generated by RT-PCR analysis. The RNA used for the cDNA synthesis was from a bulk of sixteen individual test plants from each genotype. The F. vesca Elongation factor 1 alpha (EF1a) gene was used as an internal control; H₂O indicates template-free PCR and RT reactions, which were used as technical negative controls. M indicates a molecular marker. The original gel images were cropped and stacked for clarity and conciseness, and the full-length gel images are presented in Supplementary Figure S4.

Figure 8

Gene presence–absence polymorphisms in strawberry genotypes that are resistant or susceptible to Phytophthora cactorum. The genes were selected based on lack of expression during infection of the susceptible genotype NCGR1218 (Supplementary Material S2). Gene fragments were amplified by PCR from genomic DNA, using up to two different primer pairs for each target gene. (a) Primer pair 1. (b) Primer pair 2 (see Supplementary Table S1). The genomic DNA used was from a bulk of ten individual test plants for each Fragaria vesca genotype or Korona, a moderately susceptible cultivar (F. x ananassa). Asterisks indicate genes detected only in the resistant F. vesca genotypes NCGR1603 and Bukammen. The F. vesca Elongation factor 1 alpha (EF1a) gene was used as an internal control; H₂O indicates template-free PCR reactions (negative control). The original gel images were cropped for clarity and conciseness, and the full-length gel images are presented in Supplementary Figure S5.