Transcriptome Analysis of the Cf-13-Mediated Hypersensitive Response of Tomato to Cladosporium fulvum Infection

Abstract

Tomato leaf mold disease caused by Cladosporium fulvum (C. fulvum) is one of the most common diseases affecting greenhouse tomato production. Cf proteins can recognize corresponding AVR proteins produced by C. fulvum, and Cf genes are associated with leaf mold resistance. Given that there are many physiological races of C. fulvum and that these races rapidly mutate, resistance to common Cf genes (such as Cf-2, Cf-4, Cf-5, and Cf-9) has decreased. In the field, Ont7813 plants (carrying the Cf-13 gene) show effective resistance to C. fulvum; thus, these plants could be used as new, disease-resistant materials. To explore the mechanism of the Cf-13-mediated resistance response, transcriptome sequencing was performed on three replicates each of Ont7813 (Cf-13) and Moneymaker (MM; carrying the Cf-0 gene) at 0, 9, and 15 days after inoculation (dai) for a total of 18 samples. In total, 943 genes were differentially expressed, specifically in the Ont7813 response process as compared to the Moneymaker response process. Gene ontology (GO) classification of these 943 differentially expressed genes (DEGs) showed that GO terms, including "hydrogen peroxide metabolic process (GO_Process)", "secondary active transmembrane transporter activity (GO_Function)", and "mismatch repair complex (GO_Component)", which were the same as 11 other GO terms, were significantly enriched. An analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that many key regulatory genes of the Cf-13-mediated resistance response processes were involved in the "plant hormone signal transduction" pathway, the "plant-pathogen interaction" pathway, and the "MAPK signaling pathway-plant" pathway. Moreover, during C. fulvum infection, jasmonic acid (JA) and salicylic acid (SA) contents significantly increased in Ont7813 at the early stage. These results lay a vital foundation for further understanding the molecular mechanism of the Cf-13 gene in response to C. fulvum infection.

Keywords: Cf-13 gene; Cladosporium fulvum; RNA-seq; Solanum lycopersicum; resistance response.

Figure 1

C. fulvum infection symptoms on the leaves of MM and Cf-13 plants. MM: Moneymaker; Cf-13: Ont7813; dai: days after inoculation.

Figure 2

Optical microscopy, scanning electron microscopy, and transmission electron microscopy images of tomato leaf samples. (a–c): Ont7813 leaf stained with lactophenol trypan blue at 9, 15, and 21 dai, as seen under an optical microscope. (d–f): Ont7813 leaf at 9, 15, and 21 dai under an electron microscope. (g,h): MM leaves stained with lactophenol trypan blue at 9 and 15 dai, as seen under an optical microscope. (i–k): MM leaf at 9, 15, and 21 dai under an electron microscope. Hy: hypha; Nl: necrotic lesion; S: spore; St: stoma; Et: epidermal trichome; Ch: chloroplast; Gr: granum.

Figure 3

The DEGs among different comparison groups. 0–9 dai: comparison between 0 and 9 dai; 9–15 dai: comparison between 9 and 15 dai; 0–15 dai: comparison between 0 and 15 dai.; dai: days after inoculation.

Figure 4

Venn diagrams of the different groups of DEGs. (a) Venn diagram of DEGs among the MM 0 vs. 9 dai, MM 9 vs. 15 dai, Cf-13 0 vs. 9 dai, and Cf-13 9 vs. 15 dai comparisons. (b) Venn diagram of DEGs among the MM 0 vs. 9 dai, MM 0 vs. 15 dai, Cf-13 0 vs. 9 dai, and Cf-13 0 vs. 15 dai comparisons. (c) Venn diagram of the 475 DEGs and 533 DEGs comparison (MM 0 vs. 9 dai, MM 9 vs. 15 dai, MM 0 vs. 15 dai, Cf-13 0 vs. 9 dai, Cf-13 9 vs. 15 dai, and Cf-13 0 vs. 15 dai). Cf-13 and MM tomatoes were inoculated with C. fulvum and sampled at 0, 9, and 15 dai. 475 DEGs: the common genes differentially expressed between 0–9 dai and 9–15 dai only in Cf-13; 533 DEGs: the common genes differentially expressed between 0–9 dai and 0–15 dai only in Cf-13.

Figure 5

Correlation coefficients between the RNA-seq and qRT–PCR results. The expression patterns for both Cf-13 (0, 9, and 15 dai) and MM (0, 9, and 15 dai) were analyzed for each gene. The results obtained from both methods (RNA-seq and qRT–PCR analysis) were used to calculate correlation coefficients (R² values). MM: Moneymaker; Cf-13: Ont7813.

Figure 6

GO enrichment of 943 genes differentially expressed specifically in the Ont7813 response process.

Figure 7

KEGG pathway enrichment of 943 genes differentially expressed specifically during the Ont7813 response process. (a) Statistics of the DEGs in different pathways. (b) Enrichment of the top 20 KEGG pathways.

Figure 8

Network diagram of key KEGG pathways. The KEGG pathways included the “plant–pathogen interaction”, “MAPK signaling pathway–plant”, and “plant hormone signal transduction” pathways, and the gray pathways are the relevant ones.

Figure 9

Expression patterns of genes a certain number of days after inoculation.

Figure 10

Fluctuations in JA and SA as a function of days after inoculation in Cf-13 plants and MM plants. JA: jasmonic acid; SA: salicylic acid; Cf-13: Ont7813; MM: Moneymaker; dai: days after inoculation.