Integrated Transcriptome and Metabolome Analysis Reveals Key Metabolites Involved in Camellia oleifera Defense against Anthracnose

Abstract

Camellia oleifera (Ca. oleifera) is a woody tree species cultivated for the production of edible oil from its seed. The growth and yield of tea-oil trees are severely affected by anthracnose (caused by Colletotrichum gloeosporioides). In this study, the transcriptomic and metabolomic analyses were performed to detect the key transcripts and metabolites associated with differences in the susceptibility between anthracnose-resistant (ChangLin150) and susceptible (ChangLin102) varieties of Ca. oleifera. In total, 5001 differentially expressed genes (DEGs) were obtained, of which 479 DEGs were common between the susceptible and resistant varieties and further analyzed. KEGG enrichment analysis showed that these DEGs were significantly enriched in tyrosine metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis and isoquinoline alkaloid biosynthesis pathways. Furthermore, 68 differentially accumulated metabolites (DAMs) were detected, including flavonoids, such as epicatechin, phenethyl caffeate and procyanidin B2. Comparison of the DEGs and DAMs revealed that epicatechin, procyanidin B2 and arachidonic acid (peroxide free) are potentially important. The expression patterns of genes involved in flavonoid biosynthesis were confirmed by qRT-PCR. These results suggested that flavonoid biosynthesis might play an important role in the fight against anthracnose. This study provides valuable molecular information about the response of Ca. oleifera to Co. gloeosporioides infection and will aid the selection of resistant varieties using marker-assisted breeding.

Keywords: Camellia oleifera; Colletotrichum gloeosporioides; anthracnose; flavonoid biosynthesis; metabolome; transcriptome.

Figure 1

(A) Growth of CL150 and CL102 in the field. (B) Phenotype of CL150 and CL102 after inoculation with Co. gloeosporioides, Scale bar = 1 cm. (C) Leaf lesion size over time of CL150 and CL102 after inoculation with Co. Gloeosporioides, n = 12; “*” and “**” indicate significant differences at p < 0.05 and p < 0.01, respectively.

Figure 2

(A) Number of up/down-regulated DEGs between the four groups (RD/SD, RD/RH, SD/SH and RH/SH); (B) GO functional enrichment analysis of DEGs; (C) Venn diagram showing down-regulated DEGs shared among RD/RH and SD/SH; (D) Venn diagram showing up-regulated DEGs shared among RD/RH and SD/SH.

Figure 3

(A) Sector graph of metabolite classification. (B) OPLS-DA scatter plot of resistant line sample infected or uninfected with Co. gloeosporioides.

Figure 4

(A) Heat map of 68 differentially accumulated metabolites (DAMs), data of each row was standardized. (B) Venn diagram showing DAMs shared among RD/RH, SD/SH and RD/SD. (C) Heat map of fold changes of 12 DAMs in four pairwise comparsions, data of each row was standardized.

Figure 5

Analysis of the correlation between the changes in levels of DEGs and DAMs. (A) Correlation heat map. Asterisks in the panels represent the significance of the correlation, with p-values of less than 0.001 (***), less than 0.01 (**), and less than 0.05 (*). (B) Associated network diagram created using Cytoscape. Lines colored in “red” and “blue” represent positive and negative correlations, yellow circles indicate metabolites and green circles indicate genes, p-value set at 0.05.

Figure 6

(A) Expression of DEGs in tyrosine metabolism pathway among the four sample groups, data of each row was standardized. (B) Relative concentration of four DAMs in tyrosine metabolism pathway among the four sample groups, different letters represent significant difference at p < 0.05 by Duncan.

Figure 7

Expression of candidate genes involved in flavonoid biosynthesis. (A) The expression profile of key genes among the four sample groups, data of each row was standardized. (B) The postulated biosynthetic pathway of flavonoids after Co. gloeosporioides infection. The four squares in each horizontal row correspond to the four sample groups. The light blue boxes indicate different enzymes. The bar graph represents the relative metabolic content.

Figure 8

The relative expression levels of six selected DEGs were compared by RNA-seq and qRT-PCR. The line chart shows the gene expression level from the transcriptome (FPKM); The qRT-PCR expression levels were calculated as a ratio relative to the level of expression of uninfected susceptible line CL102 (SH), which was set as 1.

Figure 9

A schematic diagram of the defense response of Ca. oleifera to the anthracnose pathogen.