Overexpression of leucoanthocyanidin reductase or anthocyanidin reductase elevates tannins content and confers cassava resistance to two-spotted spider mite

Abstract

The two-spotted spider mite (TSSM) is a destructive cassava pest. Intensive demonstration of resistance mechanism greatly facilitates the creation of TSSM-resistant cassava germplasm. Gene to metabolite network plays a crucial role in modulating plant resistance, but little is known about the genes and related metabolites which are responsible for cassava resistance to TSSM. Here, a highly resistant (HR) and a highly susceptible (HS) cassava cultivar were used, integrative and comparative transcriptomic and metabolomic analyses between these two cultivars after TSSM infestation revealed that several genes and metabolites were closely related and significantly different in abundance. In particular, the expression of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes showed a high positive correlation with most of the metabolites in the tannin biosynthesis pathway. Furthermore, transgenic cassava lines overexpressing either of the genes elevated tannin concentrations and conferred cassava resistance to TSSM. Additionally, different forms of tannins possessed distinct bioactivity on TSSM, of which total condensed tannins (LC₅₀ = 375.68 mg/l) showed maximum lethal effects followed by procyanidin B1 (LC₅₀ = 3537.10 mg/l). This study accurately targets LAR, ANR and specific tannin compounds as critical genes and metabolites in shaping cassava resistance to TSSM, which could be considered as biomarkers for evaluation and creation of pest-resistant cassava germplasm.

Keywords: anthocyanidin reductase; cassava (Manihot esculenta Crantz); leucoanthocyanidin reductase; resistance mechanism; tannins (condensed); two-spotted spider mite (Tetranychus urticae Koch).

Figure 1

Comparative transcriptome and metabolome analysis of HR and HS cassava cultivars under TSSM infestation. (A) The overview of differently expressed genes (DEGs) between HR and HS cultivars at different infestation time points [0 d (HR0 and HS0), 1 d (HR1 and HS1), and 8 d (HR8 and HS8)]. (B) Heatmap cluster analysis of DEGs in the “HR8 vs. HS8” group. (C) The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs in the “HR8 vs. HS8” group, the pathways highlighted with red font indicate either abundant genes or high rich factors. (D) The overview of differently expressed metabolites (DEMs) between HR and HS cultivars at different infestation time points (0, 1, and 8 d). (E) Heatmap cluster analysis of DEMs in the “HR8 vs. HS8” group. (F) The KEGG enrichment analysis of DEMs in the “HR8 vs. HS8” group. The pathways highlighted with red font indicate either abundant genes or high rich factors. (G–I) The representative DEMs classes enriched with most flavonoids and tannins compounds in different samples.

Figure 2

Changes in expression of genes and abundances of metabolites related to flavonoid/phenylpropanoid biosynthesis during TSSM infestation on HR and HS cultivars at different infestation time points [0 d (HR0 and HS0), 1 d (HR1 and HS1), and 8 d (HR8 and HS8)]. (A) Summary of pathways of flavonoid/phenylpropanoid biosynthesis. Heatmaps are shown where the abundance of the metabolite changed significantly between the infestation times of HR and HS samples. Genes that were identified as being differentially expressed are indicated in red, definitions of the groups are presented in the black and white frames. (B) Heatmap of flavonoid/phenylpropanoid biosynthesis genes significantly affected during TSSM infestation. (C) Correlation between the DEGs and DEMs in the tannin biosynthesis pathway. Single asterisk and double asterisk indicated significant (p < 0.05) and extremely significant level (p < 0.01), respectively.

Figure 3

RT-qPCR and enzymatic validation of tannin biosynthesis genes. (A–F) Transcription and activity changes of tannin biosynthesis genes in HS and HR cassava plants, different lowercase letters above standard error bars indicate significant differences in gene transcription of different time points within a cultivar, and capital letters indicate significant differences in enzyme activity across cultivars, all analysis was based on one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) multiple comparison test (p < 0.05): (A) F3H, (B) DFR, (C) ANS, (D) ANR, (E) LAR. (F) The diagram of condensed tannin biosynthesis. (G) Correlation between RT-qPCR and RNA-Seq results. (H) Correlation between the transcription and activity of enzymes involved in tannin biosynthesis. (I) Correlation among the transcription of tannin biosynthesis genes.

Figure 4

Performance of transgenic cassava lines against TSSM infestation. (A) Alterations in the relative transcription and enzyme activities of LAR and ANR at different time points of TSSM infestation in HR, HS, WT, and transgenic cassava plants (ML1 and ML2 stand for transgenic lines overexpressing LAR gene, and MA1 and MA2 stand for transgenic lines overexpressing ANR gene, respectively), different capital and lowercase letters above standard error bars indicate significant differences in transcription and activity of LAR and ANR across different cassava plants based on one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) multiple comparison test (p < 0.05), respectively. (B) The TSSM infestation symptom of HR, HS, WT, and transgenic cassava plants. The “zoom in” areas of plants before mite infestation and mite infestation for 8 days were indicated by white dashed boxes and red dashed boxes, respectively. (C) The leaf infestation rate of cassava plants after TSSM feeding, different letters indicate significant differences based on one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) multiple comparison test (p < 0.05). (D) Effect on mortality of TSSM after feeding HR, HS, WT, and transgenic cassava plants. (E) Effect on the fecundity of TSSM after feeding on HR, HS, WT, and transgenic plants. (F) Effect on the hatchability of TSSM after feeding on HR, HS, WT, and transgenic plants.

Figure 5

The bioactivity of different forms of tannins on TSSM and the concentration changes in cassava plants under TSSM infestation. (A) The concentration of terminal and extension units of condensed tannins of cassava leaves was determined using HPLC following thiolysis degradation. (B,C) The probit mortality analysis of (B) total condensed tannins and (C) proanthocyanidin B1 on TSSM. (D,E) The mortality of TSSM treated with different concentrations of (D) epigallocatechin and (E) gallocatechin. (F–I) The concentration changes of different forms of tannins in different cassava plants under TSSM infestation: (F) The total condensed tannins, (G) Proanthocyanidin B1, (H) Epigallocatechin, and (I) Gallocatechin. Legends and columns with the same color indicate the concentrations of tested compounds in cassava plants are equal to their toxicity indexes (i.e., LC₅, LC₁₀, LC₂₀ or LC₃₀). In addition, different letters indicate significant differences based on one-way analysis of variance (ANOVA) with Tukey’s honestly significant difference (HSD) multiple comparison test (p < 0.05).

Figure 6

Summary of the dissection of candidate TSSM resistant genes MeLAR and MeANR and validation of their role in overproducing tannins and confers cassava resistance to TSSM.