DkWRKY transcription factors enhance persimmon resistance to Colletotrichum horii by promoting lignin accumulation through DkCAD1 promotor interaction

Abstract

Persimmon anthracnose, a severe disease caused by the hemibiotrophic fungus Colletotrichum horii, poses a substantial threat to China's persimmon industry. Previous research showed that 'Kangbing Jianshi' cultivar exhibits strong resistance to anthracnose. Notably, 'Kangbing Jianshi' branches exhibit greater lignification compared with the susceptible 'Fuping Jianshi' cultivar. In this study, higher lignin content was observed in 'Kangbing Jianshi' compared with 'Fuping Jianshi', and this difference was associated with disease resistance. Transcriptome and metabolome analyses revealed that the majority of differentially expressed genes and differentially accumulated metabolites were primarily enriched in the phenylpropanoid biosynthesis and lignin synthesis pathways. Furthermore, significant upregulation of DkCAD1, a pivotal gene involved in lignin metabolism, was observed in the resistant cultivar when inoculated with C. horii. Transient overexpression of DkCAD1 substantially increased lignin content and improved resistance to C. horii in a susceptible cultivar. Furthermore, through yeast one-hybrid (Y1H) assays, we identified two WRKY transcription factors, DkWRKY8 and DkWRKY10, which interacts with the DkCAD1 promoter and induces its activity. Overexpression of DkWRKY8 and DkWRKY10 not only increased leaf lignin content but also enhanced persimmon tolerance to C. horii. Moreover, the expression levels of DkCAD1, DkWRKY8, and DkWRKY10 were significantly increased in response to salicylic acid and jasmonic acid in the resistant cultivar. These findings enhance our understanding of the molecular functions of DkWRKY8, DkWRKY10, and DkCAD1 in persimmons, as well as their involvement in molecular breeding processes in persimmons.

Keywords: Colletotrichum horii; DkCAD1; DkWRKY; JA; Lignin; Persimmon; SA.

Fig. 1

Morphological characteristics of persimmon anthracnose C. horii in various persimmon tissues. A-E Morphological characteristics of persimmon anthracnose C. horii in the new shoot (A), perennial branch (B), leaf (C), petiole (D), and fruit (E). F Stereo structure of C. horii conidia on the fruit surface of the ‘Fuping Jianzhi’ variety, as observed under the anatomical microscope. G and (H) Images from above (G) and below (H) of C. horii anthracnose cultured on the PDA plate for 10 days. I Conidia. J-M Microscopic view of C. horii invading the back of ‘Fuping Jianzhi’ leaves under scanning electron microscope. Scale bar = 10 μm

Fig. 2

Characteristics and physiological changes in persimmon anthracnose in two persimmon cultivars. A Characteristic of the highly resistant cultivar ‘Kangbing Jianshi’ (R var.) and the highly susceptible cultivar ‘Fuping Jianshi’ (S var.) following inoculation with C. horii. Images were taken at 0, 1-, 2-, 3-, 5-, 10-days post-inoculation (dpi). Scale bars = 1 cm. B Lignin staining on the cross sections of R var. and S var. branches. Samples were collected from the apical region of young shoots, young shoots, and semi-lignified branches. Scale bars = 10 µm. C Lignin content in branches of R var. and S var. cultivars following inoculation with C. horii. D Endogenous salicylic acid (SA) and (E) jasmonic acid (JA) contents in R var. and S var. Error bars represent standard deviation (n = 3)

Fig. 3

Transcriptome and metabolome analysis in ‘Fuping Jianshi’ (S var.) and ‘Kangbing Jianshi’ (R var.) infected by C. horii. A, B The number of DEGs and DAMs under control and C. horii treatment. The bar chart shows the numbers of DEGs and DAMs. DEG, differentially expressed genes. DAM, differentially accumulated metabolites. 1, 3, and 5 represent the time points for collecting samples. C, D Mapman function enrichment analysis of DEGs and DAMs in S var. and R var. The histograms illustrated the –log10 of P-value of each term of enrichment. E, F Heatmaps of Metabolic profiles of DAMs, which illustrated the log₂fold change Normalized Metabolic value

Fig. 4

The lignin synthesis pathways and the expression level of related genes. A The simplified roadmap for the lignin synthesis pathway. B Gene expression pattern in lignin synthesis pathways from RNA-seq data (Normalized gene profile with Z-score value of Log2 (TPM+1)). C Metabolic profiling in lignin synthesis pathways (Normalized metabolite value was Log2fold change between metabolite content and detection-minimum). D, E The correlation networks in lignin synthesis pathways (Orange nodes represented DEGs in R var. or S var.; Blue nodes represented DAMs in R var. or S var.; Lines illustrated correlation relationship with Pearson coefficient > 0.8)

Fig. 5

Enhanced resistance to C. horii in S var. through DkCAD1 overexpression promoting lignin accumulation. A Relative expression of DkCAD1 in inoculated persimmon branches. The samples were collected at 0, 1-, 2-, 3-, 5-, and 10-days post incubation (dpi). B Expression level of DkCAD1 in persimmon leaves after transient overexpression of DkCAD1 for 2 d. C Disease resistance of pMV-empty and DkCAD1 overexpressing leaves. Scale bar = 1 cm. D Quantification of the data shown in (A), images were taken at 5 dpi. E Relative disease index in DkCAD1-overexpressing leaves after inoculation for 5 d. pMV2-GFP was used as a control. F The lignin content in DkCAD1-overexpressing leaves after inoculation for 5 d. Error bars indicate the standard deviation (n=3). The letters indicate significant differences according to one-way ANOVA (Tukey’s test; p < 0.05)

Fig. 6

Interaction between the DkCAD1 promoter and DkWRKY8 and DkWRKY10. A Self-activation of the DkCAD1 promoter was detected on SD/−Ura medium with AbA (200 ng/mL). B Y1H assay of DkCAD1 promoter with DkWRKY8 and DkWRKY10. The interaction was determined on the medium of SD/−Leu + AbA (200 ng/mL). C Relative expression of DkWRKY8 and DkWRKY10 in inoculated persimmon branches. The samples were collected at 0, 1-, 2-, 3-, 5-, and 10-days post incubation (dpi). D Ratios of LUC/REN on the promoter fragments of DkCAD1 to DkWRKY8 and DkWRKY10. The LUC/REN ratio of the empty vector (EV) plus promoter was set as 1. Error bars represent the standard deviation (n=5). The letters indicate significant differences according to one-way ANOVA (Tukey’s test; p < 0.05)

Fig. 7

Enhanced resistance to C. horii in persimmon through DkWRKY8 and DkWRKY10 overexpression. A, B The expression level of DkWRKY8 and DkWRKY10 in persimmon leaves after transient overexpression. C Disease resistance of pMV-empty, DkWRKY8, and DkWRKY10-overexpressing leaves. Bars = 1 cm. D Quantification of the data shown in (C). E, F Relative disease index in the DkWRKY8 and DkWRKY10 overexpressing leaves. ‘Fuping Jianshi’ leaves infiltrated with DkWRKY8 and DkWRKY10 and collected leaves after eight days of agroinfiltration. OE1, OE2, and OE3 represent three different lines infiltrated with pMV2-DkWRKY8 and pMV2-DkWRKY10 vectors. The pMV2-GFP was used as a control. G, H Lignin content in the DkWRKY8 and DkWRKY10 overexpressing leaves. Error bars indicate the standard deviation (n=3). I, J The expression level of DkCAD1 in transiently overexpressed lines of DkWRKY8 and DkWRKY10 in persimmon leaves. The letters indicate significant differences according to one-way ANOVA (Tukey’s test; p < 0.05)

Fig. 8

Positive roles of DkCAD1, DkWRKY8, and DkWRKY10 in C. horii resistance modulated by SA. A-C Expression levels of DkCAD1, DkWRKY8 and DkWRKY10 in two persimmon cultivars inoculated with C. horii after 2 days of spraying 0.1 mM SA. D-F Expression levels of DkCAD1, DkWRKY8 and DkWRKY10 in two persimmon cultivars inoculated with C. horii after 2 days of spraying 0.1 mM JA. Samples were collected at 0, 1-, 2-, 3-, 5-, and 10-days post-inoculation (dpi). Error bars indicate the standard deviation (n=3, p < 0.05)