Panax notoginseng transcription factor WRKY15 modulates resistance to Fusarium solani by up-regulating osmotin-like protein expression and inducing JA/SA signaling pathways

Abstract

Background: Panax notoginseng (Burk) F. H. Chen is a valuable traditional Chinese medicinal plant, but its commercial production is seriously affected by root rot caused by some pathogenic fungi, including Fusarium solani. Nevertheless, the genetic breeding for disease resistance of P. notoginseng remains limited. The WRKY transcription factors have been revealed to play important roles in plant defense responses, which might provide an inspiration for resistance improvement in P. notoginseng.

Results: In this study, the regulatory mechanism of transcription factor PnWRKY15 on P. notoginseng resistance to F. solani infection was revealed. The suppressed expression of PnWRKY15 via RNA interference increased the sensitivity of P. notoginseng to F. solani and decreased the expression levels of some defense-related genes, including PnOLP1, which encodes an osmotin-like protein that confers resistance to F. solani. Ectopic expression of PnWRKY15 in the model plant tobacco significantly enhanced the resistance to F. solani. Moreover, the transcriptome sequencing analysis discovered that some pathogenesis-related genes were expressed at higher levels in the PnWRKY15-overexpressing tobacco than that in the wild-type tobacco. In addition, the jasmonic acid (JA) and salicylic acid (SA) signaling pathways were evidently induced by PnWRKY15-overexpression, that was evidenced by that the JA and SA contents were significantly higher in the PnWRKY15-overexpressing tobacco than that in the wild-type. Furthermore, PnWRKY15, which was localized in the nucleus, can trans-activate and up-regulate PnOLP1 expression according to the EMSA, yeast one-hybrid and co-expression assays.

Conclusions: PnWRKY15 contributes to P. notoginseng resistance to F. solani by up-regulating the expression of resistance-related gene PnOLP1 and activating JA/SA signaling pathways. These findings will help to further elucidate the transcriptional regulatory mechanism associated with the P. notoginseng defense response to F. solani.

Keywords: Fusarium solani; Jasmonic acid; Osmotin; Panax notoginseng; Salicylic acid; WRKY transcription factor.

Fig. 1

The sequence analysis and subcellular localization of PnWRKY15. (A) The multiple sequence alignment of PnWRKY15 and three homologous WRKYs. (B) The expression of PnWRKY15-GFP and GFP empty vector in onion epidermal cells. GFP: green fluorescent protein; PI: propidium iodide; Bright: white light field; Merged: superposition of fluorescent field and white light field

Fig. 2

Analysis of transient expression of phellsgate2-PnWRKY15 in P. notoginseng leaves. (A) The symptoms of P. notoginseng leaves after Fusarium solani inoculation, in which the PnWRKY15 RNAi vector and the empty RNAi vector were expressed, respectively; (B) The analysis of diseased area in P. notoginseng leaves; (C, D) The expression levels of PnWRKY15 and defense related genes in PnWRKY15-RNAi and control P. notoginseng leaves. The results were shown as average values calculated from three replicates and the significance was determined by the Student’ s t-test (*: P < 0.05; **: P < 0.01)

Fig. 3

Resistance analysis of PnWRKY15-OE tobacco lines. (A) Transcription level of PnWRKY15 in transgenic tobacco lines. WT: wild-type tobacco; 15 - 2/3/4/6/7/9/10/11/12/14/15/21/22/23/24: PnWRKY15-OE lines. (B) The root inoculation assay revealed the enhanced resistance of PnWRKY15 transgenic tobacco lines to Fusarium solani infection. (C) The symptoms in tobacco leaves after inoculation with F. solani for one week. (D) The analysis of diseased area in tobacco leaves. The results were shown as average values calculated from three replicates and the significance was determined by the Student’ s t-test (*: P < 0.05; **: P < 0.01)

Fig. 4

Enrichment analysis of KEGG pathway based on the RNA-seq data of PnWRKY15 transgenic tobacco with the wild-type tobacco as a control. (A) KEGG bubble map; (B-E) The heat maps of differentially expressed genes in the pathways including plant pathogen interaction, plant hormone signal transduction, phenylpropanol biosynthesis, and photosynthesis. WT-1/-2/-3: wild-type tobacco; 15-2-1/-2/-3: PnWRKY15 transgenic line 15 -2

Fig. 5

Transcription levels of defense related genes and determination of JA/SA content in PnWRKY15 transgenic tobacco. WT: wild-type tobacco; 15 - 2/-21/-22/-24: PnWRKY15 transgenic tobacco lines. The results were shown as average values calculated from three replicates and the significance was determined by the Student’s t-test. *: P < 0.05; **: P < 0.01

Fig. 6

PnWRKY15 specifically bound to W-box from PnOLP1 promoter and has transcriptional activation on PnOLP1. (A) The recombinant PnWRKY15 specifically bound to W-box from PnOLP1 promoter. Lane 1: reaction solution containing only biotin labeled probes;Lane 2: reaction solution containing biotin labeled probes and recombinant PnWRKY15; Lane 3: reaction solution containing biotin labeled probes, unlabeled competitive probes and recombinant PnWRKY15; Lane 4: reaction solution containing biotin labeled mutant probes and recombinant PnWRKY15. Original image of Fig. 6A was shown in the Supplementary Material 4. (B) Analysis of the trans-activation of PnWRKY15 on PnOLP1 promoter (PPnOLP1). The pGADT7-PnWRKY15 prey vector and pAbAi-PPnOLP1 bait vector were co-transformed into yeast cells, which grew on SD/−Leu/AbA (200 ng/mL) medium, indicating that PnWRKY15 trans-activated PPnOLP1. PnWRKY15 + PPnOLP1: pGADT7-PnWRKY15 and PPnOLP1-pAbAi co-transformed yeast cells; Positive control: pGADT7-Rec-p53 and p53-pAbAi co-transformed yeast cells; Negative control: pGADT7 and pAbAi-PPnOLP1 co-transformed yeast cells

Fig. 7

Analysis of GUS activity in transgenic tobacco. (A) Transcription activation of PPnOLP1 induced by plant hormone. PPnOLP-1/-2/-3: three pBI121-PPnOLP-GUS transgenic tobacco. (B) GUS activity analysis of PPnOLP1-GUS transgenic tobacco and PPnOLP1-GUS/PnWRKY15 transgenic tobacco. pBI121-35S-GUS: pBI121-35S-GUS transgenic tobacco; pBI121-35S-GUS/PnWRKY15: pBI121-35S-GUS/PnWRKY15 transgenic tobacco; pBI121-PPnOLP1-GUS-1/-2/-3: pBI121-PPnOLP1-GUS transgenic tobacco lines; pBI121-PPnOLP1-GUS/PnWRKY15-1/-2/-3: pBI121-PPnOLP1-GUS/PnWRKY15 transgenic tobacco. The results were shown as average values calculated from three replicates and the significance was determined by the Student’ s t-test. a ~ f: significance level at P < 0.05

Fig. 8

Model diagram about regulatory mechanism of PnWRKY15 in P. notoginseng during response to F. salani infection. After P. notoginseng was infected by the root rot pathogen, PnWRKY15 activated the JA/SA signaling pathways. Moreover, the expression of JA/SA responsive genes including the PnOLP1 was up-regulated and then enhanced the defense response to F. solani