A bZIP transcription factor, PqbZIP1, is involved in the plant defense response of American ginseng

Abstract

American ginseng (Panax quinquefolius L.) is a perennial medicinal plant that has a long usage history in China. However, root rot, which is mainly caused by Fusarium solani can severely reduce the yield and quality of American ginseng, but no disease-resistant variety of American ginseng exists, and the resistance against this disease is not yet well understood. Thus, it is very urgent to analyze the interaction mechanism regulating the interactions between American ginseng and F. solani to mine disease resistance genes. Using transcriptome data and quantitative polymerase chain reaction (qPCR), we screened the transcription factor PqbZIP1 in response to induction by chitin. Yeast self-activation and subcellular localization experiments proved that PqbZIP1 showed transcriptional activity and was localized in the plant nucleus. In addition, qPCR showed that the highest relative expression level was in the roots, wherein chitin and F. solani inhibited and activated the expression of PqbZIP1, respectively, in American ginseng. Additionally, PqbZIP1 significantly inhibited the growth of the Pseudomonas syringae pv. tomato D36E strain in Nicotiana benthamiana, where expressing PqbZIP1 in N. benthamiana increased the jasmonic acid, salicylic acid, and abscisic acid content. Furthermore, PqbZIP1 expression was continually increased upon inoculation with F. solani. Hence, this study revealed that the PqbZIP1 transcription factor might mediate multiple hormonal signaling pathway to modulate root rot disease resistance in American ginseng, and provided important information to breed disease-resistant American ginseng.

Keywords: American ginseng; Disease resistance genes; Fusarium solani; Immune response; Panax quinquefolius L.; PqbZIP1; Root rot disease.

Figure 1. Volcano plot and Gene Ontology (GO) database of differential expressed genes (DEGs).

(A) Volcano plot displaying differential expression between water- and chitin-treated American ginseng. The y-axis corresponds to the mean expression value of log (P value), and the x-axis displays the log2 fold-change value. The yellow dots represent transcripts whose expression was up-regulated, and the blue dots represent the transcripts whose expression was down-regulated. (B) GO annotation of the different expression transcripts of American ginseng affected by chitin.

Figure 2. Transcription factors of American ginseng.

(A) The transcription factors of American ginseng according to the full-length transcriptome and iTAK software. (B) The relative expression level of American ginseng PqbZIP1 after being treated with chitin. Error bars represent standard error of the means. Bars with different letter represent results that are significantly different (P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). The experiment was repeated twice with similar results.

Figure 3. Phylogenetic tree for PqbZIP1 and its homologs from A. thaliana.

Figure 4. Relative expression level of PqbZIP1 in different tissues of American ginseng.

Different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). The error bars represent standard error of the means. The experiment was repeated three times with similar results.

Figure 5. Transcriptional profiling of PqbZIP1 in response to chitin treatment.

Different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). The error bars represent standard error of the means. The experiment was repeated three times with similar results.

Figure 6. Transcriptional profiling of PqbZIP1 in response to F. solani infection.

Different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). The error bars represent standard error of the means. The experiment was repeated three times with similar results.

Figure 7. Transactivation assay of PqbZIP1.

The yeast-two-hybrid (Y2H) Gold strain expressing pCL1, BD, and PqbZIP1-BD grew on YPDA or the selective medium SD-Trp-His. The pCL1 encoding the full-length GAL4 and the empty vector pGBKT7 (BD) were used as positive and negative controls, respectively.

Figure 8. Subcellular location of PqbZIP1 of American ginseng.

The subcellular location experiment of PqbZIP1 revealed that PqbZIP1-GFP was located in the nucleus of N. benthamiana leaves, while GFP was detected throughout the whole cell. Scale bar = 20 mm.

Figure 9. PqbZIP1 increased the growth ability of D36E in N. benthamiana.

Different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). The error bars represent standard error of the means. The experiment was repeated twice with similar results.

Figure 10. Agrobacterium tumefaciens strain GV3101 carrying PqbZIP1-GFP advanced the burst time of active oxygen induced by flg22 in Nicotiana benthamiana compared with that carrying GFP.

Different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). The error bars represent standard error of the means. The experiment was repeated twice with similar results.

Figure 11. PqbZIP1 increased plant jasmonic acid (A), salicylic acid (B) and abscisic acid (C) content.

Different letters indicate statistically significant differences (P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test). The error bars represent standard error of the means. The experiment was repeated three times with similar results.