Chrysanthemum WRKY15-1 promotes resistance to Puccinia horiana Henn. via the salicylic acid signaling pathway

Abstract

Chrysanthemum white rust disease, which is caused by the fungus Puccinia horiana Henn., severely reduces the ornamental quality and yield chrysanthemum. WRKY transcription factors function in the disease-resistance response in a variety of plants; however, it is unclear whether members of this family improve resistance to white rust disease in chrysanthemum. In this study, using PCR, we isolated a WRKY15 homologous gene, CmWRKY15-1, from the resistant chrysanthemum cultivar C029. Real-time quantitative PCR (RT-qPCR) revealed that CmWRKY15-1 exhibited differential expression patterns between the immune cultivar C029 and the susceptible cultivar Jinba upon P. horiana infection. In addition, salicylic acid (SA) treatment strongly induced CmWRKY15-1 expression. Overexpression of CmWRKY15-1 in the chrysanthemum-susceptible cultivar Jinba increased tolerance to P. horiana infection. Conversely, silencing CmWRKY15-1 via RNA interference (RNAi) in C029 increased sensitivity to P. horiana infection. We also determined that P. horiana infection increased both the endogenous SA content and the expression of salicylic acid biosynthesis genes in CmWRKY15-1-overexpressing plants, whereas CmWRKY15-1 RNAi plants exhibited the opposite effects under the same conditions. Finally, the transcript levels of pathogenesis-related (PR) genes involved in the SA pathway were positively associated with CmWRKY15-1 expression levels. Our results demonstrated that CmWRKY15-1 plays an important role in the resistance of chrysanthemum to P. horiana by influencing SA signaling.

Fig. 1. Bioinformatic analysis of CmWRKY15-1.

a PCR amplification product of CmWRKY15-1. b Multiple amino acid sequence alignment between CmWRKY15-1 and several related WRKY proteins from the Asteraceae. The box indicates the WRKYGQK heptapeptide sequence; the triangle indicates the zinc-finger motif. c Secondary structure of the predicted CmWRKY15-1. Blue, alpha-helix; red, folding and extending chain; green, beta-turn; purple, random coil. d Three-dimensional structure of predicted CmWRKY15-1. e Phylogenetic analysis of CmWRKY15-1. M Marker, La Lupinus angustifolius; Vr Vigna radiata var. radiata; Mn Morus notabilis; Si Sesamum indicum; Vv Vitis vinifera; Ga Gossypium aridum; Bn Brassica napus; Dc Daucus carota subsp. Sativus; Cc Cynara cardunculus L.; Ha Helianthus annuus L.; Cm Chrysanthemum morifolium.

Fig. 2. Expression patterns of CmWRKY15-1 under various treatments.

a CmWRKY15-1 transcript levels in chrysanthemum leaves during infection with P. horiana in C029 and Jinba. b CmWRKY15-1 expression levels after water and exogenous hormone treatments. SA, 0.1 mM salicylic acid; MeJA, 50 μM methyl jasmonate; ETH, 0.5 g L⁻¹ ethephon (ETH) in C029. The error bars indicate the standard deviations of three replicates. The different letters mean significant differences according Duncan’s multiple range test at p < 0.05; the same scheme applies below.

Fig. 3. Acquisition of transgenic plants.

a Diagram of the pBI121-CmWRKY15-1 and pRNAi-CmWRKY15-1 vectors. CaMV 35 S, promoter; NOS, nopaline synthase terminator; nptII, neomycin phosphotransferase gene; CmWRKY15-1, target fragment; LB and RB, left and right borders of the T-DNA; SacI, XbaI, BamHI, and HindIII, cloning sites; A1, antisense fragment; S1, sense fragment; PDK, intron. b PCR-based analysis of kanamycin-resistant transgenic plants. M, DNA ladder (DL 2000); OE-1 and OE-2, overexpression transgenic plants; RNA1 and RNA2, RNA interference transgenic plants. c Relative expression levels of CmWRKY15-1.

Fig. 4. Phenotypes of plants in which CmWRKY15-1 was overexpressed or silenced upon infection with P. horiana.

a, b Leaf phenotypes at 2 weeks after infection. c Disease index of plants.

Fig. 5. Endogenous hormone contents.

a SA levels in transgenic plant leaves during 0–24 h after P. horiana infection. FW, fresh weight. b Expression analysis of the SA biosynthesis genes ICS1 and PAL.

Fig. 6. Expression of pathogenesis-related genes involved in the SA signaling pathway.

a Pathogenesis-related gene expression in the control (Jinba) and overexpression plants. b Pathogenesis-related gene expression in the control (C029) and silenced plants.