Overexpression of BnWRKY33 in oilseed rape enhances resistance to Sclerotinia sclerotiorum

Abstract

Sclerotinia sclerotiorum causes a devastating disease in oilseed rape (Brassica napus) resulting in a tremendous yield loss worldwide. Studies on various host-pathogen interactions have shown that plant WRKY transcription factors are essential for defence. For the B. napus-S. sclerotiorum interaction, little direct evidence has been found with regard to the biological roles of specific WRKY genes in host resistance. In this study, we isolated a B. napus WRKY gene, BnWRKY33, and found that the gene is highly responsive to S. sclerotiorum infection. Transgenic B. napus plants overexpressing BnWRKY33 showed markedly enhanced resistance to S. sclerotiorum, constitutive activation of the expression of BnPR1 and BnPDF1.2, and inhibition of H2 O2 accumulation in response to pathogen infection. Further, we isolated a mitogen-activated protein (MAP) kinase substrate gene, BnMKS1, and found that not only can BnWRKY33 interact with BnMKS1, which can also interact with BnMPK4, using the yeast two-hybrid assay, consistent with their collective nuclear localization, but also BnWRKY33, BnMKS1 and BnMPK4 are substantially and synergistically expressed in response to S. sclerotiorum infection. In contrast, the three genes showed differential expression in response to phytohormone treatments. Together, these results suggest that BnWRKY33 plays an important role in B. napus defence to S. sclerotiorum, which is most probably associated with the activation of the salicylic acid (SA)- and jasmonic acid (JA)-mediated defence response and inhibition of H2 O2 accumulation, and we propose a potential mechanism in which BnMPK4-BnMKS1-BnWRKY33 exist in a nuclear localized complex to regulate resistance to S. sclerotiorum in oilseed rape.

Keywords: Brassica napus; Sclerotinia sclerotiorum; WRKY33 transcription factor; disease resistance.

Figure 1

Response of BnWRKY33 to Sclerotinia sclerotiorum infection. Relative expression levels of BnWRKY33 in Brassica napus were determined by real‐time quantitative polymerase chain reaction at 0, 12, 24, 36 and 48 h post S. sclerotiorum inoculation. Values are means of three replicates. The error bars show the standard deviation. The significances of the gene expression differences between each time point and the 0‐h time point are indicated: **(Student's t‐test, P > 0.01) or *(Student's t‐test, P > 0.05).

Figure 2

Characterization of BnWRKY33‐overexpressing lines. WT, untransformed wild‐type control; #6, #8 and #10, three independent BnWRKY33 transgenic T2 lines. (a) Diagram of the plasmids used in this study. CaMV35S, cauliflower mosaic virus 35S promoter; NOS, terminator. (b) Validation of BnWRKY33‐overexpressing lines at transcription levels revealed by reverse transcription‐polymerase chain reaction (RT‐PCR). (c) Disease responses of inoculated plants at 48 h post‐inoculation (hpi). Photographs were taken of leaves from three plants of WT and three hygromycin‐ and PCR‐positive plants of line 8. (d) Disease progression is shown from 24 to 72 hpi. Error bars indicate standard deviations. Differences in susceptibility between WT and the transgenic lines were significant (P < 0.05) from 36 to 60 hpi. (e) Relative expression levels of BnWRKY33, BnPDF1.2 and BnPR1 were quantified by real‐time qPCR. Values are means of three replicates. The error bars show the standard deviation. The significances of the gene expression differences between line 8 and WT (CK) are indicated: **(Student's t‐test, P > 0.01) or *(Student's t‐test, P > 0.05).

Figure 3

Transgenic plants overexpressing BnWRKY33 inhibit H₂O₂ accumulation in response to Sclerotinia sclerotiorum infection. In situ detection of H₂O₂ was performed using 3,3‐diaminobenzidine staining in the untransformed wild‐type (WT) control and the transgenic line 8 at 0, 6, 12, 18 and 24 h post‐inoculation (hpi).

Figure 4

Interaction and subcellular localization of BnWRKY33, BnMKS1 and BnMPK4 proteins. (a) Interaction of BnWRKY33, BnMKS1 and BnMPK4 in yeast cells. The Gal4 DNA binding domain (BD)‐BnMKS1 fusion bait vectors were co‐transformed with the activation domain (AD)‐BnWRKY33 or BnMPK4 fusion prey vectors into yeast cells, and the transformant cells were assayed for LacZ reporter gene expression on high‐stringency (SD/–Ade/–His/–Leu/–Trp) plates: A, pGBKT7‐BnMKS1 + pGDAT7‐BnWRKY33; B, pGBKT7‐BnMKS1 + pGDAT7‐BnMPK4; C, BnMKS1 (pGBKT7‐BnMKS1) and SV40 large T‐antigen (pGADT7‐T) co‐transformants were used as negative controls. (b) Subcellular localization of BnWRKY33, BnMKS1 and BnMPK4. In planta localization in Nicotiana benthamiana leaves of protein‐green fluorescent protein (GFP), nuclear marker protein‐red fluorescent protein (RFP) and merged fluorescence from RFP and GFP.

Figure 5

Expression of BnWRKY33, BnMKS1 and BnMPK4 during activation of plant defence responses. Expression of BnWRKY33, BnMKS1 and BnMPK4 in response to Sclerotinia sclerotiorum and treatment with chemicals. Plants were inoculated or treated with the various chemicals as described in Experimental procedures. SA, salicylic acid; MeJA, methyl jasmonate; ABA, abscisic acid. Values are means of three replicates. Error bars indicate standard deviations. The significances of the gene expression differences between each time point and the 0‐h time point are indicated: **(Student's t‐test, P > 0.01) or *(Student's t‐test, P > 0.05).