BnaMPK3 Is a Key Regulator of Defense Responses to the Devastating Plant Pathogen Sclerotinia sclerotiorum in Oilseed Rape.

The disease caused by Sclerotinia sclerotiorum has traditionally been difficult to control, resulting in tremendous economic losses in oilseed rape (Brassica napus). Identification of important genes in the defense responses is critical for molecular breeding, an important strategy for controlling the disease. Here, we report that a B. napus mitogen-activated protein kinase gene, BnaMPK3, plays an important role in the defense against S. sclerotiorum in oilseed rape. BnaMPK3 is highly expressed in the stems, flowers and leaves, and its product is localized in the nucleus. Furthermore, BnaMPK3 is highly responsive to infection by S. sclerotiorum and treatment with jasmonic acid (JA) or the biosynthesis precursor of ethylene (ET), but not to treatment with salicylic acid (SA) or abscisic acid. Moreover, overexpression (OE) of BnaMPK3 in B. napus and Nicotiana benthamiana results in significantly enhanced resistance to S. sclerotiorum, whereas resistance is diminished in RNAi transgenic plants. After S. sclerotiorum infection, defense responses associated with ET, JA, and SA signaling are intensified in the BnaMPK3-OE plants but weakened in the BnaMPK3-RNAi plants when compared to those in the wild type plants; by contrast the level of both H2O2 accumulation and cell death exhibits a reverse pattern. The candidate gene association analyses show that the BnaMPK3-encoding BnaA06g18440D locus is a cause of variation in the resistance to S. sclerotiorum in natural B. napus population. These results suggest that BnaMPK3 is a key regulator of multiple defense responses to S. sclerotiorum, which may guide the resistance improvement of oilseed rape and related economic crops.

Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions.

Data normalization is essential for reliable output of quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assays, as the unsuitable choice of reference gene(s), whose expression might be influenced by exogenous treatments in plant tissues, could cause misinterpretation of results. To date, no systematic studies on reference genes have been performed in stressed Brassica napus. In this study, we investigated the expression variations of nine candidate reference genes in 40 samples of B. napus leaves subjected to various exogenous treatments. Parallel analyses by geNorm and NormFinder revealed that optimal reference genes differed across the different sets of samples. The best-ranked reference genes were PP2A and TIP41 for salt stress, TIP41 and ACT7 for heavy metal (Cr(6+)) stress, PP2A and UBC21 for drought stress, F-box and SAND for cold stress, F-box and ZNF for salicylic acid stress, TIP41, ACT7, and PP2A for methyl jasmonate stress, TIP41 and ACT7 for abscisic acid stress, and TIP41, UBC21, and PP2A for Sclerotinia sclerotiorum stress. Two newly employed reference genes, TIP41 and PP2A, showed better performances, suggesting their suitability in multiple conditions. To further validate the suitability of the reference genes, the expression patterns of BnWRKY40 and BnMKS1 were studied in parallel. This study is the first systematic analysis of reference gene selection for qRT-PCR normalization in B. napus, an agriculturally important crop, under different stress conditions. The results will contribute toward more accurate and widespread use of qRT-PCR in gene analysis of the genus Brassica.

Overexpression of BnWRKY33 in oilseed rape enhances resistance to Sclerotinia sclerotiorum.

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.