Molecular Mechanism Of Barley Oxalate Oxidase And WRKY Transcription Factors Participating In The Resistance Against S. Sclerotiorum

Sclerotinia stem rot(SSR),is one of the major disease in oilseed rape,which severely impacts the rapeseed production in China. Studies on the mechanism of the response to sclerotinia sclerotiorum by oilseed rape are available to realiz the interaction between sclerotinia sclerotiorum and oilseed rape, which has significance for resistance breeding in oilseed rape. Our study is focus on two perspective as follows.1. Sclerotinia sclerotiorum(S. sclerotiorum), causes SSR disease, resulting in the secretion of Oxalic oxalic acid(OA) which can be further degraded to carbon dioxide(CO2) and hydrogen peroxide(H2O2) by oxalate oxidase(OXO). In the present investigations and observations, the barley oxalate oxidase(BOXO, Y14203) gene was introduced into oilseed rape by agrobacterium-mediated transformation to investigate the mechanism by which oxalate oxidase promotes the resistance in Brassica napus to S. sclerotiorum. As compared to the control, the transgenic oilseed rape showed reduced lesions on leaves by approximately 15%-61% after 72 h of post-inoculation, thus exhibiting a detectable level of partial resistance in leaf tissue resistance to S. sclerotiorum. Transgenic oilseed rape also showed decreased oxalate and increased hydrogen peroxide levels compared to the control, and the expression of defense response genes involved in the hydrogen peroxide signaling pathway was also induced. Therefore, the major factors involved in the improved resistance of oilseed rape could be attributed to the enhanced OA metabolism, production of hydrogen peroxide and the hydrogen peroxide-mediated defense levels during infection.2. Base on the results of suppression subtractive hybridization(SSH) in our laboratory, a gene induced by S. sclerotiorum was detected and the full length of its CDS was also isolated and ecoded Bn WRKY33 transcription factor. It was mapped on A5 linkage group by intron polymorphism marker. Overexpression Bn WRKY33 could increase the resistance to S. sclerotiorum in the cultivar Westar. The genes involved in synthesis of camalexin showed up-regulated expression in the transgenic plants. Promoter analysis showed that Bn WRKY33 was induced by pathogen responsive molecule salicylic acid and S. sclerotiorum. Promoter truncation analysis was performed and a region containing three W-box was found to be responsive to the infection of S. sclerotiorum. Yeast-one-hybrid was conducted used this region to screen the transcription factors upstream of Bn WRKY33, and another WRKY transcription factor homologous to At WRKY15, which was named as Bn WRKY15, was obtained. The binding between Bn WRKY15 and this region was confirmed by EMSA and Arabidopsis protoplast transient expression, also Bn WRKY33 binding to this region was verified by the two methods. Transcriptional activation activity analysis indicated that Bn WRKY15 is a weak transcriptional activator, and Bn WRKY33 is a typical transcriptional activator. Two transcriptional activation domains in Bn WRKY33 and one transcriptional repression domain in Bn WRKY15 were found by transcriptional activation/repression domain analysis. It was found that Bn WRKY15 could reduce the transactivation of Bn WRKY33 by itself using Arabidopsis protoplast transient expression. Additionally, the expression of Bn WRKY33 and its downsteam genes involved in camalexin synthesis was lowered in the Bn WRKY15 overexpression plants. Also, Bn WRKY15 overexpression plants showed reduced ressitance to S. sclerotiorum. Therefore, the effects of Bn WRKY15 on the transcriptional activation of Bn WRKY33 by itself, which were studied using transcriptional activation analysis and competitive EMSA, were attributed to two aspects. First, Bn WRKY15 could repress the transcriptional activation activity of Bn WRKY33. Second, Bn WRKY15 and Bn WRKY33 would bind to the W-box competitively, and Bn WRKY15 has higher W-box binding activity compared with Bn WRKY33. In addition, Bn WRKY28 and Bn WRKY40 were found to bind to the promoter region containing three W-box of Bn WRKY33, which might indicated that Bn WRKY33 located at the center of regulation in the responses to environmental stimulus of plants.