| Brassica napus L. is an important oilseed and energy crop in China. In the recent years, the industry has suffered from serious economic losses due to rapeseed diseases, especially Brassica Powdery Mildew. Therefore, it is significant to study resistance genes in B. napus. The genome of B. napus (AACC,2n=38) is originated from a spontaneous hybridization between B.rapa L.(AA,2n=20) and B.oleracea L.(CC,2n=18) and comprises two sets of homoeologous chromosomes from the two species. Both Arabidopsis thaliana and B. napus belong to brassica genus in cruciferae family. According to literatures, Arabidopsis thaliana has high similarity with Brassica in genome sequence, suggesting close functions of homologues in Brassica napus. Arabidopsis RPW8 gene locus confers resistance to powdery mildew disease. However, it is largely uncertain whether these homologues in B. napus have similar functions to Arabidopsis RPW8. Seven RPW8 homologues exist in B. napus, i.e. BoHRa, BoHRb, BoHRc from B. oleracea; and BrHRa, BrHRb, BrHRc and BrHRd originated from B.rapa.In this study, we performed investigation on RPW8 homologues in B.napus, including DNA polymorphism and phylogenetic analyses, and ectopic expression in transgenic Arabidopsis. The main results are as follows.The data showed that BoHRa, BoHRb,BoHRc and BrHRa, BrHRb, BrHRc were highly homologous, respectively, with sequence identities ranged from 95% to 99% at the nucleotide level. Compared with RPW8.2, the amino acid sequences of B. napus homologues had identities about 50% and all shared a RPW8 Pfam family domain. The transcriptional levels of the RPW8 homologues in four B. napus accessions were examined. The transcription levels of the RPW8 homologues were the highest in R7 and RS-2. After inoculation of powdery mildew pathogen, the expression level of BnHRa was up-regulated; while BnHRc was constitutively expressed at low level. Furthermore, polymorphysim analysis using DnaSP software demonstrated that BrHRb (S= 29, Pi= 0.01135,0= 0.01103, K= 8.54) and BrHRc (S= 29, Pi= 0.01148,9= 0. 01266, K= 8.65) displayed the most abundant genetic diversity, indicating that the two homologues may play a more important role in evolution of resistance gene in B. napus.To investigate the subcellular localization of RPW8 homologous, we constructed transgenic plants expressing RPW8 homologous fused to yellow fluorescent protein (YFP) from the RPW8.2 promoter in Arabidopsis. All the RPW8 homologous except BrHRd-eYFP was expression in Arabidopsis. Specifically, BoHRa-eYFP, BnHRa-Br-eYFP and the C-terminal deletion of BnHRa-br (i.e. BnHRat-Br-eYFP) were strongly expressed, and specifically target to the extra-haustorial membrane of powdery mildew; while BnHRb-Br-eYFP and the C-terminal deletion of BnHRb-Br (i.e. BnHRbt-Br-eYFP) showed polarized distribution, preferentially localized on the front of haustorium. BoHRc-eYFP and BnHRc-Br-eYFP and C-terminal deletion of BnHRc (i.e. BnHRct-Br-eYFP) were accumulated in the cytoplasm and and may be not to target to the extra-haustorial membrane.Disease resistance assay revealed that transgenic plants expressing BnHRat-Br-eYFP and BnHRbt-Br-eYFP had strong HR cell death and powdery mildew resistance. However, transgenic plants expressing BnHRa-Br-eYFP, BnHRb-Br-eYFP and BnHRc-Br-eYFP had phenotypes similar to Col-gl.In conclusion, our data indicate that some RPW8 homologues in B. napus possess similar functions as RPW8 in Arabidopsis, and could confer resistance to powdery mildew. This study opens an avenue to explore information from model plant to identify and characterize important genes in crops, e.g. disease resistance genes in B. napus, which could promote genetic improvement in these crops. The research stragety in this work could also be applied to study disease resistance in other cruciferae plants. |
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