稻瘟病菌F-box蛋白MoFbr7生物学功能分析
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摘要
F-box蛋白在泛素-蛋白酶体途径(ubiquitin-proteasome pathway,UPP)中参与调控胞内蛋白降解、受体识别、信号传导等生物学过程。本研究从稻瘟病菌中克隆了F-box基因MoFbr7,序列分析表明,该基因编码产物具有1个F-box结构域(N-端)和8个连续的WD40重复序列(C-端),在丝状真菌中高度保守。利用基因敲除方法,获得4个MoFbr7基因敲除突变体,同时构建了回补菌株。表型分析结果显示,MoFbr7基因缺失突变体在产孢量、附着胞形态、原生质体释放、致病性等方面均无异常。突变体在MM、RDC培养基上生长速率下降;对细胞壁胁迫因子CFW(Calcofluor white)、刚果红敏感。以上结果表明MoFbr7参与稻瘟病菌的营养生长与细胞壁完整性,为进一步揭示其生物学功能奠定基础。
F-box protein is involved in the regulation of intracellular protein degradation, receptor recognition, signal transduction and other biological processes in the ubiquitin-proteasome pathway(UPP). In this study, we identified an F-box protein MoFbr7 in the rice blast fungus. Sequence analysis showed that MoFbr7 had an F-box domain at the N-terminal and a WD40 repeats at the C-terminal, and was highly conserved among filamentous fungi based on the phylogenetic analysis. Four knockout mutants of MoFbr7 were obtained by gene knockout method and a complementary strain was constructed based on the deletion mutant. Phenotypic analysis showed that there were no significant differences in sporulation, conidial production, protoplast release and pathogenicity compared with the wild-type stain Guy11. However, the growth rate of ΔMoFbr7 was significantly decreased on MM and RDC media. The knockout strains were sensitive to the cell wall stressors Calcofluor white(CFW) and Congo Red(CR). The above results indicated that MoFbr7 was involved in vegetative growth and cell wall integrity, supplying the foundation for further revealing its biological functions.
F-box protein is involved in the regulation of intracellular protein degradation, receptor recognition, signal transduction and other biological processes in the ubiquitin-proteasome pathway(UPP). In this study, we identified an F-box protein MoFbr7 in the rice blast fungus. Sequence analysis showed that MoFbr7 had an F-box domain at the N-terminal and a WD40 repeats at the C-terminal, and was highly conserved among filamentous fungi based on the phylogenetic analysis. Four knockout mutants of MoFbr7 were obtained by gene knockout method and a complementary strain was constructed based on the deletion mutant. Phenotypic analysis showed that there were no significant differences in sporulation, conidial production, protoplast release and pathogenicity compared with the wild-type stain Guy11. However, the growth rate of ΔMoFbr7 was significantly decreased on MM and RDC media. The knockout strains were sensitive to the cell wall stressors Calcofluor white(CFW) and Congo Red(CR). The above results indicated that MoFbr7 was involved in vegetative growth and cell wall integrity, supplying the foundation for further revealing its biological functions.
引文
[1] DEAN R A, TALBOT N J, EBBOLE D J, et al. The genome sequence of the rice blast fungus Magnaporthe grisea[J]. Nature, 2005, 434 (7036): 980-986.
[2] SAKULKOO W, OSES-RUIZ M, OLIVEIRA GARCIA E, et al. A single fungal MAP kinase controls plant cell-to-cell invasion by the rice blast fungus [J]. Science,2018, 359(6382): 1399-1403.
[3] LI Xiao, GAO Chuyun, LI Lianwei,et al. MoEnd3 regulates appressorium formation and virulence through mediating endocytosis in rice blast fungus Magnaporthe oryzae [J/OL]. PLoS Pathogens, 2017, 13(6): e1006449.
[4] LECHNER E, ACHARD P, VANSIRI A, et al. F-box proteins everywhere [J]. Current Opinion in Plant Biology, 2006, 9(6): 631-638.
[5] KIPREOS E T, MICHELE P. The F-box protein family [J]. Genome Biology, 2000, 1(5): 1-7.
[6] 杨杨杨, 李淑晶, 伍会健. F-box蛋白家族在肿瘤发生中的作用[J]. 中国细胞生物学学报, 2015, 37(8): 1180-1186.
[7] JIN J, ANG X L, SHIROGANE T, et al. Identification of substrates for F-Box proteins[J]. Methods in Enzymology,2005, 399(12): 287-309.
[8] WANG Yupeng, WANG Xiyin, PATERSON A H. Genome and gene duplications and gene expression divergence: a view from plants [J].Annals of the New York Academy of Sciences,2012, 1256(1): 1-14.
[9] SMET R D, ADAMS K L, VANDEPOELE K, et al. Convergent gene loss following gene and genome duplications creates single-copy families in flowering plants [J]. Proceedings of the National Academy of Sciences,2013, 110(8): 2898-2903.
[10] IBARRA-LACLETTE E, LYONS E, HERNáNDEZ-GUZMáN G, et al. Architecture and evolution of a minute plant genome [J]. Nature, 2013, 498(7452): 94-98.
[11] KURODA H,TAKAHASHI N, SHIMADA H, et al. Classification and expression analysis of Arabidopsis F-box-containing protein genes [J].Plant & Cell Physiology,2002,43(10):1073-1085.
[12] MAJEE M, KUMAR S, KATHARE P K, et al. KELCH F-BOX protein positively influences Arabidopsis seed germination by targeting PHYTOCHROME-INTERACTING FACTOR1 [J]. Proceedings of the National Academy of Science of the united states of America,2018, 115(17): E4120-E4129.
[13] PRAKASH C, MANJREKAR J, CHATTOO B B. Skp1, a component of E3 ubiquitin ligase, is necessary for growth, sporulation, development and pathogenicity in rice blast fungus (Magnaporthe oryzae)[J]. Molecular Plant Pathology,2016, 17(6): 903-919.
[14] LANG P, CHRISTIE M D, DOGAN E S, et al. A role for the F-box protein HAWAIIAN SKIRT in plant microRNA function [J]. Plant Physiology, 2018, 176(1): 730-741.
[15] BARRA Y, JENTSCH S, MANN C. G1 cyclin turnover and nutrient uptake are controlled by a common pathway in yeast[J]. Genes & Development, 1995, 9(4): 399-409.
[16] 喻海峰,高飞,朱小蕾,等.稻瘟病菌Mofbox基因缺失突变体构建及生物学表型分析[J].安徽农业大学学报,2013,40(5):752-757.
[17] GUO M, GAO F, ZHU X, et al. MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae [J]. Applied Microbiology and Biotechnology,2015,99(19):8075-8088.
[18] MATAR K, CHEN X, CHEN D, et al. WD40-repeat protein MoCreC is essential for carbon repression and is involved in conidiation, growth and pathogenicity of Magnaporthe oryzae [J]. Current Genetics,2017, 63(4): 685-696.
[19] FERNáNDEZ-ALVAREZ A, ELíAS-VILLALOBOS A, IBEAS J I. The O-mannosyltransferase PMT4 is essential for normal appressorium formation and penetration in Ustilago maydis[J]. Plant Cell, 2009, 21(10): 3397-3412.
[2] SAKULKOO W, OSES-RUIZ M, OLIVEIRA GARCIA E, et al. A single fungal MAP kinase controls plant cell-to-cell invasion by the rice blast fungus [J]. Science,2018, 359(6382): 1399-1403.
[3] LI Xiao, GAO Chuyun, LI Lianwei,et al. MoEnd3 regulates appressorium formation and virulence through mediating endocytosis in rice blast fungus Magnaporthe oryzae [J/OL]. PLoS Pathogens, 2017, 13(6): e1006449.
[4] LECHNER E, ACHARD P, VANSIRI A, et al. F-box proteins everywhere [J]. Current Opinion in Plant Biology, 2006, 9(6): 631-638.
[5] KIPREOS E T, MICHELE P. The F-box protein family [J]. Genome Biology, 2000, 1(5): 1-7.
[6] 杨杨杨, 李淑晶, 伍会健. F-box蛋白家族在肿瘤发生中的作用[J]. 中国细胞生物学学报, 2015, 37(8): 1180-1186.
[7] JIN J, ANG X L, SHIROGANE T, et al. Identification of substrates for F-Box proteins[J]. Methods in Enzymology,2005, 399(12): 287-309.
[8] WANG Yupeng, WANG Xiyin, PATERSON A H. Genome and gene duplications and gene expression divergence: a view from plants [J].Annals of the New York Academy of Sciences,2012, 1256(1): 1-14.
[9] SMET R D, ADAMS K L, VANDEPOELE K, et al. Convergent gene loss following gene and genome duplications creates single-copy families in flowering plants [J]. Proceedings of the National Academy of Sciences,2013, 110(8): 2898-2903.
[10] IBARRA-LACLETTE E, LYONS E, HERNáNDEZ-GUZMáN G, et al. Architecture and evolution of a minute plant genome [J]. Nature, 2013, 498(7452): 94-98.
[11] KURODA H,TAKAHASHI N, SHIMADA H, et al. Classification and expression analysis of Arabidopsis F-box-containing protein genes [J].Plant & Cell Physiology,2002,43(10):1073-1085.
[12] MAJEE M, KUMAR S, KATHARE P K, et al. KELCH F-BOX protein positively influences Arabidopsis seed germination by targeting PHYTOCHROME-INTERACTING FACTOR1 [J]. Proceedings of the National Academy of Science of the united states of America,2018, 115(17): E4120-E4129.
[13] PRAKASH C, MANJREKAR J, CHATTOO B B. Skp1, a component of E3 ubiquitin ligase, is necessary for growth, sporulation, development and pathogenicity in rice blast fungus (Magnaporthe oryzae)[J]. Molecular Plant Pathology,2016, 17(6): 903-919.
[14] LANG P, CHRISTIE M D, DOGAN E S, et al. A role for the F-box protein HAWAIIAN SKIRT in plant microRNA function [J]. Plant Physiology, 2018, 176(1): 730-741.
[15] BARRA Y, JENTSCH S, MANN C. G1 cyclin turnover and nutrient uptake are controlled by a common pathway in yeast[J]. Genes & Development, 1995, 9(4): 399-409.
[16] 喻海峰,高飞,朱小蕾,等.稻瘟病菌Mofbox基因缺失突变体构建及生物学表型分析[J].安徽农业大学学报,2013,40(5):752-757.
[17] GUO M, GAO F, ZHU X, et al. MoGrr1, a novel F-box protein, is involved in conidiogenesis and cell wall integrity and is critical for the full virulence of Magnaporthe oryzae [J]. Applied Microbiology and Biotechnology,2015,99(19):8075-8088.
[18] MATAR K, CHEN X, CHEN D, et al. WD40-repeat protein MoCreC is essential for carbon repression and is involved in conidiation, growth and pathogenicity of Magnaporthe oryzae [J]. Current Genetics,2017, 63(4): 685-696.
[19] FERNáNDEZ-ALVAREZ A, ELíAS-VILLALOBOS A, IBEAS J I. The O-mannosyltransferase PMT4 is essential for normal appressorium formation and penetration in Ustilago maydis[J]. Plant Cell, 2009, 21(10): 3397-3412.