基于报告基因敲入构建果生刺盘孢细胞核荧光标记菌株
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摘要
运用基因敲入技术,将GFP、mCherry整合到果生刺盘孢Colletotrichum fructicola组蛋白histone H1位点,实现融合表达,获得细胞核荧光标记菌株。基于标记菌株可以对分生孢子、营养菌丝、附着胞、侵染菌丝等结构中的细胞核进行实时活体观察。果生刺盘孢存在性亲和的"+"、"-"型菌株分化,GFP"+"型菌株和m Cherry"-"型菌株接触形成明显杂交线,杂交线上单子囊内含红绿两种孢子,表明"+"、"-"型菌株间发生杂交;杂交线上子囊壳壁表达m Cherry,表明由"-"型菌株发育而来。本研究构建的核荧光标记菌株将是研究果生炭疽菌细胞周期调控和有性繁殖过程的重要材料。
Based on a gene knock-in strategy,the reporter gene GFP(or m Cherry) was fused with a histone H1 gene and the cassettes were transformed and incorporated into the genome of Colletotrichum fructicola for in-fusion expression and nuclear labeling.Based on the obtained strains,live image of nuclear behavior was possible for various fungal structures such as conidium,vegetative hyphae,appressorium,and infectious hyphae.C.fructicola contains sexually compatible ‘plus' and ‘minus' strains,GFP-labeled ‘plus' strain and m Cherry-labeled ‘minus' strain formed a ‘mating line' at the contact zone.Both m Cherry-labeled and GFP-labeled ascospores could be observed within the same ascus at the ‘mating line',demonstrating crossing events between the ‘plus' and ‘minus' strains.The cell walls of ‘mating line' perithecia expressed m Cherry,demonstrating their ‘minus' strain derivation.The nuclear-labeled strains reported here allow for convenient and direct observation of nuclear behaviors,and will be important materials for studying cell cycle regulations and sexual reproductions of C.fructicola.
Based on a gene knock-in strategy,the reporter gene GFP(or m Cherry) was fused with a histone H1 gene and the cassettes were transformed and incorporated into the genome of Colletotrichum fructicola for in-fusion expression and nuclear labeling.Based on the obtained strains,live image of nuclear behavior was possible for various fungal structures such as conidium,vegetative hyphae,appressorium,and infectious hyphae.C.fructicola contains sexually compatible ‘plus' and ‘minus' strains,GFP-labeled ‘plus' strain and m Cherry-labeled ‘minus' strain formed a ‘mating line' at the contact zone.Both m Cherry-labeled and GFP-labeled ascospores could be observed within the same ascus at the ‘mating line',demonstrating crossing events between the ‘plus' and ‘minus' strains.The cell walls of ‘mating line' perithecia expressed m Cherry,demonstrating their ‘minus' strain derivation.The nuclear-labeled strains reported here allow for convenient and direct observation of nuclear behaviors,and will be important materials for studying cell cycle regulations and sexual reproductions of C.fructicola.
引文
Andes JO,1941.Experiments on the inheritance of the“plus”and“minus”characters in Glomerella cingulata.Bulletin of the Torrey Botanical Club,9:609-614
Cisar CR,Tebeest DO,1999.Mating system of the filamentous ascomycete,Glomerella cingulata.Current Genetics,35(2):127-133
Edgerton CW,1908.The physiology and development of some anthracnoses.International Journal of Plant Sciences,45(6):367-408
Edgerton CW,1914.Plus and minus strains in the genus Glomerella.American Journal of Botany,1(163):244-254
Freitag M,Hickey PC,Raju NB,Selker EU,Read ND,2004.GFPas a tool to analyze the organization,dynamics and function of nuclei and microtubules in Neurospora crassa.Fungal Genetics&Biology,41(10):897-910
Han XL,Bai JK,Zhang W,Zhang R,Sun GY,2016.PEG-mediated transformation of Colletotrichum fructicola.Acta Agriculturae Boreali-occidentalia Sinica,25(3):442-449(in Chinese)
Hokyoung S,Min K,Lee J,Raju NB,Lee YW,2011.Meiotic silencing in the homothallic fungus Gibberella zeae.Fungal Biology,115(12):1290-1302
Li H,Zhou GY,Liu JA,Xu J,2016.Population genetic analyses of the fungal pathogen Colletotrichum fructicola on tea-oil trees in China.PLo S One,11(6):e0156841
Lucas GB,Chilton SJP,Edgerton CW,1944.Genetics of Glomerella.I.Studies on the behavior of certain strains.American Journal of Botany,31(4):233-239
Nesher I,Barhoom S,Sharon A,2008.Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen Colletotrichum gloeosporioides.BMC Biology,6(1):9
Prihastuti H,Mckenzie EHC,Hyde KD,Cai L,Mckenzie H,Hyde EHCA,2009.Characterization of Colletotrichum species associated with coffee berries in Chiangmai,Thailand.Fungal Diversity,39(2):89-109
Rodríguez-Guerra R,Ramírez-Rueda MT,Cabral-Enciso M,García-Serrano M,Lira-Maldonado Z,Guevara-González RG,2005.Heterothallic mating observed between Mexican isolates of Glomerella lindemuthiana.Mycologia,97(4):793-803
Rojas EI,Rehner SA,Samuels GJ,Bael SAV,Herre EA,Cannon P,2010.Colletotrichum gloeosporioides s.l.associated with Theobroma cacao and other plants in Panamá:multilocus phylogenies distinguish host-associated pathogens from asymptomatic endophytes.Mycologia,102(6):1318-1338
Roper M,Simonin A,Hickey PC,Leeder A,Glass NL,2013.Nuclear dynamics in a fungal chimera.Proceedings of the National Academy of Sciences,110(32):12875-12880
Ruiz-Roldán MC,K?hli M,Roncero MI,Philippsen P,Di PA,Espeso EA,2010.Nuclear dynamics during germination,conidiation,and hyphal fusion of Fusarium oxysporum.Eukaryotic Cell,9(8):1216-1224
Saunders DG,Aves SJ,Talbot NJ,2010.Cell cycle-mediated regulation of plant infection by the rice blast fungus.Plant Cell,22(22):497-507
Veneault-Fourrey C,Barooah M,Egan M,Wakley G,Talbot NJ,2006.Autophagic fungal cell death is necessary for infection by the rice blast fungus.Science,312(5773):580-583
Wang W,Fu DD,Zhang R,Sun GY,2015.Etiology of apple leaf spot caused by Colletotrichum spp.Mycosystema,34(1):13-25(in Chinese)
Weir BS,Johnston PR,Damm U,2012.The Colletotrichum gloeosporioides species complex.Studies in Mycology,73:115-180
Zhang L,Wang CX,Li BH,Li BD,2015.Sexual reproduction,biological characteristics and pathogenicity of ascospores of Glomerella cingulata.Mycosystema,34(6):1101-1110(in Chinese)
韩小路,白静科,张玮,张荣,孙广宇,2016.PEG介导的苹果果生刺盘孢Colletotrichum fructicola原生质体转化.西北农业学报,25(3):442-449
王薇,符丹丹,张荣,孙广宇,2015.苹果炭疽叶枯病病原学研究.菌物学报,34(1):13-25
张路,王彩霞,李保华,李宝笃,2015.围小丛壳Glomerella cingulata子囊孢子交配繁殖、生物学特性及致病性.菌物学报,34(6):1101-1110
Cisar CR,Tebeest DO,1999.Mating system of the filamentous ascomycete,Glomerella cingulata.Current Genetics,35(2):127-133
Edgerton CW,1908.The physiology and development of some anthracnoses.International Journal of Plant Sciences,45(6):367-408
Edgerton CW,1914.Plus and minus strains in the genus Glomerella.American Journal of Botany,1(163):244-254
Freitag M,Hickey PC,Raju NB,Selker EU,Read ND,2004.GFPas a tool to analyze the organization,dynamics and function of nuclei and microtubules in Neurospora crassa.Fungal Genetics&Biology,41(10):897-910
Han XL,Bai JK,Zhang W,Zhang R,Sun GY,2016.PEG-mediated transformation of Colletotrichum fructicola.Acta Agriculturae Boreali-occidentalia Sinica,25(3):442-449(in Chinese)
Hokyoung S,Min K,Lee J,Raju NB,Lee YW,2011.Meiotic silencing in the homothallic fungus Gibberella zeae.Fungal Biology,115(12):1290-1302
Li H,Zhou GY,Liu JA,Xu J,2016.Population genetic analyses of the fungal pathogen Colletotrichum fructicola on tea-oil trees in China.PLo S One,11(6):e0156841
Lucas GB,Chilton SJP,Edgerton CW,1944.Genetics of Glomerella.I.Studies on the behavior of certain strains.American Journal of Botany,31(4):233-239
Nesher I,Barhoom S,Sharon A,2008.Cell cycle and cell death are not necessary for appressorium formation and plant infection in the fungal plant pathogen Colletotrichum gloeosporioides.BMC Biology,6(1):9
Prihastuti H,Mckenzie EHC,Hyde KD,Cai L,Mckenzie H,Hyde EHCA,2009.Characterization of Colletotrichum species associated with coffee berries in Chiangmai,Thailand.Fungal Diversity,39(2):89-109
Rodríguez-Guerra R,Ramírez-Rueda MT,Cabral-Enciso M,García-Serrano M,Lira-Maldonado Z,Guevara-González RG,2005.Heterothallic mating observed between Mexican isolates of Glomerella lindemuthiana.Mycologia,97(4):793-803
Rojas EI,Rehner SA,Samuels GJ,Bael SAV,Herre EA,Cannon P,2010.Colletotrichum gloeosporioides s.l.associated with Theobroma cacao and other plants in Panamá:multilocus phylogenies distinguish host-associated pathogens from asymptomatic endophytes.Mycologia,102(6):1318-1338
Roper M,Simonin A,Hickey PC,Leeder A,Glass NL,2013.Nuclear dynamics in a fungal chimera.Proceedings of the National Academy of Sciences,110(32):12875-12880
Ruiz-Roldán MC,K?hli M,Roncero MI,Philippsen P,Di PA,Espeso EA,2010.Nuclear dynamics during germination,conidiation,and hyphal fusion of Fusarium oxysporum.Eukaryotic Cell,9(8):1216-1224
Saunders DG,Aves SJ,Talbot NJ,2010.Cell cycle-mediated regulation of plant infection by the rice blast fungus.Plant Cell,22(22):497-507
Veneault-Fourrey C,Barooah M,Egan M,Wakley G,Talbot NJ,2006.Autophagic fungal cell death is necessary for infection by the rice blast fungus.Science,312(5773):580-583
Wang W,Fu DD,Zhang R,Sun GY,2015.Etiology of apple leaf spot caused by Colletotrichum spp.Mycosystema,34(1):13-25(in Chinese)
Weir BS,Johnston PR,Damm U,2012.The Colletotrichum gloeosporioides species complex.Studies in Mycology,73:115-180
Zhang L,Wang CX,Li BH,Li BD,2015.Sexual reproduction,biological characteristics and pathogenicity of ascospores of Glomerella cingulata.Mycosystema,34(6):1101-1110(in Chinese)
韩小路,白静科,张玮,张荣,孙广宇,2016.PEG介导的苹果果生刺盘孢Colletotrichum fructicola原生质体转化.西北农业学报,25(3):442-449
王薇,符丹丹,张荣,孙广宇,2015.苹果炭疽叶枯病病原学研究.菌物学报,34(1):13-25
张路,王彩霞,李保华,李宝笃,2015.围小丛壳Glomerella cingulata子囊孢子交配繁殖、生物学特性及致病性.菌物学报,34(6):1101-1110