Hydrophobin Protein from Trichoderma harzianum Induced Maize Resistance to Maize Leaf Spot Pathogen Curvularia lunata
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- 英文论文题名：Hydrophobin Protein from Trichoderma harzianum Induced Maize Resistance to Maize Leaf Spot Pathogen Curvularia lunata
- 论文作者：YU Chuan-jin ; LU Zhi-xiang ; XIA Hai ; GAO Jin-xin ; DOU Kai ; WANG Meng ; LI Ya-qian ; CHEN Jie
- 英文论文作者：YU Chuan-jin ; LU Zhi-xiang ; XIA Hai ; GAO Jin-xin ; DOU Kai ; WANG Meng ; LI Ya-qian ; CHEN Jie ; Key Laboratory of Urban Agriculture(South) ; Ministry of Agriculture ; School of Agriculture and Biology ; Shanghai Jiao Tong University ; State Key Laboratory of Microbial Metabolism ; Shanghai Jiao Tong University
- 年：2016
- 作者机构：Key Laboratory of Urban Agriculture(South),Ministry of Agriculture,School of Agriculture and Biology,Shanghai Jiao Tong University;State Key Laboratory of Microbial Metabolism,Shanghai Jiao Tong University;
- 英文论文关键词：Trichoderma harzianum ; Hydrophobin ; Ubiquilinl ; MAMPs ; Protein interaction ; Induce systemic resistance
- 会议召开时间：2016-08-05
- 会议录名称：中国植物病理学会2016年学术年会论文集
- 英文会议录名称：Proceedings of the Annual Meeting of Chinese Society for Plant Pathology(2016)
- 语种：英文
- 分类号：S435.13
- 学会代码：ZGVS
- 会议名称：中国植物病理学会2016年学术年会
- 会议地点：中国江苏南京
- 主办单位：中国植物病理学会
- 学会名称：中国植物病理学会
- 页数：2
- 文件大小：93k
- 原文格式：O
- 会议级别：全国
摘要
Corn Curvularia leaf spot disease was a frequently occurring disease to maize leaf,which caused by Curvularia lunata leaf spot pathogen,this disease was very common in corn planting area in China.Therefore,the prevention and treatment of the disease was of great significance.Hydrophobins,low molecular mass(about 10 kDa) secreted proteins of fungi,were characterized by moderate to high levels of hydrophobic material(hydrophobic substances) to cover on the surface of the microbial cells and the presence of eight conserved cysteine(Cys) residues and formed 4 disulfide bond.A class Ⅱ hydrophobin gene hyd1 was cloned.A 2kb fragment of the hyd1 promoter sequence upstream hyd1,hyd1 and DsRed were ligated together.The expression cassete P_(hyd1):DsRed and P_(hyd1):hyd1:DsRed were introduced into the KO 16(hyd1 disruption transformant) and showed that hyd1 was localized in the plasma membrane by the lazer confocal fluorescense microscope.B73 seeds that treated with the conidia of different hyd1 transformants and grew in greenhouse,the 4-leaf stage leaves were inoculated C.lunata in vitro and in vivo.The results demonstrated that the maize leaf lesion size that treated by KO 16 was larger than WT(wild type),the disease index of the overexpression treatment(OE3 and OE5) maize was lowest.The callose accumulation was increased in the maize leaves infiltrated with in vitro expressed Hyd1 protein comparied with control.It also indicated that hyd1 influenced Trichoderma colonization in maize roots by the asssy of SEM and qPCR.Meanwhile,Hyd1 protein was detected on the plasma membrane of maize root by the IEM and it was demonstrated that hyd1 acted as MAMPs(Microbial-associated molecular patterns) to induce the maize resistance.The yeast bait plasmid pGBKT7-hyd1 was constructed and screened the maize root cDNA Library and found that hyd1 interacted with ubiquilin1.We verified the two interaction proteins again by BiFC and pulll down method and also identified ubiquilinl and hyd1 interaction motif in yeast cells.At the same time,it was proved that the signal peptide sequence and the cysteine amino acid of Hyd1 were necessary for the interaction between Ubiquilin1-N and Hyd1.The hyd1 and ubiquilinl were expressed in A.thiana and then promoted the plant resistance synergistically.RNA-seq analyzed maize leaf global genes expression that regulated by the hyd1 at the time of spraying C.lunata for 24 h,and the result showed that it probably regulated by BAK1 of BR(brassinosteroids,BR)pathway signaling transduction and it was preliminarily verified by the mutant BANK1-4.Furthermore,it indicated that the genes of potassium channe protein,CaM and WRKY differentiated notably.We also detected the CaM in different maize leaves,the CaM concentration in KO 16 treatment maize leaves was lower than WT,and the OE3 and 0E5 was highest of all.The hyd1 and ubiquilinl were localized on the plasma membrane and Hyd1 promoted the stability of the Ubiquilin1 in Nicotiana benthamiana.Interaction between Hyd1 and Ubiquilin1,perhaps the Ubiquilin1 might interact with plant receptor BAK1 and together with Hyd1 to form a complex,thereby stimulating the relevant resistance of the plant.This study was to lay a foundation about molecular mechanism of hyd1 acted as MAMPs interacted with plant.
Corn Curvularia leaf spot disease was a frequently occurring disease to maize leaf,which caused by Curvularia lunata leaf spot pathogen,this disease was very common in corn planting area in China.Therefore,the prevention and treatment of the disease was of great significance.Hydrophobins,low molecular mass(about 10 kDa) secreted proteins of fungi,were characterized by moderate to high levels of hydrophobic material(hydrophobic substances) to cover on the surface of the microbial cells and the presence of eight conserved cysteine(Cys) residues and formed 4 disulfide bond.A class Ⅱ hydrophobin gene hyd1 was cloned.A 2kb fragment of the hyd1 promoter sequence upstream hyd1,hyd1 and DsRed were ligated together.The expression cassete P_(hyd1):DsRed and P_(hyd1):hyd1:DsRed were introduced into the KO 16(hyd1 disruption transformant) and showed that hyd1 was localized in the plasma membrane by the lazer confocal fluorescense microscope.B73 seeds that treated with the conidia of different hyd1 transformants and grew in greenhouse,the 4-leaf stage leaves were inoculated C.lunata in vitro and in vivo.The results demonstrated that the maize leaf lesion size that treated by KO 16 was larger than WT(wild type),the disease index of the overexpression treatment(OE3 and OE5) maize was lowest.The callose accumulation was increased in the maize leaves infiltrated with in vitro expressed Hyd1 protein comparied with control.It also indicated that hyd1 influenced Trichoderma colonization in maize roots by the asssy of SEM and qPCR.Meanwhile,Hyd1 protein was detected on the plasma membrane of maize root by the IEM and it was demonstrated that hyd1 acted as MAMPs(Microbial-associated molecular patterns) to induce the maize resistance.The yeast bait plasmid pGBKT7-hyd1 was constructed and screened the maize root cDNA Library and found that hyd1 interacted with ubiquilin1.We verified the two interaction proteins again by BiFC and pulll down method and also identified ubiquilinl and hyd1 interaction motif in yeast cells.At the same time,it was proved that the signal peptide sequence and the cysteine amino acid of Hyd1 were necessary for the interaction between Ubiquilin1-N and Hyd1.The hyd1 and ubiquilinl were expressed in A.thiana and then promoted the plant resistance synergistically.RNA-seq analyzed maize leaf global genes expression that regulated by the hyd1 at the time of spraying C.lunata for 24 h,and the result showed that it probably regulated by BAK1 of BR(brassinosteroids,BR)pathway signaling transduction and it was preliminarily verified by the mutant BANK1-4.Furthermore,it indicated that the genes of potassium channe protein,CaM and WRKY differentiated notably.We also detected the CaM in different maize leaves,the CaM concentration in KO 16 treatment maize leaves was lower than WT,and the OE3 and 0E5 was highest of all.The hyd1 and ubiquilinl were localized on the plasma membrane and Hyd1 promoted the stability of the Ubiquilin1 in Nicotiana benthamiana.Interaction between Hyd1 and Ubiquilin1,perhaps the Ubiquilin1 might interact with plant receptor BAK1 and together with Hyd1 to form a complex,thereby stimulating the relevant resistance of the plant.This study was to lay a foundation about molecular mechanism of hyd1 acted as MAMPs interacted with plant.
Corn Curvularia leaf spot disease was a frequently occurring disease to maize leaf,which caused by Curvularia lunata leaf spot pathogen,this disease was very common in corn planting area in China.Therefore,the prevention and treatment of the disease was of great significance.Hydrophobins,low molecular mass(about 10 kDa) secreted proteins of fungi,were characterized by moderate to high levels of hydrophobic material(hydrophobic substances) to cover on the surface of the microbial cells and the presence of eight conserved cysteine(Cys) residues and formed 4 disulfide bond.A class Ⅱ hydrophobin gene hyd1 was cloned.A 2kb fragment of the hyd1 promoter sequence upstream hyd1,hyd1 and DsRed were ligated together.The expression cassete P_(hyd1):DsRed and P_(hyd1):hyd1:DsRed were introduced into the KO 16(hyd1 disruption transformant) and showed that hyd1 was localized in the plasma membrane by the lazer confocal fluorescense microscope.B73 seeds that treated with the conidia of different hyd1 transformants and grew in greenhouse,the 4-leaf stage leaves were inoculated C.lunata in vitro and in vivo.The results demonstrated that the maize leaf lesion size that treated by KO 16 was larger than WT(wild type),the disease index of the overexpression treatment(OE3 and OE5) maize was lowest.The callose accumulation was increased in the maize leaves infiltrated with in vitro expressed Hyd1 protein comparied with control.It also indicated that hyd1 influenced Trichoderma colonization in maize roots by the asssy of SEM and qPCR.Meanwhile,Hyd1 protein was detected on the plasma membrane of maize root by the IEM and it was demonstrated that hyd1 acted as MAMPs(Microbial-associated molecular patterns) to induce the maize resistance.The yeast bait plasmid pGBKT7-hyd1 was constructed and screened the maize root cDNA Library and found that hyd1 interacted with ubiquilin1.We verified the two interaction proteins again by BiFC and pulll down method and also identified ubiquilinl and hyd1 interaction motif in yeast cells.At the same time,it was proved that the signal peptide sequence and the cysteine amino acid of Hyd1 were necessary for the interaction between Ubiquilin1-N and Hyd1.The hyd1 and ubiquilinl were expressed in A.thiana and then promoted the plant resistance synergistically.RNA-seq analyzed maize leaf global genes expression that regulated by the hyd1 at the time of spraying C.lunata for 24 h,and the result showed that it probably regulated by BAK1 of BR(brassinosteroids,BR)pathway signaling transduction and it was preliminarily verified by the mutant BANK1-4.Furthermore,it indicated that the genes of potassium channe protein,CaM and WRKY differentiated notably.We also detected the CaM in different maize leaves,the CaM concentration in KO 16 treatment maize leaves was lower than WT,and the OE3 and 0E5 was highest of all.The hyd1 and ubiquilinl were localized on the plasma membrane and Hyd1 promoted the stability of the Ubiquilin1 in Nicotiana benthamiana.Interaction between Hyd1 and Ubiquilin1,perhaps the Ubiquilin1 might interact with plant receptor BAK1 and together with Hyd1 to form a complex,thereby stimulating the relevant resistance of the plant.This study was to lay a foundation about molecular mechanism of hyd1 acted as MAMPs interacted with plant.
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