CmWRKY15增加菊花对黑斑病的敏感性
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- 英文论文题名：CmWRKY15 Facilitates Alternariatenuissima Infection of Chrysanthemum
- 论文作者：范青青 ; 宋爱萍 ; 辛静静 ; 陈素梅 ; 蒋甲福 ; 王银杰 ; 陈发棣
- 英文论文作者：FAN Qing-qing ; SONG Ai-ping ; XIN Jing-jing ; CHEN Su-mei ; JIANG Jia-fu ; WANG Yin-jie ; CHEN Fa-di ; College of Horticulture ; Nanjing Agricultural University
- 年：2016
- 作者机构：南京农业大学园艺学院;
- 论文关键词：菊花 ; CmWRKY15 ; 黑斑病
- 英文论文关键词：Chrysanthemum morifolium ; CmWRKY15 ; Alternariatenuissima
- 会议召开时间：2016-07-19
- 会议录名称：中国观赏园艺研究进展2016
- 语种：中文
- 分类号：S436.8
- 学会代码：EGYP
- 会议名称：2016年中国观赏园艺学术研讨会
- 会议地点：中国湖南长沙
- 主办单位：中国园艺学会观赏园艺专业委员会、国家花卉工程技术研究中心
- 学会名称：中国园艺学会
- 页数：9
- 文件大小：1559k
- 原文格式：O
- 会议级别：全国
摘要
菊花(Chrysanthemum morifolium)隶属菊科菊属,原产我国,是我国十大传统名花和世界四大切花之一,具有很高的观赏与经济价值。由链格孢属真菌侵染引起的黑斑病是菊花广泛发生的重要叶部病害,在菊花露地栽培生产中,植株或插穗发病较重。因此,研究与调控胁迫相关的基因功能可以为菊花抗性基因工程育种提供坚实的理论基础。本研究在前期克隆获得菊花CmWRKY15基因基础上,通过转基因技术对该基因功能进行了初步研究,主要研究结果如下:通过对CmWRKY15序列分析及系统进化树构建,发现CmWRKY15与拟南芥AtWRKY40同源性较高;通过遗传转化菊花‘神马'、转基因菊花黑斑病表型鉴定,以及病情指数调查,发现两个转基因菊花株系的发病程度比野生型植株更加严重,两个转基因菊花株系的病情指数为57.01和56.73,其寄主反应表现为感病,而野生型菊花的病情指数为14.63,其寄主反应表现为抗病,说明CmWRKY15转基因菊花增加对黑斑病的敏感性;通过转基因菊花和野生型菊花的内源ABA含量测定,发现转基因菊花的内源ABA含量比野生型菊花低,说明CmWRKY15可能参与ABA合成的调控;通过对ABA合成与响应ABA信号的基因的表达特性进行研究,发现CmWRKY15抑制了ABF4、ABI4、ABI5和RAB18等ABA促进气孔关闭过程中起到正调控作用的基因及NCED3A和NCED3B等ABA合成相关基因的表达,推测CmWRKY15通过抑制ABA正调控作用的基因以及合成相关基因的表达,从而降低转基因株系中ABA浓度以及抑制ABA参与调控气孔关闭的作用,促进转基因植株气孔开放,使黑斑病菌容易侵入,这可能是CmWRKY15参与菊花对黑斑病菌响应的作用机制。
Chrysanthemum(Chrysanthemum morifolium) is one of the most famous cut flowers globally,has a high ornamental value,and occupies an irreplaceable position in the international commerce of flowers.Black spot disease,one of the most harmful diseases of chrysanthemum,is caused by the necrotrophic fungus Alternaria.Hence,it is essential to improve chrysanthemum tolerance to achieve sustainable production.In this study,we place emphasis on the mechanism through which CmWRKY modulates the ABA-mediated pathway in response toAltemariatenuissima in chrysanthemum.The main results are as follows:Phylogenetic analysis showed that CmWRKY15 showed high similarity to AtWRKY40 in Arabidopsis.We used transgenic chrysanthemum lines overexpressing CmWRKY15,After exposure to A.tenuissima for 2 weeks,the surface area of the lesions on transgenic plants was much larger compared to the control.Significant differences in A.tenuissima infection were clearly observable between the transgenic lines and wild type plants.The disease severity indexes(DSIs) of transgenic chrysanthemum lines and wild type plants were 57.01,56.73 and 14.63,respectively.The data revealed that the transgenic lines overexpressing CmWRKYXS were susceptible(S) to black spot disease,while the non-transgenic plants were resistant(R).Necrosis was much more evident in the transgenic plants compared to the WT plants.Our results suggest that CmWRKYlS overexpression enhanced the susceptibility of chrysanthemum to A.tenuissima attack.Endogenous ABA content was measured in the transgenic lines and wild type plants under normal conditions and 24 h after exposure to A.tenuissima.The results show that WT plants had a higher ABA content than transgenic lines overexpressing CmWRKY15 under normal conditions and after exposure to A.tenuissima.These data indicate that the ABA content increased in response to A.tenuissima and that CmWRKYlS might inhibit endogenous ABA synthesis in transgenic plants.To identify the mechanisms by which CmWRKYl5 responds to A.tenuissima-induced stress,the expression levels of a set of ABA-related genes,including ABF4,ASI4,ABI5,RAB18,DREB1 A,DREB2A,PP2 C,SnRK2.2,SnRK2.3,RCARl,MYB2,PYL2,NCED3 A,NCED3Band GTG1,were compared between transgenic and WT plants.Following A.tenuissima exposure,the transcription of the ABA-upregulated geneswas impaired to varying degrees in CmWRKY15-overexpressing lines compared to the WT plants.In summary,our data suggest that CmWRKYlS might facilitate A.tenuissima infection by directly or indirectly antagonistically regulating the expression of ABA-responsive genes.
Chrysanthemum(Chrysanthemum morifolium) is one of the most famous cut flowers globally,has a high ornamental value,and occupies an irreplaceable position in the international commerce of flowers.Black spot disease,one of the most harmful diseases of chrysanthemum,is caused by the necrotrophic fungus Alternaria.Hence,it is essential to improve chrysanthemum tolerance to achieve sustainable production.In this study,we place emphasis on the mechanism through which CmWRKY modulates the ABA-mediated pathway in response toAltemariatenuissima in chrysanthemum.The main results are as follows:Phylogenetic analysis showed that CmWRKY15 showed high similarity to AtWRKY40 in Arabidopsis.We used transgenic chrysanthemum lines overexpressing CmWRKY15,After exposure to A.tenuissima for 2 weeks,the surface area of the lesions on transgenic plants was much larger compared to the control.Significant differences in A.tenuissima infection were clearly observable between the transgenic lines and wild type plants.The disease severity indexes(DSIs) of transgenic chrysanthemum lines and wild type plants were 57.01,56.73 and 14.63,respectively.The data revealed that the transgenic lines overexpressing CmWRKYXS were susceptible(S) to black spot disease,while the non-transgenic plants were resistant(R).Necrosis was much more evident in the transgenic plants compared to the WT plants.Our results suggest that CmWRKYlS overexpression enhanced the susceptibility of chrysanthemum to A.tenuissima attack.Endogenous ABA content was measured in the transgenic lines and wild type plants under normal conditions and 24 h after exposure to A.tenuissima.The results show that WT plants had a higher ABA content than transgenic lines overexpressing CmWRKY15 under normal conditions and after exposure to A.tenuissima.These data indicate that the ABA content increased in response to A.tenuissima and that CmWRKYlS might inhibit endogenous ABA synthesis in transgenic plants.To identify the mechanisms by which CmWRKYl5 responds to A.tenuissima-induced stress,the expression levels of a set of ABA-related genes,including ABF4,ASI4,ABI5,RAB18,DREB1 A,DREB2A,PP2 C,SnRK2.2,SnRK2.3,RCARl,MYB2,PYL2,NCED3 A,NCED3Band GTG1,were compared between transgenic and WT plants.Following A.tenuissima exposure,the transcription of the ABA-upregulated geneswas impaired to varying degrees in CmWRKY15-overexpressing lines compared to the WT plants.In summary,our data suggest that CmWRKYlS might facilitate A.tenuissima infection by directly or indirectly antagonistically regulating the expression of ABA-responsive genes.
引文
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2.Huang S.,Gao Y.,Liu J.,Peng X.,Niu X.,Fei Z.,Cao S.,Liu Y.(2012)Genome-wide analysis of WRKY transcription factors in Solarium lycopersicum[J].Molecular Genetics and Genomics,287(6),495-513.
3.Fan X.,Guo Q.,Xu P.,Gong Y.,Shu H.,Yang Y.,Ni W.,Zhang X.,Shen X.(2015)Transcriptome-wide identification of salt-responsive members of the WRKY gene family in Gossypium aridum[J].PLoS One 10:e0 126148.
4.Gonzalez-Guzman M.,Pizzio GA.,Antoni R.,Vera-Sirera F.,Merilo E.,BasselGW.,Fernandez MA.,Holdsworth MJ.,Perez-Amador MA.,Kollist H.,Rodriguez PL(2012)ArabidopsisPYR/PYL/RCAR receptors play a major role in quantitative regulation of stomatal aperture and transcriptional response to abscisic acid[J].The Plant Cell 24:2483-2496.
5.Ishiguro S.,Nakamura K.(1994)Characterization of a cdnaencoding a novel dna-binding protein.,SPF1.,that recognizes SP8 sequences in the 5'upstream regions of genes-coding for sporamin and beta-amylase from sweet-potato[J].Molecular&General Genetics 244:563-571.
6.Jalali B.,Bhargava S.,Kamble A.(2006)Signal transduction and transcriptional regulation of plant defence responses[J].Journal Phytopathol 154(2):65-74.
7.Larkin MA.,Blackshields G.,Brown NP.,Chenna R.,McGettigan PA.,McWilliam H.,Valentin F.,WallacelM.,Wilm A.,Lopez R.,Thompson JD.,Gibson TJ.,Higgins DG.(2007)Clustal Wand clustal X version 2.0[J].Bioinformatics 23:2947-2948.
8.Li P.,Song A.,Gao C.,Wang L,Wang Y.,Sun J.,Jiang J.,Chen F.,Chen S.(2015)Chrysanthemum WRKY gene CmWRKYH negatively regulates salt stress tolerance in transgenic chrysanthemum and Arabidopsis plants[J].Plant Cell Reports 34:1365-1378.
9.Livak KJ.,Schmittgen TD.(2001)Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods 25:402-408.
10.Pandey S.,Nelson D C.,Assmann S M.(2009)Two novel GPCR-type G proteins are abscisic acidreceptors in Arabidopsis[J].Celll36:136-148.
11.Pandey SP.,Somssich IE.(2009)The role of WRKY transcription factors in plant immunity[J].Plant Physiology150:1648-1655.
12.Rushton DL,Tripathi P.,Rabara RC.,Lin J.,Ringler P.,Boken AK.,LangumTJ.,SmidtL.,BoomsmaDD.,Emme NJ.,Chen X.,Finer JJ.,Shen,QJ.,RushtonPJ.(2012)WRKY transcription factors;key components in abscisic acid signalling[J].Plant Biotechnology Journal 10:2-11.
13.Rushton PJ.,Torres JT.,Parniske M.,Wernert P.,Hahlbrock K.,Somssich I.(1996)Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes[J].EMBO Journal15(20):5690.
14.Tamura K.,Stecher G.,Peterson D.,Filipski A.,Kumar S.(2013)MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution 30:2725-2729.
15.Shang Y.,Yan L.,Liu ZQ.,Cao Z.,MeiC.,XinQ.,WuFQ.,WangXF.,DuSY.,JiangT.,Zhang XF.,ZhaoR.,Sun HL,Liu R.,YuYT.,Zhang DP.(2010)The Mg-chelatase H subunit of Arabidopsis antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition[J].The Plant Cell 22:1909-1935.
16.Song A.,Zhu X.,Chen F.,Gao H.,Jiang J.,Chen S.(2014)A chrysanthemum heat shock protein confers tolerance to abiotic stress[J].International Journal Molecular Science 15:5063-5078.
17.(U|¨)lker B.,Somssich IE.(2004)WRKY transcription factors:from DNA binding towards biological function[J].Current Opinion Plant Biology 7(5):491-498.
18.Vogel JT.,Zarka DG.,Van Buskirk HA.,Fowler SG.,Thomashow MF.(2005)Roles of the CBF2 andZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis[J].Plant Journal 41:195-211.
19.Wang X.,Yan Y.,Li Y.,Chu X.,Wu C.,Guo X.(2014)GhWRKY40,a multiple stress-responsive cotton wrky gene,plays an important role in the wounding response and enhances susceptibility to Ralstonia salanacearum infection in transgenic Niotiana benthamiana[J].PLoS One 9.
20.Wang Q.,Wang M.,Zhang X.,Hao B.,Kaushik SK.,Pan Y.(2011)WRKY gene family evolution in Arabidopsis thaliana[J].Genetica 139:973-983.
21.Xu G.,Liu Y.,Chen S.,Chen F.(2011)Potential structural and biochemical mechanisms of compositae wild species resistance to Altemaria tenuissima[J].Russian Journal of Plant Physiology 58:491-497.
22.Yang B.,Jiang Y.,Rahman MH.,Deyholos MK.,Kav NN.(2009)Identification and expression analysis of WRKY transcription factor genes in canola(Brassica napus L.)in response to fungal pathogens and hormone treatments[J].BMC Plant Biology 9:68.
23.陈远平,杨文钰.2005.卵叶韭休眠芽中GA3、IAA、ABA和ZT的高效液相色谱法测定[J].四川农业大学学报,4:498-500.
24.李会云.2014.菊花黑斑病抗性鉴定及抗黑斑病分子机理研究[D].南京农业大学博士论文.
25.许高娟(2009)部分菊花近缘种属植物黑斑病苗期抗性及hrfa基因转化菊花的研究[D].南京农业大学博士论文.
26.张国斌,张喜贤,王云月,杨红玉.(2013)拟南芥灰霉病抗性相关转录因子[J].遗传,35(8):971-982.
2.Huang S.,Gao Y.,Liu J.,Peng X.,Niu X.,Fei Z.,Cao S.,Liu Y.(2012)Genome-wide analysis of WRKY transcription factors in Solarium lycopersicum[J].Molecular Genetics and Genomics,287(6),495-513.
3.Fan X.,Guo Q.,Xu P.,Gong Y.,Shu H.,Yang Y.,Ni W.,Zhang X.,Shen X.(2015)Transcriptome-wide identification of salt-responsive members of the WRKY gene family in Gossypium aridum[J].PLoS One 10:e0 126148.
4.Gonzalez-Guzman M.,Pizzio GA.,Antoni R.,Vera-Sirera F.,Merilo E.,BasselGW.,Fernandez MA.,Holdsworth MJ.,Perez-Amador MA.,Kollist H.,Rodriguez PL(2012)ArabidopsisPYR/PYL/RCAR receptors play a major role in quantitative regulation of stomatal aperture and transcriptional response to abscisic acid[J].The Plant Cell 24:2483-2496.
5.Ishiguro S.,Nakamura K.(1994)Characterization of a cdnaencoding a novel dna-binding protein.,SPF1.,that recognizes SP8 sequences in the 5'upstream regions of genes-coding for sporamin and beta-amylase from sweet-potato[J].Molecular&General Genetics 244:563-571.
6.Jalali B.,Bhargava S.,Kamble A.(2006)Signal transduction and transcriptional regulation of plant defence responses[J].Journal Phytopathol 154(2):65-74.
7.Larkin MA.,Blackshields G.,Brown NP.,Chenna R.,McGettigan PA.,McWilliam H.,Valentin F.,WallacelM.,Wilm A.,Lopez R.,Thompson JD.,Gibson TJ.,Higgins DG.(2007)Clustal Wand clustal X version 2.0[J].Bioinformatics 23:2947-2948.
8.Li P.,Song A.,Gao C.,Wang L,Wang Y.,Sun J.,Jiang J.,Chen F.,Chen S.(2015)Chrysanthemum WRKY gene CmWRKYH negatively regulates salt stress tolerance in transgenic chrysanthemum and Arabidopsis plants[J].Plant Cell Reports 34:1365-1378.
9.Livak KJ.,Schmittgen TD.(2001)Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods 25:402-408.
10.Pandey S.,Nelson D C.,Assmann S M.(2009)Two novel GPCR-type G proteins are abscisic acidreceptors in Arabidopsis[J].Celll36:136-148.
11.Pandey SP.,Somssich IE.(2009)The role of WRKY transcription factors in plant immunity[J].Plant Physiology150:1648-1655.
12.Rushton DL,Tripathi P.,Rabara RC.,Lin J.,Ringler P.,Boken AK.,LangumTJ.,SmidtL.,BoomsmaDD.,Emme NJ.,Chen X.,Finer JJ.,Shen,QJ.,RushtonPJ.(2012)WRKY transcription factors;key components in abscisic acid signalling[J].Plant Biotechnology Journal 10:2-11.
13.Rushton PJ.,Torres JT.,Parniske M.,Wernert P.,Hahlbrock K.,Somssich I.(1996)Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes[J].EMBO Journal15(20):5690.
14.Tamura K.,Stecher G.,Peterson D.,Filipski A.,Kumar S.(2013)MEGA6:molecular evolutionary genetics analysis version 6.0[J].Molecular Biology and Evolution 30:2725-2729.
15.Shang Y.,Yan L.,Liu ZQ.,Cao Z.,MeiC.,XinQ.,WuFQ.,WangXF.,DuSY.,JiangT.,Zhang XF.,ZhaoR.,Sun HL,Liu R.,YuYT.,Zhang DP.(2010)The Mg-chelatase H subunit of Arabidopsis antagonizes a group of WRKY transcription repressors to relieve ABA-responsive genes of inhibition[J].The Plant Cell 22:1909-1935.
16.Song A.,Zhu X.,Chen F.,Gao H.,Jiang J.,Chen S.(2014)A chrysanthemum heat shock protein confers tolerance to abiotic stress[J].International Journal Molecular Science 15:5063-5078.
17.(U|¨)lker B.,Somssich IE.(2004)WRKY transcription factors:from DNA binding towards biological function[J].Current Opinion Plant Biology 7(5):491-498.
18.Vogel JT.,Zarka DG.,Van Buskirk HA.,Fowler SG.,Thomashow MF.(2005)Roles of the CBF2 andZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis[J].Plant Journal 41:195-211.
19.Wang X.,Yan Y.,Li Y.,Chu X.,Wu C.,Guo X.(2014)GhWRKY40,a multiple stress-responsive cotton wrky gene,plays an important role in the wounding response and enhances susceptibility to Ralstonia salanacearum infection in transgenic Niotiana benthamiana[J].PLoS One 9.
20.Wang Q.,Wang M.,Zhang X.,Hao B.,Kaushik SK.,Pan Y.(2011)WRKY gene family evolution in Arabidopsis thaliana[J].Genetica 139:973-983.
21.Xu G.,Liu Y.,Chen S.,Chen F.(2011)Potential structural and biochemical mechanisms of compositae wild species resistance to Altemaria tenuissima[J].Russian Journal of Plant Physiology 58:491-497.
22.Yang B.,Jiang Y.,Rahman MH.,Deyholos MK.,Kav NN.(2009)Identification and expression analysis of WRKY transcription factor genes in canola(Brassica napus L.)in response to fungal pathogens and hormone treatments[J].BMC Plant Biology 9:68.
23.陈远平,杨文钰.2005.卵叶韭休眠芽中GA3、IAA、ABA和ZT的高效液相色谱法测定[J].四川农业大学学报,4:498-500.
24.李会云.2014.菊花黑斑病抗性鉴定及抗黑斑病分子机理研究[D].南京农业大学博士论文.
25.许高娟(2009)部分菊花近缘种属植物黑斑病苗期抗性及hrfa基因转化菊花的研究[D].南京农业大学博士论文.
26.张国斌,张喜贤,王云月,杨红玉.(2013)拟南芥灰霉病抗性相关转录因子[J].遗传,35(8):971-982.