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High-throughput screening system of citrus bacterial cankerassociated transcription factors and its application to the regulation of citrus canker resistance

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Journal of Integrative Agriculture 2024年第1期23卷 155-165页

作者： Jia Fu Jie Fan chenxi Zhang Yongyao Fu Baohang Xian Qiyuan Yu Xin Huang Wen Yang shanchun chen Yongrui He Qiang Li Integrative Science Center of Germplasm Creation in Western China(Chongqing)Science City Citrus Research InstituteSouthwest UniversityChongqing 400712China School of Advanced Agriculture and Bioengineering Yangtze Normal UniversityChongqing 408100China National Citrus Engineering Research Center Chongqing 400712China

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker(CBC),caused by the bacteria Xanthomonas citri ***(Xcc).Response to CBC is a complex process,with both proteinDNA as well as protein–protein interactions for the regulatory *** detect such interactions in CBC resistant regulation,a citrus high-throughput screening system with 203 CBC-inducible transcription factors(TFs),were *** the upstream regulators of target by yeast-one hybrid(Y1H)methods was also performed.A regulatory module of CBC resistance was identified based on this *** TF(CsDOF5.8)was explored due to its interactions with the 1-kb promoter fragment of CsPrx25,a resistant gene of CBC involved in reactive oxygen species(ROS)homeostasis *** mobility shift assay(EMSA),dual-LUC assays,as well as transient overexpression of CsDOF5.8,further validated the interactions and transcriptional *** CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via *** high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance *** addition,it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

关键词： citrus bacterial canker(CBC) high-throughput screening system transcription factor(TF) yeast-one hybrid(Y1H) CsPrx25

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茉莉酸在植物抗逆性中的研究进展

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生物工程学报 2024年第1期40卷 15-34页

作者：张乐欢邹昌玉朱天翔杜美霞邹修平何永睿陈善春龙琴西南大学中国农业科学院柑桔研究所重庆400712

茉莉酸(jasmonic acid,JA)是一种植物内源合成的脂类激素,在植物响应胁迫的调控中发挥着重要作用。本文概括了JA的生物合成与代谢途径及其调控机制;总结了JA信号的传导通路;系统归纳了JA在植物响应生物和非生物胁迫应答中的作用机制和... 详细信息

茉莉酸(jasmonic acid,JA)是一种植物内源合成的脂类激素,在植物响应胁迫的调控中发挥着重要作用。本文概括了JA的生物合成与代谢途径及其调控机制;总结了JA信号的传导通路;系统归纳了JA在植物响应生物和非生物胁迫应答中的作用机制和调控网络,重点关注了最新的研究进展。此外,本文梳理了JA与其他植物激素在植物抗逆性调节过程中的信号交流。最后讨论了JA信号通路介导的植物抗逆性研究中亟待解决的问题,并展望了新的分子生物学技术在调控JA信号通路增强作物抗性中的应用前景,以期为植物的抗逆性研究和改良提供参考。

关键词：茉莉酸生物胁迫非生物胁迫植物抗逆性

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Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system

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Horticultural Plant Journal 2023年第3期9卷 473-480页

作者： Yongrui He Lanzhen Xu Aihong Peng Tiangang Lei Qiang Li Lixiao Yao Guojin Jiang shanchun chen Zhengguo Li Xiuping Zou Key Laboratory of Plant Hormones and Development Regulation of Chongqing School of Life SciencesChongqing UniversityChongqing 401331China National Citrus Engineering Research Center(NCERC) Citrus Research InstituteSouthwest University/Chinese Academy of Agricultural SciencesChongqing 400712China

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciensmediated transformation for generation of marker-free transgenic plants from navel orange(Citrus sinensis Osbeck) mature stems using a CreloxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1-0.5(1-2 weeks), > 0.5 cm(3-4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange(Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase(IPT) and an anti-bacterial peptide(PR1aCB).Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were *** blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants(deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange(Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

关键词： Citrus sinensis Osbeck Mature stem segment Marker-free Transgene Bud micrografting

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Comparative analysis of Wanjincheng orange leaf and root responses to ‘Candidatus liberibacter asiaticus’ infection using leaf-disc grafting

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Horticultural Plant Journal 2021年第5期7卷 401-410页

作者： Zhu Xie Ke Zhao Junhong Long Lin Zhen Xiuping Zou shanchun chen Chinese Academy of Agricultural Sciences and National Center for Citrus Variety Improvement Citrus Research InstituteSouthwest UniversityChongqing 400716China

Citrus Huanglongbing,associated with Candidatus Liberibacter asiaticus(Las),is the serious disease of citrus ***,we compared differences between leaf and root responses in Wanjincheng Orange(Citrus sinensis Osbeck)to Las infection using leaf-disc *** had no obvious symptoms in the first two months after grafting(MAG),but yellowing leaves,thickened midribs and decayed roots began to appear at 6 *** Las growth rate in roots was greater than inmidribs from 2 to 6 MAG;however,by 10 MAG,it was significantly lower in roots than in *** analysis revealed that starch accumulation,callose deposition,cellwall thickness and the number of cell layers increased in the phloem of infected *** Las infection,the starch content in the leaves was significantly higher than that in the roots,while the callose deposition in the midribs was 3–19 times that in the roots.A gene expression analysis showed that transcripts of callose-and starch metabolism-related genes were obviously affected by Las infection,and the pathogenesis-related genes PR1,PR2 and PR5 were significantly upregulated and downregulated in midrib and root,*** results indicated that the PR-mediated resistance response was repressed in root but activated in midrib,which may result in the more rapid Las growth in roots than in midribs during the early infection stage.

关键词： Huanglongbing Leaf-disc grafting Starch Ultrathin sections qPCR Callose

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CsPrx25,a class III peroxidase in Citrus sinensis,confers resistance to citrus bacterial canker through the maintenance of ROS homeostasis and cell wall lignification

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Horticulture Research 2020年第1期7卷 130-140页

作者： Qiang Li Xiujuan Qin Jingjing Qi Wanfu Dou Christophe Dunand shanchun chen Yongrui He Citrus Research Institute Southwest University/Chinese Academy of Agricultural SciencesChongqing 400712China Laboratoire de Recherche en Sciences Végétales Universitéde ToulouseCNRSUPSAuzeville-Tolosane 31320France Key Laboratory of Plant Hormones and Development Regulation of Chongqing School of Life SciencesChongqing University401331 ChongqingChina

Citrus bacterial canker(CBC)results from Xanthomonas citri ***(Xcc)infection and poses a grave threat to citrus *** III peroxidases(CIII Prxs)are key proteins to the environmental adaptation of citrus plants to a range of exogenous pathogens,but the role of CIII Prxs during plant resistance to CBC is poorly ***,we explored the role of CsPrx25 and its contribution to plant defenses in molecular *** on the expression analysis,CsPrx25 was identified as an apoplast-localized protein that is differentially regulated by Xcc infection,salicylic acid,and methyl jasmone acid in the CBC-susceptible variety Wanjincheng(***)and the CBC-resistant variety Calamondin(***).Transgenic Wanjincheng plants overexpressing CsPrx25 were generated,and these transgenic plants exhibited significantly increased CBC resistance compared with the WT *** addition,the CsPrx25-overexpressing plants displayed altered reactive oxygen species(ROS)homeostasis accompanied by enhanced H_(2)O_(2) levels,which led to stronger hypersensitivity responses during Xcc ***,the overexpression of CsPrx25 enhanced lignification as an apoplastic barrier for Xcc *** together,the results highlight how CsPrx25-mediated ROS homeostasis reconstruction and cell wall lignification can enhance the resistance of sweet orange to CBC.

关键词： sinensis citrus Citrus

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CsWAKL08,a pathogen-induced wall-associated receptor-like kinase in sweet orange,confers resistance to citrus bacterial canker via ROS control and JA signaling

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Horticulture Research 2020年第1期7卷 2053-2067页

作者： Qiang Li Anhua Hu Jingjing Qi Wanfu Dou Xiujuan Qin Xiuping Zou Lanzhen Xu shanchun chen Yongrui He Citrus Research Institute Southwest University/Chinese Academy of Agricultural SciencesChongqing 400712China

Citrus bacterial canker(CBC)is a disease resulting from Xanthomonas citri ***(Xcc)infection and poses a grave threat to citrus production ***-associated receptor-like kinases(WAKLs)are proteins with a central role in resisting a range of fungal and bacterial *** roles of WAKLs in the context of CBC resistance,however,remain ***,we explored the role of CsWAKL08,which confers resistance to CBC,and we additionally analyzed the molecular mechanisms of CsWAKL08-mediated CBC *** on systematic annotation and induced expression analysis of the CsWAKL family in Citrus sinensis,CsWAKL08 was identified as a candidate that can be upregulated by Xcc infection in the CBC-resistant ***08 can also be induced by the phytohormones salicylic acid(SA)and methyl jasmonic acid(MeJA)and spans the plasma *** of CsWAKL08 resulted in strong CBC resistance in transgenic sweet oranges,whereas silencing of CsWAKL08 resulted in susceptibility to *** peroxidase(POD)and superoxide dismutase(SOD)activities were significantly enhanced in the CsWAKL08-overexpressing plants compared to the control plants,thereby mediating reactive oxygen species(ROS)homeostasis in the transgenic ***,the JA levels and the expression of JA biosynthesis and JA responsive genes were substantially elevated in the CsWAKL08 overexpression plants relative to the controls upon Xcc *** on these findings,we conclude that the wall-associated receptor-like kinase CsWAKL08 positively regulates CBC resistance through a mechanism involving ROS control and JA *** results further highlight the importance of this kinase family in plant pathogen resistance.

关键词： resistance orange citrus

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CsBZIP40 confers resistance against citrus bacterial canker by repressing CsWRKY43-CsPrx53/CsSOD13 cascade mediated ROS scavenging

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Horticulture Research 2023年第8期10卷 265-279页

作者： Qiang Li Xiujuan Qin Miao Zhang Qiyuan Yu Ruirui Jia Jie Fan Xin Huang Jia Fu chenxi Zhang Baohang Xian Wen Yang Qin Long Aihong Peng Lixiao Yao shanchun chen Yongrui He Citrus Research Institute Southwest University/Chinese Academy of Agricultural SciencesBeibeiChongqing 400712China National Citrus Engineering Research Center BeibeiChongqing 400712China National Citrus Improvement Center Southwest UniversityChongqing 400712China

As the bacterial etiologic agent causing citrus bacterial canker(CBC),Xanthomonas citri ***(Xcc)seriously impacts citrus plantation and fruit production *** an earlier study,we demonstrated that CsBZIP40 can positively impact CBC resistance in the sweet orange(Citrus sinensis).However,the mechanistic basis for the protective benefits conferred by CsBZIP40 is yet to be ***,we show that CsBZIP40 positively regulates CBC resistance and reactive oxygen species(ROS)homeostasis in transgenic sweet orange overexpressing ***40 directly binds to the TGA-box of the CsWRKY43 promoter to repress its transcriptional ***43 overexpression induces CBC susceptibility in transgenic sweet *** contrast,its inhibition produces strong resistance to ***43 directly binds to the W-boxes of the CsPrx53 and CsSOD13 promoters to positively regulate the activities of these antioxidant enzymes,resulting in the negative regulation of ROS homeostasis and CBC resistance in sweet orange ***53/CsSOD13 knockdown enhances ROS accumulation and CBC ***,our results outline a regulatory pathway through which CsBZIP40 transcriptionally represses CsWRKY43-CsPrx53/CsSOD13 cascade-mediated ROS scavenging in a manner conducive to CBC *** mechanisms underscore the potential importance of CsBZIP40,CsWRKY43,CsPrx53,and CsSOD13,providing promising strategies for the prevention of CBC.

关键词： citrus resistance orange

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CitGVD:a comprehensive database of citrus genomic variations

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Horticulture Research 2020年第1期7卷 2529-2536页

作者： Qiang Li Jingjing Qi Xiujuan Qin Wanfu Dou Tiangang Lei Anhua Hu Ruirui Jia Guojin Jiang Xiuping Zou Qin Long Lanzhen Xu Aihong Peng Lixiao Yao shanchun chen Yongrui He Citrus Research Institute Southwest University/Chinese Academy of Agricultural Sciences400712 ChongqingChina Key Laboratory of Plant Hormones and Development Regulation of Chongqing School of Life SciencesChongqing University401331 ChongqingChina

Citrus is one of the most important commercial fruit crops *** the vast genomic data currently available for citrus fruit,genetic relationships,and molecular markers can be assessed for the development of molecular breeding and genomic selection *** this study,to permit the ease of access to these data,a web-based database,the citrus genomic variation database(CitGVD,http://***/home)was developed as the first citrusspecific comprehensive database dedicated to genome-wide variations including single nucleotide polymorphisms(SNPs)and insertions/deletions(INDELs).The current version(V1.0.0)of CitGVD is an open-access resource centered on 1,493,258,964 high-quality genomic variations and 84 phenotypes of 346 organisms curated from in-house projects and public *** integrates closely related information on genomic variation annotations,related gene annotations,and details regarding the organisms,incorporating a variety of built-in tools for data accession and *** an example,CitGWAS can be used for genome-wide association studies(GWASs)with SNPs and phenotypic data,while CitEVOL can be used for genetic structure *** features make CitGVD a comprehensive web portal and bioinformatics platform for citrus-related *** also provides a model for analyzing genome-wide variations for a wide range of crop varieties.

关键词： database analysis comprehensive

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柑橘CsMYB41和CsMYB63响应溃疡病菌侵染的表达

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园艺学报 2023年第3期50卷 495-507页

作者：刘语诺曹亚王帅杜美霞郑林陈善春邹修平西南大学/中国农业科学院柑桔研究所国家柑桔工程技术研究中心国家柑桔品种改良中心重庆400712

MYB转录因子不仅在植物生长发育中起着重要作用,且在植物抗病反应中承担着重要的调节功能。本研究中以溃疡病感病品种纽荷尔脐橙(Citrus sinensis Osbeck)和抗病品种四季橘(***)为材料,基于前期构建的溃疡病诱导柑橘转录组数据库,筛选获... 详细信息

MYB转录因子不仅在植物生长发育中起着重要作用,且在植物抗病反应中承担着重要的调节功能。本研究中以溃疡病感病品种纽荷尔脐橙(Citrus sinensis Osbeck)和抗病品种四季橘(***)为材料,基于前期构建的溃疡病诱导柑橘转录组数据库,筛选获得2个受柑橘溃疡病菌Xanthomonas citri ***(Xcc)显著诱导的MYB基因:CsMYB41(Cs1g06220)和CsMYB63(Cs4g12760)。利用PCR技术克隆了纽荷尔脐橙的CsMYB41和CsMYB63,并对其结构特征及表达特性进行分析。PCR克隆测序分析发现,这2个基因的全长分别为1304 bp、1398 bp,开放阅读框(ORF)为1047 bp、1170 bp,各编码349、390个氨基酸。蛋白质同源序列比对和结构分析表明,这2个基因属于MYB类转录因子家族中的R2R3-MYB亚家族;进化树分析表明,CsMYB41和CsMYB63蛋白分别与可可、蓖麻等MYB41和MYB63蛋白高度同源。亚细胞定位结果显示,CsMYB41和CsMYB63定位在细胞核中。实时荧光定量PCR(qRT-PCR)分析表明,CsMYB41和CsMYB63均受Xcc诱导显著上调表达。激素诱导试验表明,在纽荷尔脐橙中,乙烯利(ETH)、水杨酸(SA)、脱落酸(ABA)诱导CsMYB41上调表达,而ETH、SA、茉莉酸(JA)、ABA诱导CsMYB63上调表达。在四季橘中,CsMYB41受ETH、SA诱导上调表达,CsMYB63仅受IAA诱导上调表达。以上结果表明,这2个MYB基因可能在应答柑橘溃疡病菌侵染中起重要作用。

关键词：柑橘溃疡病 MYB转录因子 qRT-PCR

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柑橘响应黄龙病菌侵染的NAC基因的克隆及表达分析

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园艺学报 2022年第7期49卷 1441-1457页

作者：郑林王帅刘语诺杜美霞彭爱红何永睿陈善春邹修平西南大学/中国农业科学院柑桔研究所重庆400712

挖掘柑橘抗黄龙病(Huanglongbing,HLB)基因是抗病育种的基础和关键。以感染黄龙病菌亚洲种Candidatus Liberibacter asiaticus(CLas)早期(2个月)锦橙根和叶片中脉比较转录组数据为基础,筛选到9个响应柑橘黄龙病侵染诱导的NAC基因,从中选... 详细信息

挖掘柑橘抗黄龙病(Huanglongbing,HLB)基因是抗病育种的基础和关键。以感染黄龙病菌亚洲种Candidatus Liberibacter asiaticus(CLas)早期(2个月)锦橙根和叶片中脉比较转录组数据为基础,筛选到9个响应柑橘黄龙病侵染诱导的NAC基因,从中选3个差异表达水平较高的基因克隆,分别命名为Cs NAC21/22、Cs NAC68和Cs NAC78。生物信息分析表明3个基因均符合NAC基因家族的特征;烟草亚细胞定位结果表明,Cs NAC68定位在细胞核,Cs NAC21/22和Cs NAC78定位在细胞核和细胞质。实时荧光定量PCR(q RT-PCR)分析表明,3个候选基因在易感HLB的锦橙、耐病的马蜂柑和高耐病的九里香的组织和病原菌诱导表达特征呈现明显差异。以健康植株为对照,Cs NAC68和Cs NAC78主要在锦橙的根中响应CLas感染,显著上调表达,Cs NAC68在九里香叶肉和马蜂柑根中响应CLas感染,显著上调表达;Cs NAC21/22主要在锦橙根和马蜂柑叶肉中显著下调表达。以‘锦橙’叶片为试材通过q RT-PCR分析候选基因响应SA、JA、ABA、ETH诱导的表达特征,结果表明,3个基因可能参与ABA的信号转导途径,Cs NAC68可能参与SA和JA的信号转导途径,而Cs NAC78可能参与ETH的信号转导途径。

关键词：柑橘黄龙病 NAC转录因子基因克隆表达分析

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