马蜂柑响应黄龙病菌不同侵染时期的生物学和转录组学分析

doi:10.3864/j.issn.0578-1752.2020.07.007

摘要/Abstract

摘要： 【目的】 柑橘黄龙病（Huanglongbing,HLB）是制约柑橘产业发展的重大病害,我国发生的黄龙病由韧皮部杆菌亚洲种（‘Candidatus Liberibacter asiaticus’,CLas）引起。本研究通过分析潜在耐黄龙病柑橘品种马蜂柑（Citrus hystrix）在感染黄龙病后不同时期的生物学症状、显微结构和转录组学差异,揭示马蜂柑不同时期对黄龙病菌具有耐性的分子机理,为进一步深入挖掘抗性基因及开展抗病育种打下基础。 【方法】 以嫁接在两年生卡里佐枳橙砧木上的马蜂柑作为供试材料,使用只感染CLas、其他常见柑橘病毒类病原均呈阴性的毒源为接毒材料对马蜂柑进行接种,并在接种毒源后每隔15 d进行定期的荧光定量PCR检测。将马蜂柑接种毒源4个月（最早检测到CLas阳性）作为感病前期处理,接种毒源14个月作为感病后期处理,以健康植株为对照,进行生物学症状和显微结构观察,分析其感病后不同时期的结构变化。结合比较转录组学分析与荧光定量PCR验证,解析其耐病相关机制。 【结果】 症状观察发现,马蜂柑在感病前期和感病后期,植株叶片几乎不显症;显微结构观察表明,马蜂柑在感病前期中脉组织结构清晰,细胞形态正常,无淀粉粒积累的现象,仅在后期出现韧皮部极少数的筛管被堵塞;对转录组测序结果进行筛选鉴定,马蜂柑感病前期共筛选鉴定到181个差异表达基因,感病后期共筛选鉴定到1 384个差异表达基因;比较转录组分析表明,马蜂柑感病前期和后期的差异表达基因主要涉及细胞壁代谢、防御反应、淀粉与蔗糖代谢、胼胝质合成以及信号转导等方面,其中在感病前期,淀粉与蔗糖代谢、细胞壁代谢相关的调控基因下调表达,在感病后期,水杨酸代谢及其信号转导途径、病程相关蛋白和谷胱甘肽转移酶相关的调控基因上调表达。 【结论】 马蜂柑响应CLas早期侵染主要表现为物理结构稳定、淀粉合成和光合作用等途径不受干扰;而水杨酸介导的抗性信号转导、效应蛋白触发免疫反应（effector-triggered immunity,ETI）激活的病程相关蛋白和谷胱甘肽转移酶参与的解毒作用是感病后期的主要耐病机制。

关键词: 柑橘黄龙病, 韧皮部杆菌亚洲种, 马蜂柑, 耐病性, 转录组

Abstract: 【Objective】 Citrus Huanglongbing (HLB), associated with phloem-colonized ‘Candidatus Liberibacter asiaticus’ (CLas), severely impedes worldwide citrus production. The objective of this study is to analyze the biological symptoms, microstructures and transcriptomes of Citrus hystrix in response to CLas infection at different stages, to reveal the tolerance mechanism of C. hystrix, and to provide a basis for further screening of resistance genes and HLB-tolerant/resistant citrus breeding. 【Method】| The budwoods of C. hystrix grafted on two-year-old Carrizo citrange rootstocks (C. sinensis× P. trifoliata) used in this study were graft-inoculated with budwoods from a CLas (strain GZBJT)-infected Guanximiyou pummelo maintained in a greenhouse at Citrus Research Institute. The budwoods used as inoculum were tested CLas positive and free of other potential phloem-limited pathogens, such as Citrus tristeza virus (CTV) or Citrus tatter leaf virus (CTLV) by PCR before grafting. Inoculated plants were kept in greenhouse along with mock-inoculated healthy control plants. Real-time quantitative PCR (qPCR) was performed every 15 days after inoculation. Four months after inoculation (the earliest establishment of CLas by qPCR) and 14 months after inoculation were defined as the early infection stage and the late infection stage, respectively. The biological symptoms and microstructures were observed to analyze the structural changes of different infection stages. Combined with comparative transcriptome and RT-qPCR validation, the response mechanism of C. hystrix against HLB was explored.【Result】 No typical symptom was observed in C. hystrix at the early and late stages of infection. Light microscopy observation from the midribs of HLB-affected and uninfected C. hystrix revealed that no significant structure change was found at the early infection stage and only a few sieves in the phloem were blocked at the late stage. By comparing the RNA-seq data, 181 and 1 384 genes were found to be differentially expressed at the early stage and late stage, respectively. Differentially expressed genes (DEGs) mainly involved in cell wall metabolism, host defense response, starch and sucrose metabolism, callose synthesis and signal transduction. Comparative transcriptome analysis showed that the expression of related genes in starch and sucrose metabolism and cell wall metabolism was down-regulated at the early infection stage, and the expression of related genes in salicylic acid metabolism, salicylic acid signal transduction pathway, pathogenesis-related protein and glutathione-S-transferases was up-regulated at the late infection stage.【Conclusion】 The early response of C. hystrix to CLas infection is mainly characterized by stable physical structure, undisturbed pathways such as starch synthesis and photosynthesis. Salicylic acid-mediated resistance signals, effector-triggered immunity (ETI), and glutathione-S-transferases mediated detoxification contribute to the tolerance of C. hystrix against CLas at the late infection stage.

Key words: citrus Huanglongbing, ‘Candidatus Liberibacter asiaticus’ (CLas);, Citrus hystrix, disease tolerance, transcriptome

滕彩玲,钟晰,吴昊娣,胡燕,周常勇,王雪峰. 马蜂柑响应黄龙病菌不同侵染时期的生物学和转录组学分析[J]. 中国农业科学, 2020, 53(7): 1368-1380.

CaiLing TENG,Xi ZHONG,HaoDi WU,Yan HU,ChangYong ZHOU,XueFeng WANG. Biologic and Transcriptomic Analysis of Citrus hystrix Responses to ‘Candidatus Liberibacter asiaticus’ at Different Infection Stages[J]. Scientia Agricultura Sinica, 2020, 53(7): 1368-1380.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2020.07.007

https://www.chinaagrisci.com/CN/Y2020/V53/I7/1368

图/表 6

表1

图1

图2

图3

图4

图5

参考文献 47

[1]	BOVÉ J M . Huanglongbing: A destructive, newly-emerging, century-old disease of citrus. Journal of Plant Pathology, 2006,88(1):7-37.
[2]	胡燕, 王雪峰, 周常勇 . 柑橘黄龙病菌亚洲种、虫媒及植物寄主互作研究进展. 园艺学报, 2016,43(9):1688-1698.
	HU Y, WANG X F, ZHOU C Y . Recent advances in interactions among ‘ Candidatus Liberibacter asiaticus’, insect vector and plant host. Acta Horticulturae Sinica, 2016,43(9):1688-1698. (in Chinese)
[3]	REINKING O A . Diseases of economic plants in Southern China. Philippine Agriculturist, 1919,8(4):109-134.
[4]	SCHNEIDER H . Anatomy of greening-diseased sweet orange shoots. Phytopathology, 1968,58(1):1155-1160.
[5]	ETXEBERRIA E, GONZALEZ P, ACHOR D, ALBRIGO G . Anatomical distribution of abnormally high levels of starch in HLB-affected Valencia orange trees. Physiological and Molecular Plant Pathology, 2009,74(1):76-83.
[6]	ALBRECHT U, BOWMAN K D. Transcriptional response of susceptible and tolerant citrus to infection with ‘Candidatus Liberibacter asiaticus’. Plant Science, 2012,185/186:118-130.
[7]	BOAVA L P, CRISTOFANI-YALY M, MACHADO M A . Physiologic, anatomic, and gene expression changes in Citrus sunki, Poncirus trifoliata and their hybrids after ‘Candidatus Liberibacter asiaticus’ infection. Phytopathology, 2017,107(5):590-599.
[8]	FOLIMONOVA S Y, ROBERTSON C J, GARNSEY S M, GOWDA S, DAWSON W O . Examination of the responses of different genotypes of citrus to Huanglongbing (citrus greening) under different conditions. Phytopathology, 2009,99(12):1346-1354.
[9]	SHOKROLLAH H, ABDULLAH T L, SIJAM K, ABDULLAH S N A, ABDULLAH N A P. Differential reaction of citrus species in Malaysia to Huanglongbing (HLB) disease using grafting method. American Journal of Agricultural and Biological Science, 2009,4(1):338.
[10]	MARTINELLI F, URATSU S L, ALBRECHT U, REAGAN R L, PHU M L, BRITTON M, BUFFALO V, FASS J, LEICHT E, ZHAO W, LIN D, D’SOUZA R, DAVIS C E, BOWMAN K D, DANDEKAR A M. Transcriptome profiling of citrus fruit response to Huanglongbing disease. PLoS ONE, 2012,7(5):e38039.
[11]	ALBRECHT U, BOWMAN K D . Gene expression in Citrus sinensis(L.) Osbeck following infection with the bacterial pathogen ‘Candidatus Liberibacter asiaticus’ causing Huanglongbing in Florida. Plant Science, 2008,175(3):291-306.
[12]	KIM J S, SAGARAM U S, BURNS J K, LI J L, WANG N . Response of sweet orange ( Citrus sinensis) to ‘Candidatus Liberibacter asiaticus’ infection: Microscopy and microarray analyses. Phytopathology, 2009,99(1):50-57.
[13]	FAN J, CHEN C, BRLANSKY R H, GMITTER F G, LI Z G . Changes in carbohydrate metabolism in Citrus sinensis infected with ‘Candidatus Liberibacter asiaticus’. Plant Pathology, 2010,59(6):1037-1043.
[14]	FAN J, CHEN C, YU Q, BRLANSKY R H, LI Z G, GMITTER F G . Comparative iTRAQ proteome and transcriptome analyses of sweet orange infected by ‘ Candidatus Liberibacter asiaticus’. Physiologia Plantarum, 2011,143(3):235-245.
[15]	ALBRECHT U, FIEHN O, BOWMAN K D . Metabolic variations in different citrus root stock cultivars associated with different responses to Huanglongbing. Plant Physiology and Biochemistry, 2016,107:33-44.
[16]	ARITUA V, ACHOR D, GMITTER F G, ALBRIGO G, WANG N . Transcriptional and microscopic analyses of citrus stem and root responses to ‘ Candidatus Liberibacter asiaticus’ infection. PLoS ONE, 2013,8(9):e73742.
[17]	MARTINELLI F, REAGAN R L, URATSU S L, PHU M L, ALBRECHT U, ZHAO W, DAVIS C E, BOWMAN K D, DANDEKAR A M . Gene regulatory networks elucidating Huanglongbing disease mechanisms. PLoS ONE, 2013,8(9):e74256.
[18]	ZHONG Y, CHENG C Z, JIANG B, JIANG N H, ZHANG Y Y, HU M L, ZHONG G Y . Digital gene expression analysis of Ponkan mandarin (Citrus reticulata Blanco) in response to Asia citrus psyllid-vectored Huanglongbing infection. International Journal of Molecular Sciences, 2016,17(7):1063.
[19]	FAN J, CHEN C, YU Q, KHALAF A, ACHOR D S, BRLANSKY R H, MOORE G A, LI Z G, GMITTER F G . Comparative transcriptional and anatomical analyses of tolerant rough lemon and susceptible sweet orange in response to ‘ Candidatus Liberibacter asiaticus’ infection. Molecular Plant-Microbe Interactions, 2012,25(11):1396-1407.
[20]	ZHONG Y, CHENG C Z, JIANG N H, JIANG B, ZHANG Y Y, WU B, HU M L, ZENG J W, YAN H X, YI G J, ZHONG G Y . Comparative transcriptome and iTRAQ proteome analyses of citrus root responses to ‘ Candidatus Liberibacter asiaticus’ infection. PLoS ONE, 2015,10(6):e0126973.
[21]	LIAO H L, BURNS J K . Gene expression in Citrus sinensis fruit tissues harvested from Huanglongbing-infected trees: Comparison with girdled fruit. Journal of Experimental Botany, 2012,63(8):3307-3319.
[22]	DUAN Y P, ZHOU L J, HALL D G, LI W B, DODDAPANENI H, LIN H, LIU L, VAHLING C M, GABRIEL D W, WILLIAMS K P, DICKERMAN A, SUN Y J, COTTWALD T . Complete genome sequence of citrus Huanglongbing bacterium, ‘ Candidatus Liberibacter asiaticus’ obtained through metagenomics. Molecular Plant-Microbe Interactions, 2009,22(8):1011-1020.
[23]	LU H, ZHANG C, ALBRECHT U, SHIMIZU R, WANG G, BOWMAN K D . Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis. Frontiers in Plant Science, 2013,4:157.
[24]	ROSALES R, BURNS J K . Phytohormone changes and carbohydrate status in sweet orange fruit from Huanglongbing-infected trees. Journal of Plant Growth Regulation, 2011,30(3):312-321.
[25]	NEHELA Y, HIJAZ F, ELZAAWELY A A, EL-ZAHABY H M, KILLINY N. Citrus phytohormonal response to ‘ Candidatus Liberibacter asiaticus’ and its vector Diaphorina citri. Physiological and Molecular Plant Pathology, 2018,102:24-35.
[26]	HU Y, ZHONG X, LIU X L, LOU B H, ZHOU C Y, WANG X F . Comparative transcriptome analysis unveils the tolerance mechanisms of Citrus hystrix in response to ‘Candidatus Liberibacter asiaticus’ infection. PLoS ONE, 2017,12(12):e0189229.
[27]	KOH E J, ZHOU L, WILLIAMS D S, PARK J, DING N, DUAN Y P, KANG B H . Callose deposition in the phloem plasmodesmata and inhibition of phloem transport in citrus leaves infected with ‘ Candidatus Liberibacter asiaticus’. Protoplasma, 2012,249(3):687-697.
[28]	FAN J, CHEN C, ACHOR D S, BRLANSKY R H, LI Z G, GMITTER F G . Differential anatomical responses of tolerant and susceptible citrus species to the infection of ‘ Candidatus Liberibacter asiaticus’. Physiological and Molecular Plant Pathology, 2013,83:69-74.
[29]	DENG H, ACHOR D S, EXTEBERRIA E, YU Q, DU D, STANTON D, LIANG G, GMITTER FG . Phloem regeneration is a mechanism for Huanglongbing-tolerance of ‘Bearss’ lemon and ‘LB8-9’ sugar Belle® mandarin. Frontiers in Plant Science, 2019,10:277.
[30]	WANG Y S, ZHOU L J, YU X Y, STOVER E, LUO F, DUAN Y P . Transcriptome profiling of Huanglongbing (HLB) tolerant and susceptible citrus plants reveals the role of basal resistance in HLB tolerance. Frontiers in Plant Science, 2016,7:933.
[31]	LIONETTI V . PECTOPLATE: The simultaneous phenotyping of pectin methylesterases, pectinases, and oligogalacturonides in plants during biotic stresses. Frontiers in Plant Science, 2015,6:331.
[32]	OWENS R A, BLACKBURN M, DING B . Possible involvement of the phloem lectin in long-distance viroid movement. Molecular Plant-Microbe Interactions, 2001,14(7):905-909.
[33]	FOLIMONOVA S Y, ACHOR D S . Early events of citrus greening (Huanglongbing) disease development at the ultrastructural level. Phytopathology, 2010,100(9):949-958.
[34]	CHISHOLM S T, COAKER G, DAY B, STASKAWICA B J . Host-microbe interactions: Shaping the evolution of the plant immune response. Cell, 2006,124(4):803-814.
[35]	JONES J D, DANGL J L . The plant immune system. Nature, 2006,444(7117):323-329.
[36]	ZOU H, GOWDA S, ZHOU L, HAJERI S, CHEN G, DUAN Y . The destructive citrus pathogen, ‘ Candidatus Liberibacter asiaticus’ encodes a functional flagellin characteristic of a pathogen-associated molecular pattern. PLoS ONE, 2012,7(9):e46447.
[37]	HAO G X, BOYLE M, ZHOU L J, DUAN Y P . The intracellular citrus Huanglongbing bacterium, ‘ Candidatus Liberibacter asiaticus’ encodes two novel autotransporters. PLoS ONE, 2013,8(7):e68921.
[38]	SHI Q C, FEBRES V J, ZHANG S J, YU F H, MCCOLLUM G, HALL D G, MOORE G A, STOVER E . Identification of gene candidates associated with Huanglongbing tolerance, using ‘ Candidatus Liberibacter asiaticus’ flagellin 22 as a proxy to challenge citrus. Molecular Plant-Microbe Interactions, 2017,31(2):200-211.
[39]	DA GRACA J V, DOUHAN G W, HALBERT S E, KEREMANE M L, LEE R F, VIDALAKIS G, ZHAO H . Huanglongbing: An overview of a complex pathosystem ravaging the world’s citrus. Journal of Integrative Plant Biology, 2016,58(4):373-387.
[40]	ZHANG Y, LI X . Salicylic acid: Biosynthesis, perception, and contributions to plant immunity. Current Opinion in Plant Biology, 2019,50:29-36.
[41]	ZHU F, XI D H, YUAN S, XU F, ZHANG D W, LIN H H . Salicylic acid and jasmonic acid are essential for systemic resistance against Tobacco mosaic virus in Nicotiana benthamiana. Molecular Plant-Microbe Interactions, 2014,27(6):567-577.
[42]	ZOU X, BAI X, WEN Q, XIE Z, WU L, PENG A, HE Y, XU L, CHEN S . Comparative analysis of tolerant and susceptible citrus reveals the role of methyl salicylate signaling in the response to Huanglongbing. Journal of Plant Growth Regulation, 2019,38:1516-1528.
[43]	VAN LOON L C, REP M, PIETERSE C M J . Significance of inducible defense-related proteins in infected plants. Annual Reviews of Phytopathology, 2006,44(1):135-162.
[44]	WU J, ALFREZ F M, JOHNSON E G, GRAHAM J H . Up-regulation of PR 1 and less disruption of hormone and sucrose metabolism in roots is associated with lower susceptibility to ‘ Candidatus Liberibacter asiaticus’. Plant Pathology, 2018,67(6):1426-1435.
[45]	FU S, SHAO J, ZHOU C, HARTUNG J S . Transcriptome analysis of sweet orange trees infected with ‘ Candidatus Liberibacter asiaticus’ and two strains of Citrus tristeza virus. BMC Genomics, 2016,17:349.
[46]	NWUGO C C, DUAN Y, LIN H . Study on citrus response to Huanglongbing highlights a down-regulation of defense-related proteins in lemon plants upon ‘ Ca. Liberibacter asiaticus’ infection. PLoS ONE, 2013,8(6):e67442.
[47]	MARTINELLI F, REAGAN R L, DOLAN D, FILECCIA V, DANDEKAR A M . Proteomic analysis highlights the role of detoxification pathways in increased tolerance to Huanglongbing disease. BMC Plant Biology, 2016,16(1):167.

基因标志 Gene symbol	基因ID Gene ID	上游引物序列 Forward primer sequence (5′-3′)	下游引物序列 Reverse primer sequence (5′-3′)	基因功能描述 Description of gene function
CESA7	102616000	GGAGTAGGAATGGATGACGAC	GGATGCTGGACTTGAGGAAG	纤维素合酶A催化亚基7 Cellulose synthase A catalytic subunit 7
EXPA5	102618439	TCCTGGAGGCTGGTGT	CAAGGCGAGCATAAGAC	扩张蛋白A5 Expansin-A5
CalS3	102611841	GGTTGTGATTGCTCTGTGG	GTGATTGAAAGCGAGACCTTAG	胼胝质合成酶3 Callose synthase 3
Susy	102616003	GTAGTGGGTGGTGATAGG	AACAGTCAAGCCAAAGG	蔗糖合成酶 Sucrose synthase
glgC	102615570	TAGGTGGTGGTGCTGG	ATTAAACTGGGTGAGGAT	葡萄糖-1-磷酸腺苷转移酶大亚基1 Glucose-1-phosphate adenylyltransferase large subunit 1-like
Rboh	102616720	AGGTACTTGCTGGCACA	CGTCGGAAATCTGGTC	呼吸爆发氧化酶同源蛋白D Respiratory burst oxidase homolog protein D
JAZ	102616825	CCCCACTTCAGTTCCC	TGGTATGGTGCTCTTGC	蛋白质TIFY 10A Protein TIFY 10A-like
UGT74G1	102613233	TTCGTCATCCTCGTCC	CCAACCAGCCCAAACT	UDP葡萄糖基转移酶74G1 UDP-glycosyltransferase 74G1
SAMT	102624842	TGAAGCACATAACCCTAG	AGAAGACCCACCACCT	水杨酸羧甲基转移酶 Salicylate carboxymethyltransferase
PR1	102627194	GACCGTATGGCGAGAA	CACCCAATGCGAACC	病程相关蛋白1 Pathogenesis-related protein 1-like
R8L3	107176058	TGCGTTGGCTCTGGT	AGTCCGTAGTCTTTGTCTCA	抗病性rpp8样蛋白3 Disease resistance RPP8-like protein 3
KTI	102621677	AACTCTTCTTCACCGAATG	AGGCTAACTCCACCACC	21 kD种子蛋白21 kD seed protein