Scientia Agricultura Sinica ›› 2020, Vol. 53 ›› Issue (7): 1368-1380.doi: 10.3864/j.issn.0578-1752.2020.07.007

• PLANT PROTECTION • Previous Articles     Next Articles

Biologic and Transcriptomic Analysis of Citrus hystrix Responses to ‘Candidatus Liberibacter asiaticus’ at Different Infection Stages

CaiLing TENG1,Xi ZHONG1,HaoDi WU1,Yan HU2,ChangYong ZHOU1,XueFeng WANG1   

  1. 1. National Citrus Engineering Research Center, Citrus Research Institute, Chinese Academy of Agricultural Sciences, Chongqing 400712
    2. Ganzhou Bureau of Fruit Industry, Ganzhou 341000, Jiangxi
  • Received:2019-09-01 Accepted:2019-10-15 Online:2020-04-01 Published:2020-04-14

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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

Table 1

The genes and primers of RT-qPCR"

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

Fig. 1

Phenotypic identification and microstructure observation of C. hystrix infected by CLas at different stages"

Fig. 2

Comparative PageMan display of perturbed pathway of DEGs in C. hystrix infected by CLas at different stages"

Fig. 3

DEGs involved in sucrose-starch metabolism (A) and photosynthesis (B)"

Fig. 4

Biotic stress-related DEGs altered in response to CLas infection at different stages"

Fig. 5

RT-qPCR for the DEGs validation A: CH-M-VS-CH-HLB-E; B: CH-M-VS-CH-HLB-L; C: CH-HLB-E-VS-CH-HLB-L"

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