Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (8): 1638-1652.doi: 10.3864/j.issn.0578-1752.2021.08.006

• PLANT PROTECTION • Previous Articles     Next Articles

Response Characteristics of Plant SAR and Its Signaling Gene CsSABP2 to Huanglongbing Infection in Citrus

ZHAO Ke(),ZHENG Lin,DU MeiXia,LONG JunHong,HE YongRui,CHEN ShanChun(),ZOU XiuPing()   

  1. Citrus Research Institute, Southwest University/Chinese Academy of Agricultural Sciences/National Citrus Engineering Research Center/National Center for Citrus Varieties Improvement, Chongqing 400712
  • Received:2020-06-19 Accepted:2020-07-24 Online:2021-04-16 Published:2021-04-25
  • Contact: ShanChun CHEN,XiuPing ZOU;;


【Background】Plant systemic acquired resistance (SAR) plays an important role in citrus against Huanglongbing (HLB). The signal exchange between salicylic acid (SA) and methyl salicylate (MeSA) is a key signaling for activating SAR, but its roles in HLB is still unclear. 【Objective】In order to understand regulation mechanisms of citrus SAR in HLB, response characteristics of SAR and its key enzyme gene CsSABP2 (salicylic acid binding protein 2) in ‘Candidatus Liberibacter asiaticus’ (CLas) infection were compared among citrus varieties with different HLB disease-tolerance. 【Method】The response characteristics of citrus SAR in CLas, SA, and MeSA inoculation were determined based on the expression of SAR Marker gene CsPR1, CsPR2, CsPR5, levels of reactive oxygen species (H2O2) and starch. Furthermore, according to comparative transcriptome data between HLB-susceptible variety Jincheng orange (JC, Citrus sinensis) and HLB-tolerant variety Sour pomelo (SP, C. grandis), the differentially expressed genes of CsSABP2s were screened and cloned. The biological function of selected genes was predicted by bioinformatics analysis. qRT-PCR was further used to analyze expression profiles of CsSABP2s induced by Clas infection in JC, SP and other HLB-tolerant variety Kaffir lime (KL, C. hystrix) and induced by exogenous SA and MeSA in JC variety. 【Result】qRT-PCR analysis showed that CsPR1, CsPR2 and CsPR5 were up-regulated in response to CLas infection, and the expression level of CsPR2 and CsPR5 in SP and KL, especially in mesophyll, was significantly higher than that in JC. On the contrast, the expression level of CsPR1 in vein was significantly higher than that in mesophyll, and its expression level in JC vein was significantly higher than that in SP and KL vein. Hormone treatment showed that MeSA treatment obviously induced up-regulated expressions of CsPR1, CsPR2 and CsPR5 in treated and non-treated sites compared to SA treatment. Exogenous MeSA induced H2O2 accumulation in non-treated sites, which was stronger than that of SA treatment. MeSA significantly reduced the accumulation of starch in Clas-infected leaves during five weeks of hormone treatment. Transcriptome data and bioinformatics analysis showed that CsSABP2-1, CsSABP2-2, CsSABP2-3, and CsSABP2-4 had significantly different expressions in response to CLas infection, and their encoded proteins contained conserved domains necessary for SABP2 hydrolysis activity. qRT-PCR showed CsSABP2-1 and CsSABP2-4 were significantly up-regulated by CLas in the HLB-resistant varieties SP and KL, and the expression level in vein was higher than that in mesophyll. The expression levels of CsSABP2-2 and CsSABP2-3 did not change significantly. Hormone induction experiments show that CsSABP2 was mainly induced by MeSA, and MeSA significantly up-regulated CsSABP2-2 expression (>10 times), but significantly down-regulated expressions of CsSABP2-1 and CsSABP2-4 (down to 15%-55% of the expression at 0 h). 【Conclusion】The SAR response to CLas infection in the HLB-tolerant varieties Sour pomelo and Kaffir lime is significantly stronger than that in HLB-susceptible variety Jincheng orange, and MeSA plays a positive role in regulating citrus SAR against HLB. Its key enzyme genes CsSABP2-1 and CsSABP2-4 play an important role in SA and MeSA signal transduction responding to CLas infection, and their high-level expressions are closely related to citrus HLB tolerance; and CsSABP2-1, CsSABP2- 2, CsSABP2-4 may play a key synergistic role in the signal conversion between SA and MeSA responding to CLas infection.

Key words: citrus Huanglongbing, Candidatus Liberibacter asiaticus’ (CLas), systemic acquired resistance (SAR), salicylic acid (SA), methyl salicylate (MeSA), SABP2, gene expression

Table 1

The sequences of common PCR primer pairs"

引物名称 Primer name 用途 Amplification 引物序列 Primer sequence (5′-3′)
OI1-F CLas普通PCR检测
CLas common PCR detection
CsSABP2-1-F 基因克隆
Gene cloning
CsSABP2-2-F 基因克隆
Gene cloning
CsSABP2-3-F 基因克隆
Gene cloning
CsSABP2-4-F 基因克隆
Gene cloning

Table 2

The sequences of qRT-PCR primer pairs"

引物名称 Primer name 用途 Amplification 引物序列 Primer sequence (5′-3′)
CsPR1-F 检测CsPR1的表达量
To detect the expression of CsPR1
CsPR2-F 检测CsPR2的表达量
To detect the expression of CsPR2
CsPR5-F 检测CsPR5的表达量
To detect the expression of CsPR5
CsSABP2-1-F 检测CsSABP2-1的表达量
To detect the expression of CsSABP2-1
CsSABP2-2-F 检测CsSABP2-2的表达量
To detect the expression of CsSABP2-2
CsSABP2-3-F 检测CsSABP2-3的表达量
To detect the expression of CsSABP2-3
CsSABP2-4-F 检测CsSABP2-4的表达量
To detect the expression of CsSABP2-4
actin-F 检测actin的表达量
To detect the expression of actin

Fig. 1

Expression analysis of PR genes in response to CLas"

Fig. 2

qRT-PCR analysis of SAR-related genes CsPR1, CsPR2 and CsPR5 induced by MeSA and SA The treatment site and non-treatment site refer to hormone treated and non-treated sites in the same isolated leaf, the same as Fig. 3. The relative expression is calculated by using water as a control. Different capital and lowercase letters on the bars indicate that the gene expression levels are significantly different under MeSA and SA treatment (P<0.05)"

Fig. 3

Detection of H2O2 accumulation induced by MeSA and SA “*” indicates significant difference with water control (P<0.05)"

Fig. 4

Effect of MeSA and SA on starch accumulation of infected leaves"

Table 3

Transcriptome comparative analysis of CsSABP2 family in response to CLas infection in Jincheng and Sour pomelo"

Gene ID
Gene name
SP CLas vs SP mock JC CLas vs JC mock 关键氨基酸分析
Analysis of key amino acids
log2 fold change 校正值padj log2 fold change 校正值padj
Cs1g23200 CsSABP2-1 3.44 1.88E-14 2.67 3.87E-16 有MeSA脂酶活性 It has MeSA lipase activity
Cs7g24820 CsSABP2-2 1.09 8.70E-05 -2.44 4.33E-35 有MeSA脂酶活性 It has MeSA lipase activity
Cs7g24830 CsSABP2-3 -2.05 3.75E-18 3.44 1.07E-12 有MeSA脂酶活性 It has MeSA lipase activity
Cs7g29470 CsSABP2-4 1.13 1.47E-03 2.17 5.33E-32 有MeSA脂酶活性 It has MeSA lipase activity
Cs2g16450 CsSABP2-5 1.29 5.14E-04 Ser突变,可能无MeSA脂酶活性
Serine is mutated, may not have MeSA lipase activity
Cs7g24810 CsSABP2-6 3.34 8.30E-04 缺失突变,可能无MeSA脂酶活性
Deletion mutation, may not have MeSA lipase activity

Fig. 5

Amino acid sequence comparison of CsSABP2 Red represents the same amino acid, black represents different amino acids, yellow represents catalytic triplets, green represents SA key binding sites, blue diamond indicates SA-binding residues, green and blue arrows identify the secondary structural elements"

Fig. 6

Gene expression of CsSABP2s in response to CLas infection in different varieties"

Fig. 7

Analysis of CsSABP2s expression induced by SA Different letters on the bars indicate significant difference at P<0.05 level. The same as Fig. 8"

Fig. 8

Analysis of CsSABP2s expression induced by MeSA"

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