Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (5): 837-848.doi: 10.3864/j.issn.0578-1752.2019.05.006

• PLANT PROTECTION • Previous Articles     Next Articles

Function of Copper-Resistant Gene copA of Ralstonia solanacearum

WANG XiaoNing,LIANG Huan,WANG Shuai,FANG WenSheng,XU JingSheng,FENG Jie,XU Jin(),CAO AoCheng()   

  1. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2018-10-30 Accepted:2018-11-29 Online:2019-03-01 Published:2019-03-12
  • Contact: Jin XU,AoCheng CAO E-mail:jinxu@ippcaas.cn;caoac@vip.sina.com

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Abstract:

【Objective】 Bacterial wilt of plants, caused by Ralstonia solanacearum , is a major soil-borne disease around the world. As an important bactericide to control bacterial diseases such as bacterial wilt, the widespread use of copper-based bactericides has led to the emergence of copper-resistant strains in a variety of plant pathogenic bacterial population. The copper-resistant coding gene copA , homologous with Pseudomonas syringae , was carried on the megaplasmid of R. solanacearum Po82 strain. The objective of this study is to investigate the biological function of copA in copper resistance and pathogenicity of Po82 strain.【Method】The phylogenetic relationship of the copper-resistant gene copA in different strains of R. solanacearum and other phytobacterial strains was analyzed based on neighbor-joining method using MEGA6.0 for constructing the phylogenetic tree of copA . By means of reverse genetics strategy, using the methods of gene homologous recombination and electroporation, copA gene deletion and complementary strains of Po82 were constructed. Copper minimal inhibition concentration (MIC) test, RT-qPCR, Biolog chip analysis, pathogenicity test and other basic biological methods were employed to clarify the relationship between copA and biological characteristics such as response to copper stress, metabolic activity, pathogenicity, and motility of R. solanacearum. 【Result】The results of homology analysis showed that the copA existed widely in the bacterial population, and the copA of R. solanacearum was most closely related to Cupriavidus metallidurans , but far genetic relationship with Xanthomonas oryzae , P. syringae and Escherichia coli . RT-qPCR analysis showed that the expression of copA was induced by copper. The expression of copA increased with the increase of CuSO4 concentration. The expression level of copA was the highest when the CuSO4 concentration was 1.0 mmol·L -1. By MIC analysis, the result showed that the sensitivity of the copA deletion strain to copper was significantly increased. The MIC value of copA deletion strain was 0.8 mmol·L -1, which decreased by 33.3% compared with that of wild-type strain (1.2 mmol·L -1). The complementary strain restored copper resistance. The results indicated that copA played an important role in copper resistance of R. solanacearum . Compared with wild-type strain, the logarithmic growth rate of copA gene deletion strain decreased in both NA medium and NA medium containing 0.6 mmol·L -1 CuSO4, indicating that copA was related to the growth rate of R. solanacearum . The absence of copA resulted in a decrease in the pathogenicity of R. solanacearum . On the 10th day of inoculation, the disease index of the copA gene deletion strain decreased by 11.7% compared with that of the wild-type strain Po82. The absence of copA resulted in a reduction of metabolic utilization rate of carbon sources such as α -D-glucose, D-trehalose and nitrogen sources such as L-alanine and glucuronide. Compared with wild-type strain Po82, the expression level of hrpB , hrpG and ripX genes, which are important components of the type Ⅲ secretion system, was also significantly down-regulated in copA gene deletion strain. 【Conclusion】 The copper-resistant gene copA plays an important role in copper stress response and pathogenicity of R. solanacearum . The results provide a theoretical basis for further analysis of copper resistance mechanism and the control of copper-resistant strains.

Key words: Ralstonia solanacearum, copper-resistant, copA, pathogenicity

Table 1

Strains and plasmids used in this study"

菌株和质粒 Strains and plasmids 特征 Characteristics 来源 Source
菌株
Strain		 R. solanacearum Po82 Wild-type potato strain; Phylotype Ⅱ, sequevar. 4 (race 3 biovar. 2) 本实验室The laboratory
E. coli DH5α mcrA φ80 lacZΔM15, recA1, endA1 本研究This study
ΔPo82copA copA deletion strain, Gmr 本研究This study
PBBR-ΔPo82copA copA complentmentary strain, Gmr, Ampr 本研究This study
质粒
Plasmid		 pKMS1-gm (+U) Cloning vector, Gmr, Kanr 本实验室The laboratory
pBBR1MCS-4 Ampr, lacZ alpha 本实验室The laboratory
pKMS1-copA-gm Gmr, for copA gene deletion 本研究This study
pBBR-copA Ampr, for copA gene complementation 本研究This study

Table 2

Primer sequences used in this study"

引物名称 Primer name 引物序列 Primer sequence (5′-3′) 用途Purpose
759f GTCGCCGTCAACTCACTTTCC 青枯菌特异性验证
Specificity verification of R. solanacearum
760r GTCGCCGTCAGCAATGCGGAATCG
copA Upf GGTCTTAAUCCCGTGAGGTTGGGAGGTGA 扩增copA 上游片段
Amplification of upstream of copA
copA Upr GGCATTAAUTGCTCGACTTTCACAGCGGA
copA Downf GGACTTAAUGATCGAGTTTTCCTTACGTAA 扩增copA 下游片段
Amplification of downstream of copA
copA Downr GGGTTTAAUTCACGCAGCGCCGTGTAGTCC
copAf ATATGGCGGCCTCATCATCG 基因缺失菌株验证
Verification of gene deletion strain
copAr CGAGAAAAGCCCTGTCCAGT
pBRRf CATTAGGCACCCCAGGCTTTAC 质粒线性化
Plasmid linearization
pBRRr CCTCTTCGCTATTACGCCAGC
copA Hf CCTCGAGGTCGACGGTATCGATATGCGCAGCAATCGTGCATCCCGCC copA 扩增
Amplification of copA
copA Hr GAACTAGTGGATCCCCCGGGCTTCAGGCCACCACCACTTCGCGGAAC
copA Testf GCCGTTTGTGATGGCTTCC 基因互补菌株验证
Verification of gene complementary strain
copA Testr CTTATTCAGGCGTAGCACCAGG
gyrBf GACCTTCCAGGGGTTGATCG RT-qPCR
gyrBr TCTCCCCCAGCCCCTTATAC
copAf ATATGGCGGCCTCATCATCG
copAr CGAGAAAAGCCCTGTCCAGT
hrpBf GAAGTGGCCGCCCATATC
hrpBr GCTTGCGGTAGCCCTTGA
hrpGf GGACACATTCCACGTTCTGCA
hrpGr CCATGAAATTCGCCGTATTGA
popAf TTCAGGAGCTTCACCAGGTCT
popAr CAACACCAATGGCAACTCCAA

Fig. 1

Constituent components of GEN Ⅲ MicroPlant"

Fig. 2

Phylogenetic tree based on copA sequence"

Fig. 3

PCR detection of copA gene deletion and complementary strains"

Fig. 4

Induced expression of copA The asterisk on the column indicates significant difference in gene expression under different inductive concentrations (P <0.05)"

Fig. 5

Growth curve test of the wild-type Po82, ΔPo82copA and pBBR-ΔPo82copA strains * indicates significant difference between the mutant strain and wild-type strain under the same treatment (0.01<P ≤0.05); ** indicates extremely significant difference between the mutant strain and wild-type strain under the same treatment (P <0.01). The same as below"

Fig. 6

Pathogenicity test of the wild-type Po82, ΔPo82copA and pBBR-ΔPo82copA strains on tomato"

Fig. 7

Expression analysis of pathogenic genes in the wild-type Po82 and ΔPo82copA strains"

Fig. 8

Biolog metabolic microarray detection Purple pore is defined as positive and pores shallower than purple are defined as boundary values. The picture indicates the metabolic results of Po82, ΔPo82copA and pBBR-ΔPo82copA strains in 96 h, respectively. The OD590 of α -D-glucose (micropore plate C1) in the three strains is 0.632, 0.521 and 0.372, respectively. The OD590 of D-trehalose (micropore plate A4) in the three strains is 0.620, 0.480 and 0.383, respectively. The OD590 of L-alanine (micropore plate E3) in the three strains is 0.786, 0.370 and 0.436, respectively. The OD590 of glucuronamide (micropore plate F6) in the three strains is 0.566, 0.388 and 0.380, respectively"

Fig. 9

The mobility of the wild-type Po82, ΔPo82copA and pBBR-ΔPo82copA strains"

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