Transgenic citrus expressing synthesized cecropin B genes in the phloem exhibits decreased susceptibility to Huanglongbing
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Expression of synthesized cecropin B genes in the citrus phloem, where Candidatus Liberibacter asiaticus resides, significantly decreased host susceptibility to Huanglongbing.
Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus bacteria, is the most destructive disease of citrus worldwide. All of the commercial sweet orange cultivars lack resistance to this disease. The cationic lytic peptide cecropin B, isolated from the Chinese tasar moth (Antheraea pernyi), has been shown to effectively eliminate bacteria. In this study, we demonstrated that transgenic citrus (Citrus sinensis Osbeck) expressing the cecropin B gene specifically in the phloem had a decreased susceptibility to HLB. Three plant codon-optimized synthetic cecropin B genes, which were designed to secrete the cecropin B peptide into three specific sites, the extracellular space, the cytoplasm, and the endoplasmic reticulum, were constructed. Under the control of the selected phloem-specific promoter GRP1.8, these constructs were transferred into the citrus genome. All of the cecropin B genes were efficiently expressed in the phloem of transgenic plants. Over more than a year of evaluation, the transgenic lines exhibited reduced disease severity. Bacterial populations in transgenic lines were significantly lower than in the controls. Two lines, in which bacterial populations were significantly lower than in others, showed no visible symptoms. Thus, we demonstrated the potential application of the phloem-specific expression of an antimicrobial peptide gene to protect citrus plants from HLB.
KeywordsCitrus Huanglongbing Cecropin B Phloem-specific promoter Disease resistance Genetic transformation
This work was supported by grants from the National Natural Sciences Foundation of China (31272150, to XZ), the Earmarked Fund for China Agriculture Research System (CARS-27, to SC), and the Fundamental Research Funds for the Central Universities (XDJK2012B023, to XZ).
XZ designed the experiments, constructed the vectors, and wrote the manuscript. LX and AP performed the citrus genetic transformations. XJ evaluated the resistance to HLB. TL and YH performed molecular analyses. LY performed the Southern blot analysis. SC analyzed the data. All of the authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
- Boscariol RL et al (2006) Attacin A gene from Tricloplusia ni reduces susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinensis ‘Hamlin’. J Am Soc Hortic Sci 131:530–536Google Scholar
- He Y, Chen S, Peng A, Zou X, Xu L, Lei T (2011) Production and evaluation of transgenic sweet orange (Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary buds. Sci Hortic 128:99–107. doi: 10.1016/j.scienta.2011.01.002 CrossRefGoogle Scholar
- Hilf ME, Sims KR, Folimonova SY, Achor DS (2013) Visualization of ‘Candidatus Liberibacter asiaticus’ cells in the vascular bundle of citrus seed coats with fluorescence in situ hybridization and transmission electron microscopy. Phytopathology 103:545–554. doi: 10.1094/PHYTO-09-12-0226-R CrossRefPubMedGoogle Scholar
- Jaynes JM, Nagpala P, Destefanobeltran L, Huang JH, Kim JH, Denny T, Cetiner S (1993) Expression of a cecropin-B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas Solanacearum. Plant Sci 89:43–53. doi:Doi: 10.1016/0168-9452(93)90169-Z CrossRefGoogle Scholar
- Miyata LY, Harakava R, Stipp LC, Mendes BM, Appezzato-da-Gloria B, de Assis Alves Mourao Filho F (2012) GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters. Plant Cell Rep 31:2005–2013. doi: 10.1007/s00299-012-1312-2 CrossRefPubMedGoogle Scholar
- Zhang Q, Zhang J, Huang Z, Tan S, Guo Z (1995) The bactericidal effect of antibacterial peptide from Chinese Oak silkworm on the pathogen of bacterial ulcer and yellow shoot disease in citrus. Acta Sericol Sin 21:77–81Google Scholar
- Zou X et al (2014a) Secreted expression of cecropin B gene enhances resistance to Xanthomonas axonopodis pv. citri in transgenic citrus sinensis‘Tarocco rsquo. Acta Hortic Sin 41:417–428Google Scholar