Transgenic citrus expressing synthesized cecropin B genes in the phloem exhibits decreased susceptibility to Huanglongbing

Abstract

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.

Can CRISPR Win the Battle against Huanglongbing?

“…Recently, three reports described citrus enhanced HLB resistance by transgenic approach [42][43][44]. NPR1 is a core factor in plant systemic acquired resistance [45] that has been widely used to enhance plant resistance to pathogens, such as wheat [46], strawberry [47], cotton [48,49], carrot [50], rice [51] etc.…”

“…A modified thionin was transformed into citrus and the transgenic citrus showed stronger resistance to citrus canker and HLB [43]. Cecropin B, an antimicrobial peptide, from Chinese oak silkworm (A. pernyi) was expressed under phloem-specific promoter to reduce the susceptibility of Tarocco blood orange to HLB [44].…”

Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana

“…Since conventional breeding cannot create HLB-tolerance/resistance citrus, the transgenic approach has been used to enhance citrus HLB tolerance. The approach of overexpression has been applied to improve citrus HLB resistance [42][43][44]. However, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR/Cas) cannot be used in citrus HLB improvement without known HLB susceptible gene [45] in spite of the advantages [46,47] and successful generation of plants that were resistant to bacterial pathogens and fungal pathogens [48] including citrus canker [49,50] by targeting LOB1 which is the susceptibility gene of citrus canker [51].…”

Bacterial Citrus Diseases: Major Threats and Recent Progress

“…Examples of transgenes or genes whose overexpression confer enhanced resistance are shown in Table 4 [124][125][126][127][128][129][130][131][132][133][134][135][136][137][138]. Nonetheless, a main limitation of the transgenic approach is that adoption of genetically modified plants takes many years because of the rigorous field testing required and the long regulation process.…”