Transgenic citrus plants expressing a ‘Candidatus Liberibacter asiaticus’ prophage protein LasP235 display Huanglongbing-like symptoms

Abstract

‘Candidatus Liberibacter asiaticus’ (Las) is one of the most destructive plant pathogens associated with citrus huanglongbing (HLB). Las is a Gram-negative, as yet uncultured, alpha-Proteobacterium and is transmitted by the Asian citrus psyllid, Diaphorina citri. A putative protein (designated as LasP235) was identified in the prophage region of the Las psy62 genome. LasP235 gene encodes a 123 amino-acid protein which is predicted to localize to the plant nucleus. Green fluorescence protein (GFP)-fused with LasP235 appeared to accumulate in Nicotiana benthamiana cell nuclei following Agrobacterium-mediated transient expression. To eliminate potential side effects of GFP protein, LasP235 alone was inserted into a binary vector for transforming Carrizo citrange (Citrus sinensis X Poncirus trifoliata). Transgenic Carrizo plants were obtained. LasP235 gene integration was confirmed by PCR and the levels of LasP235 expression were compared by RT-quantitative PCR (RT-qPCR). Some LasP235 expressing Carrizo plants displayed HLB-like symptoms, including leaf chlorosis and plant growth retardation. LasP235 gene expression levels, determined by RT-qPCR, correlated with HLB-like symptoms. Furthermore, The expression of LasP235 was upregulated in chlorotic tissue compared to green tissue of Las-infected, blotchy mottled leaves of lemon and grapefruit. Transcriptome analysis revealed that metabolic pathways and biosynthesis of secondary metabolites were significantly altered in transgenic citrus expressing the LasP235 effector. Further investigation of LasP235, especially research focused on identifying its binding protein in citrus, may provide a way to block Las infection.