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

‘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.