Cloning, overexpression, and purification of a gene of unknown function of prophage loci from 'Candidatus Liberibacter asiaticus,' the destructive bacterial pathogen of huanglongbing disease in citrus plants.

Citrus Huanglongbing (HLB) or citrus greening is one of the most destructive diseases affecting citrus industry worldwide. The causal agent in Asia is a phloem-limited, Gram-negative bacterium, 'Candidatus Liberibacter asiaticus' (CLas). Within the genome of CLas lies prophage regions, classified as Type-A, B, C, and D. In particular, Type-D has been indicated to correlate with the blotchy-mottle symptoms of citrus trees. Here we reported the cloning, overexpression, and purification of the ORF1, an open reading frame from the partial Type-D region of CLas obtained from an infected lime tree (Citrus aurantifolia Swingle). Overexpression of the ORF1 was toxic to the E. coli BL21(DE3), and the transient expression of ORF1 in Arabidopsis seedlings by Agrobacterium-mediated transformation exhibited rapid and total chlorosis of the seedlings within two days post-transformation. The native-PAGE of the purified protein showed multiple bands, indicative of various conformations in solution. The ESI-TOF mass spectrum confirmed the molecular weight of the purified ORF1 to be 15,364.3150 Da, corresponding to the [M+1]+ of the ORF1 without an N-terminal methionine. The protein predominantly consisted of α-helix as evidenced by circular dichroism (CD), and the transition toward random coil structure upon heating was reversible. The template-based modeling (I-TASSER) of the ORF1 indicated eight α-helices connected through variable loops. The simulated CD spectrum, generated from the atomic coordinates of the I-TASSER model, was notably similar to the experimental spectrum. Our report offers the basis for understanding the contributions of genes within Type-D prophage region toward the disease pathogenicity of citrus HLB.