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Title: The 'Candidatus Liberibacter asiaticus' effector LasP235 targeting the citrus chloroplast induces Huanglongbing-like symptoms in transgenic citrus

Author
item Hao, Guixia
item AMMAR, DESOUKY - Orise Fellow
item Duan, Ping
item Stover, Eddie

Submitted to: AGRI GENE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/20/2019
Publication Date: 3/23/2019
Citation: Hao, G., Ammar, D., Duan, Y., Stover, E.W. 2019. The 'Candidatus Liberibacter asiaticus' effector LasP235 targeting the citrus chloroplast induces Huanglongbing-like symptoms in transgenic citrus. AGRI GENE. 12:100085. https://doi.org/10.1016/j.aggene.2019.100085.
DOI: https://doi.org/10.1016/j.aggene.2019.100085

Interpretive Summary: Huanglongbing (HLB or citrus greening disease) caused by a bacterial pathogen is the most devastating disease of citrus and threatens citrus production globally. Though there has been some progress in understanding HLB, details of pathogen and citrus interactions are still not clear. In this study, we identified a pathogen-produced molecule that binds to the citrus chloroplast, and verified that this molecule could be identified in infected citrus. When this molecule was produced transgenically in citrus, HLB-like symptoms were observed and citrus metabolism was significantly altered. This is the first report of an HLB pathogen molecule directly inducing HLB-like symptoms in transgenic citrus. By identifying the citrus protein that first interacts with this pathogen molecule, we may be able to intervene through therapeutic compounds or biotechnology to provide resistance to HLB.

Technical Abstract: ‘Candidatus Liberibacter asiaticus’ (Las) is the most destructive agent causing citrus huanglongbing (HLB). A putative protein (designated as LasP235) with a eukaryotic nuclear localization signal (NLS) was identified in the prophage region of the Las psy62 genome. LasP235 encodes a 123 amino-acid protein which was predicted to localize to the plant nucleus by PSORT. N-terminal green fluorescence protein (GFP)-fused with LasP235 appeared to accumulate in Nicotiana benthamiana cell nuclei following Agrobacterium-mediated transient expression, however, LasP235 fused with C-terminal GFP protein in transgenic citrus was observed to target the citrus chloroplast. To eliminate potential side effects of GFP protein, LasP235 alone was inserted into a binary vector for citrus transformation. Over 40 independent transgenic Carrizo plants were obtained. Gene integration and expression were confirmed by molecular analysis. We observed that some LasP235 expressing Carrizo plants displayed HLB-like symptoms, including leaf chlorosis and plant growth retardation. LasP235 expression levels, determined by Real-time quantitative polymerase chain reaction (RT-qPCR), correlated with HLB-like symptoms. Furthermore, we showed that the expression of LasP235 was upregulated in chlorotic tissue compared to green tissue of HLB 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. This is the first report that ‘Candidatus Liberibacter asiaticus’ encodes a protein that targets the chloroplast of citrus and alters plant-host phenotypes.