Control citrus HLB by blocking the functions of two critical effectors encoded by ‘Candidatus Liberibacter asiaticus’

Control citrus HLB by blocking the functions of two critical effectors encoded by 'Candidatus Liberibacter asiaticus'

Report Date: 01/20/2016
Project: 717   Year: 2015
Category: Other
Author: Yong-Ping Duan
Sponsor: Citrus Research and Development Foundation

The objectives of this project are to characterize the molecular interactions between the effectors and the host mitochondrial proteins; to screen for molecules that inhibit the effector functions; and to control HLB using the inhibitor(s) and/or other related molecules. Transgenic Arabidopsis plants expressing lasAI or lasAII showed a different degree of impaired growth. In particular, the LasAI contains domains responsible for abnormal growth of the root and/or meristem. Trangenic citrus plants expressing Las AI also display growth retardation. Meanwhile, to further study the function of LasAI in citrus, transgenic citrus were generated to express LasAI, LasAI N-terminal, LasAI C-terminal, LasAI repeat region, LasAII and GFP control, respectively. We have obtained transgenic citrus plants transformed with different domains of LasAI, Interestingly, transgenic plants show different degree of growth retardation, in particular the full length LasA1 and LasA1 C-terminal shows slower growth compared to the other constructs. Using RNAseq and RT-qPCR, we were able to identify the up- and down- regulations of some important genes involved in host-pathogen interactions and biosynthesis of secondary metabolites in these transgenic plants. Transient expression of LasAI and three different LasAI domains, LasAI-N-terminal, LasAI-repeat, LasAI-C-terminal allowed us to visualize the sub-cellular localizations of different domains. Because of high level expression of these effector proteins, we developed a novel in vitro screening system that evaluates small molecules against these Las effectors. The library consists of more than 30 million compounds obtained from the small molecule libraries of the TPIMS (Torrey Pines Institute for Molecular Studies). Interestingly, a few groups of compounds showed interference activity against the mitochondrial localization of LasAI. Meanwhile, to concert this screening, we developed another in vitro screening system in conjunction with the culture screening using Liberibacter cresence (Lcr). From these screening of 65 scaffold chemicals, we identified a number of chemical groups that disrupted the interaction between LasA1 and mitochondria and inhibit both Las and Lcr growth. We are narrowing down to individual compounds that inhibit the function of the Las AI effector or kill Las bacteria via other pathways, and measured the dosage effect of these potential candidate. The selected chemicals (individual or small groups) are in the evaluation process with graft-based assay. In addition, another hypothetical protein has been expressed in planta via transient and stable transformation, and founded to affect host resistance to a bacterial pathogen. The antibody against this protein was able to detect this antigen both in the transgenic plants and in the Las-infected plants. Meanwhile, the Western blot results revealed unique formation of this protein in E. coli and plants. Citrus plants with high level expression of this transgene displayed HLB-like symptoms, yellow shoot and impaired growth. Further characterization of this effector revealed its unique sub-cellular localizations. We also analyzed the expression of LasA1 in Las-infected citrus plants. RT-PCR results indicate that LasA1 expression is correlated with severity of HLB symptoms. In particular, LasA1 was expressed more in the yellow leaves or the yellow spots than green spots of the symptomatic leaves with blotchy mottle. Since transgenic plants expressing Las A1 showed up-regulation of defense-related genes, these transgenic plants were propagated and graft-inoculated with Las bacteria. We detected LasA1 in Las infected citrus leaves using antibody-based tissue printing. The results indicate that LasA1 was abundant and diffusible in Las-infected vascular tissues. To further characterize LasA1, we identified several citrus proteins that interacted with LasA1 using Yeast Two-Hybrid screening system. It is worth noting that a couple of these proteins were also identified as up-regulated genes in our RNA-seq data as mentioned above.

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