Title : Understanding of plant innate immune response and engineering of novel innate immunity for pathogen clearance and disease protection.
Abstract:
A national consortium has been set up to study pathogenesis and develop diagnosis and therapy of citrus greening (aka Huanglongbing or HLB), the most devastating disease of citrus in the US and worldwide. HLB is caused by gram-negative Liberibacter, which is transmitted by insect vector, psyllid. There is no cure for HLB. Psyllid control and tree removal are the only disease management options. Psyllid control, which does not eliminate the causative Liberibacter, is only partially successful. The problem is further exacerbated by the fact that the disease symptoms appear 2-5 years after the initial Liberibacter exposure. By then disease is often too widespread to be effective by the current disease management protocols. Therefore, robust strategies for pre-symptomatic diagnosis and therapy are urgently needed for effective HLB management.
It is our hypothesis that quantitative knowledge of citrus innate immune response induced early upon Liberibacter exposure would guide the development of both pre-symptomatic diagnosis and therapy. In support of our hypothesis, we have carried out genome-wide transcriptomic analysis in the greenhouse to identify citrus mRNA and microRNAs that are induced early (within 4-24 weeks of Liberibacter exposure) and belong to the five coupled gene networks: PTI (PAMP-triggered immunity), ETI (Effector-triggered immunity), and Salicylic Acid (SA)/ Jasmonic Acid (JA)/ Ethylene (ET) signaling.
The final output of the coupled networks is the production of citrus defense proteins that can clear or block infection. We identified key genes that are induced in these networks and determine the disease outcome, i.e., Liberibacter modifies the expression pattern of these genes, which, in turn, abrogates the citrus innate immune defense allowing Liberibacter to escape killing, cause infection, and develop HLB. We identified informative microRNAs that also determine the disease outcome by regulating post-transcriptionally specific genes belonging to the five coupled PTI/ETI/SA/JA/ET networks. We believe that a combination of citrus mRNA and microRNA, induced early during infection, will serve as the biomarkers for pre-symptomatic diagnosis of HLB.
From our transcriptome analysis, we identified citrus thionin, an antimicrobial or lytic peptide and an end-product of the PTI/ETI/SA/JA/ET networks, to be severely down-regulated. We argued that thionin expression can be rescued by removing the control of the PTI/ETI/SA/JA/ET networks and putting it under the control of a constitutive promoter. In addition, we made targeted modifications in the endogenous thionin amino acid sequence to improve activity and reduce toxicity. Transgenic citrus expressing the modified thionin showed Liberibacter clearance and protection against HLB. We are in the process of designing a chimera, in which we have added a recognition domain to the modified thionin (a lysis domain). Transgenic citrus expressing the chimera of recognition and lysis domains is expected to be more effective in Liberibacter clearance and disease protection. Introduction of a modified thionin or a chimera introduces a novel innate immune defense in citrus.
In summary, we propose a platform technology that combines transcriptome analysis and protein engineering (i) to identify informative plant mRNA and microRNA for pre-symptomatic disease diagnosis and (ii) to engineer novel innate immunity for pathogen clearance and disease protection.
