Title: Outcome driven discussion: Model systems for studying HLB Author
Submitted to: Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: October 24, 2012
Publication Date: October 24, 2012
Citation: Stover, E.W. 2012. Outcome driven discussion: Model systems for studying HLB. Citrus Health Research Forum, August 27-30,2012, Ft. Collins, Colorado, Available: http://www.aphis.usda.gov/plant_health/cphst/meetings/CitrusHealthResearch/downloads/Useofmodelsystems.pdf Interpretive Summary: Even after intentional inoculation of citrus with the huanglongbing (HLB) pathogen, it takes 6 weeks to as long as 6 months to have detectable pathogen and 10-24 months for good symptom development. Even then, a typical response would be that 60-80% of the inoculated plants show infection. Since the citrus / HLB (huanglongbing) pathosystem is challenging and slow, it would be extremely advantageous to have a faster more efficient model system, and a number of approaches to model systems have been explored. Details of various model systems are outlined in this presentation.
Technical Abstract: After intentional inoculation of citrus with C. Liberibacter asiaticus (CLas), through grafting with infected budwood or exposure to infected psyllids, it takes 6 weeks to as long as 6 months to have readily detectable pathogen and 10-24 months for good symptom development. Even then, a typical response would be that 60-80 percent of the inoculated plants show infection. Since the citrus/HLB (huanglongbing) pathosystem is challenging and slow, it would be extremely advantageous to have a faster more efficient model system, and a number of approaches to model systems have been explored. The discovery of C. Liberibacter solanacearum (aka L. psyllaurous; CLsol) as a pathogen in solanaceous plants has provided the most extensively studied model system for HLB. It provides almost an exact parallel to HLB in citrus, with plant hosts displaying phloem infection following inoculation with Liberibacter through a psyllid vector. It has the additional advantages that it is an important disease in its own right, it can be studied in areas where concerns of inadvertent spread prevent direct study of HLB, and host plants are more quickly produced following transformation with strong genomic resources for some. Manjunath et al. report that symptoms appear in tomato 2-3 weeks after infection. They further report identification of resistant and tolerant varieties, that plant infection is uniformly systemic, and that psyllids are approximately 100 percent infected. Mirkov has studied tobacco/CLsol as a model system for evaluating transgenics and demonstrated resistant transgenics within 2 weeks and discrimination in level of transgenic resistance at 4 weeks. Belknap and Munyaneza used potato/CLsol as a model HLB system for evaluating transgenics and found no effect. They suspect they overwhelmed the potatoes with excessive inoculum and are repeating their experiments at lower CLsol load in the psyllid vectors. Roose and Patne explored Arabidopsis as a host for CLsol, to harness the enormous information known in this model plant. Screening of chemicals that induce resistance/tolerance was impeded by inability to infected cultured plants with psyllids and high cost of chemical for soil-grown plants. They have transitioned to a tomato system. Lin and Mwugo studied the proteomic and nutritional response of potatoes to CLsol infection: 50+ proteins were differentially expressed with up-regulation of proteins involved in pathogen-response/defense, molecular chaperones, and energy production/general metabolism. Down-regulation of protease inhibitors in potato tubers and photosynthesis-related proteins in potato leaves, correlated with a reduction in Mg, Ca, and Zn concentrations. Wuriyanghan et al. studied RNA silencing in the CLsol psyllid vector. They targeted ubiquitously expressed and gut-abundant mRNAs with double-stranded RNAs and siRNAs and were able to induce mortality in psyllids, with knockdown of target mRNAs, and accumulation of BC-Actin mRNA-specific siRNAs. Zhang et al. studied a periwinkle model system vs. citrus itself as materials for screening chemicals with therapeutic suppression of CLas. While the periwinkle system required a shorter incubation time and permitted rapid propagation, they conclude that the citrus system is superior as a direct assay in the system of interest. The Dawson lab has worked extensively with Citrus tristeza virus (CTV)"expression vectors, both as therapeutic delivery agents and tools to screen potential transgenes. The Shatters lab has worked with screening therapeutic materials against CLas or specific step in the pathosystem through artificial feeding to Asian citrus psyllid (ACP), which would ultimately be implemented as transgenic product or applied exogenously in-planta. Early results show that feeding of ACP on RNAi directed at blocking CLas uptake shows significant differences from controls, but appears to underestimate effectiveness when delivered in-planta. The Stover lab has worked with evaluating antimicrobial peptides (AMPs) using in-vitro cultures of Agrobacterium and Sinorhizobium which are related to Liberibacter but easy to culture. Results have been used by Jaynes to refine design of AMPs that show promise in CTV expression vectors and may have value expressed transgenically. Ammar, Walter and Hall used an excised-leaf assay to speed up assessment of ACP transfer of CLas into leaves from 3-12 months to 2-3 weeks. Leaf stems were inserted into small tubes containing water and leaves were maintained in ventilated tubes with known infected ACP. After 1-2 weeks of exposure to 5-10 ACP an average of approximately 40 percent of leaves were CLas+. This procedure is relevant to just a few steps in the pathosystem, but could be useful in evaluating whether genotypes or treatments prevent feeding or transfer of CLas from ACP. A tremendously powerful model system would be incorporation of therapeutic agents into reliable cultures of Liberibacter. This remains intractable to date, but merits further effort.