Jose Francisco L. Figueiredo's research while affiliated with University of Florida and other places

Xanthomonas citri subsp. citri (X. citri), causal agent of citrus canker, uses Transcription Activator-Like Effectors (TALEs) as major pathogenicity factors. TALEs, which are delivered into plant cells through the type III secretion system (T3SS), interact with Effector Binding Elements (EBEs) in host genomes to activate expression of downstream susceptibility genes to promote disease. Predictably, TALEs bind EBEs in host promoters via known combinations of TALE amino acids to DNA bases known as the TALE code. We introduced 14 EBEs, matching to distinct X. citri TALEs, into the promoter of the pepper Bs3 gene (ProBs31EBE ) and fused this engineered promoter with multiple EBEs (ProBs314EBE ) to either the GUS reporter gene or the coding sequence (cds) of the pepper gene, Bs3. TALE induced expression of the Bs3 cds in citrus leaves resulted in no visible HR. Therefore, we utilized a different approach in which ProBs31EBE and ProBs314EBE were fused to the Xanthomonas gene, avrGf1, which encodes a bacterial effector that elicits a hypersensitive response (HR) in grapefruit and sweet orange. We demonstrate in transient assays that activation of ProBs314EBE by X. citri TALEs is T3SS dependent and that expression of AvrGf1 triggers HR and correlates with reduced bacterial growth. We further demonstrated that all tested virulent X. citri strains from diverse geographic locations activated ProBs314EBE . TALEs are essential for virulence of X. citri strains and, because the engineered promoter traps are activated by multiple TALEs, this concept has potential to confer broad-spectrum, durable resistance to citrus canker in stably transformed plants. This article is protected by copyright. All rights reserved.

Acibenzolar-S-methyl (ASM), a plant activator known to induce systemic acquired resistance, has demonstrated an ability to manage a number of plant diseases, including bacterial spot on tomato caused by four distinct Xanthomonas spp. The aim of this study was to evaluate application rate and frequency of ASM in order to optimize field efficacy against bacterial spot in Florida, while minimizing its impact on marketable yields. ASM was applied biweekly (once every 2 weeks) as a foliar spray at a constant concentration of 12.9, 64.5, and 129 μM throughout four field experiments during 2007-08. A standard copper program and an untreated control were also included. Overall, biweekly applications of ASM did not significantly reduce disease development or the final disease severity of bacterial spot compared with the copper-mancozeb standard or the untreated control. Only one experiment showed a significant reduction in the final disease severity on plants treated with ASM at 129 μM compared with the untreated control. Three additional field trials conducted during 2009-10 to evaluate the effects of weekly and biweekly applications of ASM at concentrations of 30.3 to 200 μM found that weekly applications provided significantly better disease control than biweekly applications. The tomato yields were not statistically improved with the use of ASM relative to the untreated control and standard copper program. Weekly ASM applications at rates as low as 75 μM (equivalent to 1.58 g a.i./ha in 100 liters of water or 0.21 oz. a.i./acre in 100 gallons of water) to 200 μM (equivalent to 4.20 g a.i./ha in 100 liters of water or 0.56 oz. a.i./acre in 100 gallons of water) were statistically equivalent in managing bacterial spot of tomato without significantly reducing yield compared with the untreated control.

Bacterial strains and plasmids used in the study.

Xanthomonas citri subsp. citri, the causal agent of citrus canker, relies extensively on a type III secretion system for infection by delivering type III effectors into host cells. In the genus Xanthomonas, two major regulators, HrpG and HrpX, are involved in the expression of genes encoding the type III secretion system. Twenty-three candidate type III effectors were identified as targets for analysis. The involvement in pathogenicity of 20 candidate effector genes in X. citri strain 306 (Xcc-306) was investigated using site-directed mutagenesis. Pathogenicity assays in grapefruit of 19 genes using site-directed mutagenesis revealed that none of the mutants demonstrated to have reduced ability to cause canker disease. A mutation in the TAL effector pthA4 – resulted in loss of hypertrophy although no changes were observed in bacterial growth in leaves. Mutations in hrpG, hrpX, or hrpA genes displayed a complete loss of pathogenicity. Moreover, all mutants maintained the ability to trigger a hypersensitive response (HR) in non-host tomato. In contrast to previous studies, hrpG – , hrpX – and hrpA – mutants also retained the ability to elicit an HR in tomato, indicating the presence of an Hrp independent elicitor in Xcc-306.

In this study, we present a method for transient expression of the type III effector AvrGf1 from Xanthomonas citri subsp. citri strain A(w) in grapefruit leaves (Citrus paradisi) via Agrobacterium tumefaciens. The coding sequence of avrGf1 was placed under the control of the constitutive CaMV 35S promoter in the binary vectors pGWB2 and pGWB5. Infiltration of grapefruit leaves with A. tumefaciens carrying these constructs triggered a hypersensitive response (HR) in grapefruit 4 days after inoculation. When transiently expressed in grapefruit leaves, two mutants, AvrGf1ΔN116 and AvrGf1ΔC83, failed to induce an HR. Moreover, using bioinformatics tools, a chloroplast transit signal was predicted at the N terminus of AvrGf1. We demonstrated chloroplast localization by using an AvrGf1::GFP fusion protein, where confocal images revealed that GFP fluorescence was accumulating in the stomatal cells that are abundant in chloroplasts. Transient expression in citrus has the potential for aiding in the development of new disease defense strategies in citrus.