Fleites, Laura A.

Abstract Delivery of therapeutic biomolecules into plant vascular tissues remains a challenge in management of vector-borne plant pathogens. The symbiont concept uses reprogrammed Agrobacterium tumefaciens galls (called symbionts) to produce biomolecules while remaining connected to host vasculature. We evaluated whether symbionts expressing antimicrobial peptides (AMPs) suppress ‘ Candidatus Liberibacter solanacearum’ (CLso), the causal agent of potato zebra chip disease. Symbionts were engineered to express a Streptococcus mutans bacteriocin associated with bacterial membrane disruption (Blp-Sm), or an AMP isolated from finger lime and associated with resistance to citrus greening disease (MaSAMP). Effects of AMP-producing symbionts on CLso titers, infection incidence, pathogen movement, and disease symptoms were evaluated in tomato and potato. In tomato, neither AMP significantly reduced CLso titers or infection incidence. However, in potato, AMP-producing symbionts reduced CLso accumulation and movement from CLso-inoculated source shoots into non-inoculated sink shoots connected through underground tubers. Blp-Sm produced the strongest reduction in CLso accumulation and infection incidence in sink tissues. In separate assays where symbionts were established directly on potato seed tubers, MaSAMP significantly reduced CLso titers in stems and tubers and reduced zebra chip symptoms in tubers, despite no reduction of CLso titers in terminal leaves. These findings demonstrate that AMP-producing symbionts suppress vascular pathogen accumulation and movement within plants and highlight the symbiont concept as a potential platform for managing diseases caused by vascular-restricted pathogens. Further, they show the potato-CLso system is a promising infection model to both refine and improve symbiont technology, and to test additional AMPs for potency against related pathogens.

AbstractThe Asian citrus psyllid,Diaphorina citri, is a vector of ‘CandidatusLiberibacter asiaticus’ (CLas), a gram-negative, obligate biotroph whose infection inCitrusspecies is associated with citrus greening disease, or Huanglongbing (HLB). Strategies to blockCLas transmission byD. citriremain the best way to prevent the spread of the disease into new citrus growing regions. However, identifying control strategies to block HLB transmission poses significant challenges, such as the discovery and delivery of antimicrobial compounds targeting the bacterium and overcoming consumer hesitancy towards accepting the treatment. Here, we computationally identified and tested a series of 20-mer nodule-specific cysteine-rich peptides (NCRs) derived from the Mediterranean legume,Medicago truncatulaGaertn. (barrelclover) to identify those peptides that could effectively prevent or reduceCLas infection in citrus leaves and/or preventCLas acquisition by the bacterium’s insect vector,D. citri. A set of NCR peptides were tested in a screening pipeline involving three distinct assays: a bacterial culture assay, aCLas-infected excised citrus leaf assay, and aCLas-infected nymph acquisition assay that includedD. citrinymphs, the only stage ofD. citri’s life-cycle that can acquireCLas leading to the development of vector competent adult insects. We demonstrate that a subset ofM. truncatula-derived NCRs inhibit bothCLas growth in citrus leaves andCLas acquisition byD. citrifromCLas-infected leaves. These findings reveal NCR peptides as a new class and source of biopesticide molecules to controlCLas for the prevention and/or treatment of HLB.

Host acquisition of a new microbial species can readily perturb the dynamics of preexisting microbial associations. Molecular cross talk between microbial associates may be necessary for efficient resource allocation and enhanced survival. Classic examples involve quorum sensing (QS), which detects population densities and is both used and coopted to control expression of bacterial genes, including host adaptation factors. We report that a 56-amino-acid repressor protein made by the resident psyllid endosymbiont Wolbachia can enter cells of Liberibacter crescens , a cultured proxy for the uncultured psyllid endosymbiont “ Ca . Liberibacter asiaticus” and repress “ Ca . Liberibacter asiaticus” phage lytic cycle genes. Such repression in “ Ca . Liberibacter asiaticus” may be critical to survival of both endosymbionts, since phage-mediated lysis would likely breach the immunogenic threshold of the psyllid, invoking a systemic and nonspecific innate immune reaction.

ABSTRACT “ Candidatus Liberibacter asiaticus” is an uncultured alphaproteobacterium that systemically colonizes its insect host both inter- and intracellularly and also causes a severe, crop-destroying disease of citrus called huanglongbing, or citrus “greening.” In planta , “ Ca . Liberibacter asiaticus” is also systemic but phloem limited. “ Ca . Liberibacter asiaticus” strain UF506 carries two predicted prophages, SC1 and SC2. Bacteriophage particles have been observed in experimentally “ Ca . Liberibacter asiaticus”-infected periwinkle but not in any other host. Comparative gene expression analysis of predicted SC1 late genes showed a much higher level of late gene expression, including holin transcripts (SC1_gp110), in “ Ca . Liberibacter asiaticus”-infected periwinkle relative to “ Ca . Liberibacter asiaticus”-infected citrus. To functionally characterize predicted holin and endolysin activity, SC1_gp110 and two predicted endolysins, one within SC1 (SC1_gp035) and another well outside the predicted prophage region (CLIBASIA_04790), were cloned and expressed in Escherichia coli . Both SC1 genes inhibited bacterial growth consistent with holin and endolysin function. The holin (SC1_gp110) promoter region was fused with a uidA reporter on pUFR071, a wide bacterial host range (repW) replicon, and used to transform Liberibacter crescens strain BT-1 by electroporation. BT-1 is the only liberibacter strain cultured to date and was used as a proxy for “ Ca . Liberibacter asiaticus.” pUFR071 was >95% stable without selection in BT-1 for over 20 generations. The reporter construct exhibited strong constitutive glucuronidase (GUS) activity in culture-grown BT-1 cells. However, GUS reporter activity in BT-1 was suppressed in a dose-dependent manner by crude aqueous extracts from psyllids. Taken together with plant expression data, these observations indicate that “ Ca . Liberibacter asiaticus” prophage activation may limit “ Ca . Liberibacter asiaticus” host range and culturability.