Sequestration of plant derived metabolites by leaf beetle larvae: phylogeny and mechanism of glucoside carrier

ANTJE BURSE
Abt. Bioorganische Chemie
Max-Planck-Institut für Chemische Ökologie
Hans-Knoell-Str.8
07745 Jena
aburse@ice.mpg.de
http://www.ice.mpg.de

References
Burse, A., Schmidt, A., Frick, S., Kuhn, J., Gershenzon, J., and W. Boland. Iridoid biosynthesis in Chrysomelina larvae: Fat body produces early terpenoid precursors. Insect Biochemistry & Molecular Biology. 2007, 37(3):255-265.
Larvae of the Chrysomelina species Phaedon cochleariae and Gastrophysa viridula produce monoterpenoids (iridoids) to defend themselves against predatory attacks by presenting the toxins upon attack as droplets on the top of nine pairs of dorsal glands. Although the conversion of 8-hydroxygeraniol-8-O-beta-d-glucoside into the iridoids in the glandular reservoir has been studied in detail, the synthesis of the glucosidically bound precursor received only limited attention. We compared larvae of the two iridoid producing species with those of Chrysomela populi, a sequestering species producing salicylaldehyde, in terms of the key enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and isoprenyl diphosphate synthases involved in the biosynthesis of the iridoid precursor. Increased HMGR transcript abundance, high HMGR activity and accumulation of geraniol indicating geranyl diphosphate synthase activity was observed only in the fat body of the iridoid producing larvae in comparison to other larval tissues and to the tested tissues of C. populi. These results correlate with the identification of glucosidically bound 8-hydroxygeraniol in the fat body of the iridoid producers. We suggest that in P. cochleariae and G. viridula glucosidically bound 8-hydroxygeraniol is produced by the fat body and transferred via the hemolymph into the glandular reservoir for further conversion into iridoids.

Castaneda, F., Burse, A., Boland W., and R. K.-H Kinne. Thioglycosides as inhibitors of hSGLT1 and hSGLT2: Potential therapeutic agents for the control of hyperglycemia in diabetes. International Journal of Medical Sciences. 2007, 4(3):131-139.
The treatment of diabetes has been mainly focused on maintaining normal blood glucose concentrations. Insulin and hypoglycemic agents have been used as standard therapeutic strategies. However, these are characterized by limited efficacy and adverse side effects, making the development of new therapeutic alternatives mandatory. Inhibition of glucose reabsorption in the kidney, mediated by SGLT1 or SGLT2, represents a promising therapeutic approach. Therefore, the aim of the present study was to evaluate the effect of thioglycosides on human SGLT1 and SGLT2. For this purpose, stably transfected Chinese hamster ovary (CHO) cells expressing human SGLT1 and SGLT2 were used. The inhibitory effect of thioglycosides was assessed in transport studies and membrane potential measurements, using alpha-methyl-glucoside uptake and fluorescence resonance energy transfer, respectively. We found that some thioglycosides inhibited hSGLT more strongly than phlorizin. Specifically, thioglycoside I (phenyl-1'-thio-beta-D-glucopyranoside) inhibited hSGLT2 stronger than hSGLT1 and to a larger extent than phlorizin. Thioglycoside VII (2-hydroxymethyl-phenyl-1'-thio-beta-D-galacto-pyranoside) had a pronounced inhibitory effect on hSGLT1 but not on hSGLT2. Kinetic studies confirmed the inhibitory effect of these thioglycosides on hSGLT1 or hSGLT2, demonstrating competitive inhibition as the mechanism of action. Therefore, these thioglycosides represent promising therapeutic agents for the control of hyperglycemia in patients with diabetes.

Kuhn, J., Pettersson, E. M., Feld, B. K., Burse, A., Termonia, A., Pasteels, J. M., and W. Boland. Selective transport systems mediate sequestration of plant glucosides in leaf beetles: A molecular basis for adaptation and evolution. Proceedings of the National Academy of Sciences of the United States of America. 2004, 101(38):13808-13813.
Chrysomeline larvae respond to disturbance and attack by everting dorsal glandular reservoirs, which release defensive secretions. The ancestral defense is based on the de novo synthesis of monoterpene iridoids. The catabolization of the host-plant O-glucoside salicin into salicylaldehyde is a character state that evolved later in two distinct lineages, which specialized on Salicaceae. By using two species producing monoterpenes (Hydrothassa marginella and Phratora laticollis) and two sequestering species (Chrysomela populi and Phratora vitellinae), we studied the molecular basis of sequestration by feeding the larvae structurally different thioglucosides resembling natural O-glucosides. Their accumulation in the defensive systems demonstrated that the larvae possess transport systems, which are evolutionarily adapted to the glycosides of their host plants. Minor structural modifications in the aglycon result in drastically reduced transport rates of the test compounds. Moreover, the ancestral iridoid-producing leaf beetles already possess a fully functional import system for an early precursor of the iridoid defenses. Our data confirm an evolutionary scenario in which, after a host-plant change, the transport system of the leaf beetles may play a pivotal role in the adaptation on new hosts by selecting plant-derived glucosides that can be channeled to the defensive system.

Burse, A., Weingart, H., and M. S. Ullrich. The phytoalexin-inducible multidrug efflux pump AcrAB contributes to virulence in the fire blight pathogen, Erwinia amylovora. Molecular Plant-Microbe Interactions. 2004, 17(1):43-54.
The enterobacterium Erwinia amylovora causes fire blight on members of the family Rosaceae, with economic importance on apple and pear. During pathogenesis, the bacterium is exposed to a variety of plant-borne antimicrobial compounds. In plants of Rosaceae, many constitutively synthesized isoflavonoids affecting microorganisms were identified. Bacterial multidrug efflux transporters which mediate resistance toward structurally unrelated compounds might confer tolerance to these phytoalexins. To prove this hypothesis, we cloned the acrAB locus from E. amylovora encoding a resistance nodulation division-type transport system. In Escherichia coli, AcrAB of E. amylovora conferred resistance to hydrophobic and amphiphilic toxins. An acrB-deficient E. amylovora mutant was impaired in virulence on apple rootstock MM 106. Furthermore, it was susceptible toward extracts of leaves of MM 106 as well as to the apple phytoalexins phloretin, naringenin, quercetin, and (+)-catechin. The expression of acrAB was determined using the promoterless reporter gene egfp. The acrAB operon was up-regulated in vitro by the addition of phloretin and naringenin. The promoter activity of acrR, encoding a regulatory protein involved in acrAB expression, was increased by naringenin. In planta, an induction of acrAB was proved by confocal laser scanning microscopy. Our results strongly suggest that the AcrAB transport system plays an important role as a protein complex required for virulence of E. amylovora in resistance toward apple phytoalexins and that it is required for successful colonization of a host plant.

Burse, A., Weingart, H., and M. S. Ullrich. NorM, an Erwinia amylovora multidrug efflux pump involved in in vitro competition with other epiphytic bacteria. Applied & Environmental Microbiology. 2004 70(2):693-703.
Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants. Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community. Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria. Here, we report that a multidrug efflux transporter, designated NorM, of E. amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms. According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family. The corresponding gene is widely distributed among E. amylovora strains and related plant-associated bacteria. NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine. A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106. However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms. The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined. The promoter activity of norM was twofold greater at 18 degrees C than at 28 degrees C. The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites. Thus, E. amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature.

Smirnova, A., Li, H. Q., Weingart, H., Aufhammer, S., Burse, A., Finis, K., Schenk, A., and M. S. Ullrich. Thermoregulated expression of virulence factors in plant-associated bacteria. Archives of Microbiology. 2001, 176(6):393-399.
Pathogenic bacteria with habitats inside and outside a given host react to changes in environmental parameters by synthesizing gene products specifically needed during pathogenic or saprophytic growth. Temperature effects have been investigated in detail for pathogens of warm-blooded hosts, and major principles governing the temperature-sensing mechanism have been uncovered. Generally, transcription of virulence genes in these pathogens is induced at higher temperatures (37-41 degrees C), which are typical for body cavities and host tissues. However, effects of temperature on virulence determinants in plant pathogenic bacteria have not been focused on in detail. Interestingly, almost all virulence genes of plant pathogenic bacteria studied with respect to temperature exhibit increased transcription at temperatures well below the respective growth optima. This includes virulence determinants such as those directing bacteria-to-plant gene transfer, plant cell-wall-degrading enzymes, phytotoxins, ice nucleation activity, exopolysaccharide production, and the type III protein secretion machinery. Although many of the studied phytopathogens cause "cold-weather" diseases, the ecological rationale for this phenomenon remains to be studied in detail. This mini-review summarizes our current knowledge on thermoregulation of cellular processes taking place in bacterial phytopathogens in response to temperature changes. Since the temperature range of interest is different from that relevant to pathogens of mammals, one envisions novel principles of thermo-sensing in bacteria interacting with plants.

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