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NRPS-derived isoquinolines and lipopetides mediate antagonism between plant pathogenic fungi and bacteria Saima Khalid, Joshua A. Baccile, Joseph E Spraker, Joanna Tannous, Muhammad Imran, Frank C. Schroeder, and Nancy P. Keller ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00731 • Publication Date (Web): 28 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017
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ACS Chemical Biology
NRPS-derived isoquinolines and lipopetides mediate antagonism between plant pathogenic fungi and bacteria
Saima Khalid1,2#, Joshua A. Baccile3#, Joseph E. Spraker4#, Joanna Tannous1, Muhammad Imran2, Frank C. Schroeder3* and Nancy P. Keller1* 1
Departments of Bacteriology, Medical Microbiology and Immunology, University of Wisconsin-
Madison, Madison, WI, United States 2
3
Department of Microbiology, Qauid-i-Azam University, Islamabad, Pakistan Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell
University, Ithaca, NY, United States 4
Department of Plant Pathology, University of Wisconsin - Madison, Madison, WI, United States
#
These authors contributed equally
*To whom correspondence should be directed *
[email protected] and
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Abstract Bacterial-fungal interactions are presumed to be mediated chiefly by small-molecule signals; however, little is known about the signaling networks that regulate antagonistic relationships between pathogens. Here we show that the ralstonins, lipopeptides produced by the plant pathogenic bacteria Ralstonia solanacearum, interfere with germination of the plant-pathogenic fungus Aspergillus flavus by down-regulating expression of a cryptic biosynthetic gene cluster (BGC), named imq. Comparative metabolomic analysis of overexpression strains of the transcription factor ImqK revealed imq-dependent production of a family of tripeptide-derived alkaloids, the imizoquins. These alkaloids are produced via a non-ribosomal peptide synthetase(NRPS-) derived tripeptide and contain an unprecedented tricyclic imidazo[2,1-a]isoquinoline ring system. We show that the imizoquins serve a protective role against oxidative stress that is essential for normal A. flavus germination. Supplementation of purified imizoquins restored wildtype germination to a ∆imqK A. flavus strain and protected the fungus from ROS damage. Whereas the bacterial ralstonins retarded A. flavus germination and suppressed expression of the imq cluster, the fungal imizoquins in turn suppressed growth of R. solanacearum. We suggest such reciprocal small molecule-mediated antagonism is a common feature in microbial encounters affecting pathogenicity and survival of the involved species.
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ACS Chemical Biology
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Soil-associated microbial populations represent complex and dynamic microbiomes that
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communicate primarily through chemical signaling. Several recent studies demonstrated the
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importance of specialized metabolites derived from biosynthetic gene clusters (BGCs), including
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non-ribosomal peptide synthetase- (NRPS), polyketide synthase- (PKS), and terpene synthase-
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based clusters, in mediating specific interactions between bacteria and fungi1,2. Expression of
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most of these BGCs is strongly regulated by specific growth conditions or confrontations with
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other organisms. In fact, some otherwise silent BGCs are activated during interkingdom-
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encounters3 whereas other are repressed during such encounters4.
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Agricultural environments may include plant pathogens otherwise not found in the
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rhizosphere. In addition to many soil dwelling fungal pathogens, the wilt bacteria Ralstonia
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solanacearum infects a multitude of crops and persists in the soil through unclear mechanisms,
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potentially by colonizing weeds that remain asymptomatic5. We recently found that R.
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solanacearum produces hybrid PKS-NRPS-derived lipopeptides, the ralstonins (originally called
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ralsolamycin), that induce chlamydospore formation across disparate taxa of fungi, which
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facilitates bacterial entry into chlamydospore tissue which may benefit bacterial survival6. The
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structures of the ralstonins were recently characterized in detail by Murai et al.7. It is not clear,
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however, whether chlamydospore development provides a competitive advantage to the
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colonized fungus. Although thick walled chlamydospores can weather harsh environmental
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conditions, bacterial proliferation inside these spores may decrease survival of the fungus. We
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therefore hypothesized that ralstonin production, while conferring a fitness benefit to Ralstonia,
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may elicit a defensive response from challenged fungi, or, conversely ralstonin may act to
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suppress mechanisms favoring fungal over bacterial success.
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Here we characterize antagonistic crosstalk mediated by ralstonin and a newly
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discovered class of fungal isoquinoline alkaloids, the imizoquins, where each compound retards
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some aspect of the growth dynamics of the other microbe. Imizoquins stimulate germination of
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Aspergillus spores whereas ralstonin production by R. solanacearum impedes spore
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germination of A. flavus and reduces expression of the imizoquin BGC. Conversely, imizoquins
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have a slight but significant effect on slowing R. solanacearum growth. Mechanistically,
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imizoquins possess ROS quenching properties that yield protective properties to fungal spores
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and exhibit conserved germination promoting properties across diverse Aspergillus spp. We
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propose that reciprocal antagonistic signaling molecules, as uncovered in this work, are likely
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representative of interactions in the microbial rhizosphere.
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Results and Discussion
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Ralstonin downregulates expression of the imq gene cluster in A. flavus. Because the
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ralstonins induce chlamydospore formation in fungi and facilitate bacterial entry into fungal
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tissues6, we hypothesized that these compounds may affect expression of fungal BGCs
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involved in defense. We thus compared RNAseq data of A. flavus treated with either R.
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solanacearum wild-type (GMI1000) extract or ∆rmyA mutant extract, which is deficient in
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ralstonin (Figure 1a)6. Monitoring A. flavus gene expression over a 30-hour period we observed
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differential expression of 65 genes (Fold change >2, FDR-corrected p-value
