Subscriber access provided by READING UNIV
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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
[email protected] ACS Paragon Plus Environment
1
ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 26
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.
ACS Paragon Plus Environment
2
Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Chemical Biology
1
Soil-associated microbial populations represent complex and dynamic microbiomes that
2
communicate primarily through chemical signaling. Several recent studies demonstrated the
3
importance of specialized metabolites derived from biosynthetic gene clusters (BGCs), including
4
non-ribosomal peptide synthetase- (NRPS), polyketide synthase- (PKS), and terpene synthase-
5
based clusters, in mediating specific interactions between bacteria and fungi1,2. Expression of
6
most of these BGCs is strongly regulated by specific growth conditions or confrontations with
7
other organisms. In fact, some otherwise silent BGCs are activated during interkingdom-
8
encounters3 whereas other are repressed during such encounters4.
9
Agricultural environments may include plant pathogens otherwise not found in the
10
rhizosphere. In addition to many soil dwelling fungal pathogens, the wilt bacteria Ralstonia
11
solanacearum infects a multitude of crops and persists in the soil through unclear mechanisms,
12
potentially by colonizing weeds that remain asymptomatic5. We recently found that R.
13
solanacearum produces hybrid PKS-NRPS-derived lipopeptides, the ralstonins (originally called
14
ralsolamycin), that induce chlamydospore formation across disparate taxa of fungi, which
15
facilitates bacterial entry into chlamydospore tissue which may benefit bacterial survival6. The
16
structures of the ralstonins were recently characterized in detail by Murai et al.7. It is not clear,
17
however, whether chlamydospore development provides a competitive advantage to the
18
colonized fungus. Although thick walled chlamydospores can weather harsh environmental
19
conditions, bacterial proliferation inside these spores may decrease survival of the fungus. We
20
therefore hypothesized that ralstonin production, while conferring a fitness benefit to Ralstonia,
21
may elicit a defensive response from challenged fungi, or, conversely ralstonin may act to
22
suppress mechanisms favoring fungal over bacterial success.
23
Here we characterize antagonistic crosstalk mediated by ralstonin and a newly
24
discovered class of fungal isoquinoline alkaloids, the imizoquins, where each compound retards
25
some aspect of the growth dynamics of the other microbe. Imizoquins stimulate germination of
26
Aspergillus spores whereas ralstonin production by R. solanacearum impedes spore
27
germination of A. flavus and reduces expression of the imizoquin BGC. Conversely, imizoquins
28
have a slight but significant effect on slowing R. solanacearum growth. Mechanistically,
29
imizoquins possess ROS quenching properties that yield protective properties to fungal spores
30
and exhibit conserved germination promoting properties across diverse Aspergillus spp. We
31
propose that reciprocal antagonistic signaling molecules, as uncovered in this work, are likely
32
representative of interactions in the microbial rhizosphere.
33
ACS Paragon Plus Environment
3
ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 26
34
Results and Discussion
35
Ralstonin downregulates expression of the imq gene cluster in A. flavus. Because the
36
ralstonins induce chlamydospore formation in fungi and facilitate bacterial entry into fungal
37
tissues6, we hypothesized that these compounds may affect expression of fungal BGCs
38
involved in defense. We thus compared RNAseq data of A. flavus treated with either R.
39
solanacearum wild-type (GMI1000) extract or ∆rmyA mutant extract, which is deficient in
40
ralstonin (Figure 1a)6. Monitoring A. flavus gene expression over a 30-hour period we observed
41
differential expression of 65 genes (Fold change >2, FDR-corrected p-value