NRPS-Derived Isoquinolines and Lipopetides Mediate Antagonism between Plant Pathogenic Fungi and Bacteria

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 nonribosomal 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 ΔimqK 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.

Conserved Responses in a War of Small Molecules between a Plant-Pathogenic Bacterium and Fungi

“…Although previous studies have described cross-kingdom induction of SMs, e.g., in Aspergillus nidulans / Streptomyces hygroscopicus ( 34 ) or Fusarium tricinctum / Bacillus subtilis ( 36 ) pairings, knowledge of mechanisms and molecular players underlying these interactions remains scarce. We recently described a unique Ralstonia - Aspergillus flavus interaction where ralsolamycin suppressed synthesis of an A. flavus germination and antibacterial peptide, imizoquin, suggestive of a bacterial advantage in this encounter ( 8 ). In contrast, our present study implicates ralsolamycin in the specific induction of antibacterial fungal SMs, including the known metabolites bikaverin and beauvericin, in F. fujikuroi and Botrytis cinerea .…”

“…It is a devastating pathogen of well over 200 plant species, including both mono- and dicotyledonous plants ( 4 ). Although R. solanacearum has been studied primarily because of its severe impacts on plant hosts, it has recently been shown to impact plant-prokaryote community composition ( 5 ), and it also interacts intimately with many soil fungi through volatile and diffusible signaling ( 6 – 8 ). The lipopeptide ralsolamycin (synonym ralstonin A), produced by a hybrid polyketide synthase/nonribosomal peptide synthetase (PKS-NRPS) biosynthetic gene cluster ( rmy ) ( 7 , 9 , 10 ), was shown to induce chlamydospore formation in a phylogenetically diverse panel of plant-associated and/or soil-inhabiting fungi representing members of the Mucoromycota, Ascomycota, and Basidiomycota ( 7 ).…”

Chemical repertoire and biosynthetic machinery of the Aspergillus flavus secondary metabolome: A review

“…Imizoquins background. Imizoquins represent a family of tripeptide-derived alkaloids possessing a unusual imidazo[2,1-a]isoquinoline heterocyclic ring system, identified for the first time in A. flavus (Khalid et al, 2017). Figure 10a).…”

“…Imizoquins biological function. Imizoquins were discovered during the course of a study looking at the interaction of the bacterium Ralstonia solanacearum with A. flavus (Khalid et al, 2017;Spraker, Sanchez, Lowe, Dorrestein, & Keller, 2016). This work eloquently illustrated a F I G U R E 1 0 Schematic representation of (a) imizoquin gene cluster imq and (b) model of biosynthesis of the imizoquins (adapted from Khalid et al, 2017) A, adenylation; C, condensation; PCP, peptidyl carrier protein; TE, thioesterase specific case where SMs serve as chemical signals to build an antagonistic relationship between the plant pathogenic bacterium and the fungus.…”

“…Imizoquins biosynthesis. The genes responsible for the biosynthesis of imizoquins are present in A. flavus imq cluster and consist of an NRPS (ImqB), a phenol 2-monooxygenase (ImqC), two transport proteins (ImqD and ImqJ), a gibberellin oxidase (ImqE), an N-methyltransferases (ImqF), an O-methyltransferases (ImqG), an FAD-dependent oxidase (ImqH), a pathwayspecific regulator (ImqK), and two other proteins of undefined role (ImqA and ImqI)(Khalid et al, 2017; …”