Accessory Chromosome-Acquired Secondary Metabolism in Plant Pathogenic Fungi: The Evolution of Biotrophs Into Host-Specific Pathogens

Abstract

Accessory chromosomes are strain- or pathotype-specific chromosomes that exist in addition to the core chromosomes of a species and are generally not considered essential to the survival of the organism. Among pathogenic fungal species, accessory chromosomes harbor pathogenicity or virulence factor genes, several of which are known to encode for secondary metabolites that are involved in plant tissue invasion. Accessory chromosomes are of particular interest due to their capacity for horizontal transfer between strains and their dynamic "crosstalk" with core chromosomes. This review focuses exclusively on secondary metabolism (including mycotoxin biosynthesis) associated with accessory chromosomes in filamentous fungi and the role accessory chromosomes play in the evolution of secondary metabolite gene clusters. Untargeted metabolomics profiling in conjunction with genome sequencing provides an effective means of linking secondary metabolite products with their respective biosynthetic gene clusters that reside on accessory chromosomes. While the majority of literature describing accessory chromosome-associated toxin biosynthesis comes from studies of Alternaria pathotypes, the recent discovery of accessory chromosome-associated biosynthetic genes in Fusarium species offer fresh insights into the evolution of biosynthetic enzymes such as non-ribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs) and regulatory mechanisms governing their expression.

Keywords: Alternaria host specific toxins; Alternaria pathotypes; Fusarium; RIP; accessory chromosomes; biosynthetic gene clusters; metabolomics; secondary metabolites.

FIGURE 1

Structure of host specific toxins (1–21) and other secondary metabolites associated with accessory chromosomes (22–24).

FIGURE 2

AAL-toxin biosynthesis. (A) The gene cluster for AAL-toxin biosynthesis from A. alternata (AB969680). (B) The proposed biosynthesis for AAL-toxin TA1 based on homology to fumonisin biosynthesis. KS, β-ketosynthase; AT-acyltransferase; KR, β-ketoreductase; DH, dehydratase; ER, enoylreductase; MT methyltransferase; A, adenylation; C, condensation.

FIGURE 3

ACR-toxin biosynthesis. (A) The ACRTS1 and ACRTS2 genes involved in ACR-toxin biosynthesis. (B) ACR-toxin is generated from ACRTS2. KS, β-ketosynthase; AT, acyltransferase; KR, β-ketoreductase; DH, dehydratase; ER, enoylreductase; MT, methyltransferase.

FIGURE 4

ACT-toxin biosynthesis. (A) The gene cluster from A. alternata Z7 (LPVP01000048). (B) The proposed biosynthesis of the ACT-, AF-, and AK-toxin core and the addition of the ACT-toxin I side chain. KS, β-ketosynthase; AT, acyltransferase; KR, β-ketoreductase; DH, dehydratase; MT, methyltransferase; A, adenylation; C, condensation; E, epimerization.

FIGURE 5

AM-toxin Biosynthesis. (A) The biosynthetic gene cluster from A. alternata strain NBRC 8984 (AB525198) (B) The biosynthetic pathway for AM-toxin I and its non-proteinogenic building blocks. A, adenylation; C, condensation.

FIGURE 6

Apicidin biosynthesis. (A) The apicidin biosynthetic gene cluster from Fusarium incarnatum. (B) The biosynthesis of apicidin and its non-proteinogeneic amino acid building blocks. A, adenylation; C, condensation; E, epimerization.

FIGURE 7

HC-toxin biosynthesis. (A) The biosynthetic gene cluster for HC-toxin from A. brassicae strain J3 scaffold 18 (SMOM01000016.1). (B) The biosynthesis of HC-toxin and its non-proteinogenic building blocks. A, adenylation; C, condensation; E, epimerization.

FIGURE 8

Koraiol biosynthesis. (A) The terpene synthase gene responsible for koraial biosynthesis. (B) Farnesyl diphosphate cyclization generates koraiol as well as additional 1,11-cyclized sequiterpene natural products.

FIGURE 9

Accessory chromosomes in Fusarium poae illustrate the potential for biosynthetic gene duplication and TE-mediated truncation from which new small molecules could be produced.

FIGURE 10

(A) Comparison of chrysogine synthetase NRPS14 homologs in F. langsethiae Fl 201059 and F. poae 2516, illustrating genomic regions predicted to be heavily influenced by repeat-induced point mutation (RIP). Gray blocks indicate syntenic regions (>90% nt identity). F. langsethiae is a known chrysogine producer, whereas F. poae is not, likely because NRPS14 has been pseudogenized via transposable element insertion. The offending transposable element was then likely subject to RIP, leaving an approximately 800 bp long region of very low GC content (3.4%). (B) Overview of surrounding genetic region on Chr3 showing numerous regions of very low GC content. Red underscores indicate sequence blocks with very low average GC content (<20%).

FIGURE 11

Using metabolomics to identify lineage-specific secondary metabolites produced by active secondary metabolite biosynthetic gene clusters on accessory chromosomes. Extracts from single spore isolates cultured in multiple media conditions are analyzed by high resolution mass spectrometry to produce metabolomes. Metabolomes are averaged across all media to create in vitro consensus chemical phenotypes for each strain. Lineage-specific signals (indicated by the asterisk) can be correlated to secondary metabolite biosynthetic gene clusters detected from isolate genomic analysis. Mass spectra can be further analyzed to compare chemical “fingerprints” (MS2 scans) to online spectral databases or in silico predictions of molecular fragmentation spectra for compound identification.