Cloning and Characterization of a Cyclic Peptide Synthetase Gene from Alternaria alternata Apple Pathotype Whose Product Is Involved in AM-Toxin Synthesis and Pathogenicity

Abstract

Afternaria afternata apple pathotype causes Alternaria blotch of susceptible apple cultivars through the production of a cyclic peptide host-specific toxin, AM-toxin. PCR (polymerase chain reaction), with primers designed to conserved domains of peptide synthetase genes, amplified several products from A. alternata apple pathotype that showed high similarity to other fungal peptide synthetases and were specific to the apple pathotype. Screening of a Lambda Zap genomic library with these PCR-generated probes identified overlapping clones containing a complete cyclic peptide synthetase gene of 13.1 kb in length with no introns. Disruption of this gene, designated AM-toxin synthetase (AMT), by transformation of wild-type A. afternata apple pathotype with disruption vectors resulted in toxin-minus mutants, which were also unable to cause disease symptoms on susceptible apple cultivars. AM-toxin synthetase is therefore a primary determinant of virulence and specificity in the A. alternata apple pathotype/apple interaction.

Horizontal Chromosome Transfer, a Mechanism for the Evolution and Differentiation of a Plant-Pathogenic Fungus

“…In Alternaria alternata plant pathogens (37), we have shown that all strains of the A. alternata pathotypes harbor small extra chromosomes of less than 1.7 Mb, whereas nonpathogenic isolates do not have these small chromosomes (5). A cyclic peptide synthetase gene, AMT, which is involved in host-specific AM toxin biosynthesis of the apple pathotype of A. alternata, was located on a small chromosome of 1.1 to 1.7 Mb, depending on the strain (22,23). The AF toxin biosynthesis gene cluster was also present on a single small chromosome of 1.05 Mb in the strawberry pathotype of A. alternata (18).…”

Functional Analysis of All Nonribosomal Peptide Synthetases in Cochliobolus heterostrophus Reveals a Factor, NPS6, Involved in Virulence and Resistance to Oxidative Stress

“…In addition to structural diversity, nonribosomal peptides have a broad spectrum of biological activities, some of which have been useful in medicine, agriculture, and biological research (24,37,40,47). Products made by nonribosomal peptide synthetases or nonribosomal peptide synthetase/polyketide synthase hybrid enzymes include well-known antibiotics (penicillin, erythromycin, and vancomycin), immunosuppressants (cyclosporin and rapamycin), antitumor agents (actinomycin, bleomycin, and epothilone) (39,47), and toxins involved in pathogenesis (HC-toxin, enniatin, AM-toxin, and probably victorin) (17,22,32,38,48,49).…”

“…Some proposed activities include roles as signal molecules for coordination of growth and differentiation (16,21,28,35), as aids in the breakdown of cellular metabolic products (9), as defense compounds that kill competing microorganisms (46), as siderophores to assist in iron uptake (5), and as virulence effectors (17,22,32,38).…”

“…Relatively fewer fungal genes encoding nonribosomal peptide synthetases have been fully sequenced and their corresponding peptide structures determined. These include Tolypocladium inflatum (niveum) simA, which controls production of the immunosuppressive drug cyclosporine (50), Penicillium chrysogenum acvA, which controls production of the antibiotic penicillin (2), Ustilago maydis sid2 and Aspergillus nidulans sidC, each of which controls production of a siderophore (55,12), Claviceps purpurea cpps1 and cpps2, both of which are involved in the synthesis of ergot alkaloids (6,43), Hypocrea virens tex1, which controls peptaibol synthesis (51), Leptosphaeria maculans SirP, which controls sirodesmin biosynthesis (12), and three genes that control production of peptide virulence effectors: HTS1, which controls production of HC toxin by C. carbonum, a maize pathogen (38), AMT1, which controls production of AM toxin by Alternaria alternata, an apple tree pathogen (22), and Esyn1, which controls production of enniatin by Fusarium scirpi and other Fusarium spp., potato and tomato pathogens (17,19).…”

A Zinc-Finger-Family Transcription Factor, AbVf19, Is Required for the Induction of a Gene Subset Important for Virulence in Alternaria brassicicola

“…Several A. alternata pathotypes produce toxic, host-specific secondary metabolites that are pathogenicity factors (Ito et al, 2004;Johnson et al, 2000;Spassieva et al, 2002;Tanaka et al, 1999). Several secondary metabolites and proteins in A. brassicicola have been identified as toxins, such as depudecin (Wight et al, 2009), brassicenes (Hashimoto et al, 2009), Brassicicolin A (Pedras et al, 2009a), and two proteins (Oka et al, 2005;Otani et al, 1998).…”