Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in important food and feed crops such as maize, peanut,
cottonseed, and tree nuts but also produces the toxic and carcinogenic secondary metabolites (SMs) known as
aflatoxins.
Polyamines (PAs) are ubiquitous
polycations that influence normal growth, development, and stress responses in living organisms and have been shown to play a significant role in fungal pathogenesis. Biosynthesis of
spermidine (Spd) is critical for cell growth as it is required for hypusination-mediated activation of
eukaryotic translation initiation factor 5A (eIF5A), and other biochemical functions. The tri-
amine Spd is synthesized from the
diamine putrescine (Put) by the
enzyme spermidine synthase (Spds). Inactivation of spds resulted in a total loss of growth and sporulation in vitro which could be partially restored by addition of exogenous Spd. Complementation of the Δspds mutant with a wild type (WT) A. flavus spds gene restored the WT phenotype. In WT A. flavus, exogenous supply of Spd (in vitro) significantly increased the production of sclerotia and SMs.
Infection of maize kernels with the Δspds mutant resulted in a significant reduction in fungal growth, sporulation, and
aflatoxin production compared to controls. Quantitative PCR of Δspds mutant infected seeds showed down-regulation of
aflatoxin biosynthetic genes in the mutant compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed significant increase in the expression of
arginine decarboxylase (Adc) and
S-adenosylmethionine decarboxylase (Samdc) genes in the maize host and PA uptake transporters in the fungus. The results presented here demonstrate that Spd biosynthesis is critical for normal development and pathogenesis of A. flavus and pre-treatment of a Δspds mutant with Spd or Spd uptake from the host plant, are insufficient to restore WT levels of pathogenesis and
aflatoxin production during seed
infection. The data presented here suggest that future studies targeting
spermidine biosynthesis in A. flavus, using RNA interference-based host-induced gene silencing approaches, may be an effective strategy to reduce
aflatoxin contamination in maize and possibly in other susceptible crops.