The Aspergillus flavus spermidine synthase (spds) gene, is required for normal development, aflatoxin production, and pathogenesis during infection of maize kernels

Authors: Majumdar, Raj; Lebar, Matthew; Mack, Brian; MINOCHA, RAKESH - Us Forest Service (FS); MINOCHA, SUBHASH - University Of New Hampshire; Carter-Wientjes, Carol; Sickler, Christine; Rajasekaran, Kanniah - Rajah; Cary, Jeffrey

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/27/2018
Publication Date: 3/20/2018
Citation: Majumdar, R., Lebar, M.D., Mack, B.M., Minocha, R., Minocha, S., Carter-Wientjes, C.H., Sickler, C.M., Rajasekaran, K., Cary, J.W. 2018. The Aspergillus flavus spermidine synthase (spds) gene, is required for normal development, aflatoxin production, and pathogenesis during infection of maize kernels. Frontiers in Plant Science. 9:317. https://doi.org/10.3389/fpls.2018.00317.

Interpretive Summary: Aspergillus flavus (A. flavus) is an opportunistic pathogen that infects several economically important crop species and produces aflatoxins that are potent carcinogens. Aflatoxins impose a significant threat to humans and livestock and substantially reduce crop value. Maize is an important food and feed crop grown worldwide and aflatoxin contamination in maize is a serious concern. Host resistance against A. flavus is a major pre-harvest control of aflatoxins in maize. While traditional breeding techniques are often used to improve aflatoxin resistance in maize, ‘Host Induced Gene Silencing’ (HIGS) through RNA interference (RNAi) is being evaluated as a potential alternative. HIGS technology does not require that the host plant express a foreign protein so food and feed produced from resistant lines of transgenic maize should be more acceptable to regulatory agencies and consumers. To implement effective RNAi against A. flavus requires identification of suitable gene target/s that are critical for fungal growth and aflatoxin production. In the current study we evaluated the importance of a polyamine (nitrogenous compound) biosynthetic gene, spermidine synthase (Spds) that is required to produce the polyamine spermidine (Spd), which is critical for living cells to survive. Disruption of the Spds gene in A. flavus halted growth and required exogenous supply of Spd for the fungus to grow. Down-regulation of Spds in the mutant altered overall nitrogen metabolism and significantly reduced fungal growth, pathogenicity, and the production of aflatoxins during maize seed infection. Spermidine also significantly increased the production of aflatoxins and other secondary metabolites in vitro. The results presented here show that targeting A. flavus Spds gene through RNAi approach in future might be effective in reducing both fungal growth and aflatoxin contamination in maize.

Technical Abstract: Aspergillus flavus is a soil-borne saprophyte and an opportunistic pathogen of both humans and plants. This fungus not only causes disease in several 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 initiation (translational) factor 5A (eIF5A). The tri-amine Spd is synthesized from the di-amine putrescine (Put) through the action of spermidine synthase (Spds). To investigate the role of Spds in A. flavus growth and toxin production, we disrupted the Spds gene (knockout). Inactivation of Spds significantly reduced mycelial growth and sporulation in vitro and addition of exogenous Spd was required to restore fungal growth and sporulation. Complementation of the 'spds 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 augmented SM production. Infection of maize kernels with the 'spds mutant resulted in a significant reduction in fungal growth, sporulation, and aflatoxin production as compared to the controls. Quantitative PCR of 'spds mutant-infected seeds showed down-regulation of aflatoxin biosynthetic genes in the mutant as compared to WT A. flavus infected seeds. Expression analyses of PA metabolism/transport genes during A. flavus-maize interaction showed up-regulation of ornithine decarboxylase (Odc) gene (Put biosynthesis) 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 'spds mutant with Spd and Spd acquisition from the host plant, are insufficient to restore WT levels of pathogenesis during seed infection.