Thanatin confers partial resistance against aflatoxigenic fungi in maize (Zea mays)

Abstract

Aflatoxin-producing fungi can contaminate plants and plant-derived products with carcinogenic secondary metabolites that present a risk to human and animal health. In this study, we investigated the effect of antimicrobial peptides on the major aflatoxigenic fungi Aspergillus flavus and A. parasiticus. In vitro assays with different chemically-synthesized peptides demonstrated that the broad-spectrum peptide thanatin from the spined soldier bug (Podisus maculiventris) had the greatest potential to eliminate aflatoxigenic fungi. The minimal inhibitory concentrations of thanatin against A. flavus and A. parasiticus were 3.13 and 12.5 µM, respectively. A thanatin cDNA was subsequently cloned in a plant expression vector under the control of the ubiquitin-1 promoter allowing the recombinant peptide to be directed to the apoplast in transgenic maize plants. Successful integration of the thanatin expression cassette was confirmed by PCR and expression was demonstrated by semi-quantitative RT-PCR in transgenic maize kernels. Infection assays with maize kernels from T1 transgenic plants showed up to three-fold greater resistance against Aspergillus spp. infections compared to non-transgenic kernels. We demonstrated for the first time that heterologous expression of the antimicrobial peptide thanatin inhibits the growth of Aspergillus spp. in transgenic maize plants offering a solution to protect crops from aflatoxin-producing fungi and the resulting aflatoxin contamination in the field and under storage conditions.

Functional Biology and Molecular Mechanisms of Host-Pathogen Interactions for Aflatoxin Contamination in Groundnut (Arachis hypogaea L.) and Maize (Zea mays L.)

“…Recently, aflC and aflR genes were targeted that encode the enzyme in Aspergillus aflatoxin biosynthetic pathway to develop aflatoxin-free transgenic kernels (Masanga et al, 2015;Thakare et al, 2017). Also, thanatin, a growth inhibitor of A. flavus, was overexpressed in maize, reducing aflatoxin contamination and increasing resistance by three to four-fold resistance (Schubert et al, 2015).…”

Advances in molecular and genomic research to safeguard food and feed supply from aflatoxin contamination

“…Molecular breeding has become a useful rapid technology for development of crops resistant to aflatoxin contamination. Prominent among them is heterologous expression of foreign antifungal gene(s), either natural or synthetic, to provide resistance to the fungus and toxin production (Rajasekaran et al, 2005b;Ruhlman et al, 2014;Schubert et al, 2015;Sharma et al, in press). RNA-interference (RNAi)-mediated host induced gene silencing (HIGS) of key fungal genes critical for fungal pathogenesis and aflatoxin production has been successfully employed to incorporate A. flavus resistance in susceptible maize or peanut varieties (Arias et al, 2015;Bhatnagar-Mathur et al, 2015;Majumdar et al, 2017a;Masanga et al, 2015;Power et al, 2017;Sharma et al, in press;Thakare et al, 2017).…”

Surveying the potential of secreted antimicrobial peptides to enhance plant disease resistance

“…While transgenic rice plants were not fully protected against M. oryzae , they had acquired partial resistance (Imamura et al, 2010 ). Very recently, thanatin gene was introduced into maize and controlled by the ubiquitin-1 promoter which targets the expressed AMP to the plant apoplastic space (Schubert et al, 2015 ; Table 3 ). Transgenic maize plants were grown until ears were fully developed and the mature ears were then exposed to the pathogenic fungus Aspergillus flavus .…”

“…Transgenic maize plants were grown until ears were fully developed and the mature ears were then exposed to the pathogenic fungus Aspergillus flavus . The expression of thanatin in maize transformants led to a significant reduction in fungal biomass of A. flavus relative to that of WT plants (Schubert et al, 2015 ). The mode-of-action of thanatin in plants has yet to be fully understood.…”