Characterization of stress-releated genes that could affect aflatoxin contamination

Authors: CHU, Y - UNIV OF GA; FAUSTINELLI, P - UNIV OF GA; RAMOS, L - UNIV OF GA; OZIAS-AKINS, P - UNIV OF GA; Holbrook, Carl - Corley; Rajasekaran, Kanniah - Rajah; Cary, Jeffrey; KOLOMIETS, M - TEXAS A&M UNIV

Submitted to: Multicrop Aflatoxin and Fumonisin Elimination and Fungal Genomics Workshop-The Peanut Foundation
Publication Type: Abstract Only
Publication Acceptance Date: 11/15/2007
Publication Date: 1/15/2008
Citation: Chu, Y., Faustinelli, P., Ramos, L., Ozias-Akins, P., Holbrook Jr, C.C., Rajasekaran, K., Cary, J.W., Kolomiets, M. 2008. Characterization of stress-releated genes that could affect aflatoxin contamination. [abstract]. In: Proceedings of the Multicrop Aflatoxin/Fumonisin Elimination and Fungal Genomics Workshop, Atlanta, GA, October 22-24, 2007. p. 105.

Interpretive Summary: not required

Technical Abstract: Aflatoxin contamination has been a major food safety concern for the peanut industry. Production of aflatoxin by Aspergillus flavus is correlated with the level of stress a plant encounters. Previous studies have shown that peanut plants subject to stresses such as drought, heat, or insect damage accumulate higher levels of aflatoxin. In order to reduce aflatoxin in peanut, we have worked on four proteins or protein families that are involved in stress responses. Chloroperoxidase (CPO) was initially isolated from Pseudomonas pyrrocinia. Expression of CPO in tobacco demonstrates significant reduction in A. flavus growth (Jacks et al., 2000). CPO was transformed into peanut through microprojectile bombardment and transgenic CPO-expressing peanut progenies exhibited antifugal activity when tested with an A. flavus strain AF70-GFP in a seed inoculation assay. The second protein introduced into peanut through genetic transformation was Bcl-xL, a human anti-apoptotic gene. Bcl-xL transgenic plants of other species show less negative responses to a wide range biotic and abiotic stresses. Our Bcl-xL transgenic peanut also demonstrated resistance to paraquat, a chloroplast-targeted herbicide. In both CPO and Bcl-xL transgenic events, transgene silencing was observed among progenies. The demethylation agent azacytidine was tested for reactivation of CPO transgene expression; however, no reactivation was observed. In addition to employing transgenes from other species, we also have studied peanut endogeneous genes: ara h 2 and lipoxygenase (lox). Ara h 2 is a major peanut allergen that can be recognized by IgE from >90% of peanut allergic individuals. Other than being an allergen and a seed storage protein, Ara h 2 also functions as a trypsin inhibitor. In maize silencing a 14 kDa trypsin inhibitor promotes the growth of A. flavus and increases production of aflatoxin (Chen et al., 2007). In an effort to produce hypoallergic peanut, we silenced ara h 2 using RNA interference. Ara h 2 silenced transgenic lines show a significant reduction in Ara h 2 protein level. Our Ara h 2 silenced lines can be a useful tool to study the role of Ara h 2 on aflaxtoxin production in peanut. Lipoxygenases in plants catalyze the oxygenation of polyunsaturated fatty acids into a variety of hydroperoxide compounds. The role of LOXs in fungal-host interaction has been clearly demonstrated in maize studies. A ZmLOX 5 knock-out mutant line shows significant reduction in A. flavus growth and inhibition of aflatoxin accumulation. A ZmLOX 3 mutant has reduced fumonisin but increased aflatoxin production (Kolomiets et al., 2007). Peanut LOX genes are also implicated in A. flavus fungal infection. PnLOX 1, a mixed-function LOX, shows increased expression upon A. flavus infection (Burow et al., 2000), whereas PnLOX2-3 (13-LOX) expression was severely reduced (Tsitsigiannis et al., 2005). Currently there is been no thorough study of the LOX gene family in peanut. We used the catalytic region of the maize LOX gene family and three other conserved regions to screen a cDNA phage library generated from peanut seeds. Eighteen positive phage clones were sequenced. A new LOX isoform that demonstrates polymorphism to published peanut LOX genes was identified. Three clones appear to be chimeras of LOX and other unrelated genes. One clone retains an intron sequence. Another clone has a 63 nt deletion in the coding region. Further sequencing and 5’ RACE are needed to complete the coding region analysis. Those LOX genes that are responsive to A. flavus challenge will be further characterized.