Submitted to: Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2009
Publication Date: 1/15/2010
Citation: Chen, Z., Brown, R.L., Damann, K.E., Cleveland, T.E. 2010. PR10 expression in maize and its effect on host resistance against Aspergillus flavus infection and aflatoxin production. Molecular Plant Pathology. 11(1):69-81. Interpretive Summary: The fungus named Aspergillus flavus produces a poison called aflatoxin when it infects corn kernels. Aflatoxin prevents the corn from being used commercially. The best strategy for controlling this problem is to develop corn that is resistant to aflatoxin contamination. Towards this aim, we isolated and identified through comparisons of resistant with susceptible corn lines, proteins that are produced in relatively higher amounts in the resistant lines. One of these proteins, pathogenesis-related protein 10 (PR-10), shown to inhibit growth of Aspergillus flavus, was studied further to enhance our understanding of its involvement in corn resistance. The pr-10 gene was knocked out using a technique called RNAi gene silencing in conjunction with corn transformation technology. Kernels with low or no production of PR-10 significantly and greatly increased in susceptibility to aflatoxin production. Further studies may determine this protein to be a marker for resistance and therefore useful to breeders developing aflatoxin-resistant commercial corn. This could lead to future savings of millions of dollars to growers, as a result of the elimination of aflatoxin contamination of corn.
Technical Abstract: Maize (Zea mays L.) is a major crop susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the potent carcinogenic secondary metabolites of the fungus. Protein profiles of maize genotypes resistant and susceptible to A. flavus infection and/or aflatoxin contamination were compared, and several resistance-associated proteins have been found, including a pathogenesis-related protein 10 (PR10). In the present study, RNAi gene silencing technology was employed to further investigate the importance of PR10. An RNAi gene silencing vector was constructed and introduced into immature Hi II maize embryos through both bombardment and Agrobacterium infection procedures. Pr10 expression was reduced by 65% to over 99% in transgenic callus lines from bombardment. The RNAi silenced callus lines also showed increased sensitivity to heat stress treatment. Similar reduction in pr10 transcript levels was observed in seedling leaf and root tissues developed from transgenic kernels. When inoculated with A. flavus, RNAi silenced mature kernels produced from Agrobacterium-mediated transformation showed significant increase in fungal colonization and aflatoxin production in ten and six, respectively, out of 11 RNAi lines compared to the non16 silenced control. Further proteomic analysis of RNAi silenced kernels revealed a significant reduction in PR10 production in eight out of 11 RNAi lines that showed positive for transformation. A significant negative correlation between the PR10 expression at either transcript or protein level and the kernel aflatoxin production was observed. Results indicate a major role for PR10 expression in maize aflatoxin resistance.