IDENTIFICATION AND ENHANCEMENT OF SEED-BASED BIOCHEMICAL RESISTANCE IN CROPS TO AFLATOXIN PRODUCING PATHOGENS
Location: Food and Feed Safety Research
Title: Identifying Aflatoxin Resistance-Related Proteins/Genes Through Proteomics and RNAi Gene Silencing
Submitted to: Peanut Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 5, 2007
Publication Date: January 12, 2009
Citation: Chen, Z.-Y, Brown, R.L., Guo, B.Z., Menkir, A., Cleveland, T.E. 2009. Identifying Aflatoxin Resistance-Related Proteins/Genes Through Proteomics and RNAi Gene Silencing. Peanut Science. 36(1):35-41.
Aflatoxins are carcinogenic secondary metabolites produced mainly by Aspergillus flavus Link ex. Fries, and A. prarasiticus Speare during infection of susceptible crops, such as maize, cottonseed, peanuts, and tree nuts. For maize, although genotypes resistant to A. flavus infection or aflatoxin production have been identified, the incorporation of resistance into commercial lines has been slow due to the lack of selectable markers and poor understanding of host resistance mechanisms. Recently, resistance-associated proteins (RAPs) were identified through proteomic comparison of constitutive protein profiles between resistant and susceptible maize genotypes. These proteins belong to three major groups based on their peptide sequence homologies: storage proteins, stress-related proteins, and antifungal proteins. Preliminary characterization of some of these RAPs suggest that they play a direct role in host resistance, such as pathogenesis-related protein 10 (PR10), or an indirect role, such as glyoxalase I (GLX I), through enhancing the host stress tolerance.
To verify whether these RAPs play a direct role in host resistance, RNA interference (RNAi) gene silencing technique was used to silence the expression of these genes in maize. RNAi vectors (GLX I RNAi and PR10 RNAi) were constructed suing Gateway technology, and then transformed into immature maize embryos using both bombardment and Agrobacterium infection. Thirty-two out of 38 and 11 out of 15 independent callus lines of glx I and pr10, respectively were confirmed positive for transformation through PCR. The extent of gene silencing in transgenic callus tissues ranged from 20% to over 99%. The RNAi silenced transgenic maize seeds have also been obtained from plants regenerated from Agrobacterium transformed callus lines. For each construct, kernels from 8 ears were germinated and genomic DNA was isolated. PCR confirmation of transformation using seedling genomic DNA found that only two out of eight and one out of eight were negative, for glx I and pr10, respectively. Kernel screen assay of the transgenic maize kernels demonstrated a significant increase in susceptibility to A. flavus colonization and aflatoxin production in some of silenced transgenic lines compared with non-silenced control kernels, suggesting the direct involvement of these two proteins in aflatoxin resistance in maize.