Doctor of Philosophy (PhD)
Plant Pathology and Crop Physiology
Maize (Zea mays L.) is susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the most potent naturally produced carcinogenic secondary metabolites. Here, the A. flavus gene aflM encoding the versicolorin dehydrogenase in aflatoxin biosynthesis and the p2c gene encoding the polygalacturonase that is involved in infection were selected as targets for suppression through host induced gene silencing (HIGS). A HIGS vector targeting these genes was constructed and introduced into immature B104 maize embryos. Thirteen out of fifteen p2c transformation events and six out of seven aflM events were confirmed positive by PCR. Kernels containing the p2c gene from four out of seven events examined from T1 generation and six events out of eleven events from T2 generation examined had less aflatoxin than those without the transgene. Field-inoculated homozygous T3 and T4 generation kernels from four events also revealed significantly lower aflatoxins (p>0.02) than the kernels from the null or B104 controls. Transferring the p2c gene to elite background also resulted in crosses with significantly less aflatoxin production (p>0.02) compared to the controls. For aflM transgenic lines, kernels containing the aflM gene from one (aflM14) out four events examined in T1 generation and from two events (aflM14 and aflM16) out of four examined had less aflatoxin (p≤0.01 and p≤0.08) than those without the transgene than those without the transgene. Homozygous T3 and T4 transgenic kernels of aflM14 and aflM16 showed significantly less aflatoxin production than kernels from the null or B104 controls under both field inoculation and laboratory kernel screening assay conditions. Transferring aflM from these events into four elite inbred lines resulted in F1 crosses with significantly less aflatoxins (p≤0.02) than the controls. P2c13, P2c17, aflM14 and aflM16 were confirmed to contain a single copy insertion of the transgene according to droplet digital PCR analysis. The enhanced aflatoxin resistance in homozygous transgenic lines is associated with high levels of gene specific small RNAs detected in the transgenic leaf and kernel tissues, indicating that it is possible to manage aflatoxin contamination in maize through HIGS targeting p2c or aflM.
Raruang, Yenjit, "Transgenic Control of Aflatoxin Contamination in Maize Through Host-Induced Gene Silencing Targeting Aspergillus flavus Genes Encoding Polygalacturonase (p2c) and Versicolorin Dehydrogenase (aflM)" (2019). LSU Doctoral Dissertations. 5102.