Transgenic Control of Aflatoxin Contamination in Maize Through Host-Induced Gene Silencing Targeting Aspergillus flavus Genes Encoding Polygalacturonase (p2c) and Versicolorin Dehydrogenase (aflM)

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\u3e0.02) 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\u3e0.02) 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

Targeting the Aspergillus flavus p2c gene through host-induced gene silencing reduces A. flavus infection and aflatoxin contamination in transgenic maize

Aspergillus flavus is an opportunistic fungal pathogen that infects maize and produces aflatoxins. Using biocontrol or developing resistant cultivars to reduce aflatoxin contamination has only achieved limited success. Here, the A. flavus polygalacturonase gene (p2c) was targeted for suppression through host-induced gene silencing (HIGS) to reduce aflatoxin contamination in maize. An RNAi vector carrying a portion of the p2c gene was constructed and transformed into maize B104. Thirteen out of fifteen independent transformation events were confirmed to contain p2c. The T2 generation kernels containing the p2c transgene had less aflatoxin than those without the transgene in six out of eleven events we examined. Homozygous T3 transgenic kernels from four events produced significantly less aflatoxins (P ≤ 0.02) than the kernels from the null or B104 controls under field inoculation conditions. The F1 kernels from the crosses between six elite inbred lines with P2c5 and P2c13 also supported significantly less aflatoxins (P ≤ 0.02) than those from the crosses with null plants. The reduction in aflatoxin ranged from 93.7% to 30.3%. Transgenic leaf (T0 and T3) and kernel tissues (T4) were also found to have significantly higher levels of p2c gene-specific small RNAs. Further, homozygous transgenic maize kernels had significantly less fungal growth (27~40 fold) than the null control kernels 10 days after fungal inoculation in the field. The calculated suppression of p2c gene expression based on RNAseq data was 57.6% and 83.0% in P2c5 and P2c13 events, respectively. These results indicate clearly that the reduced aflatoxin production in the transgenic kernels is due to RNAi-based suppression of p2c expression, which results in reduced fungal growth and toxin production