Dataset Information

Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene.

ABSTRACT: Aspergillus ?avus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B(1 )(AFB(1)), and aflatoxin G(1) (AFG(1)) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB(1) production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB(1 )production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB(1) production by A. flavus EGP9 and AFG(1 )production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG(1) production by A. parasiticus SSWT 2999. Changes in AFB(1) production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.

SUBMITTER: Abdel-Hadi AM

PROVIDER: S-EPMC3202845 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene.

Abdel-Hadi Ahmed M AM Caley Daniel P DP Carter David R F DR Magan Naresh N

Toxins 20110617 6

Aspergillus ﬂavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (r ...[more]

PMID: 22069731

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Project description:Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the most potent naturally produced carcinogenic secondary metabolites. This pathogen can pose serious health concerns and cause severe economic losses due to the Food and Drug Administration (FDA) regulations on permissible levels of aflatoxins in food and feed. Although biocontrol has yielded some successes in managing aflatoxin contamination, enhancing crop resistance is still the preferred choice of management for long-term sustainability. Hence, host induced gene silencing (HIGS) strategy was explored in this study. The A. flavus gene aflM encoding versicolorin dehydrogenase, a key enzyme involved in the aflatoxin biosynthetic pathway, was selected as a possible target for suppression through HIGS. An RNAi vector containing a portion of the aflM gene was constructed and introduced into immature B104 maize zygotic embryos through Agrobacterium transformation. PCR analysis of the genomic DNA from T0 leaf tissue confirmed the presence of the transgene in six out of the seven events. The seeds from the lines that showed reduced aflatoxin production in laboratory aflatoxin kernel screening assay (KSA) have been increased from T1 to T4 generation in the past four years. Changes in aflatoxin resistance in these transgenic kernels have been evaluated under both field and laboratory conditions. The T2 generation kernels containing the transgene from two events out of four examined had less aflatoxin (P ≤ 0.01 and P ≤ 0.08) than those without the transgene. Field-inoculated homozygous T3 and T4 transgenic kernels also revealed lower levels of aflatoxins (P ≤ 0.04) than kernels from the null (segregated non-transgenic samples) or B104 controls. A similar result was observed when the harvested T3 and T4 homozygous transgenic kernels were evaluated under KSA conditions without inoculation (P ≤ 0.003-0.05). These two events were crossed with LH195, LH197, LH210, and PHW79 elite breeding lines and the resulting crosses supported less aflatoxin (P ≤ 0.02) than the crosses made with non-transgenic lines. In addition, significantly higher levels of aflM gene-specific small RNAs were detected in the transgenic leaf and kernel tissues, indicating that the enhanced aflatoxin resistance in the homozygous transgenic kernels is likely due to suppression of aflM expression through HIGS.

Dataset Information

Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene.

Publications

Control of aflatoxin production of Aspergillus flavus and Aspergillus parasiticus using RNA silencing technology by targeting aflD (nor-1) gene.

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