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Identification of maize genes associated with host plant resistance or susceptibility to Aspergillus flavus infection and aflatoxin accumulation.

Kelley RY, Williams WP, Mylroie JE, Boykin DL, Harper JW, Windham GL, Ankala A, Shan X - PLoS ONE (2012)

Bottom Line: Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35.A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35.A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, Mississippi, United States of America.

ABSTRACT

Background: Aspergillus flavus infection and aflatoxin contamination of maize pose negative impacts in agriculture and health. Commercial maize hybrids are generally susceptible to this fungus. Significant levels of host plant resistance have been observed in certain maize inbred lines. This study was conducted to identify maize genes associated with host plant resistance or susceptibility to A. flavus infection and aflatoxin accumulation.

Results: Genome wide gene expression levels with or without A. flavus inoculation were compared in two resistant maize inbred lines (Mp313E and Mp04:86) in contrast to two susceptible maize inbred lines (Va35 and B73) by microarray analysis. Principal component analysis (PCA) was used to find genes contributing to the larger variances associated with the resistant or susceptible maize inbred lines. The significance levels of gene expression were determined by using SAS and LIMMA programs. Fifty candidate genes were selected and further investigated by quantitative RT-PCR (qRT-PCR) in a time-course study on Mp313E and Va35. Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E.

Conclusion: Maize genes associated with host plant resistance or susceptibility were identified by a combination of microarray analysis, qRT-PCR analysis, and QTL mapping methods. Our findings suggest that multiple mechanisms are involved in maize host plant defense systems in response to Aspergillus flavus infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation.

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Bar-graph showing the comparison of the grand mean relative expression levels for each candidate gene in Mp313E and Va35 lines over all time points.Thirty-one of the 50 genes were found to be significantly differentially expressed (P<0.05) by a paired t-test between the Mp313E and Va35 samples. The error bars represent 95% CI values.
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pone-0036892-g002: Bar-graph showing the comparison of the grand mean relative expression levels for each candidate gene in Mp313E and Va35 lines over all time points.Thirty-one of the 50 genes were found to be significantly differentially expressed (P<0.05) by a paired t-test between the Mp313E and Va35 samples. The error bars represent 95% CI values.

Mentions: The maize microarray analysis provided a list of candidate genes possibly contributing to the host plant resistance and susceptibility. To verify and study the selected candidate genes in more details, we conducted a separate experiment for a time course quantitative study using qRT-PCR method. The resistant maize inbred line Mp313E and the susceptible maize inbred line Va35 were selected for this study. The field experimental design was a randomized complete block design with three replications for each genotype. Kernel samples were collected in the field over eight time points. The first collection was performed within three hours after the fungal inoculation on the same day (0) and the other collecting time points were at 1, 2, 3, 4, 7, 14, and 21 days after inoculation. There were three replications collected for each time point. We prepared RNA samples and performed qRT-PCR analysis to evaluate the expression levels of 50 candidate genes (Table 2) in Mp313E and Va35. The maize GAPDH gene was used for normalization. The qRT-PCR data were analyzed using the software GenEx 5. 0. 1 [20]. Figure 2 is a bar-graph showing the comparison on the grand mean expression levels for each gene between Mp313E and Va35 lines. Thirty-one of the 50 genes were found to be significantly differentially expressed (P<0.05) by a paired t-test between the Mp313E and Va35 samples (Table 3). PCA analysis on the samples showed that Mp313E and Va35 samples were classified into two distinct groups (Figure 3), which indicated that the criteria we used for candidate gene selection from the microarray data were effective in reflecting host plant specific responses to the fungal infection. Figure 4 is a plot from a PCA analysis on the 50 candidate genes to visualize group of genes differentially expressed in Mp313E versus Va35. Genes highly expressed in Mp313E were present in the area with positive y axis coordinates in this plot, whereas genes located in the area with negative y axis coordinates in this plot were those expressed more inVa35 (Figure 4).


Identification of maize genes associated with host plant resistance or susceptibility to Aspergillus flavus infection and aflatoxin accumulation.

Kelley RY, Williams WP, Mylroie JE, Boykin DL, Harper JW, Windham GL, Ankala A, Shan X - PLoS ONE (2012)

Bar-graph showing the comparison of the grand mean relative expression levels for each candidate gene in Mp313E and Va35 lines over all time points.Thirty-one of the 50 genes were found to be significantly differentially expressed (P<0.05) by a paired t-test between the Mp313E and Va35 samples. The error bars represent 95% CI values.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351445&req=5

pone-0036892-g002: Bar-graph showing the comparison of the grand mean relative expression levels for each candidate gene in Mp313E and Va35 lines over all time points.Thirty-one of the 50 genes were found to be significantly differentially expressed (P<0.05) by a paired t-test between the Mp313E and Va35 samples. The error bars represent 95% CI values.
Mentions: The maize microarray analysis provided a list of candidate genes possibly contributing to the host plant resistance and susceptibility. To verify and study the selected candidate genes in more details, we conducted a separate experiment for a time course quantitative study using qRT-PCR method. The resistant maize inbred line Mp313E and the susceptible maize inbred line Va35 were selected for this study. The field experimental design was a randomized complete block design with three replications for each genotype. Kernel samples were collected in the field over eight time points. The first collection was performed within three hours after the fungal inoculation on the same day (0) and the other collecting time points were at 1, 2, 3, 4, 7, 14, and 21 days after inoculation. There were three replications collected for each time point. We prepared RNA samples and performed qRT-PCR analysis to evaluate the expression levels of 50 candidate genes (Table 2) in Mp313E and Va35. The maize GAPDH gene was used for normalization. The qRT-PCR data were analyzed using the software GenEx 5. 0. 1 [20]. Figure 2 is a bar-graph showing the comparison on the grand mean expression levels for each gene between Mp313E and Va35 lines. Thirty-one of the 50 genes were found to be significantly differentially expressed (P<0.05) by a paired t-test between the Mp313E and Va35 samples (Table 3). PCA analysis on the samples showed that Mp313E and Va35 samples were classified into two distinct groups (Figure 3), which indicated that the criteria we used for candidate gene selection from the microarray data were effective in reflecting host plant specific responses to the fungal infection. Figure 4 is a plot from a PCA analysis on the 50 candidate genes to visualize group of genes differentially expressed in Mp313E versus Va35. Genes highly expressed in Mp313E were present in the area with positive y axis coordinates in this plot, whereas genes located in the area with negative y axis coordinates in this plot were those expressed more inVa35 (Figure 4).

Bottom Line: Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35.A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35.A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, Mississippi State University, Mississippi State, Mississippi, United States of America.

ABSTRACT

Background: Aspergillus flavus infection and aflatoxin contamination of maize pose negative impacts in agriculture and health. Commercial maize hybrids are generally susceptible to this fungus. Significant levels of host plant resistance have been observed in certain maize inbred lines. This study was conducted to identify maize genes associated with host plant resistance or susceptibility to A. flavus infection and aflatoxin accumulation.

Results: Genome wide gene expression levels with or without A. flavus inoculation were compared in two resistant maize inbred lines (Mp313E and Mp04:86) in contrast to two susceptible maize inbred lines (Va35 and B73) by microarray analysis. Principal component analysis (PCA) was used to find genes contributing to the larger variances associated with the resistant or susceptible maize inbred lines. The significance levels of gene expression were determined by using SAS and LIMMA programs. Fifty candidate genes were selected and further investigated by quantitative RT-PCR (qRT-PCR) in a time-course study on Mp313E and Va35. Sixteen of the candidate genes were found to be highly expressed in Mp313E and fifteen in Va35. Out of the 31 highly expressed genes, eight were mapped to seven previously identified quantitative trait locus (QTL) regions. A gene encoding glycine-rich RNA binding protein 2 was found to be associated with the host hypersensitivity and susceptibility in Va35. A nuclear pore complex protein YUP85-like gene was found to be involved in the host resistance in Mp313E.

Conclusion: Maize genes associated with host plant resistance or susceptibility were identified by a combination of microarray analysis, qRT-PCR analysis, and QTL mapping methods. Our findings suggest that multiple mechanisms are involved in maize host plant defense systems in response to Aspergillus flavus infection and aflatoxin accumulation. These findings will be important in identification of DNA markers for breeding maize lines resistant to aflatoxin accumulation.

Show MeSH
Related in: MedlinePlus