ABSTRACT: Aflatoxin contamination caused by the opportunistic pathogen A. flavus is a major concern in maize production prior to harvest and during storage. Previous studies indicate that both constitutive and induced resistance are involved in maize kernel defense against A. flavus infection, little is known about molecular mechanisms of mature kernels in response to fungal infection. The purpose of this study is to determine gene expression differences in maize kernels between resistant and susceptible lines in response to A. flavus challenge. To avoid the environmental effects in the field inoculation, a laboratory based inoculation technique Kernel Screening Assay (KSA) was used to challenge kernels with A. flavus. After 72 hours incubation of inoculated and noninculated mature kernels, gene expression profiles of two Near Isogenic Lines (NIL) of Eyl25 (A. flavus resistant) and Eyl31 (A. flavus susceptible) were compared using oligonucleotide array. Direct comparisons were designed. The comparisons of NIL Eyl25 and Eyl31 include: Treated/Control, Control/Control and Treated/Treated. After 72 hours incubation under KSA conditions, forty seeds used in each A. flavus inoculated and noninculated group were bulked to extract total RNA. Four technical replications were performed in each comparison including two dye-swaps, total 16 hybridization reactions.
Project description:Aflatoxin contamination occurring after infection by Aspergillus flavus is a major concern in maize production both pre- and post-harvest. A recent gene expression study of imbibed kernels highlighted induced resistance and gene regulation in kernels. In the present study, stored mRNA was profiled using oligo-nucleotide array. This comparison of stored mRNAs would enhance our understanding of the difference between resistant and susceptible lines at the kernel storage phase. To minimize the effect of different genetic backgrounds on the differential genes between resistant and susceptible lines, two closely related inbred lines were used. Of the two lines, Eyl25 is aflatoxin-resistant (R), and Eyl31 is –susceptible (S). These were derived from a cross between two resistant lines, 1368 and GT-MASK:gk, in the IITA and USDA- ARS collaborative breeding project. Direct comparisons were designed between Eyl25 and Eyl31. Dry kernels from the two lines were selected randomly into four groups to extract total RNA. Four biological replications were performed in the comparison including two dye-swaps.
Project description:Aflatoxin contamination caused by the opportunistic pathogen A. flavus is a major concern in maize production prior to harvest and during storage. Previous studies indicate that both constitutive and induced resistance are involved in maize kernel defense against A. flavus infection, little is known about molecular mechanisms of mature kernels in response to fungal infection. The purpose of this study is to determine gene expression differences in maize kernels between resistant and susceptible lines in response to A. flavus challenge. To avoid the environmental effects in the field inoculation, a laboratory based inoculation technique Kernel Screening Assay (KSA) was used to challenge kernels with A. flavus. After 72 hours incubation of inoculated and noninculated mature kernels, gene expression profiles of two Near Isogenic Lines (NIL) of Eyl25 (A. flavus resistant) and Eyl31 (A. flavus susceptible) were compared using oligonucleotide array.
Project description:Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understand host response to infection by the fungus, transcription of approximately 9,000 maize genes were monitored during the host-pathogen interaction with a custom-designed Affymetrix GeneChip® DNA array. More than 1,000 maize genes were found differentially expressed at a fold change of 2 or greater. This included the up regulation of defense-related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development. Maize kernels were mock inoculated at the blister (R2) or dough (R4) stage or inoculated with A. flavus at the blister (R2), milk (R3), dough (R4), or dent (R5) stage, and harvested 4 days later. Each treatment consisted of three biological replications. For each biological replication, 8 kernels were ground and RNA was isolated and further processed.
Project description:Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understand host response to infection by the fungus, transcription of approximately 9,000 maize genes were monitored during the host-pathogen interaction with a custom-designed Affymetrix GeneChip® DNA array. More than 1,000 maize genes were found differentially expressed at a fold change of 2 or greater. This included the up regulation of defense-related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development.
Project description:Maize kernel is an important source of food, feed and industrial raw materials. The illustration of the molecular mechanisms of maize kernel development will be helpful for the manipulation of maize improvements. Although a great many researches based on molecular biology and gecetics have greatly increased our understanding on the kernel development, many of the mechanisms controlling this important process remain elusive. In current study, a microarray with approximately 58,000 probes was used to study the dynamic gene expression during kernel development from the fertilization to physiological maturity. Samples from two consecutive time-points were paired and labeled using different fluorescent dyes (Cy3 and Cy5) and hybridized in the same array. Hybridization of slides was performed according to the manufacturer’s instructions (http://www.maizearray.org/). The hybridized slides were scanned by a Genepix 4000B (Axon, USA). A loop design was applied for running the microarray. Two replicates of each pair of samples were carried out to test both the reproducibility and quality of the chip hybridizations. By comparing six consecutive time-points, namely 1, 5, 10, 15, 25 and 35 days after pollination (DAP), 3,445 differentially expressed genes were identified. These genes were then grouped into 10 clusters showing specific expression patterns using a K-means clustering algorithm. An investigation of function and expression patterns of genes expanded our understanding of the regulation mechanism underlying the important developmental processes, cell division and kernel filling. The differential expression of genes involved in plant hormone signaling pathways suggested that phytohormone might play a critical role in the kernel developmental process. Moreover, regulation of some transcription factors and protein kinases might be involved in the whole developmental process. To obtain the global gene expression profile during maize kernel development, a microarray with approximately 58,000 probes was used. The maize inbred line X178 was planted on the field. Each plant was self-pollinated by hand. The ears were harvested from healthy plants at 1, 5, 10, 15, 25 and 35 days after pollination (DAP), respectively. In order to increase the consistency & uniformity of the isolated kernels, the upper half and about one sixth of ears from the bottom were cut and discarded, the kernels were isolated from the rest part of the ears. Samples at each time-point were collected from at least thirty ears and pooled to represent the line characteristics of X178. Two sub-samples for replication in the microarray analysis were randomly drawn. Samples from two consecutive time-points were paired and labeled using different fluorescent dyes (Cy3 and Cy5) and hybridized in the same array. A loop design was applied for running the microarray. Two replicates of each pair of samples were carried out to test both the reproducibility and quality of the chip hybridizations.
Project description:The experiment consisted of a field design containing two maize inbred lines Va35 and Mp313E. Va35 has yellow kernels and is susceptible to kernel infection by Aspergillus flavus (A. flavus). Mp313E is a white dent maize inbred line and was released primarily as a source of resistance to kernel infection by A. flavus. The test ears were inoculated with Aspergillus flavus and collected two days after inoculation. Non-inoculated ears collected 16 days after pollination were also used as a control. The microarray experimental design was a randomized complete block design with three replications. One microarray slide was used for each field plot. Each slide contained the inoculated and non-inoculated sub-treatments. A second slide for one replication of each genotype contained a dye swap for the inoculation treatment and three subsamples (dots) within the slide represented each contig. Keywords: Direct comparison
Project description:Quality Protein Maize (QPM) was created by selecting for genetic modifiers that convert the starchy endosperm of an opaque2 (o2) mutant to a hard, vitreous phenotype. Genetic analysis has shown there are multiple, unlinked o2 modifiers (Opm), but their identity and mode of action are unknown. A microarray hybridization performed with RNA obtained from true breeding o2 progeny with vitreous and opaque kernel phenotypes identified a small group of differentially expressed genes, some of which map at or near the Opm QTLs. Compared 18 days after pollination endosperm transcript profiles in true breeding modified (vitreous; V) and non-modified (opaque; O) opaque 2 kernels. Used four vitreous biological reps (V1-V4) and four opaque biological reps (O1-O4). Each biological rep is a pool of 25 kernels representing 5 kernels form each of 5 uniform phenotype ears (vitreous or opaque). Four arrays used: Array 1= V1 vs O1, Array 2 = V2 vs O2, Array 3 = V3 vs O3, Array 4 = V4 vs O4
Project description:Fusarium verticillioides is a detrimental fungus that can contaminate maize grains with mycotoxins that are harmful to human and animal health. Breeding and growing resistant genotypes is one alternative to reduce contamination and subsequent production of mycotoxins by this fungus. However, little is known about the resistant mechanism relevant to breeding in this pathosystem. Therefore, our aim was to identify genes and metabolites that may be related to Fusarium ear rot resistance using resistant and susceptible maize inbreds. Kernels of the resistant inbred showed significantly reduced disease severity, and reduced levels of total fumonisin and ergosterol content compared with the susceptible one. Gene expression data were obtained from microarray hybridizations using F. verticillioides inoculated and non inoculated maize kernels. Differentially expressed sequences were identified and classified into 36 functional categories. Most of the differentially expressed genes were assigned to the categories “protein, RNA, DNA, stress, transport, signaling and cell metabolism”. These genes encode for PR proteins, detoxification and primary metabolism enzymes. Fungal inoculation did not produce considerable changes in gene expression and metabolites in the resistant L4637 inbred, probably due to a preformed or constitutive resistance mechanism. Defense-related genes were induced or repressed in kernels of the susceptible inbred L4674, responding specifically to the pathogen infection. The qRT-PCR in infected silks showed that glucanase, lipid transfer, xylanase inhibitor, PR1 and 26S proteosome transcripts had higher expression ratios in the susceptible line compared to the resistant one in response to fungal infection. Through this study, a global view of differential genes expressed and metabolites concentration during resistance and susceptibility to F. verticillioides inoculation has been obtained, giving additional information about the mechanisms and pathways conferring resistance to this important disease in maize. Global view of differential genes expressed during resistance and susceptibility to F. verticillioides inoculation. Two maize inbred lines : one resistant (L4637) and one susceptible (L4674) to F. verticillioides infection. Two-condition experiment, Inoculated (I) vs. non-inoculated (NI) lines. Biological replicates: 3 . One replicate per array.
Project description:The prolamin-box binding factor-1 (pbf1) gene encodes a transcription factor that controls the expression of seed storage protein (zein) genes in maize. Prior studies show that pbf1 underwent selection during maize domestication, although how it affected trait change during domestication is unknown. To assay how pbf1 affects phenotypic differences between maize and teosinte, we compared isogenic lines (NILs) that differ for a maize vs.and teosinte alleles of pbf1. Kernel weight for the teosinte NIL (162 mg) is slightly and significantly greater than that for the maize NIL (156 mg). RNAseq data for developing kernels show that the teosinte allele of pbf1 is expressed at about twice the level of the maize allele. However, RNA and protein assays showed no difference in zein profiles between the two NILs. The lower expression for the maize pbf1 allele suggests that selection may have favored this change, however, how reduced pbf1 expression alters phenotype remains unknown. One possibility is that pbf1 regulates genes other than zeins and thereby a domestication trait. The observed drop in seed weight associated with the maize allele of pbf1 is counterintuitive, but could represent a negative pleiotropic effect of selection on some other aspect of kernel composition.
Project description:Expression profiling analyses for eight maize inbreds reveals extensive transcriptional variation. This is a companion dataset to an Affymetrix profiling experiment (GEO Series GSE10237). Keywords: Genotype comparison series Expression profiling was used to study gene expression in aerial tissue from 11-day seedlings of maize. Three biological replicates were performed for eight different inbred lines; B37, B73, B84, Mo17, Oh43, B14a, Wf9 and W22.