Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional profiles uncover Aspergillus flavus induced resistance in mature maize kernels

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.

ORGANISM(S): Zea mays

SUBMITTER: Meng Luo

PROVIDER: E-GEOD-19883 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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: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.

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Transcriptional profiles uncover Aspergillus flavus induced resistance in mature maize kernels

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