Wageningen Academic

World Mycotoxin Journal, February 2008; 1(1): 3-12 P u b l i s h e r s

The ‘omics’ tools: genomics, proteomics, metabolomics and their potential for solving
the aflatoxin contamination problem

D. Bhatnagar1, K. Rajasekaran1, G.A. Payne2, R.L. Brown1, J. Yu1 and T.E. Cleveland1

1United States Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, 1100
Robert E. Lee Blvd., New Orleans, LA 70124, USA; 2North Carolina State University, Raleigh, North Carolina, USA;
dbhatnag@srrc.ars.usda.gov

Received: 12 December 2007 / Accepted: 6 January 2008

© 2008 Wageningen Academic Publishers

Abstract

Aflatoxins are highly carcinogenic secondary metabolites produced primarily by the fungi Aspergillus flavus and
Aspergillus parasiticus. Aflatoxin contamination of food and feed is an age old problem of particular concern over
the last four decades. Now, for the first time control measures for this problem appear within reach. For practical and
sustainable control of aflatoxin contamination to be realised, however, additional information is needed rather rapidly,
particularly for understanding the specific molecular factors (both in the plant and the fungus) involved during host
plant-fungus interaction. The information derived from the use of novel tools such as genomics, proteomics and
metabolomics provides us with the best and the quickest opportunity to achieve a clear understanding of the survival
of toxigenic fungi in the field, the ability of the fungus to invade crops, and the process of toxin contamination under
various environmental conditions. Significant progress has been made recently in understanding the genomic make-
up of the most significant aflatoxin producing field fungus, namely Aspergillus flavus. Progress also has been made
in the study of host crop resistance to fungal invasion through the use of proteomics. The information available on
production of aflatoxin and other metabolites by Aspergillus flavus is reasonably extensive, although the application
of metabolomics as a tool in this study is relatively new. In this review there is a discussion of the use of genomics,
proteomics and metabolomics in deriving the requisite information for developing effective strategies to interrupt
the machinery in the fungus for production of these toxins, as well as to assist in the development of host-resistance
against fungal invasion and aflatoxin contamination of crops.

Keywords: Aspergillus flavus, aflatoxins, genomics, proteomics, metabolomics

1. Introduction
Aflatoxins are highly toxic and carcinogenic secondary
metabolites produced mainly by three anamorphic species
of the genus Aspergillus: A. flavus, A. parasiticus and A.
nomius (Ehrlich et al., 2003). They are the most potent,
naturally-occurring carcinogens known and have been
linked to liver cancer and several other maladies in animals
and humans (Turner et al., 2003; Valdivia et al., 2001; Otim
et al., 2005). Among the aflatoxin-producing fungi, the
ubiquitous A. flavus is the known pathogen of cotton, corn,
peanuts and other oil-seed crops, producing toxin both
in the field and during storage (Payne and Brown, 1998).
Aflatoxin contamination continues to be a serious problem
in many parts of the world (Richard and Payne, 2003).
Initially studied because of their negative impact on human
and animal health (Eaton and Gallagher, 1994), aflatoxins
are perhaps the most well-known class of mycotoxins,
serving as a model system for the study of the genetics
of mycotoxin biosynthesis and secondary metabolism
in general (Cary et al., 2000; Bhatnagar et al., 2003; Yu
et al., 2004a; Yu and Keller, 2005). Due to the acute and
chronic toxicity of aflatoxins, nearly a hundred countries

ISSN 1875-0710 print, ISSN 1875-0796 online

D. Bhatnagar et al.
are known to have regulations limiting mycotoxin levels,
with 61 having specific regulatory levels for total aflatoxins
in foodstuffs and 39 having regulations for aflatoxins in
feedstuffs (FAO, 2004; Van Egmond et al., 2007). The U.S.
Food and Drug Administration has set limits of 20 μg/kg
total aflatoxins for interstate commerce of food and feed
and 0.5 μg/kg of aflatoxin M1 for sale of milk, and in the
European Union these limits are even lower.
Chronic, as well as sporadic, aflatoxin contamination
in a variety of field crops and agricultural commodities
worldwide has had a serious impact on the economics and
food safety of these products (Jelinek et al., 1989; Henry et
al., 2002). Vardon et al. (2003) estimated the annual cost
from aflatoxin contamination in the USA at roughly $500
million through two categories of loss, market rejection
and animal health impacts. Wu (2004) has suggested
that the total economic impact of aflatoxins should take
into account many other factors such as export market
loss, sampling and testing costs, costs to food processors,
grocery markets and consumers, and human health effects.
Therefore, it is imperative that researchers across the globe
develop strategies for the effective control of aflatoxin
contamination of crops. New biotechnologies such as (1)
the use of disarmed, non-toxigenic biocompetitive strains
of A. flavus in biocontrol of aflatoxin contamination,
and (2) identification of plant constituents that disrupt
aflatoxin biosynthesis or fungal growth and their use in new
biochemical marker-based breeding strategies to enhance
resistance in crops to aflatoxin, could potentially save the
agricultural industry in the U.S. alone hundreds of millions
of dollars. However, functional genomics is needed to speed
up our understanding of the field biology of the fungus in
relationship to its interaction with the host plant. But this
insight into the behaviour of these biological systems first
requires the sequence information (structural genomics)
of the aflatoxigenic fungus of interest. Metabolic profiling
along with structural and functional genomics could provide
significant information on the fungal responses to various
alterations in its ecology. Understanding the complex
interrelationships of plant and fungal gene products during
the host plant-A. flavus interaction is the key to developing
strategies to interrupt the aflatoxin contamination process
through enhancing host-plant resistance. Plant factors have
been discovered through the use of proteomics and natural
product chemistry that may influence fungal processes
involved in invasion and aflatoxin contamination. These
factors can also be divided into three categories: (1) seed
proteins/inhibitors of fungal cell wall degrading enzymes,
(2) seed/kernel natural products which may influence
fungal growth and/or aflatoxin synthesis, and (3) plant
stress responsive proteins that have been shown to have
an indirect effect on aflatoxin contamination.
Subsequently, the fundamental strategy in a genomics,
metabolomics and proteomics approach is to expand the

scope of biological investigation from studying single genes,
individual compounds (fungal secondary metabolites)
or plant proteins to studying all genes, metabolites and
proteins at once in a systematic fashion. A. flavus genomics
and proteomics of seed-based resistance provide the best
investigative tools for simultaneous discovery and analysis
of the biochemical function and genetic regulation of the
critical genes governing fungal development, plant fungal
interaction and aflatoxin biosynthesis.
2. Genomics
Since 1995, the genomes, or genetic make-up, of dozens of
bacteria and a few model eukaryotes have been completely
sequenced, but fungal genomics was not a priority in these
early efforts. Despite the slow start, however, genomic
sequencing has now been completed for dozens of fungi,
including species that are of fundamental biological interest,
species that are important to industry and agriculture,
and species that cause opportunistic human infections
(Bennett and Arnold, 2001; Desjardins and Bhatnagar, 2003;
Fakhoury and Payne, 2003; Yu et al., 2004a; Payne et al.,
2006; Machida et al., 2005; Nierman et al., 2005, Galagan
et al., 2005). Genomic studies on several Aspergillus and
Fusarium fungal species are well underway. Among these
are structural, functional and comparative genomics of
the toxin-producing species Fusarium graminearum
(trichothecene producer), F. verticillioides (fumonisin
producer), and A. flavus (aflatoxin producer).
The term ‘genome’ has existed for over 75 years and
refers to an organism’s complete set of chromosomes
and the genes contained therein. The term ‘genomics’,
now universally accepted, was coined in 1986 by Thomas
Roderick (Kuska, 1998) to describe the scientific discipline
of mapping, sequencing and analysing genomes. As more
and more genomes are sequenced, the emphasis has shifted
from just mapping and sequencing to understanding the
functions of genome with a comparison to other sequenced
genomes. Genomics combined with bioinformatics enables
the identification of all of the genes in an organism and
the study of their functions. Therefore, genome analysis is
now being divided into ‘structural genomics’, ‘comparative
genomics’ and ‘functional genomics’. In addition, novel
terminology, new acronyms, and innovative techniques
have been added to the scientific vocabulary (Bennett
and Arnold, 2001; Desjardins and Bhatnagar, 2003).
Structural genomics has been defined as the ‘initial phase
of genomic analysis: with a clear end point that results in
the construction of high resolution genetic, physical and
transcript maps of an organism’ (Hieter and Boguski, 1997).
Comparative or evolutionary genomics is the comparison
of DNA sequences of related organisms through advanced
computer technologies, or bioinformatics. Functional
genomics is the identification of the functions of each
coding sequence through analysis of gene expression by
World Mycotoxin Journal 1 (1)
 The ‘omics’ tools: genomics, proteomics, metabolomics and their potential for solving the aflatoxin contamination problem
using libraries of Expressed Sequence Tags (ESTs) and
microarray technologies, by targeted gene knock-out
experiments, and transgenic expression.
EST technology allows rapid identification of the majority,
if not all, of the genes expressed in the fungal genome and
contributes to a better understanding of their functions,
regulation, and coordination of gene expression in
response to internal and external factors, the relationship
between primary and secondary metabolism, plant-fungal
interaction and fungal pathogenicity, as well as evolutionary
biology. For a detailed discussion of this technique refer to
Bohnert et al. (2001) or Ohlrogge and Benning (2000).
Microarray or chip analysis is a DNA hybridisation-based
approach (Donson et al., 2002). The technique enables
the monitoring of the expression of thousands of genes in
parallel, making it ideal for gene profiling in a genomics
context (reviewed in Desjardins and Bhatnagar, 2003). DNA
microarrays consist of an ‘immobile phase’, either PCR
amplified genomic sequences, cDNAs, or oligonucleotides
embedded onto a solid support such as glass or plastic
slides, silicon wafers or nylon membranes (Baldwin et al.,
1999; Drăghici, 2003).
With the application of genomics and, in particular, of
EST and microarray technologies, we have witnessed a
revolution in our understanding of biological processes.
Aspergillus flavus genomics
A. flavus genomics is aimed at understanding the genetic
control and regulation of toxin production by this
important aflatoxigenic fungus as well as the evolutionary
process in Aspergillus section Flavi (Bhatnagar et al.,
2002). More importantly, we need to understand what
is the mechanism of toxin production in response to
environmental influences on the fungus, i.e. simultaneously
to environmental conditions like nutrition status of crops,
temperature, water stress, pH, and volatile compounds
from plants. We also need to understand the ecological/
evolutionary significance of A. flavus propagation, fungal
virulence, and aflatoxin formation as manifested by changes
in gene expression profiles and global signal transduction
within the fungus. These parameters are now being rapidly
analysed using the genomic information that has been
recently obtained.
Aspergillus flavus expressed sequence tags
An A. flavus EST project has been completed using the wild
type strain NRRL 3357 (ATCC# 20026). Over 26,110 cDNA
clones from a normalised cDNA expression library were
sequenced at The Institute for Genomic Research (TIGR;
currently J. Craig Venter Institute, JCVI). A total of 19,618
A. flavus ESTs were generated, from which 7218 unique
World Mycotoxin Journal 1 (1)
EST sequences were identified (Yu et al., 2004b, 2007b).
These EST sequences are available to the public at the
NCBI GenBank Database (http://www.ncbi.nlm.nih.gov/).
The A. flavus Gene Index was constructed at TIGR and
is currently maintained and curated by The Dana Farber
Cancer Institute (http://compbio.dfci.harvard.edu/tgi). The
identified genes have been categorised according to their
potential involvement, directly or indirectly, in aflatoxin
production based on function such as in global regulation,
signal transduction, pathogenicity, virulence, and fungal
development (Yu et al., 2004b, 2007b).
Whole genome sequencing of Aspergillus flavus
The A. flavus whole genome sequencing project was
funded by a United States Department of Agriculture /
National Research Initiative (USDA/NRI) grant awarded
to Prof. Gary A. Payne and Prof. Ralph Dean, North
Carolina State University, Raleigh, North Carolina. The
Food and Feed Safety Research Unit of the Southern
Regional Research Center of the Agricultural Research
Service (USDA/ARS/SRRC) provided funding for fine
finishing and gene calling. The sequencing was completed
at TIGR under the supervision of Dr. William C. Nierman
by a shotgun approach and Sanger sequencing protocol.
Primary assembly indicated that the A. flavus genome
consists of 8 chromosomes and the genome size is about
36.8 Mb with over 12,000 functional genes in the A. flavus
genome similar to that found in related Aspergillus species
(Galagan et al., 2005; Machida et al., 2005; Nierman et al.,
2005; Payne et al., 2006; Yu et al., 2007a). The annotation of
the A. flavus genome sequence data has been achieved by
using the A. flavus EST database, A. oryzae EST database,
and the A. oryzae whole genome sequence. The availability
of the A. oryzae whole genome sequence (Machida et al.,
2005) was vital in developing the chromosomal structure of
the A. flavus genome as well. The sequence data have been
deposited in the NCBI GenBank database (http://www.
ncbi.nlm.nih.gov) and are also available at the A. flavus
website (http://www.aspergillusflavus.org).
Microarrays as tools for functional genomics studies
Several types of microarrays for A. flavus have been
constructed within the last few years. The first cDNA
microarray consisting of 753 gene features including
known aflatoxin pathway genes and regulatory gene aflR
was constructed by the laboratory of Prof. Gary A. Payne,
North Carolina State University. The unique ESTs identified
from a cDNA library constructed using A. flavus RNA
under aflatoxin-producing condition were spotted on a
Telechem SuperAldehyde glass slide using an Affymetrix
417 Arrayer (O’Brian et al., 2003). A 5,031 gene-elements
A. flavus EST based amplicon microarray was constructed
by the Food and Feed Safety Research Unit of the USDA/
ARS/SRRC at TIGR. Using genomic DNA as template,
D. Bhatnagar et al.

the specific gene sequences were amplified using primers
synthesised based on A. flavus unique EST sequences
assembled. A comprehensive whole genome A. flavus
oligo microarray has also been constructed at TIGR by
the Food and Feed Safety Research Unit. All of the 11,820
genes unique to A. flavus, the unique genes present in A.
oryzae but absent in A. flavus, and 10 genes cloned from
corn that show resistance to A. flavus infection, have been
printed on this whole genome microarray. An Affymetrix
microarray funded with a grant from USDA/NRI awarded
to a consortium led by Prof. Gary A. Payne contains all of
the A. flavus genes, A. oryzae unique genes, plus additional
genes of interest from corn, Fusarium species, mouse
and human genomes. Additionally, a peanut/A. flavus
combined microarray, funded by the Crop Protection and
Management Laboratory of the South Atlantic Area of the
Agricultural Research Service (USDA/ARS/SAA), Tifton,
Georgia, is under construction at JCVI. This crop/fungus
combined array will contain oligonucleotides representing
over 10,000 peanut ESTs, and all of the annotated A. flavus
and A. oryzae unique genes (reviewed in Yu et al., 2007a).
Expression profiling of genes involved in aflatoxin
formation using those microarrays, performed at USDA
laboratories, the laboratories of North Carolina State
University, TIGR and JCVI, identified hundreds of genes
that are significantly up or down regulated under various
nutritional and environmental conditions that either
support or prevent aflatoxin formation (Cary et al., 2007;
Chang et al., 2007; Kim et al., 2006; O’Brian et al., 2003,
2007; Price et al., 2005, 2006; Wilkinson et al., 2007a,b;
Yu et al., 2007b). Further studies using these microarray
resources for a genome-wide gene profiling and functional
analysis in relation to aflatoxin formation are expected to
reveal the regulatory elements (global or specific) required
for aflatoxin production.
Comparative genomics
Genomics of crop plants infected by Aspergillus flavus
With the advent of modern advances in plant sciences
and genomic sequencing, it is encouraging to forecast
that many plant genome sequences will be made available
shortly. The importance of gene expression profiling by
genomic and proteomic analyses will play a major role
in the increasing power of molecular breeding that will
help the breeders and biotechnologists to harness the
maximum potential of crop genomes to provide higher
yield, novel value-added traits along with increased food
and feed safety. So far, the sequences of two small plant
genomes, Arabidopsis and rice have been completed in
2000 and 2005, respectively (The Arabidopsis Genome
Initiative, 2000; International Rice Genome Sequencing
Project, 2005). Even with the currently available technology,
whole genome sequencing of several important crops is
often a laborious and expensive process because of the large
size of genomes (Table 1). As a result, several laboratories
have resorted to sequencing of large numbers of ESTs that
can deliver substantial amounts of genetic information
on protein-coding genes for comparative and functional
genomic studies. The latest available information indicates
that among all characterised genomes to date, the maize
genome is exceptionally dynamic and with over 2.3 million
cDNA sequences from a variety of tissues and physiological
regimens, the maize transcriptome is now the third best
studied after mouse and man (Wessler, 2006; Rabinowicz
and Bennetzen, 2006; Messing and Dooner, 2006; Table 1).
Other crops such as cotton, peanut and tree crops are
lagging behind and it is unlikely that their genome will be
sequenced completely in the near future due to the large
size of their genomes.
Table 1. Comparative genome size of selected cultivated crops.
For comparison, genome sizes of Arabidopsis and human are
also given.

Results from the whole genome sequencing project show Crop Approx. Ploidy EST1
that the genome of A. flavus (36.3 Mb) is larger than that genome level
of A. nidulans (30.1 Mb) or A. fumigatus (29.4 Mb) and size1 (Mbp)
thus capable of a more complex pattern of secondary
metabolites. The expanded genome of A. flavus over other Arabidopsis thaliana 128 2n=10 1,276,692
Aspergillus species suggests that A. flavus is adapted to Oryza japonica (rice) 420 2n=24 1,214,083
growing in complex environments. An analysis of the Gossypium hirsutum (cotton) 2,700 2n=4x=52 177,202
function of these extra genes may reveal those genes that Arachis hypogea (peanut) 2,800 2n=4x=40 40,627
make this fungus a successful saprophyte as well and a Zea mays (maize) 2,500 2n=20 1,159,264
pathogen of plants and animals. Interestingly, comparative Glycine max (soybean) 1,115 2n=2x=40 392,337
genomic studies show that A. flavus is highly similar to A. Hordeum vulgare (barley) 5,000 2n=6x=42 437,713
oryzae (37.6 Mb) with respect to genome size and number Triticum aestivum (wheat) 16,500 2n=6x=42 1,050,932
of genes for secondary metabolism. These results support human 2,800 2n=46 8,134,112
the conclusions of others that A. oryzae is not a separate other mammals 1,400-3,700
species, but rather a domesticated ecotype of A. flavus
(Payne et al., 2006). 1http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.html; Accessed:

14 October 2007.

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 The ‘omics’ tools: genomics, proteomics, metabolomics and their potential for solving the aflatoxin contamination problem
Improved methods for gene and genomic sequencing, and
analysis of gene expression, promise to allow rapid strides
in the identification and understanding of gene function
and phenotype. Availability of genetic linkage maps and
quantitative trait loci (QTL) maps will also be useful in
mapping traits and marker-assisted selection (MAS),
anchoring physical maps from large insert libraries and
positional cloning of important genes, comparisons of
synteny within and across species and ordered genome
sequencing. Gene expression assays using macroarray
(nylon-based) or microarray (glass slide-based) screening
methods are powerful in associating genes with proper
phenotypes and to associate genes with functions or
specific physiological conditions due to biotic or abiotic
stress factors.
Identification of specific genes and critical genetic
pathways is essential for conventional and transgenic
improvement programs. Analysis of gene function in
native plant environment is possible using efficient genetic
transformation technology and it has been practised
routinely for cotton, peanut and corn (Rajasekaran 2004;
Ozias-Akins et al., 1993; Ishida et al., 2007). In conventional
breeding, MAS may not be preferable for traits controlled
by a large number of QTLs, because the individual gene’s
contribution may be too small to measure since many genes
with small effects are involved (Morgante and Salamini,
2003). For a review of the power and limitations of MAS
for resistance to mycotoxin accumulation see review by
Robertson et al. (2005).
The new genomics resources that are becoming available
for A. flavus as well as the crops infected by the fungus
will greatly aid our understanding of the ecology and
metabolism of this fungus as well.
3. Proteomics
Proteomics is the study of a complete set of proteins in a
cell and their structures and functions in the physiological
pathways of cells. The term ‘proteomics’ is analogous to
genomics, the study of the complete set of genes. The word
‘proteome’ is derived from a combination of ‘protein’ and
‘genome’. The proteome of an organism is the set of proteins
produced by it during its life, based on its genome, i.e. its
set of genes (for details see Twyman, 2004; Westermeier
and Naven, 2002; Liebler, 2002).
Proteomics is often seen as the next step in the study of
biological systems, after genomics (Wilkins et al., 1997).
It is much more complicated than genomics, mostly
because an organism’s genome is rather constant, but a
proteome changes from cell to cell based on its biochemical
interactions with the genome and environmental factors
that the cell encounters. The complexity of proteins relative
to nucleic acids is also due to the fact that proteins can
World Mycotoxin Journal 1 (1)
be processed in the cell by alternative splicing, protein
modification (glycosylation, phosphorylation) and protein
degradation. A prime example is that there are more than
500,000 proteins in human cells derived from about 25,000
identified genes.
Current research in proteomics requires first that proteins
be resolved, sometimes on a massive scale. Protein
separation can be performed using two-dimensional gel
electrophoresis, which usually separates proteins first by
isoelectric point and then by molecular weight (Wilkins
et al., 1996; Pennington et al., 1997). This approach
takes advantage of the increased reproducibility, map
resolution, reliability and accuracy that two-dimensional
polyacrylamide gel electrophoresis (2-D PAGE) offers
over 1-dimensional gel electrophoresis (Gorg et al., 1988)
to detect post- and cotranslational modifications that
cannot be predicted from a DNA sequence. Individual
spots are cut out of the gel and cleaved into peptides with
proteolytic enzymes. These peptides can then be identified
by mass spectrometry, specifically matrix-assisted laser
desorption-ionisation time-of-flight (MALDI-TOF) mass
spectrometry.
Proteomics of Aspergillus flavus
Very few reports of studies on the proteomics of A. flavus
are available in the literature. The first extensive proteomic
study conducted to identify the secreted proteins from a
filamentous fungus was reported by Medina et al. (2004).
In this study, a proteome analysis was used to differentiate
and identify the extracellular rutin-induced and non-
induced proteins secreted by A. flavus. In a continuation
study Medina et al. (2005) reported a total of 51 unique
A. flavus secreted proteins from three growth conditions.
Ten proteins were unique to rutin-, five to glucose- and
one to potato dextrose-grown A. flavus. Sixteen secreted
proteins were common to all three media. Fourteen
identifications were hypothetical proteins or proteins of
unknown functions.
Identification of host resistance-associated proteins
through proteomics
Developing resistance to fungal infection in wounded and
intact maize kernels would accelerate solving the aflatoxin
problem (Payne, 1992). Studies of subpericarp (wounded
kernel) resistance in maize have identified corresponding
resistance mechanisms. In a number of studies resistance
associated proteins (RAPs) were identified (Brown et al.,
2004) using proteomics. By comparing lines of maize
resistant and susceptible to aflatoxin accumulation, proteins
associated with resistance, as well as the genes encoding
them, can be identified thereby facilitating marker-assisted
breeding and/or genetic engineering efforts. Endosperm
and embryo proteins from several resistant and susceptible
D. Bhatnagar et al.
genotypes were compared and over a dozen constitutively
expressed protein spots, either unique or 5-fold up-
regulated in resistant lines (Mp420 and Mp313E), were
identified and analysed with electrospray ionisation tandem
mass spectrometry (ESI-MS/MS) after in-gel digestion with
trypsin (Chen et al., 2000, 2002).
Both constitutive and induced proteins are required for
kernel resistance to aflatoxin production (Chen et al.,
2001). A major difference between resistant and susceptible
maize genotypes is the relatively high level of antifungal
proteins constitutively expressed by the resistant lines.
These constitutive proteins may delay fungal invasion, and
subsequent aflatoxin formation, until infection-induced
antifungal proteins are synthesised. The identified proteins
can be grouped into three categories based on their peptide
sequence homology: (1) storage proteins, e.g. globulins and
late embryogenesis abundant proteins, (2) stress-responsive
proteins, e.g. aldose reductase, a glyoxalase I protein and a
16.9 kDa heat shock protein, and (3) antifungal proteins,
including the pathogenesis-related protein PR-10 (Chen
et al., 2006).
The proteomes of near-isogenic maize lines from the
same backcross in a collaborative project between the
International Institute for Tropical Agriculture (IITA) and
the USDA differing significantly in aflatoxin accumulation
were analysed and proteins identified in all three of the
predicted categories (Brown et al., 2003). RAPs are more
easily identified in these lines as the confounding effects
of differences in the genetic backgrounds of the lines are
absent.
Examples of some of the resistance-associated proteins,
identified via proteomics of maize kernels, with
demonstrated antifungal activities against A. flavus are
given in Table 2. Increased temperature and drought,
which often occur together, are major factors associated
with aflatoxin contamination of maize kernels (Payne,
1992, 1998). If drought stress is imposed during grain
filling, then dry matter accumulation is reduced in kernels
(Payne, 1992, 1998) which often results in cracks in the
Table 2. Examples of resistance-associated proteins (RAP) against Aspergillus flavus identified through proteomics.
Protein Resistant genotype1
Trypsin inhibitor (14 kDa) MI82; CI2; T115
Glyoxalase I Mp420; Mp313E
Pathogenesis related protein (PR-10) Mp420; Mp313E; GT-MAS:gk
Peroxiredoxin antioxidant (PER1) Mp420; Mp313E
Zeamatin GT-MAS:gk
1Maize genotypes in which an association with resistance has been identified; other resistant genotypes are involved as well.

seed and provides an easy entry site for fungi and insects.
Possession of unique or increased levels of hydrophilic
storage or stress-related proteins by resistant lines could
increase protein synthesis and host defence under stress
conditions. Thus, commercially-useful, aflatoxin-resistant
maize lines may include not only antifungal proteins but
also high-level expression of stress-related proteins.
Further studies including physiological and biochemical
characterisation, genetic mapping, plant transformation
using RAP genes, gene silencing through RNA interference
(RNAi) and marker-assisted breeding should clarify the
roles of stress-related RAPs in kernel resistance.
The direct involvement of glyoxalase I, a purported stress-
related aflatoxin resistance protein, has been evaluated
(Chen et al., 2004). The substrate for glyoxalase I,
methylglyoxal, is a potent cytotoxic compound produced
from glycolysis and photosynthesis intermediates,
glyceraldehyde-3-phosphate and dihydroxyacetone
phosphate. Methylglyoxal induces aflatoxin production
through up-regulation of aflatoxin biosynthetic pathway
transcripts, including the qflR regulatory gene. Thus,
glyoxalase I may directly affect resistance by removing the
methylglyoxal aflatoxin-inducing substrate.
4. Metabolomics
Metabolites are small molecules that are either
intermediates or final products of metabolism. Metabolites
directly involved in the normal growth, development, and
reproduction are primary metabolites, whereas those not
involved in these processes such as aflatoxins are called
secondary metabolites. Secondary metabolites may have
important ecological significance. Therefore, metabolomics
can be defined as the ‘systematic study of the unique
chemical fingerprints that specific cellular processes leave
behind’, i.e. the study of their small-molecule metabolite
profiles (Daviss, 2005; Preti, 2005). The metabolome
represents the collection of all metabolites in a biological
organism (such as metabolic intermediates, hormones and
other signalling molecules, and secondary metabolites),
which are the end products of its gene expression (Fiehn,
Putative function
Antifungal; anti-amylase
Stress-related; antitoxigenic
Antifungal; ribonucleolytic
Peroxidase; stress-related
Antifungal
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 The ‘omics’ tools: genomics, proteomics, metabolomics and their potential for solving the aflatoxin contamination problem
2001). Thus, while mRNA gene expression data and
proteomic analyses do not tell the whole story of what
might be happening in a cell, metabolic profiling can give
an instantaneous snapshot of the physiology of that cell.
The metabolome is also dynamic, and constantly changing
because it is a reflection of the metabolic reactions, where
outputs from one enzymatic chemical reaction are inputs
to other chemical reactions (hypercycles).
The chromatographic separation techniques developed
in the late 1960’s, which made the initial detection of
metabolites possible, can be called the beginning of
metabolomics. However, modern day metabolomics began
in 1970 with the work of Arthur Robinson, although it was
not called metabolomics then. The name metabolomics
was coined twenty years later in the 1990s when the first
paper using the word metabolome was published (Oliver
et al., 1998).
Separation and detection of metabolites are two issues
that need to be addressed in obtaining metabolic profile
(Dunn and Ellis, 2005). Detection of the analytes, following
separation by chromatographic or other methods can be
achieved through the use of capillary electrophoresis (CE),
high performance liquid chromatography (HPLC), gas
chromatography, especially when interfaced with mass
spectrometry (GC-MS) and nuclear magnetic resonance
(NMR).
Metabolomics can be an excellent tool for determining the
phenotype caused by a genetic manipulation, such as gene
deletion or insertion. Sometimes this can be a sufficient
goal in itself, for instance, to detect any phenotypic changes
in a genetically-manipulated fungus. More exciting is the
prospect of predicting the function of unknown genes by
comparison with the metabolic perturbations caused by
deletion/insertion of known genes (Weckwerth, 2003).
Metabolomics of Aspergillus flavus
The sec- (secondary metabolite minus) mutant of A.
parasiticus SRRC 143 produces fewer conidia on complete
and minimum media and fails to produce aflatoxin or
to convert labelled aflatoxin pathway precursors into
aflatoxin. Transcripts of the aflatoxin pathway genes are
barely detectable and transcripts of the pathway regulatory
gene aflR are reduced 5-10 fold compared to wild type
(Kale et al., 2003). Although the sec- mutant has impaired
sporulation, it grows and produces an amount of biomass
comparable to WT on a minimal medium, indicating that
its core metabolism is intact.
In an attempt to better characterise the changes in a sec-
mutant of A. flavus, metabolic profiling was conducted on
the wild type (WT) and mutant strains (Glassbrook et al.,
2006). An initial comparison of the metabolic profile of
World Mycotoxin Journal 1 (1)
the two strains grown under conducive or non-conductive
temperatures, 28 °C and 37 °C, respectively, revealed large
differences in the small molecule compositions of the two
strains. Although the mutant strain appeared to have some
unique components when compared to the wild type,
chromatograms of the mutant samples lacked many of
the prominent peaks present in the wild type (Glassbrook
and Payne, 2005). Thus the sec- mutant appears to be
altered in several pathways of secondary metabolism, not
just aflatoxin biosynthesis. These findings suggest that the
mutation in the sec- strain may be in a global regulatory
of secondary metabolism. Further experiments are in
progress, using mass spectrometry and pulse/pulse-chase
techniques, to identify specific compounds and pathways
impacted by the mutation. Additionally, this metabolic
profile will be compared with a transcriptional profile
(using an EST microarray) to determine the changes in
gene expression resulting from the sec- mutation.
Research is underway to place the identified metabolites
into metabolic pathways and thus better understand the
metabolic changes associated with the sec- mutant. This
type of pathway mapping provides a framework for sorting
large quantities of metabolic profiling data and formulating
testable hypotheses.
5. Conclusion
One of the challenges of systems biology is the integration
of genomic, proteomic and metabolomic information
to give a more complete picture of living organisms.
The results of genomic analysis are expected to provide
important information for developing novel strategies to
control aflatoxin contamination by identifying targets for
inhibiting fungal growth or toxin production, as well as
developing ‘designer’ biological control agents. With the
large volume of information being reported with respect
to proteomics of host and genomics and field ecology
of the fungus, novel strategies will also emerge based
on a clear understanding of the aflatoxin contamination
process, especially at the molecular level. If the interaction
between the plant and the fungus can be better understood
through the use of proteomics, genomics and natural
product chemistry, particularly in how plant factors may
influence fungal processes contributing to virulence and
aflatoxin contamination, this information could accelerate
the development of breeding through marker selection
and/or gene insertion technologies for enhancing host
plant resistance.
D. Bhatnagar et al.
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