3.1. Molecular genetic investigations of aflatoxin-resistant lines
Chromosome regions associated with resistance to A. flavus and inhibition of aflatoxin production in maize have been identified through Restriction Fragment Length Polymorphism (RFLP) analysis in three “resistant” lines (R001, LB31, and Tex6) in an Illinois breeding program, after mapping populations were developed using B73 and/or Mo17 elite inbreds as the “susceptible” parents [20, 21]. Chromosome regions associated with inhibition of aflatoxin in studies considering all 3 resistant lines demonstrated that there are some regions in common. Regions on chromosome arms 2L, 3L, 4S, and 8S may prove promising for improving resistance through marker assisted breeding into commercial lines . In some cases, chromosomal regions were associated with resistance to Aspergillus ear rot and not aflatoxin inhibition, and vice versa, whereas others were found to be associated with both traits. This suggests that these two traits may be at least partially under separate genetic control. QTL studies involving other populations have identified chromosome regions associated with low aflatoxin accumulation.
In a study involving 2 populations from Tex6 x B73, conducted in 1996 and 1997, promising QTLs for low aflatoxin were detected in bins 3.05-6, 4.07-8, 5.01-2, 5.05-5, and 10.05-10.07 . Environment strongly influenced detection of QTLs for lower toxin in different years; QTLs for lower aflatoxin were attributed to both parental sources. In a study involving a cross between B73 and resistant inbred Oh516, QTL associated with reduced aflatoxin were identified on chromosomes 2, 3 and 7 (bins 2.01 to 2.03, 2.08, 3.08, and 7.06) . QTLs contributing resistance to aflatoxin accumulation were also identified using a population created by B73 and resistant inbred Mp313E, on chromosome 4 of Mp313E . This confirmed the findings of an earlier study involving Mp313E and susceptible Va35 . Another QTL in this study, which has similar effects to that on chromosome 4, was identified on chromosome 2 . A recent study to identify aflatoxin-resistance QTL and linked markers for marker-assisted breeding was conducted using a population developed from Mp717, an aflatoxin-resistant maize inbred, and NC300, a susceptible inbred adapted to the southern U.S. QTL were identified on all chromosomes, except 4, 6, and 9; individual QTL accounted for up to 11% of phenotypic variance in aflatoxin accumulation . Lastly, in a study of population of F2:3 families developed from resistant Mp715 and a southern-adapted susceptible, T173, QTL with phenotypic effects up to 18.5% were identified in multiple years on chromosomes 1, 3, 5, and 10 .
A number of genes corresponding to resistance-associated proteins (RAPs), that were identified in proteomics studies (see section 3.5.1 below) have been mapped to chromosomal location using the genetic sequence of B73 now available online (http://archive.maizesequence.org/index.html) . Using the DNA sequence of the RAPs and blasting them against the B73 sequence allowed us to place each gene into a virtual bin, allowing us to pinpoint the chromosomal location to which each gene maps. The chromosomes involved include the above-mentioned chromosomes 1, 2, 3, 7, 8 and 10, some in bins closely located to those described above. Another study also mapped RAPs to bins on the above-chromosomes as well as chromosomes 4 and 9 .
3.4. Comparing fungal growth to toxin production
When selected resistant Illinois maize inbreds (MI82, CI2, and T115) were examined by the KSA, modified to include an A. flavus GUS transformant (a strain genetically engineered with a gene construct consisting of a β-glucuronidase reporter gene linked to an A. flavus beta-tubulin gene promoter for monitoring fungal growth) , kernel resistance to fungal infection in nonwounded and wounded kernels was demonstrated both visually and quantitatively, as was a positive relationship between the degree of fungal infection and aflatoxin levels [14, 33]. This made it possible assess fungal infection levels and to determine if a correlation exists between infection and aflatoxin levels in the same kernels. A. flavus GUS transformants with the reporter gene linked to an aflatoxin biosynthetic pathway gene could also provide a way to indirectly measure aflatoxin levels [34-36], based on the extent of the expression of the pathway gene.
Recently, It was demonstrated, using the KSA and an F. moniliforme strain, genetically transformed with a GUS reporter gene linked to an A. flavus β-tubulin gene promoter, that the aflatoxin-resistant genotype, GT-MAS:gk, inhibits growth of F. moniliforme as well . This indicates that some resistance mechanisms may be generic for ear rotting/mycotoxigenic fungi.
A more recent use of reporter genes was performed on cotton using a green fluorescent protein reporter; a GFP-expressing A. flavus strain to successfully monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins . This strain provides for an easy, potentially non-destructive, rapid and economical assay which can be done in real time, and may constitute an advance over GUS transformants.
3.5. Resistance-associated proteins
Developing resistance to fungal infection in wounded as well as intact kernels would go a long way toward solving the aflatoxin problem . Studies demonstrating subpericarp (wounded-kernel) resistance in maize kernels have led to research for identification of subpericarp resistance mechanisms. Examinations of kernel proteins of several genotypes revealed differences between genotypes resistant and susceptible to aflatoxin contamination . Imbibed susceptible kernels, for example, showed decreased aflatoxin levels and contained germination-induced ribosome inactivating protein (RIP) and zeamatin . Both zeamatin and RIP have been shown to inhibit A. flavus growth in vitro . In another study, two kernel proteins were identified from a resistant corn inbred (Tex6) which may contribute to resistance to aflatoxin contamination . One protein, 28 kDa in size, inhibited A. flavus growth, while a second, over 100 kDa in size, primarily inhibited toxin formation. When a commercial corn hybrid was inoculated with aflatoxin and nonaflatoxin-producing strains of A. flavus at milk stage, one induced chitinase and one ß-1,3-glucanase isoform was detected in maturing infected kernels, while another isoform was detected in maturing uninfected kernels .
In another investigation, an examination of kernel protein profiles of 13 maize genotypes revealed that a 14 kDa trypsin inhibitor protein (TI) is present at relatively high concentrations in seven resistant maize lines, but at low concentrations or is absent in six susceptible lines . The mode of action of TI against fungal growth may be partially due to its inhibition of fungal -amylase, limiting A. flavus access to simple sugars  required not only for fungal growth, but also for toxin production . TI also demonstrated antifungal activity against other mycotoxigenic species . The identification of these proteins may provide markers for plant breeders, and may facilitate the cloning and introduction of antifungal genes through genetic engineering into other aflatoxin-susceptible crops.
An investigation into maize kernel resistance  determined that both constitutive and induced proteins are required for resistance to aflatoxin production. It also showed that one major difference between resistant and susceptible genotypes is that resistant lines constitutively express higher levels of antifungal proteins compared to susceptible lines. The real function of these high levels of constitutive antifungal proteins may be to delay fungal invasion, and consequent aflatoxin formation, until other antifungal proteins can be synthesized to form an active defense system.
3.5.1. Proteomic analysis
Two-dimensional (2-D) gel electrophoresis, which sorts proteins according to two independent properties, isoelectric points and then molecular weights, has been recognized for a number of years as a powerful biochemical separation technique. Improvements in map resolution and reproducibility [48, 49], rapid analysis of proteins, analytical soft ware and computers, and the acquisition of genomic data for a number of organisms has given rise to another application of 2-D electrophoresis: proteome analysis. Proteome analysis or “proteomics” is the analysis of the protein complement of a genome [50, 51]. This involves the systematic separation, identification, and quantification of many proteins simultaneously. 2-D electrophoresis is also unique in its ability to detect post- and cotranslational modifications, which cannot be predicted from the genome sequence.
Through proteome analysis and the subtractive approach, it may be possible to identify important protein markers associated with resistance, as well as genes encoding these proteins. This could facilitate marker-assisted breeding and/or genetic engineering efforts. Endosperm and embryo proteins from several resistant and susceptible genotypes have been compared using large format 2-D gel electrophoresis, and over a dozen such protein spots, either unique or 5-fold upregulated in resistant maize lines (Mp420 and Mp313E), have been identified, isolated from preparative 2-D gels and analyzed using ESI-MS/MS after in-gel digestion with trypsin [52, 53]. These proteins, all constitutively expressed, can be grouped into three categories based on their peptide sequence homology: (1) storage proteins, such as globulins and late embryogenesis abundant proteins; (2) stress-responsive proteins, such as aldose reductase, a glyoxalase I protein and a 16.9 kDa heat shock protein, and (3) antifungal proteins, including the above-described TI.
During the screening of progeny developed through the IITA-USDA/ARS collaborative project, near-isogenic lines from the same backcross differing significantly in aflatoxin accumulation were identified, and proteome analysis of these lines is being conducted . Investigating corn lines from the same cross with contrasting reaction to A. flavus should enhance the identification of RAPs clearly without the confounding effect of differences in the genetic backgrounds of the lines.
Heretofore, most RAPs identified have had antifungal activities. However, increased temperatures and drought, which often occur together, are major factors associated with aflatoxin contamination of maize kernels . It has also been found that drought stress imposed during grain filling reduces dry matter accumulation in kernels . This often leads to cracks in the seed and provides an easy entry site to fungi and insects. Possession of unique or of higher levels of hydrophilic storage or stress-related proteins, such as the aforementioned, may put resistant lines in an advantageous position over susceptible genotypes in the ability to synthesize proteins and defend against pathogens under stress conditions. Further studies including physiological and biochemical characterization, genetic mapping, plant transformation using RAP genes, and marker-assisted breeding should clarify the roles of stress-related RAPs in kernel resistance. RNAi gene silencing experiments involving RAPs may also contribute valuable information. .
3.5.2. Further characterization of RAPs
A literature review of the RAPs identified above indicates that storage and stress-related proteins may play important roles in enhancing stress tolerance of host plants. The expression of storage protein GLB1 and LEA3 has been reported to be stress-responsive and ABA-dependant . Transgenic rice overexpressing a barley LEA3 protein HVA1 showed significantly increased tolerance to water deficit and salinity . The role of GLX I in stress-tolerance was first highlighted in an earlier study using transgenic tobacco plants overexpressing a Brassica juncea glyoxalase I . The substrate for glyoxalase I, methylglyoxal, is a potent cytotoxic compound produced spontaneously in all organisms under physiological conditions from glycolysis and photosynthesis intermediates, glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. Methylglyoxal is an aflatoxin inducer even at low concentrations; experimental evidence indicates that induction is through upregulation of aflatoxin biosynthetic pathway transcripts including the AFLR regulatory gene . Therefore, glyoxalase I may be directly affecting resistance by removing its aflatoxin-inducing substrate, methylglyoxal. PER1, a 1-cys peroxiredoxin antioxidant identified in a proteomics investigation , was demonstrated to be an abundant peroxidase, and may play a role in the removal of reactive oxygen species. The PER1 protein overexpressed in Escherichia coli demonstrated peroxidase activity in vitro. It is possibly involved in removing reactive oxygen species produced when maize is under stress conditions . Another RAP that has been characterized further is the pathogenesis-related protein 10 (PR10). It showed high homology to PR10 from rice (85.6% identical) and sorghum (81.4% identical). It also shares 51.9% identity to intracellular pathogenesis-related proteins from lily (AAF21625) and asparagus (CAA10720), and low homology to a RNase from ginseng . The PR10 overexpressed in E. coli exhibited ribonucleolytic and antifungal activities. In addition, an increase in the antifungal activity against A. flavus growth was observed in the leaf extracts of transgenic tobacco plants expressing maize PR10 gene compared to the control leaf extract . This evidence suggests that PR10 plays a role in kernel resistance by inhibiting fungal growth of A. flavus. Further, its expression during kernel development was induced in the resistant line GT-MAS:gk, but not in susceptible Mo17 in response to fungal inoculation . Recently, a new PR10 homologue was identified from maize (PR10.1) . PR10 was expressed at higher levels in all tissues compared to PR10.1, however, purified PR10.1 overexpressed in E. coli possessed 8-fold higher specific RNase activity than PR10 . This homologue may also play a role in resistance. Evidence supporting a role for PR10 in host resistance is also accumulating in other plants. A barley PR10 gene was found to be specifically induced in resistant cultivars upon infection by Rhynchosporium secalis, but not in near-isogenic susceptible plants . In cowpea, a PR10 homolog was specifically up-regulated in resistant epidermal cells inoculated with the rust fungus Uromyces vignae Barclay . A PR10 transcript was also induced in rice during infection by Magnaporthe grisea .
To directly demonstrate whether selected RAPs play a key role in host resistance against A. flavus infection, an RNA interference (RNAi) vector to silence the expression of endogenous RAP genes (such as PR10, GLX I and TI) in maize through genetic engineering was constructed [59, 66]. The degree of silencing using RNAi constructs is greater than that obtained using either co-suppression or antisense constructs, especially when an intron is included . Interference of double-stranded RNA with expression of specific genes has been widely described [68, 69]. Although the mechanism is still not well understood, RNAi provides an extremely powerful tool to study functions of unknown genes in many organisms. This posttranscriptional gene silencing (PTGS) is a sequence-specific RNA degradation process triggered by a dsRNA, which propagates systemically throughout the plant, leading to the degradation of homologous RNA encoded by endogenous genes, and transgenes. Both particle bombardment and Agrobacterium-mediated transformation methods were used to introduce the RNAi vectors into immature maize embryos. The former was used to provide a quick assessment of the efficacy of the RNAi vector in gene silencing. The latter, which can produce transgenic materials with fewer copies of foreign genes and is easier to regenerate, was chosen for generating transgenic kernels for evaluation of changes in aflatoxin-resistance. It was demonstrated using callus clones from particle bombardment that PR10 expression was reduced by an average of over 90% after the introduction of the RNAi vector . The transgenic kernels also showed a significant increase in susceptibility to A. flavus infection and aflatoxin production. The data from this RNAi study clearly demonstrated a direct role for PR10 in maize host resistance to A. flavus infection and aflatoxin contamination . RNAi vectors to silence other RAP genes, such as GLX I and TI, have also been constructed, and introduced into immature maize embryos through both bombardment and Agrobacterium infection . It will be very interesting to see the effect of silencing the expression of these genes in the transgenic kernels on host resistance to A. flavus infection and aflatoxin production.
ZmCORp, a protein with a sequence similar to cold-regulated protein and identified in the above-proteomic studies, was shown to exhibit lectin-like hemagglutination activity against fungal conidia and sheep erythrocytes . When tested against A. flavus, ZmCORp inhibited germination of conidia by 80% and decreased mycelial growth by 50%, when germinated conidia were incubated with the protein. Quantitative real-time RT-PCR revealed ZmCORp to be expressed 50% more in kernels of a resistant maize line versus a susceptible.
ZmTIp, a 10 kDa trypsin inhibitor, had an impact on A. flavus growth, but not as great as the previously-mentioned 14 kDa TI .
3.5.3. Proteomic studies of rachis and silk tissue
A study was conducted to investigate the proteome of rachis tissue, maternal tissue that supplies nutrients to the kernels . An interesting finding in this study is that after infection by A. flavus, rachis tissue of aflatoxin-resistant genotypes did not up-regulate PR proteins as these were already high in controls where they had strongly and constitutively accumulated during maturation. However, rachis tissue of aflatoxin-susceptible lines did not accumulate PR proteins to such an extent during maturation, but increased them in response to fungal infection. Given the relationship of the rachis to kernels, these results confirm findings of a previous investigation , which demonstrated levels of proteins in resistant versus susceptible kernels was a primary factor that determined kernel genetic resistance to aflatoxin contamination. Another study was conducted to identify proteins in maize silks that may be contributing to resistance against A. flavus infection/colonization . Antifungal bioassays were performed using silk extracts from two aflatoxin-resistant and two–susceptible inbred lines. Silk extracts from resistant inbreds showed greater anti-fungal activity compared to susceptible inbreds. Comparative proteomic analysis of the two resistant and susceptible inbreds led to the identification of antifungal proteins including three chitinases that were differentially-expressed in resistant lines. When tested for chitinase activity, silk proteins from extracts of resistant lines also showed significantly higher chitinase activity than that from susceptible lines. Differential expression of chitinases in maize resistant and susceptible inbred silks suggests that these proteins may contribute to resistance.
3.5.4. Transcriptomic analyses
To investigate gene expression in response to A. flavus’ infection and to more thoroughly identify factors potentially involved in the regulation of RAP genes, a transcriptomic profile was conducted on maize kernels of two inbred lines that were genetically closely-related . Similar work had previously been performed using Tex6 as the resistant line and B73 as the susceptible , however, in the study using closely-related lines, imbibed mature kernels were used (for the first time) and proved to be a quicker and easier approach than traditional approaches. The involvement of certain stress-related and antifungal genes previously shown to be associated with constitutive resistance was demonstrated here; a kinase-binding protein, Xa21 was highly up-regulated in the resistant line compared to the susceptible, both constitutively and in the inducible state.