Aflatoxins, the toxic and highly carcinogenic secondary metabolites of Aspergillus flavus, A. parasiticus and a number of fungi in the genus Aspergillus, are the most widely investigated of all mycotoxins due to their role in establishing the significance of mycotoxins in animal diseases, and to the regulation of their presence in food [1,2]. Aflatoxins pose serious health hazards to humans and domestic animals, because they frequently contaminate agricultural commodities [3,4]. Presently, a significant number of countries have established or proposed regulations for controlling aflatoxins in food and feeds [5]; the US Food and Drug Administration (FDA) has limits of 20 ppb, total aflatoxins, on interstate commerce of food and feed, and 0.5 ppb of aflatoxin M₁ on the sale of milk. However, many countries, especially in the developing world, experience contamination of domestic-grown commodities to alarmingly greater levels than does the US A study revealed a strong association between exposure to aflatoxin and both stunting (a reflection of chronic malnutrition) and being underweight (a reflection of acute malnutrition) in West African children [6]. A 2004 outbreak of acute aflatoxicosis in Kenya, due to ingestion of contaminated maize, resulted in 125 deaths [7].

Recognition of the need to control aflatoxin contamination of food and feed grains has elicited various approaches from researchers to eliminate this toxin from maize and other susceptible crops. The approach to enhance host resistance through conventional or molecular breeding has gained renewed attention following the discovery of natural resistance to A. flavus infection and aflatoxin production in maize [8,9,10,11,12,13,14].

During the past two decades, maize genotypes with natural preharvest resistance to aflatoxin production have been identified through field screening [11,12,15]. The poor agronomic quality of these lines, however, renders them of little direct commercial value [14]. The lack of identified markers has slowed the incorporation of resistance into lines with commercially-acceptable genetic backgrounds.

The expression of maize kernel proteins has been implicated in kernel resistance to A. flavus infection/aflatoxin production [16,17,18,19]. Using reverse genetics to identify genes that are associated with aflatoxin-resistance may lead to the discovery of breeding markers. These protein/gene markers could be used to transfer resistance to good genetic backgrounds while excluding undesirable traits. The purpose of this review is to highlight the discovery of resistance-associated proteins (RAPs) and their characterization as potential breeding markers.

The development of a laboratory kernel screening assay (KSA) by Brown et al. [13] facilitated the verification of maize kernel resistance under laboratory conditions in a short time. This accelerated the discovery of knowledge surrounding host resistance mechanisms. Using this assay, Brown et al. [20] discovered the existence of subpericarp resistance in maize kernels and that the expression of this resistance requires a live embryo, indicating a potential role for kernel proteins in resistance. Guo et al. [18] found that imbibition of kernels, before inoculation with A. flavus, significantly increased aflatoxin-resistance of susceptible maize genotypes. Further investigation revealed that susceptible genotypes were able to induce antifungal proteins upon fungal infection [18], suggesting that susceptible lines have the ability to induce an active defense mechanism after fungal infection. The usefulness of the KSA as an investigative tool is aided by the fact that KSA results correlate well with field results [13] and that aflatoxin buildup occurs after kernel maturity, a developmental phase where constitutive factors required for kernel resistance are highlighted by the KSA [21]. However, resistance must be confirmed in the field; here, agronomic factors contributing to resistance can also be investigated.

Examination of kernel proteins of several maize genotypes revealed differences between genotypes resistant or susceptible to aflatoxin contamination [22,23]. Imbibed susceptible kernels showed decreased aflatoxin levels and contained germination-induced ribosome inactivating protein (RIP) and zeamatin; both proteins have demonstrated growth-inhibitory activity in vitro against A. flavus [22]. In another study, two kernel proteins were identified from a resistant corn inbred line (Tex6), which may contribute to resistance to aflatoxin contamination [19]. When a commercial maize hybrid was inoculated with toxgenic and atoxigenic strains of A. flavus at milk stage, one chitinase and one β-1,3-glucanase isoform were detected in maturing infected kernels, while another isoform was detected in maturing uninfected kernels [24]. Lazovoya [25] reported that the presence of A. flavus caused an increase in β-1,3-glucanase activity in callus tissues from a resistant genotype, but not from a susceptible one. A more rapid and stronger induction of the PR-1 and PR-5 genes in maize leaves has also been observed in an incompatible interaction when compared to a compatible interaction upon pathogen infection [26]. A 14 kDa trypsin inhibitor protein (TI) was found to express at high levels in resistant lines but at low levels or is missing in susceptible ones [27]. This protein demonstrated antifungal activity against A. flavus and several other pathogenic fungi [28], possibly through inhibition of fungal α-amylase activity and production [29]. This could limit the availability of simple sugars needed for fungal growth and aflatoxin production [30].

The above-studies indicate an important role for kernel proteins in disease resistance. Further investigation, supporting earlier work by Guo [18], found that both constitutive and inducible proteins are required for kernel resistance to A. flavus infection and aflatoxin production [21]. This work 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. Therefore, research on resistance genes/proteins has focused heavily on the identification of constitutively-produced kernel resistance-associated proteins (RAPs).

To increase protein resolution and detection sensitivity by 10 to 20 fold and, thus, enhance ability to identify more constitutively-expressed RAPs, proteomics approaches have been employed. Kernel proteins from several resistant and susceptible genotypes were compared using large format 2-D gel electrophoresis. A number of protein spots, either unique or 5-fold up-regulated in resistant lines, were detected, isolated from preparative 2-D gels and identified using ESI-MS/MS after in-gel digestion with trypsin [31,32]. These proteins can be grouped into three categories based on their peptide sequence homology: (1) storage proteins, such as globulins (GLB1, GLB2), and late embryogenesis abundant proteins (LEA3, LEA14); (2) stress-responsive proteins, such as aldose reductase (ALD), glyoxalase I (GLX I) and heat shock proteins, and (3) antifungal proteins, including TI. In total, approximately 21 proteins upregulated in resistant versus susceptible lines have been identified using comparative proteomics (Table 1).

No investigation has been conducted to determine the possible direct involvement of stress-related proteins in host fungal resistance. However, increased temperatures and drought, which often occur together, are major factors associated with aflatoxin contamination of corn kernels [34]. Unique or higher levels of hydrophilic storage or stress-related proteins, such as the aforementioned, may put resistant lines at an advantage for the ability to synthesize proteins and defend against pathogens while under stress. Further studies including physiological and biochemical characterization, genetic mapping, plant transformation using RAP genes, RNAi gene silencing experiments [35] and marker-assisted breeding should clarify the roles of stress-related RAPs in kernel resistance.

To conduct the above-described comparative proteome studies, composite profiles for resistance and for susceptibility were developed from 2 D gels of several resistant or susceptible maize lines. This was done to homogenize nonresistance-related differences among lines within each group, and, therefore, facilitate the identification of resistance-related proteins. In using the composite gel approach, only those proteins that were five-fold upregulated in resistant versus susceptible lines were studied to minimize the chance of identifying proteins unrelated to host resistance.

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 [42]. Transgenic rice overexpressing a barley LEA3 protein HVA1 showed significantly increased tolerance to water deficit and salinity [43].

The role of GLX I (Table 2) in stress-tolerance was first highlighted in an earlier study using transgenic tobacco plants overexpressing a Brassica juncea glyoxalase I [44]. 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 [45]. 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 [32], 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 [32].

Another RAP that has been characterized further is the pathogenesis-related protein 10 (PR10) (Table 2). 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 [46]. 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 [46]. 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 [46]. Recently, a new PR10 homologue was identified from maize (PR10.1) [47]. 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 [47]. 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 [48]. In cowpea, a PR10 homolog was specifically up-regulated in resistant epidermal cells inoculated with the rust fungus Uromyces vignae Barclay [49]. A PR10 transcript was also induced in rice during infection by Magnaporthe grisea [50].

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 [51,52]. 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 [53]. Interference of double-stranded RNA with expression of specific genes has been widely described [54,55]. 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 [52]. 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 [52].

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 [56]. 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 [57]. 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 [58].

Host resistance as a strategy for eliminating aflatoxin contamination of maize is closer to being a reality due to the identification of genotypes with natural resistance to aflatoxin accumulation and the development of new inbred lines through breeding. However, to exploit this resistance for the benefit of maize growers, markers have to be identified to facilitate the transfer of resistance to elite proprietary backgrounds currently in commercial use. The identification of resistance-associated proteins goes a long way towards providing the novel markers that will be indispensible to any commercial breeding undertaking. Characterization studies including RNAi gene silencing and gene mapping are instrumental in building a case for the involvement of selected RAPs in kernel resistance to aflatoxin contamination.

Several publications by the current authors have profiled many of the RAPs listed in the present review. Here, however, the most complete listing of RAPs identified through comparative proteomics is presented along with available evidence of their potential as breeding markers. Investigations of RAPs, as discussed above, not only impact the development of commercially-useful resistant maize lines, but provide an expanding base of knowledge concerning nature’s requirements for creating a durable resistance against the opportunistic pathogen, A. flavus. It remains to be determined, how the different categories of proteins, antifungal, stress-related, storage and others contribute to the total picture of resistance. Future investigations (e.g., proteomics and microarray analysis) may also impact aflatoxin-resistance through the discovery of RAPs down-regulated in resistant lines, RAPs induced upon fungal infection and also factors involved in the regulation of RAPs. These discoveries will not only contribute to the development of aflatoxin-resistant maize lines, they may aid other susceptible crops, assist in meeting the challenges of other mycotoxin-producing fungi, while enhancing our understanding of host plant interactions with fungi.