**3. Investigations of resistance mechanisms/traits in maize lines**

#### **3.1. Molecular genetic investigations of aflatoxin-resistant lines**

Chromosome regions associated with resistance to *A. flavus* and inhibition of aflatoxin pro‐ duction in maize have been identified through Restriction Fragment Length Polymorphism (RFLP) analysis in three "resistant" lines (R001, LB31, and Tex6) in an Illinois breeding pro‐ gram, after mapping populations were developed using B73 and/or Mo17 elite inbreds as the "susceptible" parents [20, 21]. Chromosome regions associated with inhibition of aflatox‐ in 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 improv‐ ing resistance through marker assisted breeding into commercial lines [21]. 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 [22]. 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) [23]. QTLs con‐ tributing resistance to aflatoxin accumulation were also identified using a population creat‐ ed by B73 and resistant inbred Mp313E, on chromosome 4 of Mp313E [24]. This confirmed the findings of an earlier study involving Mp313E and susceptible Va35 [25]. Another QTL in this study, which has similar effects to that on chromosome 4, was identified on chromo‐ some 2 [24]. 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 [26]. Lastly, in a study of population of F2:3 families developed from resistant Mp715 and a southern-adapted suscep‐ tible, T173, QTL with phenotypic effects up to 18.5% were identified in multiple years on chromosomes 1, 3, 5, and 10 [27].

**3.3. Two levels of resistance**

and susceptible genotypes [31, 32].

expression of the pathway gene.

mycotoxigenic fungi.

**3.4. Comparing fungal growth to toxin production**

The KSA employs a very simple and inexpensive apparatus involving bioassay trays, petri dishes, vial caps as seed containers, and chromatography paper for holding moisture [14]. Kernels screened by the KSA are maintained in 100% humidity, at a temperature favoring *A. flavus* (31° C) growth and aflatoxin production, and are usually incubated for seven days. Aflatoxin data from KSA experiments can be obtained two to three weeks after experiments are initiated. KSA experiments confirmed GT-MAS:gk resistance to aflatoxin production and demonstrated that it is maintained even when the pericarp barrier, in otherwise viable ker‐ nels, is breached [13]. Penetration through the pericarp barrier was achieved by wounding the kernel with a hypodermic needle down to the endosperm, prior to inoculation. Wound‐ ing facilitates differentiation between different resistance mechanisms in operation, and the manipulation of aflatoxin levels in kernels for comparison with other traits (e.g. fungal growth; protein induction). The results of this study indicate the presence of two levels of resistance: at the pericarp and at the subpericarp level. The former was supported by the above-studies which demonstrated a role for pericarp waxes in kernel resistance [30], and highlighted quantitative and qualitative differences in pericarp wax between GT-MAS:gk

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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) [14], kernel resistance to fungal infection in nonwounded and wounded kernels was demonstrated both visually and quan‐ titatively, 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 cor‐ relation 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

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 [37]. This indicates that some resistance mechanisms may be generic for ear rotting/

A more recent use of reporter genes was performed on cotton using a green fluorescent pro‐ tein reporter; a GFP-expressing *A. flavus* strain to successfully monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins [38]. 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.

A number of genes corresponding to resistance-associated proteins (RAPs), that were identi‐ fied in proteomics studies (see section 3.5.1 below) have been mapped to chromosomal loca‐ tion using the genetic sequence of B73 now available online (http:// archive.maizesequence.org/index.html) [28]. Using the DNA sequence of the RAPs and blasting them against the B73 sequence allowed us to place each gene into a virtual bin, al‐ lowing us to pinpoint the chromosomal location to which each gene maps. The chromo‐ somes 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 [29].

#### **3.2. Kernel pericarp wax**

Kernel pericarp wax of maize breeding population GT-MAS:gk has been associated with re‐ sistance to *Aspergillus flavus* infection /aflatoxin production. Previously, kernel wax of GT-MAS:gk was compared to that of 3 susceptible genotypes. Thin layer chromatography (TLC) of wax from these genotypes showed a band unique to GT-MAS:gk and a band unique to the three susceptible lines [30]. GT-MAS:gk kernel wax also was shown to inhibit *A. flavus* growth. A later investigation compared GT-MAS:gk wax resistance-associated traits to that of twelve susceptible maize genotypes [31]. TLC results of wax from these lines confirmed findings of the previous investigation, demonstrating both the unique GT-MAS:gk TLC band and the unique 'susceptible' band. Gas chromatography/mass spectroscopy (GC/MS) analysis of the whole wax component showed a higher percentage of phenol-like com‐ pounds in the resistant genotype than in the susceptibles. Alkylresorcinol content was dra‐ matically higher in GT-MAS:gk wax than in susceptible lines. An alkylresorcinol, 5 methylresorcinol, also inhibited *in vitro* growth of *A. flavus*. Further research is needed for a clear identification of the component(s) responsible for kernel wax resistance and to deter‐ mine its expression level in other maize lines.

#### **3.3. Two levels of resistance**

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) [23]. QTLs con‐ tributing resistance to aflatoxin accumulation were also identified using a population creat‐ ed by B73 and resistant inbred Mp313E, on chromosome 4 of Mp313E [24]. This confirmed the findings of an earlier study involving Mp313E and susceptible Va35 [25]. Another QTL in this study, which has similar effects to that on chromosome 4, was identified on chromo‐ some 2 [24]. 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 [26]. Lastly, in a study of population of F2:3 families developed from resistant Mp715 and a southern-adapted suscep‐ tible, T173, QTL with phenotypic effects up to 18.5% were identified in multiple years on

A number of genes corresponding to resistance-associated proteins (RAPs), that were identi‐ fied in proteomics studies (see section 3.5.1 below) have been mapped to chromosomal loca‐ tion using the genetic sequence of B73 now available online (http:// archive.maizesequence.org/index.html) [28]. Using the DNA sequence of the RAPs and blasting them against the B73 sequence allowed us to place each gene into a virtual bin, al‐ lowing us to pinpoint the chromosomal location to which each gene maps. The chromo‐ somes 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

Kernel pericarp wax of maize breeding population GT-MAS:gk has been associated with re‐ sistance to *Aspergillus flavus* infection /aflatoxin production. Previously, kernel wax of GT-MAS:gk was compared to that of 3 susceptible genotypes. Thin layer chromatography (TLC) of wax from these genotypes showed a band unique to GT-MAS:gk and a band unique to the three susceptible lines [30]. GT-MAS:gk kernel wax also was shown to inhibit *A. flavus* growth. A later investigation compared GT-MAS:gk wax resistance-associated traits to that of twelve susceptible maize genotypes [31]. TLC results of wax from these lines confirmed findings of the previous investigation, demonstrating both the unique GT-MAS:gk TLC band and the unique 'susceptible' band. Gas chromatography/mass spectroscopy (GC/MS) analysis of the whole wax component showed a higher percentage of phenol-like com‐ pounds in the resistant genotype than in the susceptibles. Alkylresorcinol content was dra‐ matically higher in GT-MAS:gk wax than in susceptible lines. An alkylresorcinol, 5 methylresorcinol, also inhibited *in vitro* growth of *A. flavus*. Further research is needed for a clear identification of the component(s) responsible for kernel wax resistance and to deter‐

chromosomes 1, 3, 5, and 10 [27].

6 Aflatoxins - Recent Advances and Future Prospects

**3.2. Kernel pericarp wax**

above-chromosomes as well as chromosomes 4 and 9 [29].

mine its expression level in other maize lines.

The KSA employs a very simple and inexpensive apparatus involving bioassay trays, petri dishes, vial caps as seed containers, and chromatography paper for holding moisture [14]. Kernels screened by the KSA are maintained in 100% humidity, at a temperature favoring *A. flavus* (31° C) growth and aflatoxin production, and are usually incubated for seven days. Aflatoxin data from KSA experiments can be obtained two to three weeks after experiments are initiated. KSA experiments confirmed GT-MAS:gk resistance to aflatoxin production and demonstrated that it is maintained even when the pericarp barrier, in otherwise viable ker‐ nels, is breached [13]. Penetration through the pericarp barrier was achieved by wounding the kernel with a hypodermic needle down to the endosperm, prior to inoculation. Wound‐ ing facilitates differentiation between different resistance mechanisms in operation, and the manipulation of aflatoxin levels in kernels for comparison with other traits (e.g. fungal growth; protein induction). The results of this study indicate the presence of two levels of resistance: at the pericarp and at the subpericarp level. The former was supported by the above-studies which demonstrated a role for pericarp waxes in kernel resistance [30], and highlighted quantitative and qualitative differences in pericarp wax between GT-MAS:gk and susceptible genotypes [31, 32].

#### **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) [14], kernel resistance to fungal infection in nonwounded and wounded kernels was demonstrated both visually and quan‐ titatively, 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 cor‐ relation 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 [37]. 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 pro‐ tein reporter; a GFP-expressing *A. flavus* strain to successfully monitor fungal growth, mode of entry, colonization of cottonseeds, and production of aflatoxins [38]. 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 [17]. Studies demonstrating subpericarp (wounded-kernel) resistance in maize kernels have led to research for identification of sub‐ pericarp resistance mechanisms. Examinations of kernel proteins of several genotypes re‐ vealed differences between genotypes resistant and susceptible to aflatoxin contamination [39]. Imbibed susceptible kernels, for example, showed decreased aflatoxin levels and con‐ tained germination-induced ribosome inactivating protein (RIP) and zeamatin [40]. Both zeamatin and RIP have been shown to inhibit *A. flavus* growth *in vitro* [40]. In another study, two kernel proteins were identified from a resistant corn inbred (Tex6) which may contrib‐ ute to resistance to aflatoxin contamination [41]. One protein, 28 kDa in size, inhibited *A. fla‐ vus* 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 detect‐ ed in maturing infected kernels, while another isoform was detected in maturing uninfected kernels [42].

electrophoresis is also unique in its ability to detect post- and cotranslational modifications,

Development of Maize Host Resistance to Aflatoxigenic Fungi

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9

Through proteome analysis and the subtractive approach, it may be possible to identify im‐ portant protein markers associated with resistance, as well as genes encoding these proteins. This could facilitate marker-assisted breeding and/or genetic engineering efforts. Endo‐ sperm 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 di‐ gestion with trypsin [52, 53]. These proteins, all constitutively expressed, can be grouped in‐ to 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

During the screening of progeny developed through the IITA-USDA/ARS collaborative project, near-isogenic lines from the same backcross differing significantly in aflatoxin accu‐ mulation were identified, and proteome analysis of these lines is being conducted [54]. In‐ vestigating 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

Heretofore, most RAPs identified have had antifungal activities. However, increased tem‐ peratures and drought, which often occur together, are major factors associated with afla‐ toxin contamination of maize kernels [55]. It has also been found that drought stress imposed during grain filling reduces dry matter accumulation in kernels [55]. 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 afore‐ mentioned, 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

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 expres‐ sion of storage protein GLB1 and LEA3 has been reported to be stress-responsive and ABAdependant [56]. Transgenic rice overexpressing a barley LEA3 protein HVA1 showed significantly increased tolerance to water deficit and salinity [57]. The role of GLX I in stresstolerance was first highlighted in an earlier study using transgenic tobacco plants overex‐ pressing a *Brassica juncea* glyoxalase I [58]. The substrate for glyoxalase I, methylglyoxal, is a potent cytotoxic compound produced spontaneously in all organisms under physiological

which cannot be predicted from the genome sequence.

proteins, including the above-described TI.

the genetic backgrounds of the lines.

*3.5.2. Further characterization of RAPs*

RAPs may also contribute valuable information. [54].

In another investigation, an examination of kernel protein profiles of 13 maize genotypes re‐ vealed 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 [43]. 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 [44] required not only for fungal growth, but also for toxin production [45]. TI also demonstrated antifungal activity against other mycotoxigenic species [46]. 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 [47] determined that both constitutive and in‐ duced proteins are required for resistance to aflatoxin production. It also showed that one major difference between resistant and susceptible genotypes is that resistant lines constitu‐ tively 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 inva‐ sion, 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 independ‐ ent properties, isoelectric points and then molecular weights, has been recognized for a number of years as a powerful biochemical separation technique. Improvements in map res‐ olution and reproducibility [48, 49], rapid analysis of proteins, analytical soft ware and com‐ puters, 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 "proteo‐ mics" is the analysis of the protein complement of a genome [50, 51]. This involves the sys‐ tematic 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 im‐ portant protein markers associated with resistance, as well as genes encoding these proteins. This could facilitate marker-assisted breeding and/or genetic engineering efforts. Endo‐ sperm 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 di‐ gestion with trypsin [52, 53]. These proteins, all constitutively expressed, can be grouped in‐ to 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 accu‐ mulation were identified, and proteome analysis of these lines is being conducted [54]. In‐ vestigating 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 tem‐ peratures and drought, which often occur together, are major factors associated with afla‐ toxin contamination of maize kernels [55]. It has also been found that drought stress imposed during grain filling reduces dry matter accumulation in kernels [55]. 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 afore‐ mentioned, 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. [54].

#### *3.5.2. Further characterization of RAPs*

**3.5. Resistance-associated proteins**

8 Aflatoxins - Recent Advances and Future Prospects

kernels [42].

to form an active defense system.

*3.5.1. Proteomic analysis*

Developing resistance to fungal infection in wounded as well as intact kernels would go a long way toward solving the aflatoxin problem [17]. Studies demonstrating subpericarp (wounded-kernel) resistance in maize kernels have led to research for identification of sub‐ pericarp resistance mechanisms. Examinations of kernel proteins of several genotypes re‐ vealed differences between genotypes resistant and susceptible to aflatoxin contamination [39]. Imbibed susceptible kernels, for example, showed decreased aflatoxin levels and con‐ tained germination-induced ribosome inactivating protein (RIP) and zeamatin [40]. Both zeamatin and RIP have been shown to inhibit *A. flavus* growth *in vitro* [40]. In another study, two kernel proteins were identified from a resistant corn inbred (Tex6) which may contrib‐ ute to resistance to aflatoxin contamination [41]. One protein, 28 kDa in size, inhibited *A. fla‐ vus* 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 detect‐ ed in maturing infected kernels, while another isoform was detected in maturing uninfected

In another investigation, an examination of kernel protein profiles of 13 maize genotypes re‐ vealed 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 [43]. 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 [44] required not only for fungal growth, but also for toxin production [45]. TI also demonstrated antifungal activity against other mycotoxigenic species [46]. The identification of these proteins may provide markers for plant breeders, and may facilitate the cloning and introduction of antifungal genes

An investigation into maize kernel resistance [47] determined that both constitutive and in‐ duced proteins are required for resistance to aflatoxin production. It also showed that one major difference between resistant and susceptible genotypes is that resistant lines constitu‐ tively 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 inva‐ sion, and consequent aflatoxin formation, until other antifungal proteins can be synthesized

Two-dimensional (2-D) gel electrophoresis, which sorts proteins according to two independ‐ ent properties, isoelectric points and then molecular weights, has been recognized for a number of years as a powerful biochemical separation technique. Improvements in map res‐ olution and reproducibility [48, 49], rapid analysis of proteins, analytical soft ware and com‐ puters, 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 "proteo‐ mics" is the analysis of the protein complement of a genome [50, 51]. This involves the sys‐ tematic separation, identification, and quantification of many proteins simultaneously. 2-D

through genetic engineering into other aflatoxin-susceptible crops.

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 expres‐ sion of storage protein GLB1 and LEA3 has been reported to be stress-responsive and ABAdependant [56]. Transgenic rice overexpressing a barley LEA3 protein HVA1 showed significantly increased tolerance to water deficit and salinity [57]. The role of GLX I in stresstolerance was first highlighted in an earlier study using transgenic tobacco plants overex‐ pressing a *Brassica juncea* glyoxalase I [58]. 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 concen‐ trations; experimental evidence indicates that induction is through upregulation of aflatoxin biosynthetic pathway transcripts including the *AFLR* regulatory gene [59]. Therefore, glyox‐ alase I may be directly affecting resistance by removing its aflatoxin-inducing substrate, methylglyoxal. PER1, a 1-cys peroxiredoxin antioxidant identified in a proteomics investiga‐ tion [60], was demonstrated to be an abundant peroxidase, and may play a role in the re‐ moval of reactive oxygen species. The PER1 protein overexpressed in *Escherichia coli* demonstrated peroxidase activity *in vitro*. It is possibly involved in removing reactive oxy‐ gen species produced when maize is under stress conditions [60]. Another RAP that has been characterized further is the pathogenesis-related protein 10 (PR10). It showed high ho‐ mology 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 as‐ paragus (CAA10720), and low homology to a RNase from ginseng [61]. The PR10 overex‐ pressed 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 trans‐ genic tobacco plants expressing maize *PR10* gene compared to the control leaf extract [61]. 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 [61]. Recently, a new *PR10* homologue was identified from maize (*PR10.1*) [62]. *PR10* was ex‐ pressed at higher levels in all tissues compared to *PR10.1*, however, purified PR10.1 overex‐ pressed in *E. coli* possessed 8-fold higher specific RNase activity than *PR10* [62]. 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 specifi‐ cally induced in resistant cultivars upon infection by *Rhynchosporium secalis*, but not in nearisogenic susceptible plants [63]. In cowpea, a *PR10* homolog was specifically up-regulated in resistant epidermal cells inoculated with the rust fungus *Uromyces vignae* Barclay [64]. A *PR10* transcript was also induced in rice during infection by *Magnaporthe grisea* [65].

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 [66]. The transgenic ker‐ nels also showed a significant increase in susceptibility to *A. flavus* infection and aflatox‐ in 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 [66]. RNAi vectors to silence other RAP genes, such as *GLX I* and *TI*, have also been con‐ structed, and introduced into immature maize embryos through both bombardment and *Agrobacterium* infection [70]. 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* infec‐

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11

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 [71]. When tested against *A. flavus*, ZmCORp inhibit‐ ed 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*

ZmTIp, a 10 kDa trypsin inhibitor, had an impact on *A. flavus* growth, but not as great as the

A study was conducted to investigate the proteome of rachis tissue, maternal tissue that supplies nutrients to the kernels [75]. An interesting finding in this study is that after infec‐ tion by *A. flavus*, rachis tissue of aflatoxin-resistant genotypes did not up-regulate PR pro‐ teins 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 re‐ sponse to fungal infection. Given the relationship of the rachis to kernels, these results con‐ firm findings of a previous investigation [47], which demonstrated levels of proteins in resistant *versus* susceptible kernels was a primary factor that determined kernel genetic re‐ sistance to aflatoxin contamination. Another study was conducted to identify proteins in maize silks that may be contributing to resistance against *A. flavus* infection/colonization [76]. Antifungal bioassays were performed using silk extracts from two aflatoxin-resistant and two–susceptible inbred lines. Silk extracts from resistant inbreds showed greater antifungal 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 chiti‐ nase activity, silk proteins from extracts of resistant lines also showed significantly higher chitinase activity than that from susceptible lines. Differential expression of chitinases in

to be expressed 50% more in kernels of a resistant maize line *versus* a susceptible.

tion and aflatoxin production.

previously-mentioned 14 kDa TI [72].

*3.5.3. Proteomic studies of rachis and silk tissue*

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 endog‐ enous 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 in‐ tron is included [67]. Interference of double-stranded RNA with expression of specific genes has been widely described [68, 69]. Although the mechanism is still not well un‐ derstood, RNAi provides an extremely powerful tool to study functions of unknown genes in many organisms. This posttranscriptional gene silencing (PTGS) is a sequencespecific RNA degradation process triggered by a dsRNA, which propagates systemically throughout the plant, leading to the degradation of homologous RNA encoded by en‐ dogenous 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 [66]. The transgenic ker‐ nels also showed a significant increase in susceptibility to *A. flavus* infection and aflatox‐ in 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 [66]. RNAi vectors to silence other RAP genes, such as *GLX I* and *TI*, have also been con‐ structed, and introduced into immature maize embryos through both bombardment and *Agrobacterium* infection [70]. 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* infec‐ tion 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 [71]. When tested against *A. flavus*, ZmCORp inhibit‐ ed 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 [72].

#### *3.5.3. Proteomic studies of rachis and silk tissue*

conditions from glycolysis and photosynthesis intermediates, glyceraldehydes-3-phosphate and dihydroxyacetone phosphate. Methylglyoxal is an aflatoxin inducer even at low concen‐ trations; experimental evidence indicates that induction is through upregulation of aflatoxin biosynthetic pathway transcripts including the *AFLR* regulatory gene [59]. Therefore, glyox‐ alase I may be directly affecting resistance by removing its aflatoxin-inducing substrate, methylglyoxal. PER1, a 1-cys peroxiredoxin antioxidant identified in a proteomics investiga‐ tion [60], was demonstrated to be an abundant peroxidase, and may play a role in the re‐ moval of reactive oxygen species. The PER1 protein overexpressed in *Escherichia coli* demonstrated peroxidase activity *in vitro*. It is possibly involved in removing reactive oxy‐ gen species produced when maize is under stress conditions [60]. Another RAP that has been characterized further is the pathogenesis-related protein 10 (PR10). It showed high ho‐ mology 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 as‐ paragus (CAA10720), and low homology to a RNase from ginseng [61]. The PR10 overex‐ pressed 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 trans‐ genic tobacco plants expressing maize *PR10* gene compared to the control leaf extract [61]. 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 [61]. Recently, a new *PR10* homologue was identified from maize (*PR10.1*) [62]. *PR10* was ex‐ pressed at higher levels in all tissues compared to *PR10.1*, however, purified PR10.1 overex‐ pressed in *E. coli* possessed 8-fold higher specific RNase activity than *PR10* [62]. 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 specifi‐ cally induced in resistant cultivars upon infection by *Rhynchosporium secalis*, but not in nearisogenic susceptible plants [63]. In cowpea, a *PR10* homolog was specifically up-regulated in resistant epidermal cells inoculated with the rust fungus *Uromyces vignae* Barclay [64]. A

10 Aflatoxins - Recent Advances and Future Prospects

*PR10* transcript was also induced in rice during infection by *Magnaporthe grisea* [65].

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 endog‐ enous 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 in‐ tron is included [67]. Interference of double-stranded RNA with expression of specific genes has been widely described [68, 69]. Although the mechanism is still not well un‐ derstood, RNAi provides an extremely powerful tool to study functions of unknown genes in many organisms. This posttranscriptional gene silencing (PTGS) is a sequencespecific RNA degradation process triggered by a dsRNA, which propagates systemically throughout the plant, leading to the degradation of homologous RNA encoded by en‐ dogenous 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 A study was conducted to investigate the proteome of rachis tissue, maternal tissue that supplies nutrients to the kernels [75]. An interesting finding in this study is that after infec‐ tion by *A. flavus*, rachis tissue of aflatoxin-resistant genotypes did not up-regulate PR pro‐ teins 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 re‐ sponse to fungal infection. Given the relationship of the rachis to kernels, these results con‐ firm findings of a previous investigation [47], which demonstrated levels of proteins in resistant *versus* susceptible kernels was a primary factor that determined kernel genetic re‐ sistance to aflatoxin contamination. Another study was conducted to identify proteins in maize silks that may be contributing to resistance against *A. flavus* infection/colonization [76]. Antifungal bioassays were performed using silk extracts from two aflatoxin-resistant and two–susceptible inbred lines. Silk extracts from resistant inbreds showed greater antifungal 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 chiti‐ nase 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.

host resistance is increased. In a preliminary proteomics comparison of constitutive protein differences between those African closely-related lines, a new category of resistance-associ‐ ated proteins (putative regulatory proteins) was identified, including a serine/threonine pro‐ tein kinase and a translation initiation factor 5A [29, 79]. The genes encoding these two resistance associated regulatory proteins are being cloned and their potential role in host re‐ sistance to *A. flavus* infection and aflatoxin production will be further investigated. Conduct‐ ing proteomic analyses using lines from this program not only enhances chances of identifying genes important to resistance, but may have immediate practical value. The II‐ TA-SRRC collaboration has registered and released six inbred lines with aflatoxin-resistance in good agronomic backgrounds, which also demonstrate good levels of resistance to south‐ ern corn blight and southern corn rust [80]. Resistance field trials for these lines on U.S. soil is being conducted; the ability to use resistance in these lines commercially will depend on having identified excellent markers, since seed companies desire insurance against the transfer of undesirable traits into their elite genetic backgrounds. The fact that this resistance is coming from good genetic backgrounds is also a safeguard against the transfer of undesir‐

Development of Maize Host Resistance to Aflatoxigenic Fungi

http://dx.doi.org/10.5772/54654

13

**Entry Aflatoxin B1 (ppb)**

Susceptible control 10197 a

GT-MAS:gk 338 de

Resistant control 76 e

**Table 1.** KSA screening of IITA-SRRC maize breeding materials which identified 2 closely related lines (87.5% genetic similarity), #22 and #25, from parental cross (GT-MASgk x Ku1414SR) x GT-MAS:gk; these contrast significantly in aflatoxin accumulation. Values followed by the same letter are not significantly different by the least significant

25\* 228 e 23 197 e

able traits.

difference test (P = 0.05).

#### *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 [73]. Similar work had previously been performed using Tex6 as the resistant line and B73 as the susceptible [74], however, in the study using closely-related lines, imbibed mature ker‐ nels were used (for the first time) and proved to be a quicker and easier approach than tradi‐ tional approaches. The involvement of certain stress-related and antifungal genes previously shown to be associated with constitutive resistance was demonstrated here; a kinase-bind‐ ing protein, Xa21 was highly up-regulated in the resistant line compared to the susceptible, both constitutively and in the inducible state.
