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PLANT GENETIC ENGINEERING

Identify Anatoxin Biosynthesis Inhibitors

MARKER-ASSISTED BREEDING

1. Vector Design

2. Plant Transformation m 3 Trjrrin eonoN

BREEDING, RELEASE AFLATOXIN RESISTANT GERMPLASM, COMMERCIALIZATION PROCESS

Figure 1 Strategy for enhancing host plant resistance to aflatoxin contamination. The research approach being used to identify and employ resistance factors, such as resistance-associated proteins (RAPs) that are identified in aflatoxin-resistant corn lines. After resistant germplasm is identified, various tools are used to characterize the expression of resistance, such as KSA-based studies, GUS/GFP reporter constructs, and seed physiology studies. These can lead to RAP identification protocols such as proteome analysis. Genes corresponding to RAPs can be cloned, and clones then used for QTL studies, plant transformation, or marker-assisted breeding.

antifungal genes through genetic engineering into other aflatoxin-susceptible crops (Figure 1).

A recent investigation into corn kernel resistance (Chen et al. 2001) 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.

2.2.1 Identification of RAPs Through Proteome Analysis

To increase protein resolution and detection sensitivity by 10-20 fold and, thus, enhance ability to identify more RAPs, a proteomics approach was recently employed. The increased reproducibility, reliability, and accuracy of 2-D gel electrophoresis is due to advances in technology, such as immobilized pH gradient (IPG) gel strips and sophisticated computerized 2-D gel analysis software (Appel et al. 1997; Gorg et al. 1998). 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 lines, have been identified, isolated from preparative 2-D gels and analyzed using ESI-MS/MS after in-gel digestion with trypsin (Chen et al. 1999a; 2000; 2002). These proteins can be grouped into three categories based on their peptide sequence homology: (a) storage proteins, such as GLB1, GLB2, and late embryogenesis abundant proteins (LEA3, LEA14); (b) stress-responsive proteins, such as aldose reductase (ALD), a glyoxalase I protein (GLX1), and a 16.9 kDa heat shock protein, and (c) antifungal proteins, which include TI.

Thus far, no investigation has been conducted to determine the possible direct involvement of stress-related proteins in host fungal resistance. 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 (Payne 1998). Other studies have 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. Therefore, the necessary requirements for developing commercially-useful, aflatoxin-resistant maize lines may include, aside from antifungal proteins, a high level of expression of stress-related proteins. 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.

2.2.2 Natural Compounds That Affect Mycotoxin Biosynthesis

Several compounds have been identified in corn which may have regulatory effects on the aflatoxin and trichothecene biosynthetic process. The compound 4-acetyl-benzoxazolin-2-one (ABOA), which was isolated from maize lines tolerant to F. graminearum, strongly inhibited acetyl-deoxynivalinol production at 5 mM, aflatoxin production at 2 mg/ml, and feeding by maize weevils at 1000ppm (Miller et al. 1996).

Steryl esters from maize significantly increased aflatoxin production by some A. flavus strains at 0.3 and 1.0mg/ml (Norton and Dowd 1996). Anthocyanins and related flavonoids, some of which occur naturally in maize kernels, inhibited aflatoxin production by more than 50% at 0.76 mM (Norton 1999). More highly glycosylated forms of the anthocyanins tended to be less effective in inhibiting aflatoxin production (Norton 1999). Carotenoids containing an alpha-ionone type ring tended to be more effective inhibitors of aflatoxin production by A. flavus, with some having an I50 of about 6 mM (Norton 1997). Although most strains of A. flavus exposed to beta-carotene at 50 mg/ml had aflatoxin production inhibited by 90% or more, some peanut derived strains were less sensitive (Wicklow et al. 1998). In vitro studies indicated plant peroxidase could greatly enhance the ability of plant chemicals to inhibit spore germination and hyphal growth of F. graminearum (Dowd et al. 1997). A. flavus was considerably more resistant to quinone products potentially produced by plant peroxidases compared to F. graminearum and F. verticillioides (moniliforme) (Dowd et al. 1997). In addition, volatile compounds from corn and cotton, which are products of the lipoxygenase pathway, were shown to have effects upon aflatoxin biosynthesis and fungal development in vitro [reviewed in Bhatnagar et al. (2001)].

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