## 1 The Concept of Phytoalexins
Six genetic constructs were thus designed: sense and antisense-oriented cDNAs of isoflavone reductase (IFR), (+) 6\(a\)-hydroxymaackiain 3-\(O\)-methyltransferase (HMM) fused to the 35S CaMV promoter, being these two enzymes involved in pisatin biosynthesis, and a pisatin demethylating activity (PDA) from _N. haematococcca_. Reduced amounts of pisatin were observed in hairy root cultures transformed with antisense-_Hmm_ which led to fewer _Hmm_ transcripts, resulting in less HMM protein and enzyme activity in response to elicitation with CuCl\({}_{2}\) or infection with the pea-pathogenic fungus, _N. haematococcca_. As a consequence of the transformation with antisense _Hmm_, roots became more susceptible to _N. haematococcca_. As expected, cultures with the lowest levels of pisatin were those containing the pisatin demethylating activity. At the time this paper was published, it appeared to be the first case of producing transgenic plant tissues with reduced capability to accumulate a given phytoalexin and demonstrating that such a tissue was less resistant to fungal infection [85]. Additionally, this loss-of-function genetic approach afforded direct evidence of the role of pterocarppan phytoalexins in plant defense mechanisms [77]. One of the most highly characterized systems for the delineation of the roles of secondary metabolites in plant disease resistance is the model pathosystem soybean (_Glycine max_)-_Phytophthora sojae_. An RNAi silencing approach was used to illustrate the role played by 5-deoxyisoflavonoid phytoalexins in the resistance and hypersensitive cell death of soybean to _P. sojae_[86]. Silencing either isoflavone synthase (IFS) or chalcone reductase (CHR) led to suppression (90%) of 5-deoxyisoflavones (daidzein and its derivatives) and glyceollin as well as disease resistance to _P. sojae_ upon treatment with a cell wall glucan elicitor (WGE) from this pathogen. Loss of resistance was accompanied by suppression of HR cell death. Thus, 5-deoxyisoflavonoid-type phytoalgebras from soybean were shown to play a critical role in the establishment of HR and race-specific resistance. Interestingly a third gene silencing RNAi approach targeting the gene _PR-2_ which encodes the elicitor-releasing endoglucanase, led to loss of HR, loss of disease resistance and most importantly to suppression of the phytoalexin response upon treatment with WGE. This underlined links existing between a general resistance elicitor (pathogen-associated molecular pattern), HR cell death, race-specific resistance and accumulation of phytoalexin compounds in a given plant--pathogen system. Loss-of-functions approaches were also used for the study of the role of 3-deoxyanthocyanidin phytoalexins in the resistance of sorghum (_Sorghum bicolor_) to anthracose leaf blight caused by the phytopathogenic fungus _Colletotrichum sublineolum_ as well as for the delineation of the biosynthesis pathway of this particular group of phytoalexins [87]. In the sorghum, it was shown that the corresponding _yellow seed1_ gene (_y1_) encoded a R2R3 type of MYB domain protein which regulates the coordinate expression of _chalcone synthase (chs1)_, _chalcone isomerase_, _dihydroflavonol reductase_ and _flavonoid 3'-hydroxylane_[88, 89] genes required for the biosynthesis of 3-deoxyflavonoids including 3-deoxyanthocyanidin phytoalexins [87, 90, 91]. Interestingly, loss-of-function alleles of _y1_ were found to be deficient in the synthesis of 3-deoxyanthocyanidins, these plants exhibiting severe symptoms characteristic of the anthracose disease [87]. This directly supported the involvement of a functional _y1_ gene in the increased resistance of sorghum to _C. sublineolum_[87]. The upper stream of the biosynthesis pathway leading to 3-deoxyflavonoids, especially formation of luteoferol from apiforol, that is, the precursor of the phytoalexin luteoninidin, was also shown to be under the control of the _red aleurone 1_ (_pr1_) gene which encodes a CYP450-dependent flavonoid 3'-hydroxylane in maize, by using a loss-of-function approach [89].