## 1 The Concept of Phytoalexins
Such apparently different regiospecicities of IOMT expressed _in vivo_ or _in vitro_ will not be discussed further but underlined potential metabolic channeling at the entry point into the isoflavoid-type phytoalexin pathway (Figure 4). In antisense IOMT transgenic lines, root tissues from those plants exhibited markedly reduced phytoalexin concentrations as a consequence of a potential downregulation of the isoflavenoid content. Ectopically overexpression of IOMT in unchallenged transgenic leaves produced 7-\(O\)-methylisoflavonoids but no 4'-\(O\)-methylisoflavonoids (see above). Conversely, abiotic elicitation using spores of _P. medicaginis_ led to the accumulation of the phytoalexins formononentin glucoside and medicarpin both in empty-vector control plants and IOMT over-expressing plants 2 days post infection (dpi), being the content of medicarpin 2.5 fold higher in IOMT overexpressing lines (67 \(\upmu\)g/g fresh WT _vs._ 30 \(\upmu\)g/g fresh WT). Interestingly, high medicarpin accumulation rates in IOMT-overexpressing plants were associated with a marked reduction in the size of the brown _P. medicaginis_ lesions on leaves measured 5 dpi (around 60% decrease as compared to empty-vector control plants), being this reduction of the disease symptoms well correlated with postinoculation increases in medicarpin concentrations, confirming the role of this compound in disease resistance in alfalfa. In contrast, authors failed to demonstrate any increased susceptibility in antisense IOMT lines with decreased amounts of medicarpin, as this may be the result of an incomplete inhibition of accumulation of the phytoalexin in those lines. Such a situation was not observed in _Arabidopsis_ mutants deficient in camalexin where susceptibility to the necrotrophic fungus _Alternaria brasicicicola_ is highly correlated with the absence of this phytoplankton [5]. These different behaviors underline limitations that can be encountered by using loss-of-function genetic approaches for the understanding of plant/pathogen interactions. In a similar approach, biosynthesis of the simple isoflavone genistein glucoside was engineered in leaves of alfalfa by constitutive expression of isoflavone synthase from _Medicago truncatula_[76] (Figure 4). The Mt_IFS1_ cDNA from _M. truncatula_ was subcloned into the pRTL2 vector and the expression cassette containing Mt_IFS1_ flanked by CaMV 35S promoter and terminator sequences was cloned into the _Hind_III site of the binary vector pCAMBIA 2300. Mt_IFS1_ was highly expressed in all organs, while genistein accumulation was limited to leaves. Other simple isoflavone-type phytoalexins were also found such as daidzein, biochanin A (4'-\(O\)-methylgenistein) and 3'-hydroxybiochanin A (pratensein) in transgenic lines with constitutive expression of Mt_IFS1_. Additionally, together with daidzein, other 5-deoxyflavone phytoalexins accumulated, for instance formonotenin, in transgenic lines irradiated with UV-B for 6h or as a result of plant infection with _P. medicaginis_. The pterocarppan phytoalexin, medicarpin, was also found to be accumulated, likely in relation to its antifungal activity [84]. MtIFS1-expressing plants contained much higher concentrations of formononetin and medicarpin than the vector control lines, being the levels of formononetin between 46 and 77 ug/g fresh WT in leaves 24 h to 72 h after infection, that is, concentrations able to inhibit the pathogen development. Loss-of-function approaches were applied to study the role of the phytoalexin pisatin in the disease resistance of hairy roots of pea (_Pisum sativum_ L.) to the fungal pathogen _Nectria haematococcca_ through the genetic manipulation of enzymes involved in both the biosynthesis and the catabolism of this compound [85] (Figure 4).