## 5 Phytoalexin Accumulation in the Upregulation of Defense-Related Marker and Elicitor Genes  
In the new concept of innate immunity for plants, defense responses are triggered after recognition by the plant cell of conserved Pathogen/Microbe-Associated Molecular Patterns (PAMPs/MAMPs) or by endogenous molecules released by pathogen ingress and called Danger-Associated Molecular Patterns (DAMPs) [134, 135, 136]. Early responses after elicitor perception are characterized by signaling processes including ion fluxes, Mitogen-Activated Protein Kinase (MAPK) cascade [137] and production of reactive oxygen species [138]. In this context, MAPKs of _Arabidopsis_, respectively, MPK3 and MPK6, were shown to be activated upon MAMP treatment or following infection by a pathogen with correlated induction of the responses further downstream such as phytoalexin accumulation by using both gain-of-function and loss-of-function transgenic systems. [13]. MPK3 and MPK6 take part in a pathogen-responsive MAPK cascade, MAPKKKa/MEKK1-MKK4/MKK5-MPK3/MPK6, involved in the up-regulation of numerous enzymes of the biosynthetic route from chorismate to camalexin via tryptophan (Trp) [139,140] (Figure 5). Constitutively active mutants of the upstream MAPKKs, that is, tobacco NtMEK2 DD and _Arabidopsis_ MKK4 DD or MKK5 DD, were overexpressed under the control of a steroid-inducible promoter. Upon treatment with the steroid dexamethasone (DEX), induction of the active MAPKs, MPK3/MPK6 activated the endogenous MAPKs, which in turn induced the responses further  
Figure 5: Biosynthetic pathways from chorismate to tryptophan and from tryptophan to camalexin (adapted from [139,140]). downstream such as accumulation of the phytoalexin camalexin. Camalexin production was associated with the induction of numerous genes encoding enzymes acting on the biosynthesis of this phytoalexin, including the route from chorismate to Trp, on one hand, and the pathway from Trp to camalexin (Figure 5). Specifically, genes encoding anthranilate synthase a and b (ASA and ASB), phosphoribosylanthranilate transferase (PAT), indole-3-glycerolphosphate synthase (IGPS) and Trp synthase b (TSB), were all highly induced with levels of induction ranging from 12-fold (PAT) to more than 35-fold (TSB) 6h after DEX treatment. Genes encoding the P450 enzymes operating in the biosynthetic pathway from Trp to camalexin, namely the one catalyzing conversion of Trp to indole-3-acetaldoxime (_CYP79B2_ gene) showed a 50-fold increase. Expression of P450 enzyme genes acting at the end of the pathway, for instance the _CYP71B15_ (_PHYTOALEXIN DEFLICIENT 3, PAD3_) gene encoding the multifunctional enzyme converting cysteine-3-indolacetonitrile to camalexin, increased by 400-fold (Figure 5). Mutations in MPK3 and MPK36 impaired _B. cinerea_-induced camalexin production and disease resistance, providing loss-of-function evidence supporting the role of this MAPK cascade in phytoalexin biosynthesis upon pathogen infection. Camalexin accumulation was reduced by 50% in _mpk3_ mutant, delayed in _mpk6_ mutant and completely abolished in _mpk3/mpk6_ double mutant, resulting in increased susceptibility to _B. cinerea_. All together, these experiments clearly showed that MPK3 and MPK6, two MAP kinases of _Arabidopsis_ play key roles in camalexin biosynthesis and resistance to fungal pathogens. Induction of camalexin after MAPK activation was considered to be independent of ethylene [13]. Phytoalexin accumulation was reported in the upregulation of defense-related marker genes in rice plants (_Oryza sativa_) exhibiting spontaneous hypersensitive response upon overexpression of the _Oryza sativa_ Accelerated Cell Death and Resistance 1 (_OsACDR1_) gene which encodes a putative Raf-like Mitogen-Activated Protein Kinase Kinase Kinase (MAPKKK) [141].