Introduction of Plant and Fungal Genes into Pea (Pisum sativum L.) Hairy Roots Reduces Their Ability to Produce Pisatin and Affects Their Response to a Fungal Pathogen

Abstract

Pisatin is an isoflavonoid phytoalexin synthesized by pea (Pisum sativum L.). Previous studies have identified two enzymes apparently involved in the synthesis of this phytoalexin, isoflavone reductase (IFR), which catalyzes an intermediate step in pisatin biosynthesis, and (+)6a-hydroxymaackiain 3-O-methyltransferase (HMM), an enzyme catalyzing the terminal step. To further evaluate the involvement of these enzymes in pisatin biosynthesis, sense- and antisense-oriented cDNAs of Ifr and Hmm fused to the 35s CaMV promoter, and Agrobacterium rhizogenes, were used to produce transgenic pea hairy root cultures. PDA, a gene encoding pisatin demethylating activity (pda) in the pea-pathogenic fungus Nectria haematococca, also was used in an attempt to reduce pisatin levels. Although hairy root tissue with either sense or antisense Ifr cDNA produced less pisatin, the greatest reduction occurred with sense or antisense Hmm cDNA. The reduced pisatin production in these lines was associated with reduced amounts of Hmm transcripts, HMM protein, and HMM enzyme activity. Hairy roots containing the PDA gene also produced less pisatin. To evaluate the role of pisatin in disease resistance, the virulence of N. haematococca on the transgenic roots that produced the lowest levels of pisatin was tested. Hairy roots expressing antisense Hmm were more susceptible than the control hairy roots to isolates of N. haematococca that are either virulent or nonvirulent on wild-type pea plants. This appears to be the first case of producing transgenic plant tissue with a reduced ability to produce a phytoalexin and demonstrating that such tissue is less resistant to fungal infection: these results support the hypothesis that phytoalexin production is a disease resistance mechanism.

Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of Plant Defense Responses

“…Indeed, silencing of the Ps HM3OMT gene by antisense or sense constructs in pea hairy root cultures not only greatly reduced the accumulation of pisatin but also resulted in undetectable amounts of 6a-hydroxymaackianin. The lack of 6a-hydroxymaackianin accumulation was most likely due to silencing of the earlier 49-O-methylation biosynthetic step possibly exploiting the dual activity of HI49OMT/HM3OMT described here (Wu and VanEtten, 2004). Further genetic, biochemical, and structure-function studies of Ps HM3OMT will be necessary to conclusively answer this question.…”

Stimulateurs des défenses naturelles des plantes : une nouvelle stratégie phytosanitaire dans un contexte d’écoproduction durable.

“…(Hipskind et Paiva 2000), respectivement. À l'inverse, des plants de pois (Pisum sativum L.) transgéniques ayant perdu leur capacité à produire la pisatine sont beaucoup moins résistants à l'infection fongique (Wu et VanEtten 2004). En utilisant la microscopie à fluorescence et la microscopie confocale, McNally et al (2003) ont montré que les phytoalexines nouvellement synthétisées s'accumulaient bien au niveau des hyphes fongiques in planta.…”

Gene Expression Modulation of Two Biosynthesis Pathways via Signal Transduction in <i>Cochliobolus heterostrophus</i>

“…This enzyme catalyzes the NADPH-dependent reduction of 2'-hydroxyformononetin to vestitone, which is the penultimate step in the synthesis of medicarpin [19]. IfR protein has been purified and characterized from several legumes, and IFR cDNA clones have been obtained from alfalfa [20], chickpea [21] and pea [16]. In unstressed alfalfa plants, transcripts from the single alfalfa IFR gene are detected mainly in roots and root nodules, consistent with the accumulation of a medicarpin conjugate, medicarpin-3-O-glucoside-6'-Omalonate, only in these organs [20].…”