Commonly, when it is desirable to replace an essential gene with an allelic series of mutated genes, or genes with altered expression patterns, the complementing constructs are introduced into heterozygous plants, followed by the selection of homozygous null segregants. To overcome this laborious and time-consuming step, the newly developed two-component system utilizes a site-specific recombinase to excise a wild-type copy of the gene of interest from transformed tissues. In the first component (the first vector), a wild-type version of the gene is placed between target sequences recognized by FLP recombinase from the yeast 2 μm plasmid. This construct is transformed into a plant heterozygous for a null mutation at the endogenous locus, and progeny plants carrying the excisable complementing gene and segregating homozygous knockout at the endogenous locus are selected. The second component (the second vector) carries the experimental gene along with the FLP gene. When this construct is introduced, FLP recombinase excises the complementing gene, leaving the experimental gene as the only functional copy. The FLP gene is driven by an egg apparatus specific enhancer (EASE) to ensure excision of the complementing cDNA in the egg cell and zygote following floral-dip transformation. The utility of this system is being tested using various experimental derivatives of the essential sucrose-proton symporter, AtSUC2, which is required for photoassimilate transport.

Le but de notre travail est de développer une stratégie de vectorisation pour conférer la mobilité phloémienne aux produits agrochimiques en appliquant le concept de prodrogue dans l'élaboration de composés phytopharmaceutiques. Le fenpiclonil, fongicide non systémique de la famille des phénylpyrroles, a été choisi comme composé parent modèle et modifié en l'associant à un acide alpha-aminé ou à un monosaccharide.La mobilité phloémienne des conjugués entre le fenpiclonil et les acides L ou D-glutamique ou le D-glucose (D-GFC) a été évaluée chez des plantules de ricin. Le test de systémie a montré que le L-aminoacide était nettement plus favorable à la mobilité phloémienne que l'acide D-aminé ou le D-glucose. Les résultats suggèrent que le conjugué fenpiclonil/L-aminoacide est reconnu et manipulé par un système de transport d'acide aminé stéréospécifique énergisé par la force proton-motrice. Des expériences complémentaires ont montré que le D-GFC était un inhibiteur puissant et sélectif de l'absorption du saccharose par les tissus foliaires et du chargement phloémien du saccharose. En raison de l'inhibition spécifique des transporteurs de saccharose (par exemple AtSUC2), le D-GFC peut être envisagé comme un nouvel outil en phloémologie.Enfin, nous avons exploré l'impact de la modification de structure de l'espaceur afin d'optimiser la stratégie de prodrogue. Après avoir introduit différentes structures d'espaceur entre le fenpiclonil et la fonction acide aminé, le conjugué qui contient un cycle triazole avec l'acide aminé comprenant la chaine la plus courte a montré la meilleure mobilité phloémienne et, par ailleurs, une systémie optimisée par rapport aux dérivés acides du fenpiclonil dans la gamme des valeurs de pH de l'apoplasme foliaire. / The purpose of our work is to develop a vectorization strategy to confer phloem mobility to agrochemicals by applying prodrug concept into agrochemical design. Fenpiclonil, a non-systemic fungicide from the phenylpyrrole family, was selected as a model parent compound and modified by associating it with an amino acid or a monosaccharide.The phloem mobility of L and D-glutamic acid and D-glucose fenpiclonil conjugates (D-GFC) was evaluated in Ricinus seedlings. The systemicity test showed that the L-amino acid promoiety was clearly more favorable to phloem mobility than D-amino acid or D-glucose. The results suggested that the transport of the L-amino acid conjugate is governed by a stereospecific amino acid carrier system energized by the proton motive force. Further investigation indicated that D-GFC was a potent and selective inhibitor of sucrose uptake by leaf tissues and sucrose phloem loading. Due to its specific inhibition of sucrose transporters (e.g. AtSUC2), D-GFC can be a candidate as a new tool in phloemology. Finally, we explored structural modifications of the spacer arm to optimize the prodrug strategy. Different structures of the spacer arm were introduced between fenpiclonil and the amino acid function. The conjugate which contains a triazole ring with the shortest amino acid chain showed the best phloem mobility and a better systemicity than fenpiclonil acidic derivatives within the pH range of the foliar apoplast.