Approccio CRISPR-Cas9 per chiarire il ruolo della proteina FHT nel meccanismo di resistenza contro Erwinia amylovora in Melo

Fire blight, caused by the bacteria Erwinia amylovora, is one of the most important apple diseases worldwide. The application of the shoot-growth regulator prohexadione-calcium (Pro-Ca) on apple plants reduces the incidence and the severity of infection by this pathogen. It causes the transient inhibition of flavanone 3-hydroxylase (FHT) activity. Apple dihydroflavonol 4-reductase (DFR), when FHT is inhibited, leads to the production of uncommon 3-deoxyflavonoids thanks to its flavanone-4-reductase (FNR) side activity. The compounds so formed, may be responsible for the reduced susceptibility to E. amylovora. Since silencing of FHT gene by RNAi did not induce the formation of 3-deoxyflavonoids, the protein may be structurally essential for their production, having a role in the formation of a macromolecular complex (metabolon) suggested to determine metabolic channelling in the flavonoid pathway. In this thesis, CRISPR-Cas9 genome editing system was applied to apple and Arabidopsis plants to mimic the inhibition effect of Pro-Ca, targeting a gene region previously shown to be crucial for the activity of FHT enzyme. Some in-vitro apple lines regenerated after transformation with CRISRP-Cas9 construct are successfully mutated in the targeted region of FHT gene. Preliminary metabolic analyses performed on these in-vitro lines gave unclear results: a shift of the flavonoids spectrum, as previously exhibit by apple leaves treated with Pro-Ca, was not clearly detectable. Accordingly, flavonoids detected in leaves of in-vitro plants were difficult to compare to the ones present in leaves extracts of apple tree sprayed with Pro-Ca in the field. Arabidopsis lines transformed with CRISPR-Cas9 construct targeting FHT were selected until T3 generation and many of them seems to be homozygotes for the insertion of the T-DNA. Apiferol and luteoferol, two of the 3-deoxyflavonoids expected to be produced due to re-channelling of the metabolic pathway after FHT inhibition thanks to FNR activity of apple DFR, were chemically synthetized and were tested to assess in-vitro inhibition activity against E. amylovora together with related flavonoids. Results indicated an antibacterial activity for luteoferol as reported in previous studies. In addition, classical engineering of Arabidopsis was performed to elucidate FHT, DFR and FNS role in 3-deoxyflavonoid biosynthesis.