, 2008; Fig. 2a). After initial screening by PCR, single spore–derived transformants were further confirmed by Southern analysis. The result showed that sahh had been deleted (Fig. 2c).
When cultured on PDA, all Δsahh isolates showed a phenotype of slower growth rate, fewer aerial hyphae, loss of orange pigmentation, lack of asexual fruiting bodies (pycnidia), and suppressed sporulation (Fig. 2b). These abnormal traits of the Δsahh strain could be Epigenetics inhibitor fully restored to the wild-type level by re-introducing a copy of the wild-type sahh gene into the knockout mutant (Fig. 2b), demonstrating that sahh is solely responsible for these traits. As shown in Fig. 3, the wild-type strain EP155 and parental strain CP80 were highly virulent and aggressively produced cankers on dormant chestnut stems (25.65 ± 0.27 cm2; 24.34 ± 0.96 cm2), whereas the hypovirus-infected strain EP713 produced much
smaller cankers (1.09 ± 0.11 cm2). Deletion of sahh resulted in a remarkable reduction in virulence (0.81 ± 0.0 cm2), and the virulence level of the Δsahh strain could be restored to the wild-type level by re-introducing a copy of the wild-type sahh gene into the mutant (24.96 ± 1.08 cm2). Quantification of transcripts revealed that genes cpga1, cpgb1, cpgc1, and ste12 that encode Gα, Gβ, Gγ and Ste12, respectively, of the heterotrimeric G-protein signaling pathway were downregulated in Δsahh by 3.4-, 2.7-, 5.7-, and 1.7-fold, respectively. The accumulation of transcript of the virulence gene cyp1 was downregulated by more than fivefold in Δsahh compared Ponatinib mw with the parental strain CP80 (Fig. 4a). Adenosine kinase, Bacterial neuraminidase MAT, and OMT are important players in the methylation pathway.
Compared with the parental strain CP80, mRNA levels of ak, mat, and omt that encode the above enzymes respectively were upregulated in Δsahh, by 2.8-, 7.7-, and 32.9-fold (Fig. 4b). As SAHH catalyzes SAH to produce ADO and HCY, we reasoned that elevated accumulation of SAH and reduced ADO level could be expected in the Δsahh strain. Indeed, SAH concentration in Δsahh was remarkably higher than that in its parental strain CP80 (3.93 nmol g−1 vs. 0.41 nmol g−1, P < 0.01), and ADO concentration in Δsahh was significantly lower than that in the strain CP80 (0.25 nmol g−1 vs. 0.52 nmol g−1, P < 0.05). Furthermore, SAM, the precursor of SAH carrying a methyl group, was almost twice as much in the Δsahh strain as in the strain CP80 in concentration (2.06 nmol g−1 vs. 1.06 nmol g−1, P < 0.01). Deletion of SAHH significantly alters the intracellular SAH/SAM ration in the Δsahh strain (0.38 in CP80 vs. 1.90 in Δsahh, P < 0.01; Fig. 5). SAHH from a wide range of eukaryotes is conserved in amino acid sequence with nine highly conserved motifs (Fig. S1).