Functional Analysis Of Genes Involved In Methionine Metabolism In Fusarium Graminearum

Fusarium graminearum Schw. is the primary causal agent of Fusarium head blight (FHB) on wheat and barley. Infection of cereal crops with F. graminearum may lead to huge yield loss in severe epidemic years. More importantly, the trichothecene mycotoxins in infected grains pose a serious threat to human and animal health. Currently, emerging fungicide resistance of F. graminearum makes it urgent to explore new compounds for FHB management. Methionine metabolism has important roles in fungal growth, pathogenicity and et al. Moreover, methionine biosynthesis pathway is absent in non-ruminant animals, research on key elements of methionine metabolism in F. graminearum shall contribute to novel fungicide development.In this study, target gene disruption and complementary strategy were employed to investigate functions of four key methionine biosynthesis elements (cystathionine y-synthase FgCGS, serine acetyltransferase FgSAT, homoserine O-acetyltransferase FgHAT and cystathionine β-lyase FgCBL) and three key elements (methionine sulfoxide reductases, FgmsrA, FgmsrB and FgfMsr) involved in methionine oxidation and reduction in F. graminearum. Results of our study demonstrated that:1) Mycelial growth was tested in different conditions. On PDA, compared with wild-type parent, FgCGS, FgSAT, FgHAT and FgCBL deletion mutants showed yellowish or fewer aerial mycelia. On FGA, FgCGS, FgHAT and FgCBL deletion mutants could not grow without supplementation of ether methionine (1mM) or homocysteine (0.5mg/ml). FgSAT deletion mutant showed fewer aerial mycelia, though the radial growth was not distinct from that of wild-type parent. Conidiation assays indicated that FgCGS, FgSAT and FgCBL deletion mutants produced as many conidia as wild-type parent, while conidiation in FgHAT deletion mutant was determined by the nutrient of media. However, compared with wild-type parent, conidial germination rate of FgCGS deletion mutant in2%sucrose solution reduced significantly, but was rescued by supplementation of methionine (1mM) or homocysteine (0.5mg/ml). Conidial germination ability of FgSAT deletion mutant reduced a little. Inoculation tests showed that FgCGS, FgHAT and FgCBL deletion mutants exhibited decreased virulence significantly on wheat heads, while the virulence of FgSAT deletion mutant was comparable to wild-type parent. FgCGS, FgSAT, FgHAT and FgCBL deletion mutants showed fewer aerial mycelia in wheat ear culture, and expression of Tri5gene was decreased significantly, compared with wild-type parent. Deoxynivalenol (DON) production of FgCGS deletion mutant was4-fold less than wild-type parent. Fungicide evaluation assays revealed that sensitivity of FgCGS, FgSAT, FgHAT and FgCBL deletion mutants to anilinopyrimidine fungicide pyrimethanil was not altered, indicating that FgCGS, FgSAT, FgHAT and FgCBL are not likely to be the target of pyrimethanil in F. graminearum. FgCGS and FgHAT deletion mutants showed increased sensitivity to sterol demethylation inhibitors (DMI), which was consistent with a low level of CYP51A/B/C expression in FgCGS deletion mutant. Sensitivity to osmotic, oxidative and cell wall (membrane) stresses revealed that FgCGS, FgSAT, FgHAT and FgCBL deletion mutants did not show observable changes in mycelia growth compared to wild-type parent, which indicate FgCGS, FgSAT, FgHAT and FgCBL are not involved in these stress responses. These results indicate that FgCGS, FgSAT, FgHAT and FgCBL play important roles in the growth, conidiation and pathogenicity of F. graminearum.2) Mycelial growth tests showed FgMsrA, FgMsrB and FgfMsr deletion mutants had normal growth on PDA and FGA, and produced as many conidia in MBL medium as wild-type parent. Inoculation tests showed that the virulence of FgMsrA、FgMsrB and FgfMsr deletion mutants was comparable to wild-type parent. Fungicide sensitivity assays revealed that FgMsrA、FgMsrB and FgfMsr deletion mutants did not change the sensitivity to pyrimethanil, DMIs and carbendazim, indicating that FgMsrA、FgMsrB and FgfMsr may not be the target of pyrimethanil in F. graminearum. Oxidative stress sensitivity tests revealed that FgMsr A、FgMsr B and FgfMsr deletion mutants did not show observable changes in mycelia growth compared to wild-type parent. However, relative expression of FgMsr A、FgMsrB and FgfMs was increased significantly under oxidative stress. These results suggest that FgMsrA、FgMsrB and FgfMsr may be involed in oxidative stress reaction process of F. graminearum, though their roles in the growth, conidiation and pathogenicity are not obvious.