Figure 1.
Phylogenetic analysis with PsMAPK1 and selected fungal MAP kinases (GenBank accession numbers in parenthesis).
Botrytis cinerea, BcBmp1 (AAG23132); Candida albicans, CaERK1 (P28869); Cryptococcus neoformans, CnCpk1 (Q8NK05); Fusarium solani, FsMAPK (AAB72017); Fusarium graminearum, FgMap1 (AAL73403) and FgMgv1 (AAM13670); Magnaporthe oryzae, MgPmk1 (AAC49521), MgMps1 (AAC63682) and MgOsm1 (AAF09475); Neurospora crassa, NcMAK2 (AAK25816); Puccinia striiformis f. sp. tritici, PsMAPK1 (HM535614); Puccinia triticina, PtMAPK1 (AAY89655); Puccinia graminis f. sp. tritici, PgMAPK (EFP88010); Saccharomyces cerevisiae, ScFus3 (CAA49292), ScHog1 (CAA97680), ScKss1 (CAA97038) and ScSlt2 (CAA41954); Sclerotinia sclerotiorum, SsSmk1 (AAQ54908); Ustilago maydis, UmKpp6 (CAD43731) and UmUbc3/Kpp2 (AAF09452). The unrooted phylogram was constructed based on NJ analysis. Confidence of groupings was estimated by using 1,000 bootstrap replicates. Numbers next to the branching point indicate the percentage of replicates supporting each branch.
Table 1.
Primers and strains used in the study.
Figure 2.
Assays for the transcript levels of PsMAPK1 during different infection stages.
RNA samples were isolated from urediniospores or leaves of wheat cultivar Suwon 11 inoculated with CYR32 and collected at the indicated time points. The expression level of PsMAPK1 was estimated by the comparative ΔΔCt method with the elongation factor gene of Pst as the endogenous reference for normalization. Relative quantification was computed with their expression levels in different stages in comparison to that in urediniospores. Means and standard errors were calculated from three biological replicates. US, urediniospores; hpi, hours post inoculation.
Figure 3.
Colony morphology of Fusarium graminearum strains.
Colonies of the wild-type (PH-1), map1 deletion mutant, and complemented strain (CF-6) grown on PDA plates for 5 days.
Figure 4.
Infection assays with flowering wheat heads.
(A) Wheat heads were drop-inoculated with sterile water or conidia from the wild-type strain PH-1, map1 mutant, and complemented map1/PsMAPK1 transformant CF-6. The inoculation sites are marked with arrows. Typical heads were photographed 14 days after inoculation. (B) Disease index scores of PH-1, map1, and CF-6. Mean and standard error were calculated from three independent infection assays.
Figure 5.
Complementation of the pmk1 mutant with the GFP-PsMAPK1 fusion construct.
(A) Appressorium formation assay. Germ tubes from the wild-type strain (Guy11) developed appressoria by 18 h, but no appressorium formation was observed in the pmk1 mutant (nn78). Under the same conditions, a transformant of nn78 expressing the GFP-PsMAPK1 fusion construct (CM-10) formed appressoria. Bar = 25 µm. (B) Barley infection assay. Left to right, barley leaves were sprayed with sterile and conidia of Guy11, nn78, or CM-10. Typical leaves were photographed at 6 days post inoculation.