Figure 1.
Phylogenetic analyses of CNA and CNB genes.
A: AfCNA (Aspergillus fumigatus, XP_753703), BfCNA (Botryotinia fuckeliana, XP_001558972), CcCNA (Coprinopsis cinerea, XP_001838986), CnCNA (Cryptococcus neoformans var. grubii, AAB97372), DmCNA (Drosophila melanogaster, NP_727985), HsCNA (Homo sapiens, NP_000936), LbCNA (Laccaria bicolor, XP_001884713), NcCNA (Neurospora crassa, XP_961193), NfCNA (Neosartorya fischeri, XP_001259754), PgCNA (Puccinia graminis f. sp. tritici, EFP89050), PpCNA (Postia placenta, XP_002470453), PsCNA (Puccinia striiformis f. sp. tritici, JX424819), PtCNA (Puccinia triticina, PTTG_07903), RnCNA (Rattus norvegicus, BAA14083), ScCNA1 (Saccharomyces cerevisiae, SCRG_04371), ScCNA2 (Saccharomyces cerevisiae, SCRG_01842), SsCNA (Sclerotinia sclerotiorum, XP_001597594), UmCNA (Ustilago maydis, AAP48999). B: AfCNB (Aspergillus flavus, XP_002378292), BfCNB (Botryotinia fuckeliana, XP_001555369), CiCNB (Coccidioides immitis, XP_001248933), CnCNB (Cryptococcus neoformans var. neoformans, XP_57033), GzCNB (Gibberella zeae, XP_387580), HsCNB (Homo sapiens, NP_000936), LbCNB (Laccaria bicolor, XP_001884421), MoCNB (Magnaporthe oryzae, ADD84607), NcCNB (Neurospora crassa, CAA73345), PbCNB (Paracoccidioides brasiliensis, XP_002795006), PgCNB, (Puccinia graminis tritici, EFP78352), PsCNB (Puccinia striiformis f. sp. tritici, JX424820), PtCNB (Puccinia triticina, PTTG_02210), RsCNB (Rattus sp., BAA03318), ScCNB (Saccharomyces cerevisiae, SCRG_03838). The unrooted phylograms were 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.
Figure 2.
Immuno-suppressive drugs assay of Pst urediospores.
Pst race CYR31 was treated with water (A, D), CsA (0.1 µM, B, E), or FK506 (1 µM, C, F). Treatment with either drug can limit the elongation of Pst germ tubes and block further differentiation. Analyses were performed using the light microscope; scale bar, 50 µm.
Table 1.
Germination rates of Pst (mean±SE).
Figure 3.
Transcript levels of PsCNA1 and PsCNB1 during Pst differentiation stages.
RNA samples were isolated from leaves of wheat cultivar Suwon 11 inoculated with Pst race CYR31 at the indicated time points. Expression levels of PsCNA1/PsCNB1 were estimated by the ΔΔCt method with the elongation factor gene of Pst as endogenous reference for normalization. S: urediospore, GS: germinated urediospore, dpi: days post inoculation.
Figure 4.
Transcript levels of PsCNA1 and PsCNB1 after HIGS during Pst differentiation stages.
RNA samples were isolated from leaves infected with BSMV of wheat cultivar Suwon 11 inoculated with Pst race CYR31. Transcript levels of PsCNA1 and PsCNB1 were estimated by the comparative ΔΔCt method with elongation factor gene of Pst as the endogenous reference for normalization. dpi: days post inoculation.
Figure 5.
Histological observation of Pst growth using calcofluor staining.
All wheat leaves were inoculated with Pst strain CYR31; fluorescence-microscopic analyses were done 2 days post inoculation. A: BSMV:γ:0-as (empty viral vector control); B: BSMV:γ:PsCNA1-as (silencing for PsCNA1); C: BSMV:γ:PsCNB1-as (silencing for PsCNB1); IH: infection hyphae; HM: haustorial mother cell; SV: substomatal vesicle. Scale bars: 50 µm.
Table 2.
Histological observation during HIGS (mean±SE).
Figure 6.
Uredia on silenced leaves 16 days after Pst inoculation.
Pst development on wheat leaves after HIGS. A: No virus and Pst (healthy leaf control); B: No virus (normal infection with Pst); C: BSMV:γ:0-as (empty viral vector control); D: BSMV:γ:PsCNA1-as (Pst infection after silencing PsCNA1); E: BSMV:γ:PsCNB1-as (Pst infection after silencing PsCNB1).
Figure 7.
SEM photograph of uredia after HIGS at 16 dpi.
Sorus of silenced PsCNA1/PsCNB1 of Pst infected leaves inoculated with CYR31. Scanning electron micrographs by 600×, scale bar: 20 µm. A: BSMV:γ:0-as (empty viral vector control); B: BSMV:γ:PsCNA1-as (silencing PsCNA1); C: BSMV:γ:PsCNB1-as (silencing PsCNB1).
Figure 8.
SEM photograph of germinated Pst urediospores after HIGS at 16 dpi.
New urediospores of silenced PsCNA1/PsCNB1 of Pst CYR31 in wheat leaves after 6 hour mist moisture. SEM micrographs for A: BSMV:γ:0-as (empty viral vector control), 1500×; B: BSMV:γ:PsCNA1-as (silencing PsCNA1), 2000×; C: BSMV:γ:PsCNB1-as (silencing PsCNB1), 2000×. Scale bars: 10 µm.
Table 3.
Primers used in this study.