Table 1.
Fungal strains used in this study.
Figure 1.
Appressorium formation in coira1 mutants of C. orbiculare on glass slides.
(A) Conidial suspensions of each strain in distilled water were incubated on multiwell glass slides at 24°C for 24 h. WT, the wild-type 104-T; DL1-1 and DL1-2, the coira1 mutants; CL1-1, the CoIRA1-complemented transformant of DL1-1; CL1-2, the CoIRA1-complemented transformant of DL1-2; EL1-1, ectopic strain. Scale bar, 10 µm. (B) Percentages of conidial germination, appressorium formation, and abnormal appressorium formation in the C. orbiculare WT and coira1 mutants on multiwell glass slides. Approximately 100 conidia of each strain were observed per well on multiwell slide glass. Three replicates were examined. Three independent experiments were conducted, and standard errors are shown. Black bar, conidial germination; gray bar, appressorium formation; white bar, abnormal appressorium formation.
Figure 2.
Penetration hyphae formation of the coira1 mutants of C. orbiculare on cellulose membranes.
Conidial suspensions of each strain in distilled water were incubated on cellulose membranes at 24°C for 48 h. WT, the wild-type 104-T; DL1-1 and DL1-2, the coira1 mutant; CL1-1, the CoIRA1-complemented transformant of DL1-1; CL1-2, the CoIRA1-complemented transformant of DL1-2; EL1-1, the ectopic strain. Scale bar, 10 µm. (B) Percentages of conidial germination, appressorium formation, penetration hyphae formation, and bulb-shaped penetration-hyphae formation of C. orbiculare WT and coira1 mutants on cellulose membranes. Approximately 200 conidia of each strain were observed on cellulose membranes. Three replicates were examined. Three independent experiments were conducted, and standard errors are shown. Black bar, conidial germination; gray bar, appressorium formation; slash bar, penetration hyphae; white bar, bulb-shape penetration formation.
Figure 3.
Pathogenicity assay and penetration ability of coira1 mutants of C. orbiculare on the cucumber cotyledons.
(A) Conidial suspensions of each strain (10 µl) placed on detached cucumber cotyledons and leaves incubated at 24°C for three days. The figure shows the leaves after incubation with the following strains: WT, the wild-type 104-T; DL1-1 and DL1-2, the coira1 mutant; CL1-1, the CoIRA1-complemented transformant of DL1-1; CL1-2, the CoIRA1-complemented transformant of DL1-2; EL1-1, the ectopic strain. (B) Penetration hyphae development of each strain on the cucumber cotyledons. Conidial suspensions (10 µl) were applied to the abaxial surface of the cucumber cotyledons and incubated at 24°C for 72 h. Scale bar, 20 µm. (C) Percentage of penetration hyphae of the coira1 mutants on the abaxial surface of cucumber cotyledons. Approximately 100 appressorium were observed per incubated site. Three replicates were examined. Three independent experiments were conducted, and standard errors are shown. Black bar, penetration hyphae. Scale bar, 20 µm.
Figure 4.
Appressorium formation by coira1 mutants on glass slide in the presence of 10-mM cAMP.
(A) Conidial suspensions of each strain in distilled water or 10-mM cAMP were incubated on the multiwell glass slide at 24°C for 24 h. WT, the wild-type 104-T; DL1-1, the coira1 mutant; CL1-1, the CoIRA1-complemented transformant of DL1-1; DC1, the cac1 mutant; DARS1, WT transformed with a dominant active form CoRAS1; DARS2, WT transformed with a dominant active form CoRAS2; iDNRS1, DL1-1 transformed with a dominant negative form CoRAS1; iDNRS2, DL1-1 transformed with a dominant negative form CoRAS2. Scale bar, 10 µm. (B) Percentages of conidial germination, appressorium formation, and abnormal appressorium formation of C. orbiculare on multiwell glass slides in the presence of 10-mM cAMP. Approximately 100 conidia of each strain were observed on multiwell glass slides. Three replicate experiments were examined. Three independent experiments were conducted, and standard errors are shown. Black bar, conidial germination; gray bar, appressorium formation that includes normal appressorium and abnormal appressorium; white bar, abnormal appressorium formation. (–) distilled water, (+) 10-mM cAMP.
Figure 5.
Intracellular cAMP levels in the coira1 mutant.
Intracellular cAMP levels were measured in tissue collection from three-day old liquid cultures of each strain. WT, the wild-type 104-T; DL1-1, the coira1 mutant; CL1-1, the CoIRA1-complemented transformant of DL1-1; DARS1, WT transformed with a dominant active form CoRAS1; DARS2, WT transformed with a dominant active form CoRAS2; iDNRS1, DI1-1 transformed with a dominant negative form CoRAS1; iDNRS2, DI1-1 transformed with a dominant negative form CoRAS2; DC1, the cac1 mutant. Three independent experiments were conducted, and standard errors are shown.
Figure 6.
Appressorium formation assay of DRS2/DAMK1 and DMK1/DARS2 on the glass slides.
(A) Conidial suspensions of each strain in distilled water incubated on multiwell glass slides at 24°C for 24 h. DRS2, the coras2 mutant; DMK1, the comekk1 mutant; DRS2/DAMK1, DRS2 transformed with a dominant active form CoMEKK1; DMK1/DARS2, DMK1 transformed with a dominant active form CoRAS2. Scale bar, 10 µm. (B) Percentages of conidial germination and appressorium formation in C. orbiculare on multiwell glass slides. Approximately 100 conidia of each strain were observed per well on the multiwell slide glass. Three replicates were examined. Three independent experiments were conducted, and standard errors are shown. Black bar, conidial germination; gray bar, appressorium formation.
Figure 7.
The phosphorylation of MAPK Cmk1 in the coira1 mutant.
(A) The total protein isolated from mycelia of each strain. WT; the wild-type, DL1; the coira1 mutant, DRS2; the coras2 mutant, DARS1, WT transformed with a dominant active form CoRAS1, DARS2; WT transformed with a dominant active form CoRAS2, iDNRS1; the coira1 mutant transformed with a dominant negative form CoRAS1, iDNRS2; the coira1 mutant transformed with a dominant negative form CoRAS2, DCK1; the cmk1 mutant The anti-phospho p44/42 MAPK antibody detected a 41-KD Cmk1 and 47-KD Maf1. The anti-actin antibody detected a 42-KD actin. (B) Relative activity of MAPK Cmk1 phosphorylation of each mutant was calculated by comparison of signal intensity with that of the wild-type, normalized by actin signal. The quantitative analysis of phosphorylated Cmk1 was performed by four replicated experiments. Asterisk represents significant differences between the wild type and each mutant. (Student’s t test: *indicate P<0.05).
Figure 8.
Localization of a functional RFP–CoRas2 fusion protein in C. orbiculare during conidial germination and appressorium formation.
Conidial suspensions of the DRS2/RFP–RS2strain in distilled water were incubated in glass slides at 24°C for 0 h, 3 h, 6 h, and 24 h. After incubation, RFP fluorescence was observed by fluorescence microscopy. DRS2/RFP–RS2, the coras2 mutant expressing RFP–CoRAS2. Scale bar, 10 µm.
Figure 9.
CoRas2 localization was regulated by CoIra1 in vegetative hyphae.
Conidia harvested from each strain were observed on glass slides by fluorescent microscopy. coras2/RFP–CoRAS2, the coras2 mutant expressing the RFP–CoRAS2; RFP–CoRas2Q65L, the wild-type strain expressing RFP–CoRAS2Q65L; and coira1/RFP–CoRas2, the coira1 mutant expressing RFP–CoRAS2. Scale bar, 10 µm.
Figure 10.
BiFC assays for CoIra1 and CoRas2 interactions in vegetative hyphae.
Conidial suspensions of Vc-CoRas2/CoIra1-n transformant in liquid PSY medium were incubated at 28°C for 24 h and BiFC fluorescence was observed by fluorescent microscopy in vegetative hyphae. Vc–CoRas2/CoIra1–Vn; the wild-type strain expressing Vc–CoRAS2 and Vn–CoIRA1. Scale bar, 10 µm.
Figure 11.
Assay for colocalization of CoIra1 and CoRas2.
Conidial suspensions of the RFP–RS2/IRA1–VENUS strain were incubated on glass slides at 24°C for 0 h, 3 h, 6 h, and 24 h and observed by fluorescent microscopy. RFP–RS2/IRA1–VENUS, the wild-type strain expressing CoIRA1–VENUS and RFP–CoRAS2. Scale bar, 10 µm.
Figure 12.
Assay for the colocalization of CoIra1 and CoRas2 in appressoria at 48 h after inoculation on cucumber leaves.
Conidial suspensions of RFP–RS2/IRA1-strain were incubated in cucumber leaves at 24°C for 48 h and observed under fluorescent microscopy. RFP–RS2/IRA1–VENUS, the wild-type strain expressing CoIRA1–VENUS and RFP–CoRAS2. Scale bar, 10 µm.
Figure 13.
Assay for colocalization of CoIra1 and F-actin.
Conidial suspensions of the LA/IRA1–VENUS strain were incubated on glass slides at 24°C for 0 h and 24 h and observed under fluorescent microscopy. LA/IRA1–VENUS, the wild-type strain expressing CoIRA1–VENUS and Lifeact–RFP. Scale bar, 10 µm.
Figure 14.
Assembly of F-actin in the appressorium pores of the coira1 mutant.
(A) Micrograph of F-actin organization in the appressorium pore visualized by the expression of Lifeact–RFP in the wild-type and in the coira1 mutant. Conidial suspensions (10 µl) of each strain were applied to the abaxial surface of the cucumber cotyledons and incubated at 24°C for 48 h. WT/RA, the wild type expressing Lifeact–RFP; iRA, the coira1 mutant expressing Lifeact–RFP (B) Percentage of the assembly of F-actin in the appressorium pore of the coira1 mutants on the abaxial surface of cucumber cotyledons. Approximately 100 appressoria of each strain were observed per incubated site for 48 h, 72 h post-inoculation. Two replicates were examined. Three independent experiments were conducted, and standard errors are shown. Black bar, WT/RA; gray bar, Ira; WT/RA, the wild type expressing LifeAct-RFP; iRA, the coira1 mutant expressing LifeAct-RFP.
Figure 15.
The hypothetical model for CoIra1 and CoRas1/2 mediated signaling transduction pathway in C. orbiculare.
The cAMP-PKA signaling pathway is involved in conidial germination, appressorium penetration and invasive hyphae formation. The MAPK CoMekk1–Cmk1 signaling pathway is involved in conidial germination and appressorium formation. The CoRas2 localization pattern in the coira1 mutant was similar to that in DARS2. Moreover, BiFC assays supported that CoIra1 interacted with CoRas2 in the plasma membrane. Therefore, CoIra1 negatively regulates CoRas2. The coira1 mutant and DARS2 significantly induced abnormal appressorium formation and the frequency of abnormal appressorium formation in iDNRS2 was lower compared with that in the coira1 mutant. Moreover, intracellular cAMP levels in the coira1 mutant and DARS2 was high compared with those in the wild type. Therefore, CoIra1 regulates cAMP-PKA signaling pathway through CoRas2. The conidia of the coras2 mutant failed to germinate, whereas DRS2/DAMK1 restored the phenotype of the coras2 mutant. Therefore, CoRas2 is an upstream regulator of the MAPK CoMekk1–Cmk1 signaling pathway. Interestingly, the phosphorylation of MAPK Cmk1 in the coras2 mutant was higher compared with that in the wild-type, although CoRas2 is an upstream regulator of CoMekk1–Cmk1. Therefore, that CoIra1 may be a key factor for regulating the crosstalk between the cAMP–PKA and CoMekk1–Cmk1 MAPK signaling pathway through CoRas2. Intracellular cAMP levels in DARS1 were higher compared with those in the wild-type in vegetative hyphae. However, DARS1 showed lower sensitivity to exogenous cAMP in appressorium development compared with DARS2. Moreover, the intensity of pathogenesis in DARS1 was similar to that in the wild-type. Therefore, CoRas1 could be involved in the cAMP–PKA signaling pathway in vegetative hyphae but not during infection related morphogenesis.