Ras GTPase Activating Protein CoIra1 Is Involved in Infection-Related Morphogenesis by Regulating cAMP and MAPK Signaling Pathways through CoRas2 in <i>Colletotrichum orbiculare</i>

<div><p><i>Colletotrichum orbiculare</i> is the causative agent of anthracnose disease on cucurbitaceous plants. Several signaling pathways, including cAMP–PKA and mitogen-activating protein kinase (MAPK) pathways are involved in the infection-related morphogenesis and pathogenicity of <i>C. orbiculare</i>. However, upstream regulators of these pathways for this species remain unidentified. In this study, <i>CoIRA1</i>, encoding RAS GTPase activating protein, was identified by screening the <i>Agrobacterium tumefaciens</i>-mediated transformation (AtMT) mutant, which was defective in the pathogenesis of <i>C. orbiculare</i>. The <i>coira1</i> disrupted mutant showed an abnormal infection-related morphogenesis and attenuated pathogenesis. In <i>Saccharomyces cerevisiae</i>, Ira1/2 inactivates Ras1/2, which activates adenylate cyclase, leading to the synthesis of cAMP. Increase in the intracellular cAMP levels in <i>coira1</i> mutants and dominant active forms of <i>CoRAS2</i> introduced transformants indicated that CoIra1 regulates intracellular cAMP levels through CoRas2. Moreover, the phenotypic analysis of transformants that express dominant active form <i>CoRAS2</i> in the <i>comekk1</i> mutant or a dominant active form <i>CoMEKK1</i> in the <i>coras2</i> mutant indicated that CoRas2 regulates the MAPK CoMekk1–Cmk1 signaling pathway. The CoRas2 localization pattern in vegetative hyphae of the <i>coira1</i> mutant was similar to that of the wild-type, expressing a dominant active form of <i>RFP</i>–<i>CoRAS2</i>. Moreover, we demonstrated that bimolecular fluorescence complementation (BiFC) signals between CoIra1 and CoRas2 were detected in the plasma membrane of vegetative hyphae. Therefore, it is likely that CoIra1 negatively regulates CoRas2 in vegetative hyphae. Furthermore, cytological analysis of the localization of CoIraI and CoRas2 revealed the dynamic cellular localization of the proteins that leads to proper assembly of F-actin at appressorial pore required for successful penetration peg formation through the pore. Thus, our results indicated that CoIra1 is involved in infection-related morphogenesis and pathogenicity by proper regulation of cAMP and MAPK signaling pathways through CoRas2.</p></div

Appressorium formation assay of DRS2/DAMK1 and DMK1/DARS2 on the glass slides.

<p>(A) Conidial suspensions of each strain in distilled water incubated on multiwell glass slides at 24°C for 24 h. DRS2, the <i>coras2</i> mutant; DMK1, the <i>comekk1</i> mutant; DRS2/DAMK1, DRS2 transformed with a dominant active form <i>CoMEKK1</i>; DMK1/DARS2, DMK1 transformed with a dominant active form <i>CoRAS2</i>. Scale bar, 10 µm. (B) Percentages of conidial germination and appressorium formation in <i>C. orbiculare</i> 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.</p

Penetration hyphae formation of the <i>coira1</i> mutants of <i>C. orbiculare</i> on cellulose membranes.

<p>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 <i>coira1</i> mutant; CL1-1, the <i>CoIRA1</i>-complemented transformant of DL1-1; CL1-2, the <i>CoIRA1</i>-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 <i>C. orbiculare</i> WT and <i>coira1</i> 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.</p

The phosphorylation of MAPK Cmk1 in the <i>coira1</i> mutant.

<p>(A) The total protein isolated from mycelia of each strain. WT; the wild-type, DL1; the <i>coira1</i> mutant, DRS2; the <i>coras2</i> mutant, DARS1, WT transformed with a dominant active form <i>CoRAS1</i>, DARS2; WT transformed with a dominant active form <i>CoRAS2</i>, iDNRS1; the <i>coira1</i> mutant transformed with a dominant negative form <i>CoRAS1</i>, iDNRS2; the <i>coira1</i> mutant transformed with a dominant negative form <i>CoRAS2,</i> DCK1; the <i>cmk1</i> 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 <i>t</i> test: *indicate <i>P</i><0.05).</p

Assay for the colocalization of CoIra1 and CoRas2 in appressoria at 48 h after inoculation on cucumber leaves.

<p>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 <i>CoIRA1</i>–VENUS and <i>RFP–CoRAS2</i>. Scale bar, 10 µm.</p

Appressorium formation by <i>coira1</i> mutants on glass slide in the presence of 10-mM cAMP.

<p>(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 <i>coira1</i> mutant; CL1-1, the <i>CoIRA1</i>-complemented transformant of DL1-1; DC1, the <i>cac1</i> mutant; DARS1, WT transformed with a dominant active form <i>CoRAS1</i>; DARS2, WT transformed with a dominant active form <i>CoRAS2</i>; iDNRS1, DL1-1 transformed with a dominant negative form <i>CoRAS1</i>; iDNRS2, DL1-1 transformed with a dominant negative form <i>CoRAS2</i>. Scale bar, 10 µm. (B) Percentages of conidial germination, appressorium formation, and abnormal appressorium formation of <i>C. orbiculare</i> 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.</p

Assembly of F-actin in the appressorium pores of the <i>coira1</i> mutant.

<p>(A) Micrograph of F-actin organization in the appressorium pore visualized by the expression of Lifeact–RFP in the wild-type and in the <i>coira1</i> 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 <i>coira1</i> mutant expressing Lifeact–RFP (B) Percentage of the assembly of F-actin in the appressorium pore of the <i>coira1</i> 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 <i>coira1</i> mutant expressing LifeAct-RFP.</p

Assay for colocalization of CoIra1 and F-actin.

<p>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 <i>CoIRA1</i>–VENUS and Lifeact–<i>RFP.</i> Scale bar, 10 µm.</p

Pathogenicity assay and penetration ability of <i>coira1</i> mutants of <i>C. orbiculare</i> on the cucumber cotyledons.

<p>(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 <i>coira1</i> mutant; CL1-1, the <i>CoIRA1</i>-complemented transformant of DL1-1; CL1-2, the <i>CoIRA1</i>-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 <i>coira1</i> 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.</p

BiFC assays for CoIra1 and CoRas2 interactions in vegetative hyphae.

<p>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–<i>CoRAS2</i> and Vn–<i>CoIRA1</i>. Scale bar, 10 µm.</p