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

Abstract

Colletotrichum orbiculare 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 C. orbiculare. However, upstream regulators of these pathways for this species remain unidentified. In this study, CoIRA1, encoding RAS GTPase activating protein, was identified by screening the Agrobacterium tumefaciens-mediated transformation (AtMT) mutant, which was defective in the pathogenesis of C. orbiculare. The coira1 disrupted mutant showed an abnormal infection-related morphogenesis and attenuated pathogenesis. In Saccharomyces cerevisiae, Ira1/2 inactivates Ras1/2, which activates adenylate cyclase, leading to the synthesis of cAMP. Increase in the intracellular cAMP levels in coira1 mutants and dominant active forms of CoRAS2 introduced transformants indicated that CoIra1 regulates intracellular cAMP levels through CoRas2. Moreover, the phenotypic analysis of transformants that express dominant active form CoRAS2 in the comekk1 mutant or a dominant active form CoMEKK1 in the coras2 mutant indicated that CoRas2 regulates the MAPK CoMekk1–Cmk1 signaling pathway. The CoRas2 localization pattern in vegetative hyphae of the coira1 mutant was similar to that of the wild-type, expressing a dominant active form of RFP–CoRAS2. 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.

MoRad6‐mediated ubiquitination pathways are essential for development and pathogenicity inMagnaporthe oryzae

“…Similar results are obtained in another fungal pathogen Colletotrichum orbiculare . Inactivation of CoIra1, the homolog of yeast RAS GTPase activating protein ScIra1/2, upregulates the intracellular cAMP level via the C. orbiculare MoRas2 homologous protein, CoRas2, which also controls the MAPK Cmk1 signaling pathway in C. orbiculare (Harata and Kubo, ).…”

Colletotrichum orbiculare Regulates Cell Cycle G1/S Progression via a Two-Component GAP and a GTPase to Establish Plant Infection

“…After the conidia germinate, the resultant germ tubes differentiate into dome-shaped appressoria and undergo nuclear division. Successful appressorium function and, thus, infection depend on the proper sequence of metabolic events, including melanin synthesis, peroxisome metabolism, establishment of cellular polarity and cell wall integrity, and signal transduction, occurring in the fungus Harata and Kubo, 2014). Importantly, a microtubule dynamics analysis revealed that a precise nuclear distribution inside cells is also required for normal appressorial development (Takano et al, 2001).…”

Threonine synthase CoTHR4 is involved in infection-related morphogenesis during the pre-penetration stage in Colletotrichum orbiculare

“…Interestingly, in contrast to the context of amino acid biosynthesis genes in M. oryzae , the majority of conidia of the cothr4 mutant produced on the threonine-supplemented PDA media were unable to germinate and the conidia required exogenously supplied threonine for efficient germination. Regulation of conidial germination in C. orbiculare is known to involve the Ras/MAPK and cAMP pathways, which serve in the transmission of environmental signals to intracellular systems for initiation of infection-related morphogenesis, but how cellular nutrients facilitate the initial infection processes remains elusive [12]. Our results suggested that threonine production regulated by threonine synthase in conidia could potentially trigger conidial germination by functioning as a cellular nutrient signal.…”

“…Kanamycin (50 μg/ml) was added to the medium as needed. A. tumefaciens C58C1 cultured in LB medium at 28 °C was used to transform C. orbiculare as previously described [12]. Total DNA extraction from the mycelia of C. orbiculare and DNA blot analysis were performed as previously described.…”

“…The infection-related morphogenesis regulated by these pathways is common to M. oryzae , Botrytis cinerea , Fusarium graminearum , and Ustilago maydis , which cause devastating diseases of cultivated crops [6–10]. Moreover, positive and negative regulators of these signaling pathways have been characterized, and a detailed signaling network for infection-related morphogenesis has been identified in C. orbiculare and M. oryzae [11, 12]. However, although intracellular signal transduction in response to environmental signals has been well documented, knowledge of the intracellular nutrient metabolism underlying the cellular differentiation processes in C. orbiculare is less understood.…”