Colletotrichum trifolii TB3 Kinase, a COT1 Homolog, Is Light Inducible and Becomes Localized in the Nucleus during Hyphal Elongation

Abstract

Colletotrichum trifolii is a fungal pathogen responsible for anthracnose disease of alfalfa. Previously, a serine/threonine protein kinase gene from this fungus (TB3), which is a functional homolog of the Neurospora crassa COT1 kinase, has been isolated in our laboratory and appears to be associated with hyphal elongation and branching. In this report we show that light treatment rapidly induces TB3 expression and hyphal branching frequency. Western analysis showed TB3 localization in both the cytoplasm and nucleus, but not in membranes. Moreover, indirect immunofluorescence indicated that TB3 levels were most abundant in the nucleus. To further evaluate the subcellular distribution of TB3, a TB3::GFP fusion construct was inserted into C. trifolii. Results indicated that the cellular location of TB3 changed during fungal growth and development. Consistent with previous observations, TB3 was localized in both the cytoplasm and the nucleus but was preferentially localized in the nucleus during extended hyphal growth. The amino terminus of TB3 contains two relatively long polyglutamine repeats. Yeast-based assays showed that these polyglutamine tracts can activate transcription. These results suggest that TB3 may be positioned in a signaling cascade regulating proper hyphal growth and development by functioning as a transcription factor.A distinctive feature of filamentous fungi is their mode of growth and development via vegetative propagagules known as hyphae. Hyphae grow in a polarized manner and account, in part, for the foraging success of fungi either as saprophytes or pathogens. Coordinated control of hyphal growth and development in filamentous fungi is achieved by activating intracellular signaling cascades in response to environmental stimuli, including nutrient availability, light, and plant host. In addition to these environmental cues that influence hyphal growth, genetic factors have also been identified. In Neurospora crassa, the serine/threonine protein kinase, COT1, has been shown to be required for hyphal elongation (29). Mutations in cot-1 are temperature sensitive; colonial growth occurs at or above 32°C, while normal radial growth is observed at or below 25°C (17). Moreover, disruption of cot-1 results in severe growth defects as disruptants grow little, if at all. cot-1 expression is also photoinducible, and light illumination increases apical hyphal cell branching frequency (16). Immunoblotting with COT1 antibody detects the COT1 kinase in the cytoplasm, membrane, and nucleus (13).Colletotrichum trifolii is the causal agent of alfalfa anthracnose (1). Pathogenicity of C. trifolii depends on proper growth and appressorium differentiation, which are believed to be regulated through the activation of intracellular signal transduction pathways in response to host stimuli (9). However, our understanding of the genes and biochemical pathways which govern hyphal growth and morphogenesis are not entirely clear. Previously, we cloned and characterized a serine/threonine protein kinase gene, tb3 (3), a...

Photostimulation of Hypocrea atroviridis growth occurs due to a cross-talk of carbon metabolism, blue light receptors and response to oxidative stress

“…Vegetative growth of fungi has been reported to be influenced by light, both negatively (Casas-Flores et al, 2006; Ambra et al, 2004;Flaherty & Dunkle, 2005;Schmoll et al, 2005) and positively (Lauter et al, 1998;Chen & Dickman, 2002;Chovanec et al, 2001). On a biochemical level, exposure of Aspergillus ornatus to a pulse of light led to protein phosphorylation and decreased glucose uptake (Hill, 1976), and in the slime mould Physarum polycephalum it stopped glucose consumption, and decreased the accumulation of glucans and D-trehalose (Schreckenbach et al, 1981).…”

“…Some authors have described effects of light on hyphal growth or branching, thereby mostly referring to inhibitory effects (Lauter et al, 1998;Chen & Dickman, 2002;Ambra et al, 2004; Casas-Flores et al, 2004;Flaherty & Dunkle, 2005; Miyake et al, 2005;Idnurm & Heitman, 2005;Brasch & Kay, 2006). For arbuscular mycorrhizal fungi, lowintensity light is also known to induce hyphal branching and the consequent increase of mycelial biomass (Nagahashi & Douds, 2003).…”

Depletion of Aspergillus nidulans cotA causes a severe polarity defect which is not suppressed by the nuclear migration mutation nudA2

“…The greatest variation in the members of this family is in the N-terminal region, which is of variable length, and differs in the presence and length of glutamine rich tracts. The function of these tracts in this family of proteins is unclear, though they are often associated with transcriptional activation (Gerber et al 1994;Chen and Dickman 2002). N. crassa and other Sordariomycetes have the most closely related genes to the Aspergillus species, but the Aspergillus glutamine tracts are not very extensive compared to the Sordariomycetes.…”

“…This difference in localisation is difficult to explain, since there is very little difference in the putative nuclear localisation signal. It was suggested that glutamine-rich N-terminal region of TB3 of C. trifolii is associated with nuclear localisation (Chen and Dickman 2002), though there as yet is no clear correlation between structure/presence of the N-terminal region and localisation in this family. As is the case with CotA, tagged-NDR2 shows a punctate distribution in the cytoplasm, and it was suggested that this might reflect presence of some of this kinase in a subcellular compartment other than the nuclei.…”

Living Colors in the Gray Mold Pathogen Botrytis cinerea: Codon-Optimized Genes Encoding Green Fluorescent Protein and mCherry, Which Exhibit Bright Fluorescence

“…(12). GFP has been used for a number of live-imaging applications with plant pathogens, such as invasion of host plant tissue (Fusarium graminearum [23] and M. oryzae [25]), the dynamics of nuclear movement and division during spore germination (M. oryzae [53], Colletotrichum gloeosporioides [35], and Fusarium oxysporum [41]), translocation of regulatory protein kinases from the cytoplasm to nuclei during development and in response to light and nutrient signals (M. oryzae [1], Colletotrichum trifolii [4], and Mycosphaerella graminicola [38]), and stage-specific expression of effector proteins in the host plants (U. maydis [11] and M. oryzae [33]). …”