Studies on genetic interaction of camk-1, camk-2, camk-3 and camk-4 genes
5.3 Discussion
Calcium (Ca2+) plays an important role in intracellular signaling process in eukaryotes including fungi (Gadd 1994; Shaw and Hoch 2001; Sanders et al. 2002). The Ca2+ signaling process primarily involves transient increase in [Ca2+]c that induce a number of effector proteins including Ca2+/CaMKs (Chin and Means 2000; Hook and Means 2001; Sorderling and Stull 2001; Zelter et al. 2004). To understand the genetic interaction of Ca2+/CaMKs, I have generated double knockout mutants of Ca2+/CaMKs in N. crassa and studied their phenotypes. The ∆camk-1::hph ∆camk-2::hph double mutant showed a severe morphological defect and reduced mycelial extension rate, suggesting a synthetic interaction of these two Ca2+/CaMKs (Figure 5.2A-B; Figure 5.3; Table 5.3). The ∆camk-4::hph ∆camk-2::hph and
∆camk-3::hph ∆camk-2::hph double mutants displayed lower growth rates than their parental single mutants and the wild-type (Figure 5.3; Table 5.3). In addition, ∆camk-1::hph ∆camk- 2::hph, ∆camk-4::hph ∆camk-2::hph and ∆camk-3::hph ∆camk-2::hph double mutant strains displayed reduced aerial hyphae (Figure 5.2C; Table 5.2). Moreover, on Vogel’s glucose medium supplemented with the CaM antagonist TFP (100 µM), the growth of the wild-type and ∆camk-2::hph were comparatively less inhibited, ∆camk-1::hph, ∆camk-3::hph, ∆camk- 4::hph and ∆camk-3::hph ∆camk-2::hph were intermediate, and ∆camk-4::hph ∆camk-2::hph and ∆camk-1::hph ∆camk-2::hph were severely inhibited (Figure 5.4; Table 5.4). I also tested if the Ca2+/CaMKs in N. crassa play any role in thermotolerance and survival in oxidative stress. The ∆camk-1::hph, ∆camk-3::hph, ∆camk-4::hph, ∆camk-1::hph ∆camk- 2::hph, ∆camk-4::hph ∆camk-2::hph and ∆camk-3::hph ∆camk-2::hph mutants showed a decreased survival percentage both in uninduced and induced thermotolerance conditions than the wild-type and ∆camk-2::hph mutant strains (Figure 5.5; Table 5.5). Moreover, survival percentage in H2O2-induced oxidative stress followed the order ∆camk-2::hph > wild-type >
∆camk-1::hph ∆camk-2::hph > ∆camk-1::hph > ∆camk-4::hph ∆camk-2::hph > ∆camk- 4::hph > ∆camk-3::hph > ∆camk-3::hph ∆camk-2::hph (Figure 5.6; Table 5.6). The ∆camk- 2::hph mutant showed an increased survival percentage than the wild-type in response to H2O2-induced oxidative stress (Figure 5.6; Table 5.6). The ∆camk-3::hph ∆camk-2::hph double mutant displayed severe sensitivity to H2O2 stress.
The camk-1 null mutant of N. crassa transiently show a slow growth phenotype immediately after germination from ascospores, indicating that CAMK-1 has important but redundant role in growth and development (Yang et al. 2001). The Ca2+/CaM possess a putative CaM-binding domain, and binding of Ca2+/CaM is essential for the activation of these kinases (Hook and Means 2001). The regulatory functions of calmodulin-dependent protein phosphorylation during conidial germination and hyphal growth in N. crassa (Praveen Rao et al. 1997). In S. cerevisiae, CaMKII was found necessary for acquisition of induced thermotolerance (Iida et al. 1995). In another study, CaMKs in human T lymphocytes appeared to have a role in H2O2-induced phosphorylation of the inhibitor of κB (IκB), indicating CaMKs as the potential therapeutic targets to minimize activation of the
transcription factor NF-κB induced by oxidative stress (Howe et al. 2002). It was found that in N. crassa, germinating spores have a higher survival percentage to the lethal shock at 52°C on pre-exposure to 44°C and this thermal protection is meditated by the heat shock proteins that are synthesized optimally at 44°C (Plesofsky-Vig and Brambl 1985). In S. cerevisiae, the acquisition of induced thermotolerance depends on CaMKII, encoded by cmk1 and cmk2 (Iida et al. 1995). The ∆cmk1 and the ∆cmk1 ∆cmk2 mutants were found slightly hypersensitive and hypersensitive, respectively, to lethal heat-shock, and showed significantly lower levels of induced thermotolerance than those of wild-type and ∆cmk2 mutant (Iida et al. 1995).
H2O2 is a deleterious agent that generates harmful and potent free •OH radicals, via the Haber-Weiss reaction, and lipid peroxides derived from H2O2 could damage the membrane (Haber and Weiss 1934; Halliwell and Gutteridge 1984). In addition, H2O2 at highest concentration induced the signs of apoptosis in Jurkat cells (Howe et al. 2002).
Crosses homozygous for the ∆camk-1::hph ∆camk-2::hph, ∆camk-4::hph ∆camk- 2::hph and ∆camk-3::hph ∆camk-2::hph mutants displayed barren phenotype like the ∆camk- 2::hph homozygous crosses (Table 5.7). In addition, viability of ascospores from some of these barren crosses was lowered than the wild-type (Figure 5.7). These results consistently suggested genetic interactions of camk-2 with camk-1, camk-3, and camk-4. The
Ca2+/CaMKs were also found important for sexual development in other organisms. The Ca2+/CaMKI homologue in C. gloeosporioides, CgCMK, might be involved in germination
and appressorium induction (Kim et al. 1998). In X. laevis, CaMKIx, activated Ca2+/CaMKI was found to phosphorylate various proteins including synapsin I, histones, and myelin basic protein in the late stages of embryogenesis (Kinoshita et al. 2004). In addition, activation of Ca2+ channels leads to Ca2+ influx that is an essential step in initiation of acrosome reaction during fertilization in human, and abnormal activity was detected in the Ca2+ -permeable channels from infertile human sperm membrane (Goodwin et al. 1997; Ma and Shi 1999).
Thus, the genetic interaction studies suggested that complex interactions of camk-1, camk-2, camk-3 and camk-4 genes regulate growth, thermotolerance, survival in oxidative stress and sexual development in N. crassa.