OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae

Somatic embryogenesis receptor kinase (SERK) proteins play pivotal roles in regulation of plant development and immunity. The rice genome contains two SERK genes, OsSerk1 and OsSerk2. We previously demonstrated that OsSerk2 is required for rice Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) and for normal development. Here we report the molecular characterization of OsSerk1. Overexpression of OsSerk1 results in a semi-dwarf phenotype whereas silencing of OsSerk1 results in a reduced angle of the lamina joint. OsSerk1 is not required for rice resistance to Xoo or Magnaporthe oryzae. Overexpression of OsSerk1 in OsSerk2-silenced lines complements phenotypes associated with brassinosteroid (BR) signaling defects, but not the disease resistance phenotype mediated by Xa21. In yeast, OsSERK1 interacts with itself forming homodimers, and also interacts with the kinase domains of OsSERK2 and BRI1, respectively. OsSERK1 is a functional protein kinase capable of auto-phosphorylation in vitro. We conclude that, whereas OsSERK2 regulates both rice development and immunity, OsSERK1 functions in rice development but not immunity to Xoo and M. oryzae.     

Phospholipase C-mediated calcium signalling is required for fungal development and pathogenicity in Magnaporthe oryzae

Calcium signalling has profound implications in the fungal infection of plants and animals, during which a series of physiological and morphological transitions are required. In this article, using a model fungal pathogen, Magnaporthe oryzae , we demonstrate that the regulation of the intracellular calcium concentration ([Ca²⁺]_int) is essential for fungal development and pathogenesis. Imaging of [Ca²⁺]_int showed that infection-specific morphogenesis is highly correlated with the spatiotemporal regulation of calcium flux. Deletion of the fungal phospholipase C gene ( M.   oryzae phospholipase C 1, MoPLC1 ) suppressed calcium flux, resulting in a fungus defective in developmental steps, including appressorium formation and pathogenicity. Surprisingly, the PLC-δ1 gene of mouse was able to functionally substitute for MoPLC1 by restoring the calcium flux, suggesting the evolutionary conservation of the phospholipase C-mediated regulation of calcium flux. Our results reveal that MoPLC1 is a conserved modulator of calcium flux that is essential for the regulation of key steps in fungal development and pathogenesis.     

RIC8 is a guanine-nucleotide exchange factor for Galpha subunits that regulates growth and development in Neurospora crassa

Heterotrimeric (αβγ) G proteins are crucial components of eukaryotic signal transduction pathways. G-protein-coupled receptors (GPCRs) act as guanine nucleotide exchange factors (GEFs) for Gα subunits. Recently, facilitated GDP/GTP exchange by non-GPCR GEFs, such as RIC8, has emerged as an important mechanism for Gα regulation in animals. RIC8 is present in animals and filamentous fungi, such as the model eukaryote Neurospora crassa, but is absent from the genomes of baker's yeast and plants. In Neurospora, deletion of ric8 leads to profound defects in growth and asexual and sexual development, similar to those observed for a mutant lacking the Gα genes gna-1 and gna-3. In addition, constitutively activated alleles of gna-1 and gna-3 rescue many defects of Δric8 mutants. Similar to reports in Drosophila, Neurospora Δric8 strains have greatly reduced levels of G-protein subunits. Effects on cAMP signaling are suggested by low levels of adenylyl cyclase protein in Δric8 mutants and suppression of Δric8 by a mutation in the protein kinase A regulatory subunit. RIC8 acts as a GEF for GNA-1 and GNA-3 in vitro, with the strongest effect on GNA-3. Our results support a role for RIC8 in regulating GNA-1 and GNA-3 in Neurospora.     

Protein phosphatase 2A regulatory subunits perform distinct functional roles in the maize pathogen Fusarium verticillioides

Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is still incomplete. We have demonstrated previously that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. In general, three PP2A subunits, structural A, regulatory B and catalytic C, make up a heterotrimer complex to perform regulatory functions. Significantly, we identified two PP2A regulatory subunits in the F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. In this study, we hypothesized that Ppr1 and Ppr2 are involved in the regulation of fumonisin biosynthesis and/or cell development in F. verticillioides, and generated a series of mutants to determine the functional role of Ppr1 and Ppr2. The PPR1 deletion strain (Δppr1) resulted in drastic growth defects, but increased microconidia production. The PPR2 deletion mutant strain (Δppr2) showed elevated fumonisin production, similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cells with a central septation site, whereas Δppr2 showed early hyphal branching during conidia germination. A kernel rot assay showed that the mutants were slow to colonize kernels, but this is probably a result of growth defects rather than a virulence defect. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out distinct roles in the regulation of fumonisin biosynthesis and fungal development.     

Implication of Ca2+ in the regulation of replicative life span of budding yeast

In eukaryotic cells, Ca(2+)-triggered signaling pathways are used to regulate a wide variety of cellular processes. Calcineurin, a highly conserved Ca(2+)/calmodulin-dependent protein phosphatase, plays key roles in the regulation of diverse biological processes in organisms ranging from yeast to humans. We isolated a mutant of the SIR3 gene, implicated in the regulation of life span, as a suppressor of the Ca(2+) sensitivity of zds1Δ cells in the budding yeast Saccharomyces cerevisiae. Therefore, we investigated a relationship between Ca(2+) signaling and life span in yeast. Here we show that Ca(2+) affected the replicative life span (RLS) of yeast. Increased external and intracellular Ca(2+) levels caused a reduction in their RLS. Consistently, the increase in calcineurin activity by either the zds1 deletion or the constitutively activated calcineurin reduced RLS. Indeed, the shortened RLS of zds1Δ cells was suppressed by the calcineurin deletion. Further, the calcineurin deletion per se promoted aging without impairing the gene silencing typically observed in short-lived sir mutants, indicating that calcineurin plays an important role in a regulation of RLS even under normal growth condition. Thus, our results indicate that Ca(2+) homeostasis/Ca(2+) signaling are required to regulate longevity in budding yeast.     

The cell cycle gene MoCDC15 regulates hyphal growth, asexual development and plant infection in the rice blast pathogen Magnaporthe oryzae

Rice blast, caused by the pathogen Magnaporthe oryzae, is a serious hindrance to rice production and has emerged as an important model for the characterization of molecular mechanisms relevant to pathogenic development in plants. Similar to other pathogenic fungi, conidiation plays a central role in initiation of M.oryzae infection and spread over a large area. However, relatively little is known regarding the molecular mechanisms that underlie conidiation in M. oryzae. To better characterize these mechanisms, we identified a conidiation-defective mutant, ATMT0225B6 (MoCDC15(T-DNA)), in which a T-DNA insertion disrupted a gene that encodes a homolog of fission yeast cdc15, and generated a second strain containing a disruption in the same allele (ΔMoCDC15(T-DNA)). The cdc15 gene has been shown to act as a coordinator of the cell cycle in yeast. Functional analysis of the MoCDC15(T-DNA) and ΔMoCDC15(T-DNA) mutants revealed that MoCDC15 is required for conidiation, preinfection development and pathogenicity in M. oryzae. Conidia from these mutants were viable, but failed to adhere to hydrophobic surface, a crucial step required for subsequent pathogenic development. All phenotypic defects observed in mutants were rescued in a strain complemented with wild type MoCDC15. Together, these data indicate that MoCDC15 functions as a coordinator of several biological processes important for pathogenic development in M. oryzae.     

The Caenorhabditis elegans ems class homeobox gene ceh-2 is required for M3 pharynx motoneuron function

Several homeobox genes, for example those of the ems class, play important roles in animal head development. We report on the expression pattern and function of ceh-2, the Caenorhabditis elegans ems/Emx ortholog. CEH-2 protein is restricted to the nuclei of one type of small muscle cell, one type of epithelial cell, and three types of neurons in the anterior pharynx in the head. We have generated a deletion allele of ceh-2 that removes the homeobox. Animals homozygous for this deletion are viable and fertile, but grow slightly slower and lay fewer eggs than wild type. We assayed the function of two types of pharynx neurons that express ceh-2, the pairs M3 and NSM. M3 activity is substantially reduced in electropharyngeograms of ceh-2 deletion mutants; this defect can account for the observed retardation in larval development, as M3 activity is known to be necessary for effective feeding. NSM function and metabolism are normal based on the assays used. All cells that express ceh-2 in wild type are present in the ceh-2 mutant and have normal morphologies. Therefore, unlike other ems/Emx genes, ceh-2 seems to be important for a late differentiation step and not for neuron specification or regional patterning. Because the CEH-2 homeodomain is well conserved, we tested whether ceh-2 can rescue ems(-) brain defects in Drosophila, despite the apparent differences in biological roles. We found that the C. elegans ems ortholog is able to substitute for fly ems in brain development, indicating that sequence conservation rather than conservation of biological function is important.     

The pseudokinase domain is required for suppression of basal activity of Jak2 and Jak3 tyrosine kinases and for cytokine-inducible activation of signal transduction

Janus (Jak) tyrosine kinases contain a tyrosine kinase (JH1) domain adjacent to a catalytically inactive pseudokinase domain (JH2). The JH2 domain has been implicated in regulation of Jak activity, but its function remains poorly understood. Here, we found that the JH2 domain negatively regulates the activity of Jak2 and Jak3. Deletion of JH2 resulted in increased tyrosine phosphorylation of the Jak2- and Jak3-JH2 deletion mutants as well as of coexpressed STAT5. In cytokine receptor signaling, the deletion of the Jak2- and Jak3-JH2 domains resulted in interferon-gamma and interleukin-2-independent STAT activation, respectively. However, cytokine stimulations did not further induce the JH2 deletion mutant-mediated STAT activation. The deletion of the Jak2 JH2 domain also abolished interferon-gamma-inducible kinase activation, although it did not affect the reciprocal Jak1-Jak2 interaction in 293T cells. Chimeric constructs, where the JH2 domains were swapped between Jak2 and Jak3, retained low basal activity and cytokine inducible signaling, indicating functional conservation between the two JH2 domains. However, the basal activity of Jak2 was significantly lower than that of Jak3, suggesting differences in the regulation of Jak2 and Jak3 activity. In conclusion, we found that the JH2 domain has a conserved function in Jak2 and Jak3. The JH2 domain is required for two distinct functions in cytokine signaling: (i) inhibition of the basal activity of Jak2 and Jak3, and (ii) cytokine-inducible activation of signaling. The Jak-JH2 deletion mutants are catalytically active, activate STAT5, and interact with another Jak kinase, but the JH2 domain is required to connect these signaling events to receptor activation. Thus, we propose that the JH2 domain contributes to both the uninduced and ligand-induced Jak-receptor complex, where it acts as a cytokine-inducible switch to regulate signal transduction.     

A type 2C protein phosphatase FgPtc3 is involved in cell wall integrity, lipid metabolism, and virulence in Fusarium graminearum

Type 2C protein phosphatases (PP2Cs) play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8) exhibited reduced aerial hyphae formation and deoxynivalenol (DON) production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum.     

The role of the NH(2)- and COOH-terminal domains of the inhibitory region of troponin I in the regulation of skeletal muscle contraction.

The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(Delta96-116)), 2) the COOH-terminal domain (TnI-(Delta105-115)), and 3) the NH(2)-terminal domain (TnI-(Delta95-106)). Measurements of Ca(2+)-regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI.TnC complex restored the Ca(2+) sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(Delta96-116).TnC and TnI-(Delta105-115).TnC. The TnI-(Delta95-106).TnC mutant retained approximately 55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca(2+) sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca(2+), and two of them (TnI-(Delta96-116) and TnI-(Delta105-115)) gave significant activation in the absence of Ca(2+). These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than the NH(2)-terminal region, consisting of residues 95-106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca(2+)-sensitive molecular switch during muscle contraction.     

The zebrafish iguana locus encodes Dzip1, a novel zinc-finger protein required for proper regulation of Hedgehog signaling

Members of the Hedgehog (Hh) family of intercellular signaling molecules play crucial roles in animal development. Aberrant regulation of Hh signaling in humans causes developmental defects, and leads to various genetic disorders and cancers. We have characterized a novel regulator of Hh signaling through the analysis of the zebrafish midline mutant iguana (igu). Mutations in igu lead to reduced expression of Hh target genes in the ventral neural tube, similar to the phenotype seen in zebrafish mutants known to affect Hh signaling. Contradictory at first sight, igu mutations lead to expanded Hh target gene expression in somites. Genetic and pharmacological analyses revealed that the expression of Hh target genes in igu mutants requires Gli activator function but does not depend on Smoothened function. Our results show that the ability of Gli proteins to activate Hh target gene expression in response to Hh signals is generally reduced in igu mutants both in the neural tube and in somites. Although this reduced Hh signaling activity leads to a loss of Hh target gene expression in the neural tube, the same low levels of Hh signaling appear to be sufficient to activate Hh target genes throughout somites because of different threshold responses to Hh signals. We also show that Hh target gene expression in igu mutants is resistant to increased protein kinase A activity that normally represses Hh signaling. Together, our data indicate that igu mutations impair both the full activation of Gli proteins in response to Hh signals, and the negative regulation of Hh signaling in tissues more distant from the source of Hh. Positional cloning revealed that the igu locus encodes Dzip1, a novel intracellular protein that contains a single zinc-finger protein-protein interaction domain. Overexpression of Igu/Dzip1 proteins suggested that Igu/Dzip1 functions in a permissive way in the Hh signaling pathway. Taken together, our studies show that Igu/Dzip1 functions as a permissive factor that is required for the proper regulation of Hh target genes in response to Hh signals.     

Deletions of any single residues in Glu40-Ser48 loop connecting a domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase result in almost complete inhibition of conformational transition and hydrolysis of phosphoenzyme intermediate

Possible roles of the Glu40-Ser48 loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca(2+)-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His32-Asn39 and Leu49-Ile54) had no or only slight effects except two specific substitutions of Asn39 found in SERCA2b in Darier's disease pedigrees. All the single deletion mutants for the Glu40-Ser48 loop and the specific Asn39 mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from Pi was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca(2+)-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu40-Ser48 loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition.