Functional characterization of heptad repeat 1 and 2 mutants of the spike protein of severe acute respiratory syndrome coronavirus

To understand the roles of heptad repeat 1(HR1) and HR2 of the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) in virus-cell interactions, the conserved Leu or Ile residues located at positions 913, 927, 941, and 955 in HR1 and 1151, 1165, and 1179 in HR2 were individually replaced with an alpha-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lower-molecular-weight S(L) form, while the wild type and all other mutants produced similar levels of both the S(L) form and the slower-migrating, higher-molecular-weight S(H) form. The wild-type S(L) form was processed to the S(H) form, whereas the S(L) form of the 913P mutant was inefficiently converted to the S(H) form after biosynthesis. None of these mutations affected cell surface expression or binding to its cognate ACE2 receptor. In a human immunodeficiency virus type 1/SARS S coexpression study, all mutants except the 913P mutant incorporated the S(H) form into the virions as effectively as did the wild-type S(H) form. The mutation at Ile-1151 did not affect membrane fusion or viral entry. The impaired viral entry of the 927P, 941P, 955P, and 1165P mutants was due to their inability to mediate membrane fusion, whereas the defect in viral entry of the 1179P mutant occurred not at the stage of membrane fusion but rather at a postfusion stage. Our study demonstrates the functional importance of HR1 and HR2 of the SARS-CoV spike protein in membrane fusion and viral entry.     

IRT1 DEGRADATION FACTOR1, a RING E3 Ubiquitin Ligase,Regulates the Degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis

Fe is an essential micronutrient for plant growth and development; plants have developed sophisticated strategies to acquire ferric Fe from the soil. Nongraminaceous plants acquire Fe by a reduction-based mechanism, and graminaceous plants use a chelation-based mechanism. In Arabidopsis thaliana, which uses the reduction-based method, IRON-REGULATED TRANSPORTER1 (IRT1) functions as the most important transporter for ferrous Fe uptake. Rapid and constitutive degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. IRT1 degradation involves ubiquitination. To identify the specific E3 ubiquitin ligases involved in IRT1 degradation, we screened a set of insertional mutants in RING-type E3 ligases and identified a mutant that showed delayed degradation of IRT1 and loss of IRT1-ubiquitin complexes. The corresponding gene was designated IRT1 DEGRADATION FACTOR1 (IDF1). Evidence of direct interaction between IDF1 and IRT1 in the plasma membrane supported the role of IDF1 in IRT1 degradation. IRT1 accumulation was reduced when coexpressed with IDF1 in yeast or Xenopus laevis oocytes. IDF1 function was RING domain dependent. The idf1 mutants showed increased tolerance to Fe deficiency, resulting from increased IRT1 levels. This evidence indicates that IDF1 directly regulates IRT1 degradation through its RING-type E3 ligase activity.     

Phenotypic Characterization of Adenovirus Type 12 Temperature-Sensitive Mutants in Productive Infection and Transformation

Eleven temperature-sensitive mutants of adenovirus type 12, capable of forming plaques in human cells at 33 C but not at 39.5 C, were isolated from a stock of a wild-type strain after treatment with either nitrous acid or hydroxyl-amine. Complementation tests in doubly infected human cells permitted a tentative assignment of eight of these mutants to six complementation groups. Temperature-shift experiments revealed that one mutant is affected early and most of the other mutants are affected late. Only the early mutant, H12ts505, was temperature sensitive in viral DNA replication. Infectious virions of all the mutants except H12ts505 and two of the late mutants produced at 33 C, appeared to be more heat labile than those of the wild type. Only H12ts505 was temperature sensitive for the establishment of transformation of rat 3Y1 cells. One of the late mutants (H12ts504) had an increased transforming ability at the permissive temperature. Results of temperature-shift transformation experiments suggest that a viral function affected in H12ts505 is required for “initiation” of transformation. Some of the growth properties of H12ts505-transformed cells were also temperature dependent, suggesting that a functional expression of a gene mutated in H12ts505 is required to maintain at least some aspects of the transformed state.     

Tomato Pto encodes a functional N-myristoylation motif that is required for signal transduction in Nicotiana benthamiana

Pto kinase of tomato (Lycopersicon esculentum) confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato expressing avrPto or avrPtoB. Pto interacts directly with these type-III secreted effectors, leading to induction of defence responses including the hypersensitive response (HR). Signalling by Pto requires the nucleotide-binding site-leucine-rich repeat (NBS-LRR) protein Prf. Little is known of how Pto is controlled prior to or during stimulation, although kinase activity is required for Avr-dependent activation. Here we demonstrate a role for the N-terminus in signalling by Pto. N-terminal residues outside the kinase domain were required for induction of the HR in Nicotiana benthamiana. The N-terminus also contributed to both AvrPto-binding and phosphorylation abilities. Pto residues 1-10 comprise a consensus motif for covalent attachment of myristate, a hydrophobic 14-carbon saturated fatty acid, to the Gly-2 residue. Several lines of evidence indicate that this motif is important for Pto function. A heterologous N-myristoylation motif complemented N-terminal deletion mutants of Pto for Prf-dependent signalling. Signalling by wild-type and mutant forms of Pto was strictly dependent on the Gly-2 residue. The N-myristoylation motif of Pto complemented the cognate motif of AvrPto for avirulence function and membrane association. Furthermore, Pto was myristoylated in vivo dependent on the presence of Gly-2. The subcellular localization of Pto was independent of N-myristoylation, indicating that N-myristoylation is required for some function other than membrane affinity. Consistent with this idea, AvrPtoB was also found to be a soluble protein. The data indicate an important role(s) for the myristoylated N-terminus in Pto signalling.     

Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana

When cultured on media containing the plant growth regulator auxin, hypocotyl explants of Arabidopsis thaliana generate adventitious roots. As a first step to investigate the genetic basis of adventitious organogenesis in plants, we isolated nine temperature-sensitive mutants defective in various stages in the formation of adventitious roots: five root initiation defective (rid1 to rid5) mutants failed to initiate the formation of root primordia; in one root primordium defective (rpd1) mutant, the development of root primordia was arrested; three root growth defective (rgd1, rgd2, and rgd3) mutants were defective in root growth after the establishment of the root apical meristem. The temperature sensitivity of callus formation and lateral root formation revealed further distinctions between the isolated mutants. The rid1 mutant was specifically defective in the reinitiation of cell proliferation from hypocotyl explants, while the rid2 mutant was also defective in the reinitiation of cell proliferation from root explants. These two mutants also exhibited abnormalities in the formation of the root apical meristem when lateral roots were induced at the restrictive temperature. The rgd1 and rgd2 mutants were deficient in root and callus growth, whereas the rgd3 mutation specifically affected root growth. The rid5 mutant required higher auxin concentrations for rooting at the restrictive temperature, implying a deficiency in auxin signaling. The rid5 phenotype was found to result from a mutation in the MOR1/GEM1 gene encoding a microtubule-associated protein. These findings about the rid5 mutant suggest a possible function of the microtubule system in auxin response.     

Phytochrome signalling modulates the SA-perceptive pathway in Arabidopsis

The interaction of phytochrome signalling with the SA signal transduction pathway has been investigated in Arabidopsis using single and multiple mutants affected in light perception (phyA and phyB deficient) and light-signal processing (psi2, phytochrome signalling). The induction of PR1 by SA and functional analogues has been found to strictly correlate with the activity of the signalling pathway controlled by both phyA and phyB photoreceptors. In darkness as well as dim light, and independently of a carbohydrate source, SA-induced PR gene expression as well as the hypersensitive response to pathogens (HR) are strongly reduced. Moreover, the initiation of HR also exhibits a strict dependence upon both the presence and the amplitude of a phytochrome-elicited signal. The growth of an incompatible strain of bacterial a pathogen (Pseudomonas syringae pv. tomato) was enhanced in phyA-phyB and decreased in psi2 mutants. While functional chloroplasts were found necessary for the development of an HR, the induction of PRs was strictly dependent on light, but independent of functional chloroplasts. Taken together, these data demonstrate that the light-induced signalling pathway interacts with the pathogen/SA-mediated signal transduction route. These results are summarized in a formalism that allows qualitative computer simulation.     

Isolation and Characterization of Temperature-Sensitive Respiratory Mutants of NEUROSPORA CRASSA

Filtration-enrichment and inositol-less death methods of mutant isolation, coupled with a screen for cyanide-insensitive respiration, proved to be highly efficient methods for isolating temperature-sensitive (ts) nuclear Neurospora mutants having defective respiration. Eighteen different ts respiratory mutants have been isolated. Most of them are pleiotropic and defective in one or more of the following phenotypes: cytochrome aa3, b, and c (individual or multiple defects); oligomycin inhibition of ATPase activity; respiration and its inhibition by KCN and salicyl hydroxamic acid; and growth rates in liquid and solid media at 25 degrees and 38 degrees. Among these mutants are the first cytochrome c mutant of Neurospora and an extranuclear ts ATPase mutant. An added bonus was the fact that over half of the mutants were affected either in ribosome assembly or in protein synthesis in the mitochondrion. We have yet to find any mutants completely lacking activities associated with the respiratory chain. However, the wide spectrum of mutants isolated here, along with those currently available, constitutes a considerable resource for investigating respiration in obligate aerobes.     

Redundant roles of four ZIP family members in zinc homeostasis and seed development in Arabidopsis thaliana

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana.     

The Jasmonic Acid Signalling Pathway Restricts the Development of the Arbuscular Mycorrhizal Association in Tomato

The role of the jasmonate signalling pathway in modulating the establishment of the arbuscular mycorrhiza (AM) symbiosis between tomato plants and Glomus intraradices fungus was studied. The consequences of AM formation due to the blockage of the jasmonate signalling pathway were studied in experiments with plant mutants impaired in JA perception. The tomato jai-1 mutant (jasmonic acid insensitive 1) failed to regulate colonization and was more susceptible to fungal infection, showing accelerated colonization. The frequency and the intensity of fungal colonization were greatly increased in the jai-1 insensitive mutant plants. In parallel, the systemic effects on mycorrhization due to the activation of the jasmonate signalling pathway by foliar application of MeJA were evaluated and histochemical and molecular parameters of mycorrhizal intensity and efficiency were measured. Histochemical determination of fungal infectivity and fungal alkaline phosphatase activity reveal that the systemic application of MeJA was effective in reducing mycorrhization and mainly affected fungal phosphate metabolism and arbuscule formation, analyzed by the expression of GiALP and the AM-specific gene LePT4, respectively. The results of the present study clearly show that JA participates in the susceptibility of tomato to infection by arbuscular mycorrhizal fungi, and it seems that arbuscular colonization in tomato is tightly controlled by the jasmonate signalling pathway.     

hxc2, an Arabidopsis mutant with an altered hypersensitive response to Xanthomonas campestris pv. campestris

A chemical mutagenized population of Arabidopsis Col-0-gl plants was screened for an altered hypersensitive response (HR) after spray inoculation with an HR-inducing isolate of Xanthomonas campestris pv. campestris (strain 147). Three classes of mutant were identified: those exhibiting an HR- phenotype or partial loss of HR; hyper-responsive mutants showing necrotic lesions rapidly leading to the collapse of leaves; and susceptible mutants. One mutant belonging to the susceptible class, hxc-2, was extensively characterized. The compatible phenotype observed several days after initiation of the interaction was confirmed by measurement of in planta bacterial growth and use of bacterial strains constitutively expressing the GUS reporter gene. In the same way, accumulation of autofluorescent compounds, salicylic acid production and defence gene expression in the mutant were found to be similar to that displayed by the susceptible ecotype. Inoculation of hxc-2 with different avirulent bacteria suggests that the mutation is specific for the interaction with the Xcc 147 strain, although the mutation has been shown to affect a single dominant locus, different from the resistance locus defined by genetic analysis of resistance to Xcc 147. Genetic mapping of the mutation indicated that it is located on chromosome III, defining a previously unknown resistance function in response to X. c. campestris.     

Multiple roles for DNA polymerase I in establishment and replication of the promiscuous plasmid pLS1

The polymerase activity of DNA polymerase I is important for the establishment of the pLS1 replicon by reconstitutive assembly in Streptococcus pneumoniae after uptake of exogenous pLS1 plasmid DNA. In polA mutants lacking the polymerase domain, such establishment was reduced at least 10-fold in frequency. Chromosomally facilitated establishment of pLS1-based plasmids carrying DNA homologous to the host chromosome was not so affected. However, both types of plasmid transfer gave mostly small colonies on initial selection, which was indicative of a defect in replication and filling of the plasmid pool. Once established, the pLS1-based plasmids replicated in polA mutants, but they showed segregational instability. This defect was not observed in strains with the wild-type enzyme or in an S. pneumoniae strain that encodes the polymerase and exonuclease domains of the enzyme on separate fragments. The role of DNA polymerase I in stably maintaining the plasmids depends on its polymerizing function in three separate steps of rolling-circle replication, as indicated by the accumulation of different replication intermediate forms in polA mutants. Furthermore, examination of the segregational stability of the pLS1 replicon in an Escherichia coli mutant system indicated that both the polymerase and the 5′-to-3′ exonuclease activities of DNA polymerase I function in plasmid replication.     

Computer aided screening of potent inhibitor compounds against inhibitor resistant TEM β-lactamase mutants from traditional Chinese medicine

Inhibitor-resistant TEM (IRT) type β-lactamase mutation is largely known. Therefore, it is of interest to identify new yet improved leads against IRT from traditional Chinese medicine. Hence, we screened more than 10,000 compounds from Chinese medicine (tcm@taiwan database) with mutant molecular IRT models through docking techniques. This exercise identified compounds affeic acid, curcumin, salvianolic acid E, ferulic acid and p-coumaric acid with high binding score with the mutants. This was further validated in vitro where salvianolic acid E combined with cefoperazone and sulbactam effectively inhibit the R244S mutant.     

A loss-of-function mutation in the nucleoporin AtNUP160 indicates that normal auxin signalling is required for a proper ethylene response in Arabidopsis

As part of a continuing effort to elucidate mechanisms that regulate the magnitude of ethylene signalling, an Arabidopsis mutant with an enhanced ethylene response was identified. Subsequent characterization of this loss-of-function mutant revealed severe hypocotyl shortening in the presence of saturating ethylene along with increased expression in leaves of a subset of ethylene-responsive genes. It was subsequently determined by map-based cloning that the mutant (sar1-7) represents a loss-of-function mutation in the previously described nucleoporin AtNUP160 (At1g33410, SAR1). In support of previously reported results, the sar1-7 mutant partially restored auxin responsiveness to roots of an rce1 loss-of-function mutant, indicating that AtNUP160/SAR1 is required for proper expression of factors responsible for the repression of auxin signalling. Analysis of arf7-1/sar1-7 and arf19-1/sar1-7 double mutants revealed that mutations affecting either ARF7 or ARF19 function almost fully blocked manifestation of the sar1-7-dependent ethylene hypersensitivity phenotype, suggesting that ARF7- and ARF19-mediated auxin signalling is responsible for regulating the magnitude of and/or competence for the ethylene response in Arabidopsis etiolated hypocotyls. Consistent with this, addition of auxin to ethylene-treated seedlings resulted in severe hypocotyl shortening, reminiscent of that seen for other eer (enhanced ethylene response) mutants, suggesting that auxin functions in part synergistically with ethylene to control hypocotyl elongation and other ethylene-dependent phenomena.     

A Trichoderma atroviride stress‐activated MAPK pathway integrates stress and light signals

Cells possess stress‐activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.