Paulraj K. Lawrence

The Role of Two F-Box Proteins, SLEEPY1 and SNEEZY, in Arabidopsis Gibberellin Signaling

The SLEEPY1 (SLY1) F-box gene is a positive regulator of gibberellin (GA) signaling in Arabidopsis (Arabidopsis thaliana). Loss of SLY1 results in GA-insensitive phenotypes including dwarfism, reduced fertility, delayed flowering, and increased seed dormancy. These sly1 phenotypes are partially rescued by overexpression of the SLY1 homolog SNEEZY (SNE)/SLY2, suggesting that SNE can functionally replace SLY1. GA responses are repressed by DELLA family proteins. GA relieves DELLA repression when the SCFSLY1 (for Skp1, Cullin, F-box) E3 ubiquitin ligase ubiquitinates DELLA protein, thereby targeting it for proteolysis. Coimmunoprecipitation experiments using constitutively expressed 35S:hemagglutinin (HA)-SLY1 and 35S:HA-SNE translational fusions in the sly1-10 background suggest that SNE can function similarly to SLY1 in GA signaling. Like HA-SLY1, HA-SNE interacted with the CULLIN1 subunit of the SCF complex, and this interaction required the F-box domain. Like HA-SLY1, HA-SNE coimmunoprecipitated with the DELLA REPRESSOR OF GA1-3 (RGA), and this interaction required the SLY1 or SNE carboxyl-terminal domain. Whereas HA-SLY1 overexpression resulted in a decrease in both DELLA RGA and RGA-LIKE2 (RGL2) protein levels, HA-SNE caused a decrease in DELLA RGA but not in RGL2 levels. This suggests that one reason HA-SLY1 is able to effect a stronger rescue of sly1-10 phenotypes than HA-SNE is because SLY1 regulates a broader spectrum of DELLA proteins. The FLAG-SLY1 fusion protein was found to coimmunoprecipitate with the GA receptor HA-GA-INSENSITIVE DWARF1b (GID1b), supporting the model that SLY1 regulates DELLA through interaction with the DELLA-GA-GID1 complex.

Crystal Structures of Pinoresinol-Lariciresinol and Phenylcoumaran Benzylic Ether Reductases and Their Relationship to Isoflavone Reductases

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 Å resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous α/β NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

Mannheimia haemolytica serotype A1 exhibits differential pathogenicity in two related species, Ovis canadensis and Ovis aries.

Mannheimia haemolytica causes pneumonia in both bighorn sheep (BHS, Ovis canadensis) and domestic sheep (DS, Ovis aries). Under experimental conditions, co-pasturing of BHS and DS results in fatal pneumonia in BHS. It is conceivable that certain serotypes of M. haemolytica carried by DS are non-pathogenic to them, but lethal for BHS. M. haemolytica serotypes A1 and A2 are carried by DS in the nasopharynx. However, it is the serotype A2 that predominantly causes pneumonia in DS. The objectives of this study were to determine whether serotype A1 exhibits differential pathogenicity to BHS and DS, and to determine whether leukotoxin (Lkt) secreted by this organism is its primary virulence factor. Three groups each of BHS and DS were intra-tracheally administered either 1 x 10(9)cfu of serotype A1 wild-type (lktA-Wt group), Lkt-deletion mutant of serotype A1-(lktA-Mt group), or saline (control group), respectively. In the lktA-Wt groups, all four BHS died within 48h while none of the DS died during the 2-week study period. In the lktA-Mt groups, none of the BHS or DS died. In the control groups, one DS died due to an unrelated cause. Necropsy and histopathological findings revealed that death of BHS in the lktA-Wt group was due to bilateral, fibrinohemorrhagic pneumonia. Although the A1-Mt-inoculated BHS were clinically normal, on necropsy, lungs of two BHS showed varying degrees of mild chronic pneumonia. These results indicate that M. haemolytica serotype A1 is non-pathogenic to DS, but highly lethal to BHS, and that Lkt is the primary virulence factor of M. haemolytica.

Mycoplasma ovipneumoniae can predispose bighorn sheep to fatal Mannheimia haemolytica pneumonia.

Mycoplasma ovipneumoniae has been isolated from the lungs of pneumonic bighorn sheep (BHS). However experimental reproduction of fatal pneumonia in BHS with M. ovipneumoniae was not successful. Therefore the specific role, if any, of M. ovipneumoniae in BHS pneumonia is unclear. The objective of this study was to determine whether M. ovipneumoniae alone causes fatal pneumonia in BHS, or predisposes them to infection by Mannheimia haemolytica. We chose M. haemolytica for this study because of its isolation from pneumonic BHS, and its consistent ability to cause fatal pneumonia under experimental conditions. Since in vitro culture could attenuate virulence of M. ovipneumoniae, we used ceftiofur-treated lung homogenates from pneumonic BHS lambs or nasopharyngeal washings from M. ovipneumoniae-positive domestic sheep (DS) as the source of M. ovipneumoniae. Two adult BHS were inoculated intranasally with lung homogenates while two others received nasopharyngeal washings from DS. All BHS developed clinical signs of respiratory infection, but only one BHS died. The dead BHS had carried leukotoxin-positive M. haemolytica in the nasopharynx before the onset of this study. It is likely that M. ovipneumoniae colonization predisposed this BHS to fatal infection with the M. haemolytica already present in this animal. The remaining three BHS developed pneumonia and died 1-5 days following intranasal inoculation with M. haemolytica. On necropsy, lungs of all four BHS showed lesions characteristic of bronchopneumonia. M. haemolytica and M. ovipneumoniae were isolated from the lungs. These results suggest that M. ovipneumoniae alone may not cause fatal pneumonia in BHS, but can predispose them to fatal pneumonia due to M. haemolytica infection.

Concerted action of two avirulent spore effectors activates Reaction to Puccinia graminis 1 (Rpg1)-mediated cereal stem rust resistance

The barley stem rust resistance gene Reaction to Puccinia graminis 1 (Rpg1), encoding a receptor-like kinase, confers durable resistance to the stem rust pathogen Puccinia graminis f. sp. tritici. The fungal urediniospores form adhesion structures with the leaf epidermal cells within 1 h of inoculation, followed by hyphae and haustorium formation. The RPG1 protein is constitutively expressed and not phosphorylated. On inoculation with avirulent urediniospores, it is phosphorylated in vivo within 5 min and subsequently degraded. Application of arginine-glycine-aspartic acid peptide loops prevented the formation of adhesion structures for spore attachment, the phosphorylation of RPG1, and germination of the viable spores. Arginine-glycine-aspartic acid affinity chromatography of proteins from the ungerminated avirulent rust spores led to the purification and identification of a protein with fibronectin type III and breast cancer type 1 susceptibility protein domains and a vacuolar protein sorting-associated protein 9 with a coupling of ubiquitin to endoplasmic reticulum degradation domain. Both proteins are required to induce in vivo phosphorylation and degradation of RPG1. Combined application of both proteins caused hypersensitive reaction on the stem rust-resistant cultivar Morex but not on the susceptible cultivar Steptoe. Expression studies indicated that mRNA of both genes are present in ungerminated urediniospores and are constitutively transcribed in sporelings, infected leaves, and haustoria in the investigated avirulent races. Evidence is presented that RPG1, in yeast, interacts with the two protein effectors from the urediniospores that activate cooperatively the stem rust resistance protein RPG1 long before haustoria formation.

Transmission of Mannheimia haemolytica from domestic sheep (Ovis aries) to bighorn sheep (Ovis canadensis): unequivocal demonstration with green fluorescent protein-tagged organisms.

Previous studies demonstrated that bighorn sheep (Ovis canadensis) died of pneumonia when commingled with domestic sheep (Ovis aries) but did not conclusively prove that the responsible pathogens were transmitted from domestic to bighorn sheep. The objective of this study was to determine, unambiguously, whether Mannheimia haemolytica can be transmitted from domestic to bighorn sheep when they commingle. Four isolates of M. haemolytica were obtained from the pharynx of two of four domestic sheep and tagged with a plasmid carrying the genes for green fluorescent protein (GFP) and ampicillin resistance (APR). Four domestic sheep, colonized with the tagged bacteria, were kept about 10 m apart from four bighorn sheep for 1 mo with no clinical signs of pneumonia observed in the bighorn sheep during that period. The domestic and bighorn sheep were then allowed to have fence-line contact for 2 mo. During that period, three bighorn sheep acquired the tagged bacteria from the domestic sheep. At the end of the 2 mo of fence-line contact, the animals were allowed to commingle. All four bighorn sheep died 2 days to 9 days following commingling. The lungs from all four bighorn sheep showed gross and histopathologic lesions characteristic of M. haemolytica pneumonia. Tagged M. haemolytica were isolated from all four bighorn sheep, as confirmed by growth in ampicillin-containing culture medium, PCR-amplification of genes encoding GFP and ApR, and immunofluorescent staining of GFP. These results unequivocally demonstrate transmission of M. haemolytica from domestic to bighorn sheep, resulting in pneumonia and death of bighorn sheep.

Genome Sequences of Mannheimia haemolytica Serotype A2: Ovine and Bovine Isolates

This report describes the genome sequences of Mannheimia haemolytica serotype A2 isolated from pneumonic lungs of two different ruminant species, one from Ovis aries, designated ovine (O), and the other from Bos taurus, designated bovine (B).

β2 integrin Mac-1 is a receptor for Mannheimia haemolytica leukotoxin on bovine and ovine leukocytes

Pneumonia caused by Mannheimia haemolytica is an important disease of cattle (BO), domestic sheep (DS, Ovis aries) and bighorn sheep (BHS, Ovis canadensis). Leukotoxin (Lkt) produced by M. haemolytica is cytolytic to all leukocyte subsets of these three species. Although it is certain that CD18, the beta subunit of beta(2) integrins, mediates Lkt-induced cytolysis of leukocytes, whether CD18 of all three beta(2) integrins, LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18) and CR4 (CD11c/CD18), mediates Lkt-induced cytolysis of BO, DS and BHS leukocytes remains a controversy. Based on antibody inhibition experiments, earlier studies suggested that LFA-1, but not Mac-1 and CR-4, serves as a receptor for M. haemolytica Lkt. PMNs express all three beta(2) integrins, and they are the leukocyte subset that is most susceptible to Lkt. Therefore we hypothesized that all three beta(2) integrins serve as the receptor for Lkt. The objective of this study was to determine whether Mac-1 of BO, DS and BHS serves as a receptor for Lkt. cDNAs for CD11b of BO, DS and BHS were transfected into a Lkt-non-susceptible cell line along with cDNAs for CD18 of BO, DS and BHS, respectively. Transfectants stably expressing BO, DS or BHS Mac-1 specifically bound Lkt. These transfectants were lysed by Lkt in a concentration-dependent manner. Increase in intracellular [Ca(2+)](i) was observed in transfectants following exposure to low concentrations of Lkt indicating signal transduction through secondary messengers. Collectively, these results indicate that Mac-1 from these three species serves as a receptor for M. haemolytica Lkt.

CD4 T Cell Antigens from Staphylococcus aureus Newman Strain Identified following Immunization with Heat-Killed Bacteria

ABSTRACT Staphylococcus aureus is a commensal bacterium associated with the skin and mucosal surfaces of humans and animals that can also cause chronic infection. The emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) and strains causing chronic intramammary infections (IMI) in cows results in severe human and livestock infections. Conventional approaches to vaccine development have yielded only a few noneffective vaccines against MRSA or IMI strains, so there is a need for improved vaccine development. CD4 T lymphocytes are required for promoting gamma interferon (IFN-γ) mediated immunoglobulin isotype switching in B lymphocytes to produce high-affinity IgG antibodies and IFN-γ-mediated phagocyte activation for an effective resolution of bacterial infection. However, the lack of known CD4 T cell antigens from S. aureus has made it difficult to design effective vaccines. The goal of this study was to identify S. aureus proteins recognized by immune CD4 T cells. Using a reverse genetics approach, 43 antigens were selected from the S. aureus Newman strain. These included lipoproteins, proteases, transcription regulators, an alkaline shock protein, conserved-domain proteins, hemolysins, fibrinogen-binding protein, staphylokinase, exotoxin, enterotoxin, sortase, and protein A. Screening of expressed proteins for recall T cell responses in outbred, immune calves identified 13 proteins that share over 80% sequence identity among MRSA or IMI strains. These may be useful for inclusion in a broadly protective multiantigen vaccine against MRSA or IMI.

A virion-associated protein kinase induces apoptosis

ABSTRACT Apoptosis and inhibition of host gene expression are often associated with virus infections. Many viral polypeptides modulate apoptosis by direct interaction with highly conserved apoptotic pathways. Some viruses induce apoptosis during late stages of the infection cycle, while others inhibit apoptosis to facilitate replication or maintain persistent infection. In previous work, we showed that Chilo iridescent virus (CIV) or CIV virion protein extract induces apoptosis in spruce budworm and cotton boll weevil cell cultures. Here, we characterize the product of a CIV gene (iridovirus serine/threonine kinase; istk) with signature sequences for S/T kinase and ATP binding. ISTK appears to belong to the superfamily, vaccinia-related kinases (VRKs). The istk gene was expressed in Pichia pastoris vectors. Purified ISTK (48 kDa) exhibited S/T kinase activity. Treatment with ISTK induced apoptosis in budworm cells. A 35-kDa cleavage product of ISTK retaining key signature sequences was identified during purification. Pichia-expressed 35-kDa polypeptide, designated iridoptin, induced apoptosis and inhibition of host protein synthesis in budworm and boll weevil cells. A mutation in the ATP-binding site eliminated both kinase and apoptosis activity of iridoptin, suggesting that kinase activity is essential for induction of apoptosis. Analysis with custom antibody confirmed that ISTK is a structural component of CIV particles. This is the first demonstration of a viral kinase inducing apoptosis in any virus-host system and the first identification of a factor inducing apoptosis or host protein shutoff for the family Iridoviridae.