Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.     

Identification of Xanthomonas citri ssp. citri host specificity genes in a heterologous expression host

We provide the first conclusive evidence that Xanthomonas axonopodis pv. citri Asiatic strain (Xac-A) and, in particular, Xac-A^w, a unique citrus canker A strain isolated from Key lime in Wellington, Florida, induces a hypersensitive reaction (HR) in grapefruit leaves. Using the heterologous tomato pathogen X. perforans , as a recipient of the Xac-A^w genomic library, we identified a 1599-bp open reading frame responsible for HR in grapefruit, but not Key lime, and designated it avrGf 1. Xac-A^wΔ avrGf 1 produced typical, although visibly reduced, citrus canker symptoms (i.e. raised pustules) in grapefruit and typical canker symptoms in Key lime. We also determined that the X. perforans transconjugant carrying an Xac-A^w hrpG elicited HR in grapefruit and Key lime leaves, and that xopA in X. perforans was partly responsible for HR. Xac-A transconjugants carrying the X. perforans xopA were reduced in ability to grow in grapefruit leaves relative to wild-type Xac-A. The X. perforans xopA appears to be a host-limiting factor. An avrBs3 homologue, which contained 18.5 repeats and induced HR in tomato, was designated avrTaw . This gene, when expressed in a pustule-minus Xac-A^w, did not complement pustule formation; however, pthA^w , a functional pthA homologue, complemented the mutant strain to produce typical pustules in Key lime, but markedly reduced pustules in grapefruit. Both avrBs3 homologues, when expressed in a typical Xac-A strain, resulted in typical citrus canker pustules in grapefruit, indicating that neither homologue suppressed pustule size in grapefruit. Xac-A^w contains other unidentified factors that suppress development in grapefruit.     

Tocopherols play a crucial role in low-temperature adaptation and Phloem loading in Arabidopsis

To test whether tocopherols (vitamin E) are essential in the protection against oxidative stress in plants, a series of Arabidopsis thaliana vitamin E (vte) biosynthetic mutants that accumulate different types and levels of tocopherols and pathway intermediates were analyzed under abiotic stress. Surprisingly subtle differences were observed between the tocopherol-deficient vte2 mutant and the wild type during high-light, salinity, and drought stresses. However, vte2, and to a lesser extent vte1, exhibited dramatic phenotypes under low temperature (i.e., increased anthocyanin levels and reduced growth and seed production). That these changes were independent of light level and occurred in the absence of photoinhibition or lipid peroxidation suggests that the mechanisms involved are independent of tocopherol functions in photoprotection. Compared with the wild type, vte1 and vte2 had reduced rates of photoassimilate export as early as 6 h into low-temperature treatment, increased soluble sugar levels by 60 h, and increased starch and reduced photosynthetic electron transport rate by 14 d. The rapid reduction in photoassimilate export in vte2 coincides with callose deposition exclusively in phloem parenchyma transfer cell walls adjacent to the companion cell/sieve element complex. Together, these results indicate that tocopherols have a more limited role in photoprotection than previously assumed but play crucial roles in low-temperature adaptation and phloem loading.     

Defensins play a crucial role in protecting mice against oral Shigella flexneri infection

An earlier study revealed that 4-day-old mice, but not older mice, were infected with invasive Shigella strains. Here we attempted to determine the underlying mechanism that induces inflammation in the intestines of neonate mice after oral Shigella infection. Wild-type BALB/c mice of different ages (7, 14, and 35 days old) were orally administered GFP-expressing Shigella flexneri 5a M90T strain (5 × 10⁹ CFU) and analyzed for colonization 6 h following infection. We found that Shigella localized in the epithelium, lamina propria, and crypt regions of the small intestines of 7-day-old BALB/c mice. Microarray analysis revealed that expression levels of cryptdin and various types of cryptdin-related mRNA (e.g., cryptrs-2, -5, -7, -12 and lysozyme) in the small intestines were significantly lower in 7-day-old than in older mice regardless of Shigella infection status. Interestingly, matrix metalloprotease-7 (matrilysin)-deficient (MAT^−/−) mice of B6 background had more colonies and more severe symptoms of inflammation in the intestines than did wild-type B6 mice after oral Shigella challenge. This suggests that cryptdin-related antimicrobial molecules are indispensable for efficient protection against oral Shigella infection.     

Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

The self-incompatibility response involves S allele-specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele-specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen-pistil self-incompatibility reaction.     

Glucose-regulated protein 78 may play a crucial role in promoting the pulmonary microvascular remodeling in a rat model of hepatopulmonary syndrome

Objective

This study is to investigate the role of glucose-regulated protein 78 (GRP78) in the pulmonary microvascular remodeling during hepatopulmonary syndrome (HPS) development.

Methods

The rat models with liver cirrhosis and HPS were induced by multiple pathogenic factors for 4 to 8 wk. The concentrations of alanine transferase (ALT) and endotoxin in plasma were detected in the models, followed by the detection of GRP78 expression. RT-PCR, quantitative real-time PCR and Western blotting were employed to assess the mRNA and protein expression levels of vascular endothelial growth factor (VEGF), respectively. Immunohistochemistry staining was used to examine the expression of a specific vascular marker, factor VIII-related antigen (FVIII-RAg), and several cell proliferation- and apoptosis-related proteins, including CHOP/GADD153, caspase-12, Bcl-2 and nuclear factor (NF)-κB.

Results

The levels of endotoxin and ALT in plasma were gradually increased as the disease progressed, so did GRP78, which were in a positive correlation. The expression levels of VEGF (both mRNA and protein) and FVIII-RAg were significantly elevated in the HPS models, indicating active angiogenesis, which was also positively correlated with GRP78 expression. Furthermore, the expression levels of the pro-apoptotic proteins of CHOP/GADD153 and caspase-12 were dramatically decreased, while the anti-apoptotic proteins of Bcl-2 and NF-κB were significantly elevated, in the HPS models. There were also close correlation between these proteins and GRP78.

Conclusions

Over-expression of GRP78 in lungs may be the critical pathogenic factor for HPS. Through promoting cell proliferation and survival and inhibiting apoptosis, GRP78 may promote the pulmonary microvascular remodeling in HPS pathogenesis. Our results provide a potential therapeutic target for clinical prevention and treatment for HPS and related complications.     

Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein

Plasmodium falciparum sporozoites invade liver cells in humans and set the stage for malaria infection. Circumsporozoite protein (CSP), a predominant surface antigen on sporozoite surface, has been associated with the binding and invasion of liver cells by the sporozoites. Although CSP across the Plasmodium genus has homology and conserved structural organization, infection of a non-natural host by a species is rare. We investigated the role of CSP in providing the host specificity in P. falciparum infection. CSP from P. falciparum, P. gallinaceum, P. knowlesi, and P. yoelii species representing human, avian, simian, and rodent malaria species were recombinantly expressed, and the proteins were purified to homogeneity. The recombinant proteins were evaluated for their capacity to bind to human liver cell line HepG2 and to prevent P. falciparum sporozoites from invading these cells. The proteins showed significant differences in the binding and sporozoite invasion inhibition activity. Differences among proteins directly correlate with changes in the binding affinity to the sporozoite receptor on liver cells. P. knowlesi CSP (PkCSP) and P. yoelii CSP (PyCSP) had 4,790- and 17,800-fold lower affinity for heparin in comparison to P. falciparum CSP (PfCSP). We suggest that a difference in the binding affinity for the liver cell receptor is a mechanism involved in maintaining the host specificity by the malaria parasite.     

Vibrio fischeri lux genes play an important role in colonization and development of the host light organ

The bioluminescent bacterium Vibrio fischeri and juveniles of the squid Euprymna scolopes specifically recognize and respond to one another during the formation of a persistent colonization within the host's nascent light-emitting organ. The resulting fully developed light organ contains brightly luminescing bacteria and has undergone a bacterium-induced program of tissue differentiation, one component of which is a swelling of the epithelial cells that line the symbiont-containing crypts. While the luminescence (lux) genes of symbiotic V. fischeri have been shown to be highly induced within the crypts, the role of these genes in the initiation and persistence of the symbiosis has not been rigorously examined. We have constructed and examined three mutants (luxA, luxI, and luxR), defective in either luciferase enzymatic or regulatory proteins. All three are unable to induce normal luminescence levels in the host and, 2 days after initiating the association, had a three- to fourfold defect in the extent of colonization. Surprisingly, these lux mutants also were unable to induce swelling in the crypt epithelial cells. Complementing, in trans, the defect in light emission restored both normal colonization capability and induction of swelling. We hypothesize that a diminished level of oxygen consumption by a luciferase-deficient symbiotic population is responsible for the reduced fitness of lux mutants in the light organ crypts. This study is the first to show that the capacity for bioluminescence is critical for normal cell-cell interactions between a bacterium and its animal host and presents the first examples of V. fischeri genes that affect normal host tissue development.     

An autonomous N-terminal transactivation domain in Fos protein plays a crucial role in transformation.

To date, three functional domains have been defined in c-Fos and v-Fos proteins and have been shown to play a role in transactivation: the leucine zipper mediating hetero-dimerization, the basic DNA contact site, and a C-terminally located transactivation domain (C-TA) harbouring the HOB1 and HOB2 motifs. While the bZip region, consisting of the leucine zipper and the DNA contact site, is indispensable for transformation, the C-TA domain is not required and is actually altered by internal deletions in the FBR-MuSV. We now show that the N-terminal regions of c-Fos and v-Fos contain a second transactivation domain (N-TA). A functionally crucial motif within the N-TA domain, termed NTM, was pinpointed to a approximately 25 amino acid stretch around positions 60-84 which is highly conserved in FosB. Analysis of LexA fusion proteins showed that the N-TA domains of both c-Fos and FosB function in an autonomous fashion in both fibroblasts and yeast. Most importantly, deletion of the NTM motif impairs the transforming properties of v-Fos. Apart from the bZip region, the N-TA domain is the only functional domain required for transformation by v-Fos, at least when its expression is driven by the strong FBR-MuSV-LTR promoter.     

Myosin II and the Gal-GalNAc lectin play a crucial role in tissue invasion by Entamoeba histolytica

Entamoeba histolytica is the human parasite responsible of amoebiasis, during which highly motile trophozoites invade the intestinal epithelium leading to amoebic colitis, and disseminate via the blood circulation causing liver abscesses. The invasive process, central to the pathogenesis, is known to be driven by parasites motility. To investigate molecules responsible for in vivo motion, we performed a high resolution dynamic imaging analysis using two-photon laser scanning microscopy. Image analysis of the parasites during invasion of Caco-2 cell monolayers, an enterocyte-like model, and hamster liver shows that E. histolytica undergoes non-Brownian motion. However, studies of movements of parasite strains dominant negative for myosin II, a central component of the cytoskeleton, and for Gal-GalNAc lectin, a major adhesion molecule, indicate that myosin II is essential for E. histolytica intercellular motility through intestinal cell monolayers and for its motility in liver. In contrast, the Gal-GalNAc lectin exclusively triggers invasion of the liver. These observations are in agreement with emerging studies that highlight marked differences in the way that cells migrate in vitro in two dimensions versus in vivo in three dimensions. The approach that we have developed should be powerful to identify adhesive complexes required for in vivo cell migration in normal and pathogenic situations and may, thereby, lead to new therapeutic drug, for pathologies based on cell motility and adhesion.     

The ORF1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants

Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided in trans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.     

Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis

Plants are sessile organisms, and their ability to adapt to stress is crucial for survival in natural environments. Many observations suggest a relationship between stress tolerance and heat shock proteins (HSPs) in plants, but the roles of individual HSPs are poorly characterized. We report that transgenic Arabidopsis plants expressing less than usual amounts of HSP101, a result of either antisense inhibition or cosuppression, grew at normal rates but had a severely diminished capacity to acquire heat tolerance after mild conditioning pretreatments. The naturally high tolerance of germinating seeds, which express HSP101 as a result of developmental regulation, was also profoundly decreased. Conversely, plants constitutively expressing HSP101 tolerated sudden shifts to extreme temperatures better than did vector controls. We conclude that HSP101 plays a pivotal role in heat tolerance in Arabidopsis. Given the high evolutionary conservation of this protein and the fact that altering HSP101 expression had no detrimental effects on normal growth or development, one should be able to manipulate the stress tolerance of other plants by altering the expression of this protein.     

Chloroplastic and mitochondrial GPX genes play a critical role in rice development

Plant glutathione peroxidases (GPX) catalyze the reduction of H₂O₂ or organic hydroperoxides to water, mitigating the toxicity of these compounds to cells. In rice plants, the GPX gene family is composed of five members that are distributed in a range of sub-cellular compartments including cytosol, mitochondria, chloroplasts, or endoplasmic reticulum. Of these, OsGPX1 and OsGPX4 are located in mitochondria and chloroplasts, respectively. To understand the role of these GPX in rice, the effect of knockdown of OsGPX1 and OsGPX4 in rice plants was evaluated. Our data show that OsGPX4 was essential for in vitro rice regeneration because no plants were obtained from calli carrying a hairpin construct against OsGPX4. Although the knockdown of OsGPX1 did not impair plant regeneration, the plants with silenced OsGPX1 (GPX1s plants) showed reduced shoot length and a reduced number of seeds compared to the non-transformed rice plants. These results indicate that OsGPX1 and OsGPX4 are essential for redox homeostasis which leads to normal growth and development of rice.     

Unexpected crucial role of residue 272 in substrate specificity of fibroblast collagenase

Degradation of type I collagen by collagenases is an important part of extracellular remodeling. To understand the role of the hinge region of fibroblast collagenase in its collagenolytic activity, we individually substituted the 10 conserved amino acid residues at positions 264, 266, 268, 296, 272, 277, 284, 289, 307, and 313 in this region of the enzyme by their corresponding residues in MMP-3, a noncollagenolytic matrix metalloproteinase. The general proteolytic and triple helicase activities of all of the enzymes were determined, and their abilities to bind to type I collagen were assessed. Among the mutants, only G272D mutant enzyme exhibited a significant change in type I collagenolysis. The alteration of the Gly(272) to Asp reduced the collagenolytic activity of the enzyme to 13% without affecting its general proteolytic activity, substrate specificity, or the collagen binding ability. The catalytic efficiency of the G272D mutant for the triple helical peptide substrate [C(6)-(GP- Hyp)(4)GPL(Mca)GPQGLRGQL(DPN)GVR(GP-HYP)(4)-NH(2)](3) and the peptide substrate Mca-PLGL(Dpa)AR-NH(2) and its dissociation constant for the triple helical collagen were similar to that of the wild type enzyme, indicating that the presence of this residue in fibroblast collagenase is particularly important for the efficient cleavage of type I collagen. Gly(272) is evidently responsible for the hinge-bending motion that is essential for allowing the COOH-terminal domain to present the collagen to the active site.     

Membrane rafts play a crucial role in receptor activator of nuclear factor kappaB signaling and osteoclast function

Membrane lipid rafts play a key role in immune cell activation by recruiting and excluding specific signaling components of immune cell surface receptors upon the receptor engagement. Despite this, the role of these microdomains in the regulation of osteoclasts as controlled by receptor activator of nuclear factor kappaB (RANK) has yet to be established. In this study, we demonstrate that the raft microdomain expression plays an essential role in osteoclast function and differentiation. Expression of raft component flotillin greatly increased during osteoclast differentiation, whereas engagement of RANK induced the translocation of tumor necrosis factor receptor-associated factor 6 to rafts where Src was constitutively resident. Disruption of rafts blocked TRAF6 translocation and Akt activation by RANK ligand in osteoclasts and further reduced the survival of osteoclasts. Actin ring formation and bone resorption by osteoclasts were also found to require the integrity of rafts. Our observations demonstrate for the first time that RANK-mediated signaling and osteoclast function are critically dependent on the expression and integrity of raft membrane microdomains.