Geminivirus C3 protein: replication enhancement and protein interactions

Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants.     

Loss of protein kinase C delta alters mammary gland development and apoptosis

As apoptotic pathways are commonly deregulated in breast cancer, exploring how mammary gland cell death is regulated is critical for understanding human disease. We show that primary mammary epithelial cells from protein kinase C delta (PKCδ) −/− mice have a suppressed response to apoptotic agents in vitro. In the mammary gland in vivo, apoptosis is critical for ductal morphogenesis during puberty and involution following lactation. We have explored mammary gland development in the PKCδ −/− mouse during these two critical windows. Branching morphogenesis was altered in 4- to 6-week-old PKCδ −/− mice as indicated by reduced ductal branching; however, apoptosis and proliferation in the terminal end buds was unaltered. Conversely, activation of caspase-3 during involution was delayed in PKCδ −/− mice, but involution proceeded normally. The thymus also undergoes apoptosis in response to physiological signals. A dramatic suppression of caspase-3 activation was observed in the thymus of PKCδ −/− mice treated with irradiation, but not mice treated with dexamethasone, suggesting that there are both target- and tissue-dependent differences in the execution of apoptotic pathways in vivo. These findings highlight a role for PKCδ in both apoptotic and nonapoptotic processes in the mammary gland and underscore the redundancy of apoptotic pathways in vivo.     

Overproduction of OsSLRL2 alters the development of transgenic Arabidopsis plants

SLR1 (SLENDER RICE 1) was thought to be the sole DELLA protein in rice considering the constitutive GA response phenotype of slr1 mutants. There were two other SLR1 homologous SLRL1 and SLRL2 (SLR1 like 1 and 2) which did not have DELLA domain but still shared high level similarity to the C-terminal region of SLR1 found after searching the whole rice genome. SLRL2 specially expressed in the embryo of immature rice seeds and the expression of SLRL2 was increased when treated with GA(3). The SLRL2 over-expressed transgenic Arabidopsis plants were semi-dwarfed, late flowering, and insensitive to GA. Moreover, the expression of AtGA20ox1 and AtGA3ox1 was increased and the expression of AtGA2ox1 decreased, indicating SLRL2 was a repressor of GA signaling. We suggested SLRL2 might function to overcome too strong GA responses and maintained a basic repression. Furthermore, a different form of DELLA family in monocots against dicots was discussed.     

The Roles of Phosphorylation and SHAGGY-Like Protein Kinases in Geminivirus C4 Protein Induced Hyperplasia

Even though plant cells are highly plastic, plants only develop hyperplasia under very specific abiotic and biotic stresses, such as when exposed to pathogens like Beet curly top virus (BCTV). The C4 protein of BCTV is sufficient to induce hyperplasia and alter Arabidopsis development. It was previously shown that C4 interacts with two Arabidopsis Shaggy-like protein kinases, AtSK21 and 23, which are negative regulators of brassinosteroid (BR) hormone signaling. Here we show that the C4 protein interacts with five additional AtSK family members. Bikinin, a competitive inhibitor of the seven AtSK family members that interact with C4, induced hyperplasia similar to that induced by the C4 protein. The Ser49 residue of C4 was found to be critical for C4 function, since: 1) mutagenesis of Ser49 to Ala abolished the C4-induced phenotype, abolished C4/AtSK interactions, and resulted in a mutant protein that failed to induce changes in the BR signaling pathway; 2) Ser49 is phosphorylated in planta; and 3) plant-encoded AtSKs must be catalytically active to interact with C4. A C4 N-myristoylation site mutant that does not localize to the plasma membrane and does not induce a phenotype, retained the ability to bind AtSKs. Taken together, these results suggest that plasma membrane associated C4 interacts with and co-opts multiple AtSKs to promote its own phosphorylation and activation to subsequently compromise cell cycle control.     

Geminivirus C2 protein might be the key player for geminiviral co-option of SCF-mediated ubiquitination

Viruses are obligate intracellular parasites, and need to create a suitable cell environment for viral propagation to complete their life cycle. In order to achieve this, viruses must usurp or interfere with the cellular machinery. Ubiquitination, a post-translational modification that controls numerous cellular processes, has proven to be a common target for viruses. Recently, geminivirus C2 protein has been shown to interact with the CSN complex and disrupt its activity over CULLIN1, interfering with the function of the CULLIN1-based SCF ubiquitin E3 ligases. Interestingly, over-expression of a given F-box protein may circumvent the general SCF malfunction caused by C2. This result raises the tantalizing idea that geminiviruses might be not only hampering, but also redirecting the activity of SCF complexes, thus co-opting the SCF-mediated ubiquitination pathway. We hypothesize that the mechanism of C2-facilitated co-option of SCF-mediated ubiquitination might not be exclusive for geminiviruses, but rather a common strategy for viruses.Key words: geminivirus, C2 protein, ubiquitination, SCF complex, CSN complex, F-box protein, pathogen co-option     

Overexpression of Arabidopsis ACK1 alters leaf morphology and retards growth and development

Cyclin dependent kinases (CDKs) play important roles in the plant cell cycle, a highly coordinated process in plant growth and development. To understand the regulatory network involving the CDKs, we have examined the role of ACK1, a gene that has significant homology to known ICKs (inhibitors of CDKs), but occupies a distinct branch of the ICK phylogenetic tree. Overexpression of ACK1 in transgenic Arabidopsis significantly inhibited growth, leading to effects such as serration of leaves, as a result of strong inhibition of cell division in the leaf meristem. ACK1 transgenic plants also differed morphologically from control Arabidopsis plants, and the cells of ACK1 transgenics were more irregular than the corresponding cells of control plants. These results suggest that ACK1 acts as a CDK inhibitor in Arabidopsis, and that the alterations in leaf shape may be the result of restricted cell division.     

DVL, a novel class of small polypeptides: overexpression alters Arabidopsis development

Small polypeptides can act as important regulatory molecules that coordinate cellular responses required for differentiation, growth, and development. In a gain-of-function genetic screen for genes that influence fruit development in Arabidopsis, we identified a novel gene -DEVIL1 (DVL1) - encoding a small protein. Overexpression of DVL1 results in pleiotropic phenotypes featured by shortened stature, rounder rosette leaves, clustered inflorescences, shortened pedicles, and siliques with pronged tips. cDNA analysis indicates that DVL1 has a 153-nucleotide (nt) open-reading frame (ORF) encoding a 51-amino acid polypeptide that shares no significant similarity to previously identified proteins. Sequence alignment shows that DVL1 belongs to a family of related genes that are limited to angiosperm plants. Ectopic overexpression of each of the five closely related Arabidopsis DVL genes causes similar phenotypic changes, suggesting overlapping function in the DVL gene family. Point mutations of conserved amino acids in the C-terminal region of the DVL1 polypeptide reveal that these conserved residues are required for DVL1-overexpression phenotypes. Our results show that the DVL family is a novel class of small polypeptides and the overexpression phenotypes suggest that these polypeptides may have a role in plant development.     

Geminivirus C4 proteins inhibit GA signaling via prevention of NbGAI degradation,to promote viral infection and symptom development in N. benthamiana

The phytohormone gibberellin (GA) is a vital plant signaling molecule that regulates plant growth and defense against abiotic and biotic stresses. To date, the molecular mechanism of the plant responses to viral infection mediated by GA is still undetermined. DELLA is a repressor of GA signaling and is recognized by the F-box protein, a component of the SCF^SLY1/GID2 complex. The recognized DELLA is degraded by the ubiquitin-26S proteasome, leading to the activation of GA signaling. Here, we report that ageratum leaf curl Sichuan virus (ALCScV)-infected N. benthamiana plants showed dwarfing symptoms and abnormal flower development. The infection by ALCScV significantly altered the expression of GA pathway-related genes and decreased the content of endogenous GA in N. benthamiana. Furthermore, ALCScV-encoded C4 protein interacts with the DELLA protein NbGAI and interferes with the interaction between NbGAI and NbGID2 to prevent the degradation of NbGAI, leading to inhibition of the GA signaling pathway. Silencing of NbGAI or exogenous GA₃ treatment significantly reduces viral accumulation and disease symptoms in N. benthamiana plants. The same results were obtained from experiments with the C4 protein encoded by tobacco curly shoot virus (TbCSV). Therefore, we propose a novel mechanism by which geminivirus C4 proteins control viral infection and disease symptom development by interfering with the GA signaling pathway.     

C4 protein of Beet severe curly top virus is a pathomorphogenetic factor in Arabidopsis

The Curtovirus C4 protein is required for symptom development during infection of Arabidopsis. Transgenic Arabidopsis plants expressing C4 from either Beet curly top virus or Beet severe curly top virus produced phenotypes that were similar to symptoms seen during infection with wild-type viruses. The pseudosymptoms caused by C4 protein alone were novel to transgenic Arabidopsis and included bumpy trichomes, severe enations, disorientation of vascular bundles and stomata, swelling, callus-like structure formation, and twisted siliques. C4 induced abnormal cell division and altered cell fate in a variety of tissues depending on the C4 expression level. C4 protein expression increased the expression levels of cell-cycle-related genes CYCs, CDKs and PCNA, and suppressed ICK1 and the retinoblastoma-related gene RBR1, resulting in activation of host cell division. These results suggest that the Curtovirus C4 proteins are involved actively in host cell-cycle regulation to recruit host factors for virus replication and symptom development.     

Low dietary protein during early pregnancy alters bovine placental development

To determine if low dietary protein concentration in the first two trimesters of pregnancy alters placental development, genetically similar heifers from closed herd were fed diets containing different levels of protein in the first and second trimesters of gestation. There were four animals per treatment group, the groups being: L/L = fed a diet containing 7% crude protein (CP) (low protein) in the first and second trimesters; H/H = fed a diet containing 14% CP (high protein) in the first and second trimesters; L/H = fed low protein in the first trimester and high in the second trimester and vice versa for the H/L group. Low protein diets in the first trimester increased dry cotyledon weight at term. Trophectoderm' volume density increased in the H/L and L/H group compared to the L/L and H/H groups. Blood vessel volume and volume density in foetal villi decreased in the H/L and L/H groups compared with the H/H and L/L groups. There was no effect of diet treatment on cotyledon number, diameter or wet weight and no effect on the volume density of connective tissue or fibroblasts in the foetal villi. These results show that a low dietary protein concentration in the first trimester of pregnancy followed by increased protein in the second trimester enhanced placental development. Further, trophectoderm volume was highly correlated with birth weight. Early protein restriction in the pregnant cow may enhance foetal growth in part by stimulating placental growth and function.     

The lack of mitochondrial AtFtsH4 protease alters Arabidopsis leaf morphology at the late stage of rosette development under short-day photoperiod

AtFtsH4 is one of four inner membrane-bound mitochondrial ATP-dependent metalloproteases in Arabidopsis thaliana , called AAA proteases, whose catalytic site is exposed to the intermembrane space. In the present study, we used a reverse-genetic approach to investigate the physiological role of the AtFtsH4 protease. We found that loss of AtFtsH4 did not significantly affect Arabidopsis growth under optimal conditions (long days); however, severe morphological and developmental abnormalities in late rosette development occurred under short-day conditions. The asymmetric shape and irregular serration of expanding leaf blades were the most striking features of the ftsh4 mutant phenotype. The severe abnormal morphology of the leaf blades was accompanied by ultrastructural changes in mitochondria and chloroplasts. These abnormalities correlated with elevated levels of reactive oxygen species and carbonylated mitochondrial proteins. We found that two classes of molecular chaperones, Hsp70 and prohibitins, were over-expressed in ftsh4 mutants during late vegetative growth under both short- and long-day conditions. Taken together, our data indicate that lack of AtFtsH4 results in impairment of organelle development and Arabidopsis leaf morphology under short-day conditions.     

Tissue-specific expression of stabilized SOLITARY-ROOT/IAA14 alters lateral root development in Arabidopsis

Auxin is important for lateral root (LR) initiation and subsequent LR primordium development. However, the roles of tissue-specific auxin signaling in these processes are poorly understood. We analyzed transgenic Arabidopsis plants expressing the stabilized mutant INDOLE-3 ACETIC ACID 14 (IAA14)/SOLITARY-ROOT (mIAA14) protein as a repressor of the auxin response factors (ARFs), under the control of tissue-specific promoters. We showed that plants expressing the mIAA14-glucocorticoid receptor (GR) fusion protein under the control of the native IAA14 promoter had the solitary-root/iaa14 mutant phenotypes, including the lack of LR formation under dexamethasone (Dex) treatment, indicating that mIAA14-GR is functional in the presence of Dex. We then demonstrated that expression of mIAA14-GR under the control of the stele-specific SHORT-ROOT promoter suppressed LR formation, and showed that mIAA14-GR expression in the protoxylem-adjacent pericycle also blocked LR formation, indicating that the normal auxin response mediated by auxin/indole-3 acetic acid (Aux/IAA) signaling in the protoxylem pericycle is necessary for LR formation. In addition, we demonstrated that expression of mIAA14-GR under either the ARF7 or the ARF19 promoter also suppressed LR formation as in the arf7 arf19 double mutants, and that IAA14 interacted with ARF7 and ARF19 in yeasts. These results strongly suggest that mIAA14-GR directly inactivates ARF7/ARF19 functions, thereby blocking LR formation. Post-embryonic expression of mIAA14-GR under the SCARECROW promoter, which is expressed in the specific cell lineage during LR primordium formation, caused disorganized LR development. This indicates that normal auxin signaling in LR primordia, which involves the unknown ARFs and Aux/IAAs, is necessary for the establishment of LR primordium organization. Thus, our data show that tissue-specific expression of a stabilized Aux/IAA protein allows analysis of tissue-specific auxin responses in LR development by inactivating ARF functions.     

Constitutive activation of a CC-NB-LRR protein alters morphogenesis through the cytokinin pathway in Arabidopsis

Possible links between plant defense responses and morphogenesis have been postulated, but their molecular nature remains unknown. Here, we introduce the Arabidopsis semi-dominant mutant uni-1D with morphological defects. UNI encodes a coiled-coil nucleotide-binding leucine-rich-repeat protein that belongs to the disease resistance (R) protein family involved in pathogen recognition. The uni-1D mutation causes the constitutive activation of the protein, which is stabilized by the RAR1 function in a similar way as in other R proteins. The uni-1D mutation induces the upregulation of the Pathogenesis-related gene via the accumulation of salicylic acid, and evokes some of the morphological defects through the accumulation of cytokinin. The rin4 knock-down mutation, which causes the constitutive activation of two R proteins, RPS2 and RPM1, induces an upregulation of cytokinin-responsive genes and morphological defects similar to the uni-1D mutation, indicating that the constitutive activation of some R proteins alters morphogenesis through the cytokinin pathway. From these data, we propose that the modification of the cytokinin pathway might be involved in some R protein-mediated responses.     

拟南芥非特异性磷脂酶C4基因表达模式的研究

拟南芥非特异性磷脂酶C4（AtNPC4）具有降解磷脂酰胆碱（PC）,产生二酰甘油（DAG）和磷酸胆碱的活性。本研究从拟南芥基因组中分离了NPC4基因起始密码子上游1 379bp的启动子序列,与GUS报告基因融合后转化拟南芥,获得转基因植株。GUS组织化学染色表明,AtNPC4基因主要在处于衰老过程中的叶片中高水平表达,在根、茎、种荚和花中也有一定程度的表达,这种表达模式与RT-PCR结果相一致。另外,通过RT-PCR发现,AtNPC4基因在转录水平上受脱落酸的诱导,但不受水杨酸和茉莉素诱导。     

Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate-limiting steps in GA plant hormone biosynthesis that control plant development. With an opposite effect, overexpression of pumpkin seed-specific GA 20-oxidase1 (CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced levels of GA4 and increased levels of physiological inactive GA17 and GA25 and unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype was accompanied by a considerable decrease in the levels of bioactive GA4 and an increase in the corresponding inactivation product GA34 in comparison to control plants. In this study, we demonstrate the potential of four pumpkin GA oxidase-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.