Artigos
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11847
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Item Identification and in silico analysis of the Citrus HSP70 molecular chaperone gene family(Genetics and Molecular Biology, 2007) Fietto, Luciano G.; Costa, Maximiller D. L.; Cruz, Cosme D.; Souza, Alessandra A.; Machado, Marcos A.; Fontes, Elizabeth P. B.The completion of the genome sequencing of the Arabidopsis thaliana model system provided a powerful molecular tool for comparative analysis of gene families present in the genome of economically relevant plant species. In this investigation, we used the sequences of the Arabidopsis Hsp70 gene family to identify and annotate the Citrus Hsp70 genes represented in the CitEST database. Based on sequence comparison analysis, we identified 18 clusters that were further divided into 5 subgroups encoding four mitochondrial mtHsp70s, three plastid csHsp70s, one ER luminal Hsp70 BiP, two HSP110/SSE-related proteins and eight cytosolic Hsp/Hsc70s. We also analyzed the expression profile by digital Northern of each Hsp70 transcript in different organs and in response to stress conditions. The EST database revealed a distinct population distribution of Hsp70 ESTs among isoforms and across the organs surveyed. The Hsp70-5 isoform was highly expressed in seeds, whereas BiP, mitochondrial and plastid HSp70 mRNAs displayed a similar expression profile in the organs analyzed, and were predominantly represented in flowers. Distinct Hsp70 mRNAs were also differentially expressed during Xylella infection and Citrus tristeza viral infection as well as during water deficit. This in silico study sets the groundwork for future investigations to fully characterize functionally the Citrus Hsp70 family and underscores the relevance of Hsp70s in response to abiotic and biotic stresses in Citrus.Item Genetic diversity of begomovirus infecting tomato and associated weeds in southeastern Brazil(Fitopatologia Brasileira, 2002-07) Ambrozevicius, Luciana P.; Calegario, Renata F.; Fontes, Elizabeth P. B.; Carvalho, Murilo G. de; Zerbini, F. MuriloThe genetic diversity of begomovirus isolates from tomato (Lycopersicon esculentum) fields in the Southeastern region of Brazil was analyzed by direct sequencing of PCR fragments amplified by using universal oligonucleotides for the begomovirus DNA-A, and subsequent computer-aided phylogenetic analysis. Samples of tomato plants and associated weeds showing typical symptoms of virus infection were collected at seven locations in the states of Minas Gerais, Espírito Santo and Rio de Janeiro. A total of 137 out of 369 samples were infected with a begomovirus based on PCR analysis. Phylogenetic analysis indicated a high degree of genetic diversity among begomoviruses infecting tomatoes in the sampled area. One species (Tomato chlorotic mottle virus, TCMV) occurs predominantly in Minas Gerais, whereas in Rio de Janeiro and Espírito Santo a distinct species, not yet fully characterized, predominates. Phylogenetic analysis further indicates the presence of an additional four possible new species. This high degree of genetic diversity suggests a recent transfer of indigenous begomovirus from wild hosts into tomatoes. The close phylogenetic relationship verified between begomovirus infecting tomato and associated weeds favors this hypothesis.Item Complete inventory of soybean NAC transcription factors: Sequence conservation and expression analysis uncover their distinct roles in stress response(Gene, 2009-09-01) Pinheiro, Guilherme L.; Marques, Carolina S.; Costa, Maximiller D.B.L.; Reis, Pedro A. B.; Alves, Murilo S.; Carvalho, Claudine M.; Fietto, Luciano G.; Fontes, Elizabeth P. B.We performed an inventory of soybean NAC transcription factors, in which 101 NAC domain-containing proteins were annotated into 15 different subgroups, showing a clear relationship between structure and function. The six previously described GmNAC proteins (GmNAC1 to GmNAC6) were located in the nucleus and a transactivation assay in yeast confirmed that GmNAC2, GmNAC3, GmNAC4 and GmNAC5 function as transactivators. We also analyzed the expression of the six NAC genes in response to a variety of stress conditions. GmNAC2, GmNAC3 and GmNAC4 were strongly induced by osmotic stress. GmNAC3 and GmNAC4 were also induced by ABA, JA and salinity but differed in their response to cold. Consistent with an involvement in cell death programs, the transient expression of GmNAC1, GmNAC5 and GmNAC6 in tobacco leaves resulted in cell death and enhanced expression of senescence markers. Our results indicate that the described soybean NACs are functionally non-redundant transcription factors involved in response to abiotic stresses and in cell death events in soybean.Item A PERK-Like receptor Kinase interacts with the geminivirus nuclear shuttle protein and potentiates viral infection(Journal of Virology, 2006-04-09) Florentino, Lilian H.; Santos, Anésia A.; Fontenelle, Mariana R.; Pinheiro, Guilherme L.; Zerbini, Francisco M.; Baracat-Pereira, Maria C.; Fontes, Elizabeth P. B.The nuclear shuttle protein (NSP) from bipartite geminiviruses facilitates the intracellular transport of viral DNA from the nucleus to the cytoplasm and acts in concert with the movement protein (MP) to promote the cell-to-cell spread of the viral DNA. A proline-rich extensin-like receptor protein kinase (PERK) was found to interact specifically with NSP of Cabbage leaf curl virus (CaLCuV) and of tomato-infecting geminiviruses through a yeast two-hybrid screening. The PERK-like protein, which we designated NsAK (for NSP-associated kinase), is structurally organized into a proline-rich N-terminal domain, followed by a transmembrane segment and a C-terminal serine/threonine kinase domain. The viral protein interacted stably with defective versions of the NsAK kinase domain, but not with the potentially active enzyme, in an in vitro binding assay. In vitro-translated NsAK enhanced the phosphorylation level of NSP, indicating that NSP functions as a substrate for NsAK. These results demonstrate that NsAK is an authentic serine/threonine kinase and suggest a functional link for NSP-NsAK complex formation. This interpretation was corroborated by in vivo infectivity assays showing that loss of NsAK function reduces the efficiency of CaLCuV infection and attenuates symptom development. Our data implicate NsAK as a positive contributor to geminivirus infection and suggest it may regulate NSP function.Item A New branch of endoplasmic reticulum stress signaling and the osmotic signal converge on Plant-specific Asparagine-rich proteins to promote cell death(The Journal of biological chemistry, 2008-05-19) Costa, Maximiller D. L.; Reis, Pedro A. B.; Valente, Maria Anete S.; Irsigler, André S. T.; Carvalho, Claudine M.; Loureiro, Marcelo E.; Aragão, Francisco J. L.; Boston, Rebecca S.; Fietto, Luciano G.; Fontes, Elizabeth P. B.NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene gly- col treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activa- tion of the UPR by ER stress inducers, but did not affect activa- tion of NRPs. We also found that this integrated pathway trans- duces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senes- cence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmen- tation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde pro- duction, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytoki- nin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.Item A naturally occurring recombinant DNA-A of a typical bipartite begomovirus does not require the cognate DNA-B to infect Nicotiana benthamiana systemically(Journal of General Virology, 2003-03-01) Galvao, Rafaelo M.; Mariano, Andrea C.; Luz, Dirce F.; Alfenas, Poliane F.; Andrade, Eduardo C.; Zerbini, Francisco M.; Almeida, Marcia R.; Fontes, Elizabeth P. B.Species of the genus Begomovirus (family Geminiviridae) found in the western hemisphere typically have a bipartite genome that consists of two 26 kb DNA genomic components, DNA-A and DNA-B. We have identified and cloned genomic components of a new tomato-infecting begomovirus from Brazil, for which the name Tomato crinkle leaf yellows virus (TCrLYV) is proposed, and a DNA-A variant of Tomato chlorotic mottle virus (ToCMV-[MG-Bt1]). Sequence analysis revealed that TCrLYV was most closely related to ToCMV, although it was sufficiently divergent to be considered a distinct virus species. Furthermore, these closely related viruses induce distinguishable symptoms in tomato plants. With respect to ToCMV-[MG-Bt1] DNA-A, evidence is presented that suggests a recombinant origin. It possesses a hybrid genome on which the replication compatible module (AC1 and replication origin) was probably donated by ToCMV- [BA-Se1] and the remaining sequences appear to have originated from Tomato rugose mosaic virus (ToRMV). Despite the high degree of sequence conservation with its predecessors, ToCMV-[MG- Bt1] differs significantly in its biological properties. Although ToCMV-[MG-Bt1] DNA-A did not infect tomato plants, it systemically infected Nicotiana benthamiana, induced symptoms of mottling and accumulated viral DNA in the apical leaves in the absence of a cognate DNA-B. The modular rearrangement that resulted in ToCMV-[MG-Bt1] DNA-A may have provided this virus with a more aggressive nature. Our results further support the notion that interspecies recombination may play a significant role in geminivirus diversity and their emergence as agriculturally important pathogens.Item Soybean chlorotic spot virus, a novel begomovirus infecting soybean in Brazil(Archives of Virology, 2012-10-10) Coco, Daniela; Calil, Iara P.; Brustolini, Otavio J. B.; Santos, Anesia A.; Inoue-Nagata, Alice K.; Fontes, Elizabeth P. B.A novel soybean-infecting begomovirus from Brazil was identified in Jaíba, in the state of Minas Gerais, and molecularly characterized. By using rolling-circle amplification-based cloning of viral DNAs, three DNA-A variants and a cognate DNA-B were isolated from infected samples. The DNA variants share more than 98 % sequence identity but have less than 89 % identity to other reported begomovirus, the limit for demarcation of new species. In a phylogenetic analysis, both DNA-A and DNA-B clustered with other Brazilian begomoviruses. Infectious cloned DNA-A and DNA-B components induced distinct symptoms in Solanaceae and Fabaceae species by biolistic inoculation. In soybean, the virus induced mild symptoms, i.e., chlorotic spots on the leaves, from which the name soybean chlorotic spot virus (SoCSV) was proposed. The most severe symptoms were displayed by common beans, which exhibited leaf distortion, blistering, interveinal chlorosis, mosaic and golden mosaic. The possibility that SoCSV may become a threat to bean production in Brazil is discussed.Item The soybean sucrose binding protein gene family: genomic organization, gene copy number and tissue‐specific expression of the SBP2 promoter(Journal of Experimental Botany, 2003-12-01) Contim, Luis Antônio S.; Waclawovsky, Alessandro J.; Delú‐Filho, Nelson; Pirovani, Carlos P.; Clarindo, Wellington R.; Loureiro, Marcelo E.; Carvalho, Carlos R.; Fontes, Elizabeth P. B.The sucrose binding protein (SBP) from soybean has been implicated as an important component of the sucrose uptake system. Two SBP genomic clones, gsS641.1 and gsS641.2, which correspond to allelic forms of the GmSBP2/S64 gene, have been isolated and characterized. As a member of the seed storage protein superfamily, it has been shown that the SBP gene structure is similar to vicilin genes with intron/exon boundaries at conserved positions. Fluores cence in situ hybridization (FISH) suggested that the soybean SBP gene family is represented by at least two non‐allelic genes corresponding to the previously isolated GmSBP1 and GmSBP2/S64 cDNAs. These two cDNAs share extensive sequence similarity but are located at different loci in the soybean genome. To investigate transcriptional activation of the GmSBP2 gene, 2 kb 5′‐flanking sequences of gsS641.1 and gsS641.2 were fused to the β‐glucuronidase (GUS) reporter gene and to the green fluorescent protein (GFP) reporter gene and inde pendently introduced into Nicotiana tabacum by Agrobacterium tumefaciens‐mediated transformation. The SBP2 promoter directed expression of both GUS and GFP reporter genes with high specificity to the phloem of leaves, stems and roots. Thus, the overall pattern of SBP–GUS or SBP–GFP expression is consistent with the involvement of SBP in sucrose translocation‐dependent physiological processes.Item GmNAC30 and GmNAC81 integrate the endoplasmic reticulum stress- and osmotic stress-induced cell death responses through a vacuolar processing enzyme(PNAS, 2013-09-30) Mendes, Giselle C.; Reis, Pedro A. B.; Calil, Iara P.; Carvalho, Humberto H.; Aragão, Francisco J. L.; Fontes, Elizabeth P. B.Prolonged endoplasmic reticulum and osmotic stress synergistically activate the stress-induced N-rich protein-mediated signaling that transduces a cell death signal by inducing GmNAC81 (GmNAC6) in soybean. To identify novel regulators of the stress-induced programmed cell death (PCD) response, we screened a two-hybrid library for partners of GmNAC81. We discovered another member of the NAC (NAM-ATAF1,2-CUC2) family, GmNAC30, which binds to GmNAC81 in the nucleus of plant cells to coordinately regulate common target promoters that harbor the core cis-regulatory element TGTG[TGC]. We found that GmNAC81 and GmNAC30 can function either as transcriptional repressors or activators and cooperate to enhance the transcriptional regulation of common target promoters, suggesting that heterodimerization may be required for the full regulation of gene expression. Accordingly, GmNAC81 and GmNAC30 display overlapping expression profiles in response to multiple environmental and developmental stimuli. Consistent with a role in PCD, GmNAC81 and GmNAC30 bind in vivo to and transactivate hydrolytic enzyme promoters in soybean protoplasts. A GmNAC81/GmNAC30 binding site is located in the promoter of the caspase-1–like vacuolar processing enzyme (VPE) gene, which is involved in PCD in plants. We demonstrated that the expression of GmNAC81 and GmNAC30 fully transactivates the VPE gene in soybean protoplasts and that this transactivation was associated with an increase in caspase-1–like activity. Collectively, our results indicate that the stress-induced GmNAC30 cooperates with GmNAC81 to activate PCD through the induction of the cell death executioner VPE.Item NSP-interacting kinase, NIK: a transducer of plant defence signalling(Journal of Experimental Botany, 2010-06-24) Santos, Anésia A.; Lopes, Kênia V. G.; Apfata, Jorge A. C.; Fontes, Elizabeth P. B.The NSP-interacting kinase, NIK, belongs to the five leucine-rich repeats-containing receptor-like serine/threonine kinase subfamily that includes members involved in plant development and defence. NIK was first identified by its capacity to interact with the geminivirus nuclear shuttle protein (NSP) and has been strongly associated with plant defence against geminivirus. Recent studies corroborate its function in transducing a defence signal against virus infection and describe components of the NIK-mediated antiviral signalling pathway. This mini-review describes the role of NIK as a transducer of a novel layer of plant innate defence, presents new data on NIK function, and discusses its possible involvement in plant development.