FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex

P>The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl-truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp citri

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and spray-inoculated with an Xcc suspension (10(6) cfu mL(-1)). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (non-transgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

Evaluation of citrus somatic hybrids for tolerance to Phytophthora nicotianae and citrus tristeza virus

Somatic hybridization is a biotechnology tool that can be used in citrus breeding programs to produce somatic hybrids with the complete genetic combination of both parents. The goal of this work was to test the reaction of citrus somatic hybrids that may be useful as rootstocks to trunk and root infections caused by Phytophthora nicotianae van Breda de Haan (P parasitica Dastur) and to citrus tristeza virus (CTV). The somatic hybrids evaluated were `Caipira` sweet orange (Citrus sinensis L. Osbeck) + `Rangpur` lime (C. limonia Osbeck), `Caipira` sweet orange + `Cleopatra` mandarin (C. reshni hort. ex Tanaka), `Caipira` sweet orange + `Volkamer` lemon (C. volkameriana V Ten. & Pasq.), `Caipira` sweet orange + rough lemon (C. jambhiri Lush.), `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange (C. aurantium L.), `Rangpur` lime + `Sunki` mandarin (C. sunki (Hayata) hort. ex Tanaka), `Ruby Blood` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, `Rohde Red` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, and `Valencia` sweet orange + Fortunella obovata hort. ex Tanaka. For P. nicotianae trunk and root infection assays, plants of the somatic hybrids, obtained from 9-month semi-hardwood cuttings, were evaluated and compared with diploid citrus rootstock cultivars after mycelia inoculation in the trunk or spore infestation in the substrate, respectively. `Cleopatra` mandarin + sour orange, `Rangpur` lime + `Sunki` mandarin, `Cleopatra` mandarin + `Volkamer` lemon, `Ruby Blood` sweet orange + `Volkamer` lemon, `Rohde Red` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Volkamer` lemon had less trunk rot occurrence, whereas the somatic hybrids `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange, `Caipira` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Rangpur` lime were tolerant to root rot. For CTV assays, plants of the somatic hybrids along with tolerant and intolerant rootstocks were budded with a mild strain CTV-infected or healthy `Valencia` sweet orange budwood. Differences in average scion shoot length indicated that the hybrids `Cleopatra` mandarin + sour orange and `Valencia` sweet orange + Fortunella obovata were intolerant to CTV (c) 2007 Elsevier B.V. All rights reserved.

Biochemistry of the Envenomation Response-a Generator Theme for Interdisciplinary Integration

The understanding of complex physiological processes requires information from many different areas of knowledge. To meet this interdisciplinary scenario, the ability of integrating and articulating information is demanded. The difficulty of such approach arises because, more often than not, information is fragmented through under graduation education in Health Sciences. Shifting from a fragmentary and deep view of many topics to joining them horizontally in a global view is not a trivial task for teachers to implement. To attain that objective we proposed a course herein described Biochemistry of the envenomation response aimed at integrating previous contents of Health Sciences courses, following international recommendations of interdisciplinary model. The contents were organized by modules with increasing topic complexity. The full understanding of the envenoming pathophysiology of each module would be attained by the integration of knowledge from different disciplines. Active-learning strategy was employed focusing concept map drawing. Evaluation was obtained by a 30-item Likert-type survey answered by ninety students; 84% of the students considered that the number of relations that they were able to establish as seen by concept maps increased throughout the course. Similarly, 98% considered that both the theme and the strategy adopted in the course contributed to develop an interdisciplinary view.

A mosaic of beach bean (Canavalia rosea) caused by an isolate of Cowpea aphid-borne mosaic virus (CABMV) in Brazil

Beach bean (Canavalia rosea) plants showing mosaic symptoms were found at Massaguacu beach, Caraguatatuba, Brazil. A potyvirus was found to be responsible for the symptoms, based on transmission assays and electron microscopy. A positive reaction in ELISA was obtained against cowpea aphid-borne mosaic (CABMV) antisera. Viral identity was confirmed by RT-PCR using specific primers to amplify part of the NIb and the entire CP coding region of the genome and the 3`NTR. Comparison of the amplified sequences with that of CABMV showed a nucleotide sequence identity of 97% for the CP coding region. Thus, the potyvirus from beach bean should be considered a CABMV isolate, referred to as CABMV-Cr.

Identification and partial characterization of a Carica papaya-infecting isolate of Alfalfa mosaic virus in Brazil

A Carica papaya plant with severe yellow leaf mosaic, leaf distortion, and systemic necrosis was found in the municipality of Piracicaba, state of So Paulo, Brazil. Transmission electron microscopy (TEM) analysis revealed the presence of potyvirus-like particles and bacilliform particles similar to those of the Alfamovirus genus. The potyvirus was identified as Papaya ringspot virus-type P (PRSV-P). Biological, serological, and molecular studies confirmed the bacilliform virus as an isolate of Alfalfa mosaic virus (AMV). Partial nucleotide and amino acid sequences of the coat protein gene of this AMV isolate shared 97-98% identity with the AMV isolates in the GenBank database. This report is the first of the natural infection of papaya plants by AMV.

Evaluation of the genetic variability of orchid fleck virus by single-strand conformational polymorphism analysis and nucleotide sequencing of a fragment from the nucleocapsid gene

The variability of a fragment of the nucleocapsid gene of orchid fleck virus (OFV) was investigated by single-strand conformational polymorphism (SSCP) analysis and nucleotide sequencing. Forty-eight samples of 18 genera of orchids were collected from Brazil, Costa Rica and Australia. The SSCP analysis yielded six different band patterns, and phylogenetic analysis based on the nucleotide fragment sequence obtained in this work and six available in GenBank showed two different groups, one with isolates 023Germany and So-Japan, and other with the rest of the isolates. None of the analyses showed geographic correlation among the Brazilian strains. The data obtained in this study showed a low genetic variation in this region of the genome; the d(N)/d(S) ratio of 0.251-0.405 demonstrated a negative selective pressure that maintains the stability of the analyzed fragments.

Semliki Forest virus as an expression vector in insect cell lines

Studies were undertaken to determine if replication-deficient Semliki Forest virus expression vectors could be successfully used to express foreign gene constructs in insect cell lines. Using green fluorescent protein (GFP) as a marker we recorded infection levels of nearly 100% in the Aedes albopictus cell lines C6/36 and Aa23T, as well as in the Ae. aegypti cell line MOS20. The virus was capable of infecting an Anopheles gambiae cell line MOS55. The amount of GFP protein produced in each cell line was quantified. Northern analysis of viral transcription revealed the presence of novel transcripts in Aa23T, C6/36, and MOS55 cell lines, but not in the BHK or MOS20. The initial characterization of these transcripts is described.

The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family

The complete nucleotide sequence of the genomic RNA from the insect picorna-like virus Drosophila C virus (DCV) was determined. The DCV sequence predicts a genome organization different to that of other RNA virus families whose sequences are known. The single-stranded positive-sense genomic RNA is 9264 nucleotides in length and contains two large open reading frames (ORFs) which are separated by 191 nucleotides. The 5' ORF contains regions of similarities with the RNA-dependent RNA polymerase, helicase and protease domains of viruses from the picornavirus, comovirus and sequivirus families. The 3' ORF encodes the capsid proteins as confirmed by N-terminal sequence analysis of these proteins. The capsid protein coding region is unusual in two ways: firstly the cistron appears to lack an initiating methionine and secondly no subgenomic RNA is produced, suggesting that the proteins may be translated through internal initiation of translation from the genomic length RNA. The finding of this novel genome organization for DCV shows that this virus is not a member of the Picornaviridae as previously thought, but belongs to a distinct and hitherto unrecognized virus family.

Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway

To evaluate an antigen delivery system in which exogenous antigen can target the major histocompatibility complex (MHC) class I pathway, a single human papillomavirus (HPV) 16 E7 cytotoxic T lymphocyte (CTL) epitope and a single HIV gp160 CTL epitope were separately fused to the C-terminus or bovine papillomavirus 1 (BPV1) L1 sequence to form hybrid BPV1L1 VLPs. Mice immunized with these hybrid VLPs mounted strong CTL responses against the relevant target cells in the absence of any adjuvants. In addition, the CTL responses induced by immunization with BPV1L1/HPV16E7CTL VLPs protected mice against challenge with E7-transformed tumor cells. Furthermore, a high titer-specific antibody response against BPV1L1 VLPs was also induced, and this antiserum could inhibit papillomavirus-induced agglutination of mouse erythrocytes, suggesting that the antibody may recognize conformational determinates relevant to virus neutralization. These data demonstrate that hybrid BPV1L1 VLPs can be used as carriers to target antigenic epitopes to both the MHC class I and class II pathways, providing a promising strategy for the design of vaccines to prevent virus infection, with the potential to elicit therapeutic virus-specific CTL responses. (C) 1998 Academic Press.

Antibody responses and protective immunity to recombinant vaccinia virus-expressed bluetongue virus antigens

The role of individual viral proteins in the immune response to bluetongue virus (BTV) is not clearly understood. To investigate the contributions of the outer capsid proteins, VP2 and VP5, and possible interactions between them, these proteins were expressed from recombinant vaccinia viruses either as individual proteins or together in double recombinants, or with the core protein VP7 in a triple recombinant. Comparison of the immunogenicity of the vaccinia expressed proteins with BTV expressed proteins was carried out by inoculation of rabbits and sheep. Each of the recombinants was capable of stimulating an anti-BTV antibody response, although there was a wide range in the level of response between animals and species. Vaccinia-expressed VP2 was poorly immunogenic, particularly in rabbits. VP5, on the whole, stimulated higher ELISA titers in rabbits and sheep and in some animals in both species was able to stimulate virus neutralizing antibodies. When the protective efficacy of VP2 and VP5 was tested in sheep, vaccinia-expressed VP2, VP5 and VP2 + VP5 were protective, with the most consistent protection being in groups immunized with both proteins. (C) 1997 Elsevier Science B.V.