Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus. The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome. Here, we analyze different putative roles of the VirE2 protein in the plant cell. In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated. We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plant cell nucleus. Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2. In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent. We conclude that the VirE2 protein of A. tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.

Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens.

The virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. However, it is not clear how the phenolic compounds are sensed by the VirA/VirG system. We tested the vir-inducing abilities of 15 different phenolic compounds using four wild-type strains of A. tumefaciens--KU12, C58, A6, and Bo542. We analyzed the relationship between structures of the phenolic compounds and levels of vir gene expression in these strains. In strain KU12, vir genes were not induced by phenolic compounds containing 4'-hydroxy, 3'-methoxy, and 5'-methoxy groups, such as acetosyringone, which strongly induced vir genes of the other three strains. On the other hand, vir genes of strain KU12 were induced by phenolic compounds containing only a 4'-hydroxy group, such as 4-hydroxyacetophenone, which did not induce vir genes of the other three strains. The vir genes of strains KU12, A6, and Bo542 were all induced by phenolic compounds containing 4'-hydroxy and 3'-methoxy groups, such as acetovanillone. By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic-sensing determinant is associated with Ti plasmid. Subcloning of Ti plasmid indicates that the virA locus determines which phenolic compounds can function as vir gene inducers. These results suggest that the VirA protein directly senses the phenolic compounds for vir gene activation.

Agrobacterium-mediated genetic transformation of 'Hamlin' sweet orange

The development and optimization of efficient transformation protocols is essential in new citrus breeding programs, not only for rootstock, but also for scion improvement. Transgenic 'Hamlin' sweet orange (Citrus sinensis (L.) Osbeck) plants were obtained by Agrobacterium tumefaciens-mediated transformation of epicotyl segments collected from seedlings germinated in vitro. Factors influencing genetic transformation efficiency were evaluated including seedling incubation conditions, time of inoculation with Agrobacterium and co-culture conditions. Epicotyl segments were adequate explants for transformation, regenerating plants by direct organogenesis. Higher percentage of transformation was obtained with explants collected from seedlings germinated in darkness, transferred to 16 hours photoperiod for 2-3 weeks, and inoculated with Agrobacterium for 15-45 min. The best co-culture condition was the incubation of the explants in darkness, for three days in culture medium supplemented with 100 muM of acetosyringone. Genetic transformation was confirmed by performing beta-glucoronidase (GUS) assays and, subsequently, by PCR amplification for the nptII and GUS genes.

Biologia molecular do processo de infecção por Agrobacterium spp.

Agrobacterium tumefaciens é o agente causal da galha-da-coroa, doença que afeta a maioria das plantas dicotiledôneas e caracteriza-se pelo crescimento de tumores na junção entre o caule e a raiz (coroa). A formação desses tumores é o resultado de um processo natural de transferência de genes de Agrobacterium spp. para o genoma da planta infetada. Esses genes estão contidos em um plasmídio de alto peso molecular (120 a 250 kb), denominado Ti ("tumor inducing"), presente em todas as linhagens patogênicas de Agrobacterium spp. Duas regiões do plasmídio Ti estão diretamente envolvidas na indução do tumor: a região-T, que corresponde ao segmento de DNA transferido para a célula vegetal, e a região de virulência (região vir), que contém genes envolvidos na síntese de proteínas responsáveis pelo processo de transferência da região-T. Esta região, uma vez transferida e integrada no genoma da célula vegetal, passa a ser denominada de T-DNA ("transferred DNA"). Os genes presentes no T-DNA codificam enzimas envolvidas na via de biossíntese de reguladores de crescimento, auxinas e citocininas. A síntese desses reguladores pelas células transformadas causa um desbalanço hormonal, levando à formação do tumor no local da infecção. Outro grupo de genes presentes no T-DNA codifica enzimas responsáveis pela síntese de opinas, que são catabolisadas especificamente pela bactéria colonizadora, como fonte de nutrientes. O conhecimento preliminar das bases moleculares envolvidas no processo de infecção de uma planta hospedeira por Agrobacterium spp., permitiu a utilização desta bactéria como vetor natural de transformação genética de plantas.

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. (C) 2011 Elsevier Inc. All rights reserved.

Transformação genética de embriões somáticos de soja [Glycine max (L.) Merr.] utilizando o bombardeamento e sistema Agrobacterium de maneira integrada

O objetivo do presente trabalho foi otimizar o sistema de transformação genética de embriões somáticos de soja [Glycine max (L.) Merr.] utilizando a biolística e o sistema Agrobacterium de maneira integrada. Os antibióticos, adicionados ao meio de cultura para supressão da bactéria após a transferência do transgene, foram o alvo do estudo. Inicialmente, comparou-se o efeito de diferentes tratamentos com antibióticos sobre o tecido embriogênico de soja e sua eficiência na supressão da linhagem LBA4404 de Agrobacterium tumefaciens durante o processo de transformação. A carbenicilina (500 mg/l) apresentou efeitos diferentes sobre o tecido vegetal das duas cultivares testadas. Os tecidos embriogênicos da cv. IAS5 não apresentaram diferenças significativas em relação ao controle, enquanto que a proliferação dos embriões somáticos da cv. Bragg foi três vezes maior com a adição deste antibiótico ao meio de cultura. Contudo, a presença da carbenicilina nas duas concentrações testadas (500 e 1000 mg/l) não foi eficiente para supressão de Agrobacterium. Por outro lado, nos tratamentos com cefotaxima sozinha (350 e 500 mg/l), ou cefotaxima (250 mg/l) + vancomicina (250 mg/l) esta bactéria foi completamente suprimida da superfície dos embriões somáticos após 49 dias de tratamento. No entanto, enquanto a presença de cefotaxima, em qualquer concentração, foi prejudicial à sobrevivência do tecido embriogênico, a combinação de cefotaxima + vancomicina não afetou significativamente os embriões somáticos de soja até os 63 dias de tratamento. Portanto, os resultados indicam que o tratamento com cefotaxima + vancomicina por um período de 49 - 63 dias é o mais adequado para a transformação genética de soja, por suprimir Agrobacterium e apresentar mínimos efeitos sobre o tecido embriogênico. Por fim, conjuntos de embriões somáticos de soja foram transformados e tratados com a combinação recomendada de antibióticos para avaliação da eficiência do método na obtenção de transformantes estáveis. Foram obtidos 48 e 232 clones higromicina-resistentes para Bragg e IAS5, respectivamente. Para cv. Bragg, 26 plantas foram obtidas de um único clone, enquanto 580 plantas foram regeneradas de 105 clones da cv. IAS5. As plantas transgênicas eram férteis e morfologicamente normais. A presença do transgene no genoma destas plantas foi confirmada por análises moleculares. Portanto, a adequação dos antibióticos permitiu o desenvolvimento de um método de transformação altamente eficiente para soja. Os resultados do presente trabalho constituem o primeiro registro (1) do efeito de antibióticos sobre tecidos de soja ou de leguminosas e (2) de obtenção de transformantes estáveis de soja utilizando a biolística e o sistema Agrobacterium de maneira integrada.

Interactions in the Agrobacterium-soybean system and capability of some Brazilian soybean cultivars to produce somatic embryos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

AGROBACTERIUM VIRB10 CONTRIBUTIONS TO TYPE IV SUBSTRATE SECRETION, T-PILUS BIOGENESIS, AND OUTER MEMBRANE PORE FORMATION

The VirB/D4 type IV secretion system (T4SS) of Agrobacterium tumefaciens functions to transfer substrates to infected plant cells through assembly of a translocation channel and a surface structure termed a T-pilus. This thesis is focused on identifying contributions of VirB10 to substrate transfer and T-pilus formation through a mutational analysis. VirB10 is a bitopic protein with several domains, including a: (i) cytoplasmic N-terminus, (ii) single transmembrane (TM) α-helix, (iii) proline-rich region (PRR), and (iv) large C-terminal modified β-barrel. I introduced cysteine insertion and substitution mutations throughout the length of VirB10 in order to: (i) test a predicted transmembrane topology, (ii) identify residues/domains contributing to VirB10 stability, oligomerization, and function, and (iii) monitor structural changes accompanying energy activation or substrate translocation. These studies were aided by recent structural resolution of a periplasmic domain of a VirB10 homolog and a ‘core’ complex composed of homologs of VirB10 and two outer membrane associated subunits, VirB7 and VirB9. By use of the substituted cysteine accessibility method (SCAM), I confirmed the bitopic topology of VirB10. Through phenotypic studies of Ala-Cys insertion mutations, I identified “uncoupling” mutations in the TM and β-barrel domains that blocked T-pilus assembly but permitted substrate transfer. I showed that cysteine replacements in the C-terminal periplasmic domain yielded a variety of phenotypes in relation to protein accumulation, oligomerization, substrate transfer, and T-pilus formation. By SCAM, I also gained further evidence that VirB10 adopts different structural states during machine biogenesis. Finally, I showed that VirB10 supports substrate transfer even when its TM domain is extensively mutagenized or substituted with heterologous TM domains. By contrast, specific residues most probably involved in oligomerization of the TM domain are required for biogenesis of the T-pilus.

Agrobacterium ParA/MinD-like VirC1 spatially coordinates early conjugative DNA transfer reactions.

Agrobacterium tumefaciens translocates T-DNA through a polar VirB/D4 type IV secretion (T4S) system. VirC1, a factor required for efficient T-DNA transfer, bears a deviant Walker A and other sequence motifs characteristic of ParA and MinD ATPases. Here, we show that VirC1 promotes conjugative T-DNA transfer by stimulating generation of multiple copies per cell of the T-DNA substrate (T-complex) through pairwise interactions with the processing factors VirD2 relaxase, VirC2, and VirD1. VirC1 also associates with the polar membrane and recruits T-complexes to cell poles, the site of VirB/D4 T4S machine assembly. VirC1 Walker A mutations abrogate T-complex generation and polar recruitment, whereas the native protein recruits T-complexes to cell poles independently of other polar processing factors (VirC2, VirD1) or T4S components (VirD4 substrate receptor, VirB channel subunits). We propose that A. tumefaciens has appropriated a progenitor ParA/MinD-like ATPase to promote conjugative DNA transfer by: (i) nucleating relaxosome assembly at oriT-like T-DNA border sequences and (ii) spatially positioning the transfer intermediate at the cell pole to coordinate substrate-T4S channel docking.

Agrobacterium VirD2 protein interacts with plant host cyclophilins

Agrobacterium tumefaciens induces crown gall tumors on plants by transferring a nucleoprotein complex, the T-complex, from the bacterium to the plant cell. The T-complex consists of T-DNA, a single-stranded DNA segment of the tumor-inducing plasmid, VirD2, an endonuclease covalently bound to the 5′ end of the T-DNA, and perhaps VirE2, a single-stranded DNA binding protein. The yeast two-hybrid system was used to screen for proteins interacting with VirD2 and VirE2 to identify components in Arabidopsis thaliana that interact with the T-complex. Three VirD2- and two VirE2-interacting proteins were identified. Here we characterize the interactions of VirD2 with two isoforms of Arabidopsis cyclophilins identified by using this analysis. The VirD2 domain interacting with the cyclophilins is distinct from the endonuclease, omega, and the nuclear localization signal domains. The VirD2–cyclophilin interaction is disrupted in vitro by cyclosporin A, which also inhibits Agrobacterium-mediated transformation of Arabidopsis and tobacco. These data strongly suggest that host cyclophilins play a role in T-DNA transfer.

Genetic transformation of HeLa cells by Agrobacterium

Agrobacterium tumefaciens is a soil phytopathogen that elicits neoplastic growths on the host plant species. In nature, however, Agrobacterium also may encounter organisms belonging to other kingdoms such as insects and animals that feed on the infected plants. Can Agrobacterium, then, also infect animal cells? Here, we report that Agrobacterium attaches to and genetically transforms several types of human cells. In stably transformed HeLa cells, the integration event occurred at the right border of the tumor-inducing plasmid's transferred-DNA (T-DNA), suggesting bona fide T-DNA transfer and lending support to the notion that Agrobacterium transforms human cells by a mechanism similar to that which it uses for transformation of plants cells. Collectively, our results suggest that Agrobacterium can transport its T-DNA to human cells and integrate it into their genome.

Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses?

Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants. The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell. The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames. The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons.

Agrobacterium vitis strains lack tumorigenic ability on in vitro grown grapevine stem segments

Grapevine stem segments were cocultivated with three different Agrobacterium tumefaciens and three different A. vitis strains. A. tumefaciens strains induced tumors at variable frequencies, while A. vitis-infected stem segments never formed crown galls. The tumorous nature of tissues grown on hormone free medium was confirmed by opine assays. Bioinformatic and PCR analysis of the virulence regions of various A. tumefaciens and A. vitis Ti plasmids showed that virH2 and virK genes are common in A. tumefaciens but they are lacking from A. vitis. Thus virH2 and virK genes may be essential for grapevine stem segment transformation, but expression of certain T-DNA genes of A. vitis may also prevent the growth of transformed cells. Our data indicate that the tumorigenic ability of A. vitis is different on intact plant and on their explants, and that the specific host association of A. vitis on grapevine is probably determined by physiological and biochemical factors (e. g., better colonizing ability) rather than by its increased tumorigenic ability. Therefore it is not reasonable to develop „helper” plasmids for grapevine transformation from A. vitis pTis, unless their avirulence on in vitro explants is determined by T-DNA gene(s). Due to the inability of A. vitis to induce tumors on grapevine stem segments, the use of in vitro explant assays cannot be reliably used to select A. vitis resistant grapevine genotypes or transgenic lines.

Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance

Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. 'Richter 110' rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twentyone transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58.

Novel insights into the genomic basis of citrus canker based on the genome sequences of two strains of Xanthomonas fuscans subsp aurantifolii

Background: Citrus canker is a disease that has severe economic impact on the citrus industry worldwide. There are three types of canker, called A, B, and C. The three types have different phenotypes and affect different citrus species. The causative agent for type A is Xanthomonas citri subsp. citri, whose genome sequence was made available in 2002. Xanthomonas fuscans subsp. aurantifolii strain B causes canker B and Xanthomonas fuscans subsp. aurantifolii strain C causes canker C. Results: We have sequenced the genomes of strains B and C to draft status. We have compared their genomic content to X. citri subsp. citri and to other Xanthomonas genomes, with special emphasis on type III secreted effector repertoires. In addition to pthA, already known to be present in all three citrus canker strains, two additional effector genes, xopE3 and xopAI, are also present in all three strains and are both located on the same putative genomic island. These two effector genes, along with one other effector-like gene in the same region, are thus good candidates for being pathogenicity factors on citrus. Numerous gene content differences also exist between the three cankers strains, which can be correlated with their different virulence and host range. Particular attention was placed on the analysis of genes involved in biofilm formation and quorum sensing, type IV secretion, flagellum synthesis and motility, lipopolysacharide synthesis, and on the gene xacPNP, which codes for a natriuretic protein. Conclusion: We have uncovered numerous commonalities and differences in gene content between the genomes of the pathogenic agents causing citrus canker A, B, and C and other Xanthomonas genomes. Molecular genetics can now be employed to determine the role of these genes in plant-microbe interactions. The gained knowledge will be instrumental for improving citrus canker control.