Comparative Effects of Different Materials on Alveolar Preservation.

OBJECTIVE: The purpose of this study was to compare different materials' effects on alveolar ridge preservation of postextraction sockets in anterior maxilla. MATERIALS AND METHOD: In this prospective, single center, randomized, controlled clinical trial, healthy patients who needed one single anterior maxillary tooth extraction (including bicuspids) were selected. After a minimally traumatic extraction without complications, 44 patients were randomly allocated into 4 groups: 1) natural socket healing (blood clot), 2) xenograft and gingival free graft, 3) dense polytetrafluoroethylene membrane, and 4) platelet rich fibrin plugs. Alveolar ridge height and width loss were evaluated in cone beam computed tomography (CBCT) and in dental casts at 3 moments: 1) preoperative (T1), 2) 7 days postoperative (T2), and 3) 120 days postoperative (T3). Height and width alveolar ridge loss detected in CBCT and in dental casts were compared among the groups (two-way analysis of variance [ANOVA]; P < .05). RESULTS: Forty patients (24 women and 16 men) ranging from 25 to 70 years old (mean of 42 years old) participated in this study. Group 2 showed the least alveolar ridge height loss results in CBCT (9.8 ± 1.9% at T3) and dental cast analysis (1.0 ± 0.2 mm). Groups 2 (12.7 ± 4.7% at T3) and 3 (15.4 ± 2.7% at T3) showed the least alveolar ridge width loss measured in CBCT compared with groups 1 and 4, but the difference between groups 2 and 3 were not statistically significant (P = .968). Group 3 (0.9 ± 0.2 mm) and group 2 (1.0 ± 0.2 mm) showed the least width loss compared with groups 1 and 4 in dental cast analysis. Again, the difference between groups 3 and 2 was not statistically significant (P = 1.000). CONCLUSION: In postextraction sockets of the anterior maxilla and bicuspid region, group 2 (xenogenous bone graft with free gingival graft) and group 3 (dense polytetrafluoroethylene) obtained the best results in alveolar preservation, with group 2 being more indicated when the vertical alveolar ridge preservation is mandatory.

Split Crest Technique with Immediate Implant to Treat Horizontal Defects of the Alveolar Ridge: Analysis of Increased Thickness and Implant Survival.

OBJECTIVE: To assess bone thickness augmentation and implant survival in ridges with horizontal atrophy managed through split crest technique with concomitant installation of dental implants. MATERIALS AND METHODS: Thirteen patients with maxillary bone atrophy underwent surgery and had their bone thickness assessed through cone beam computed tomography 6 months pre- and postoperatively. Comparative measurements of initial and final bone height and thickness were taken using Dolphin Imaging® 11.5 software. The distance between the nasal fossa floor or the maxillary sinus and the alveolar crest determined the bone height, while the measurement of bone thickness took into account the distance between the vestibular cortical bone and the palatal cortical bone at the crest level, and at 5 mm and 10 mm from it. RESULTS: The bone height loss of 0.68 mm was statistically significant (p = 0.01). The average horizontal bone gain was 3.45 mm at ridge level, 3.03 mm at 5 mm from it and 2.42 mm at 10 mm from it. The mean horizontal gain for the three regions was 2.97 mm, and the values were statistically significant for all three regions assessed (p < 0.01). No complications were associated with the surgical procedures, and 23 implants were installed following the surgical expansion. No implants were lost (100% survival). CONCLUSION: The split crest technique proved to be viable and predictable, enabling a significant increase in ridge thickness and a high percentage of implant survival.

Uncovering Bax inhibitor-1 dual role in the legume-rhizobia symbiosis in common bean roots.

Bax-inhibitor 1 (BI-1) is a cell death suppressor conserved in all eukaryotes that modulates cell death in response to abiotic stress and pathogen attack in plants. However, little is known about its role in the establishment of symbiotic interactions. Here, we demonstrate the functional relevance of an Arabidopsis thaliana BI-1 homolog (PvBI-1a) to symbiosis between the common bean (Phaseolus vulgaris) and Rhizobium tropici. We show that the changes in expression of PvBI-1a observed during early symbiosis resemble those of some defence response-related proteins. By using gain- and loss-of-function approaches, we demonstrate that the overexpression of PvBI-1a in the roots of common bean increases the number of rhizobial infection events (and therefore the final number of nodules per root), but induces the premature death of nodule cells, affecting their nitrogen fixation efficiency. Nodule morphological alterations are known to be associated with changes in the expression of genes tied to defence, autophagy, and vesicular trafficking. Results obtained in the present work suggest that BI-1 has a dual role in the regulation of programmed cell death during symbiosis, extending our understanding of its critical function in the modulation of host immunity while responding to beneficial microbes.

Molecular Features and mRNA Expression of the Receptor for Activated C Kinase 1 from Symbiodinium microadriaticum ssp. microadriaticum During Growth and the Light/Dark cycle.

Two genes of the RACK1 homolog from the photosynthetic dinoflagellate Symbiodinium microadriaticum ssp. microadriaticum (SmicRACK1), termed SmicRACK1A and SmicRACK1B, were found tandemly arrayed and displayed a single synonymous substitution (T/C) encoding threonine. They included two exons of 942 bp each, encoding 313 amino acids with seven WD-40 repeats and two PKC-binding motifs. The protein theoretical mass and pI were 34,200 Da and 5.9, respectively. SmicRACK1 showed maximum identities with RACK1 homologs at the amino acid and nucleotide level, respectively, of 92 and 84% with S. minutum, and phylogenetic analysis revealed clustered related RACK1 sequences from the marine dinoflagellates S. minutum, Heterocapsa triquetra, Karenia brevis, and Alexandrium tamarense. Interestingly, light-dependent regulatory elements were found both within the 282 bp SmicRACK1A promotor sequence, and within an intergenic sequence of 359 nucleotides that separated both genes, which strongly suggest light-related functions. This was further supported by mRNA accumulation analysis, which fluctuated along the light and dark phases of the growth cycle showing maximum specific peaks under either condition. Finally, qRT-PCR analysis revealed differential SmicRACK1 mRNA accumulation with maxima at 6 and 20 d of culture. Our SmicRACK1 characterization suggests roles in active growth and proliferation, as well as light/dark cycle regulation in S. microadriaticum.

Preservação da crista alveolar através da utilização de membrana densa de politetrafluoretileno / Preservation of the alveolar crest through the use of polytetrafluoroethylene dense membrane

O processo de remodelação óssea que inicia-se após a extração de um dente pode dificultar a reabilitação através de implantes dentários. Nesse sentido, a técnica de regeneração óssea guiada (ROG), com o uso de membrana não reabsorvível, busca minimizar estes efeitos, favorecendo a cicatrização do alvéolo e diminuindo a necessidade de enxertos ósseos. Sendo assim, o objetivo deste trabalho foi avaliar a utilização da membrana densa de politetrafluoretileno (d-PTFE) em alvéolos pós-extração. Para isso, 8 pacientes que foram submetidos à remoção de elemento dentário receberam em seus alvéolos a colocação da membrana d-PTFE. A mesma foi posicionada sobre o alvéolo imediatamente após a extração e deixada no local por 21 dias. Para avaliar a preservação do rebordo alveolar, tomografias foram realizadas no pré-operatório e no pós-operatório de 90 dias. Os resultados mostraram uma efetiva preservação do rebordo alveolar proporcionado pelo uso da membrana. A perda óssea em espessura dos alvéolos foi de apenas 0,32 mm, em média. Já a perda óssea em altura foi de 0,79 mm, em média. Oito implantes foram instalados, sendo que nenhuma complicação ou perda de implantes foi observada. A membrana de PTFE denso mostrou-se efetiva na manutenção da arquitetura alveolar, minimizando a perda óssea em altura e espessura (AU).

The process of bone remodeling that begins after the extraction of a tooth can make rehabilitation difficult through dental implants. In this sense, the technique of guided bone regeneration (ROG), with the use of a non-resorbable membrane, seeks to minimize these effects, favoring healing of the alveolus and reducing the need for bone grafts. Therefore, the objective of this work was to evaluate the use of polytetrafluoroethylene dense membrane (d-PTFE) in post-extraction alveoli. For this, 8 patients who were submitted to tooth removal, received in their alveoli the placement of the d-PTFE membrane. It was placed on the alveolus immediately after extraction and left in place for 21 days. To evaluate the preservation of the alveolar ridge, CT scans were performed preoperatively and postoperatively for 90 days. The results showed an effective preservation of the alveolar ridge provided by the use of the membrane. The bone loss in the alveoli thickness was only 0.32 mm on average. The bone loss in height was 0.79 mm on average. Eight implants were installed, and no complications or loss of implants were observed. The dense PTFE membrane was effective in maintaining the alveolar architecture, minimizing bone loss in height and thickness (AU).

qPCR and HRM-based diagnosis of SNPs on growth differentiation factor 9 (GDF9), a gene associated with sheep (Ovis aries) prolificacy.

Prolificacy is a desirable trait for genetic improvement of sheep flocks, since it holds the potential to improve productivity. Animals carrying single-nucleotide polymorphisms (SNPs) in genes associated with this trait can be identified and employed to increase prolificacy in flocks. In this study, we report a diagnostic method based on quantitative PCR and high-resolution melting curves to detect different SNPs in the prolificacy-associated gene growth differentiation factor 9 (GDF9). The diagnostic method was validated using artificial sequences representing known SNPs in GDF9, then applied to a real flock comprising four breeds and admixed animals (n = 306). Five different SNPs were identified in this flock, as was a low or null frequency of occurrence of SNPs positively associated with prolificacy. This indicates a need to implement a breeding strategy for recovering or reintroducing such SNPs. Our method provides a genotyping strategy for identifying individuals with SNPs of interest for prolificacy, which will help producers plan a breeding strategy for this trait. This method can be adapted and expanded for the diagnosis of other traits of interest.

Nodulin 22, a novel small heat-shock protein of the endoplasmic reticulum, is linked to the unfolded protein response in common bean.

The importance of plant small heat shock proteins (sHsp) in multiple cellular processes has been evidenced by their unusual abundance and diversity; however, little is known about their biological role. Here, we characterized the in vitro chaperone activity and subcellular localization of nodulin 22 of Phaseolus vulgaris (PvNod22; common bean) and explored its cellular function through a virus-induced gene silencing-based reverse genetics approach. We established that PvNod22 facilitated the refolding of a model substrate in vitro, suggesting that it acts as a molecular chaperone in the cell. Through microscopy analyses of PvNod22, we determined its localization in the endoplasmic reticulum (ER). Furthermore, we found that silencing of PvNod22 resulted in necrotic lesions in the aerial organs of P. vulgaris plants cultivated under optimal conditions and that downregulation of PvNod22 activated the ER-unfolded protein response (UPR) and cell death. We also established that PvNod22 expression in wild-type bean plants was modulated by abiotic stress but not by chemicals that trigger the UPR, indicating PvNod22 is not under UPR control. Our results suggest that the ability of PvNod22 to suppress protein aggregation contributes to the maintenance of ER homeostasis, thus preventing the induction of cell death via UPR in response to oxidative stress during plant-microbe interactions.

Detailed analysis of putative genes encoding small proteins in legume genomes.

Diverse plant genome sequencing projects coupled with powerful bioinformatics tools have facilitated massive data analysis to construct specialized databases classified according to cellular function. However, there are still a considerable number of genes encoding proteins whose function has not yet been characterized. Included in this category are small proteins (SPs, 30-150 amino acids) encoded by short open reading frames (sORFs). SPs play important roles in plant physiology, growth, and development. Unfortunately, protocols focused on the genome-wide identification and characterization of sORFs are scarce or remain poorly implemented. As a result, these genes are underrepresented in many genome annotations. In this work, we exploited publicly available genome sequences of Phaseolus vulgaris, Medicago truncatula, Glycine max, and Lotus japonicus to analyze the abundance of annotated SPs in plant legumes. Our strategy to uncover bona fide sORFs at the genome level was centered in bioinformatics analysis of characteristics such as evidence of expression (transcription), presence of known protein regions or domains, and identification of orthologous genes in the genomes explored. We collected 6170, 10,461, 30,521, and 23,599 putative sORFs from P. vulgaris, G. max, M. truncatula, and L. japonicus genomes, respectively. Expressed sequence tags (ESTs) available in the DFCI Gene Index database provided evidence that ~one-third of the predicted legume sORFs are expressed. Most potential SPs have a counterpart in a different plant species and counterpart regions or domains in larger proteins. Potential functional sORFs were also classified according to a reduced set of GO categories, and the expression of 13 of them during P. vulgaris nodule ontogeny was confirmed by qPCR. This analysis provides a collection of sORFs that potentially encode for meaningful SPs, and offers the possibility of their further functional evaluation.

Common bean (Phaseolus vulgaris L.) PvTIFY orchestrates global changes in transcript profile response to jasmonate and phosphorus deficiency.

BACKGROUND: TIFY is a large plant-specific transcription factor gene family. A subgroup of TIFY genes named JAZ (Jasmonate-ZIM domain) has been identified as repressors of jasmonate (JA)-regulated transcription in Arabidopsis and other plants. JA signaling is involved in many aspects of plant growth/development and in defense responses to biotic and abiotic stresses. Here, we identified the TIFY genes (designated PvTIFY) from the legume common bean (Phaseolus vulgaris) and functionally characterized PvTIFY10C as a transcriptional regulator. RESULTS: Nineteen genes from the PvTIFY gene family were identified through whole-genome sequence analysis. Most of these were induced upon methyl-JA elicitation. We selected PvTIFY10C as a representative JA-responsive PvTIFY gene for further functional analysis. Transcriptome analysis via microarray hybridization using the newly designed Bean Custom Array 90 K was performed on transgenic roots of composite plants with modulated (RNAi-silencing or over-expression) PvTIFY10C gene expression. Data were interpreted using Gene Ontology and MapMan adapted to common bean. Microarray differential gene expression data were validated by real-time qRT-PCR expression analysis. Comparative global gene expression analysis revealed opposite regulatory changes in processes such as RNA and protein regulation, stress responses and metabolism in PvTIFY10C silenced vs. over-expressing roots. These data point to transcript reprogramming (mainly repression) orchestrated by PvTIFY10C. In addition, we found that several PvTIFY genes, as well as genes from the JA biosynthetic pathway, responded to P-deficiency. Relevant P-responsive genes that participate in carbon metabolic pathways, cell wall synthesis, lipid metabolism, transport, DNA, RNA and protein regulation, and signaling were oppositely-regulated in control vs. PvTIFY10C-silenced roots of composite plants under P-stress. These data indicate that PvTIFY10C regulates, directly or indirectly, the expression of some P-responsive genes; this process could be mediated by JA-signaling. CONCLUSION: Our work contributes to the functional characterization of PvTIFY transcriptional regulators in common bean, an agronomically important legume. Members from the large PvTIFY gene family are important global transcriptional regulators that could participate as repressors in the JA signaling pathway. In addition, we propose that the JA-signaling pathway involving PvTIFY genes might play a role in regulating the plant response/adaptation to P-starvation.

Transcript profiling of common bean nodules subjected to oxidative stress.

Several environmental stresses generate high amounts of reactive oxygen species (ROS) in plant cells, resulting in oxidative stress. Symbiotic nitrogen fixation (SNF) in the legume-rhizobia symbiosis is sensitive to damage from oxidative stress. Active nodules of the common bean (Phaseolus vulgaris) exposed to the herbicide paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride hydrate), which stimulates ROS accumulation, exhibited reduced nitrogenase activity and ureide content. We analyzed the global gene response of nodules subjected to oxidative stress using the Bean Custom Array 90K, which includes probes from 30,000 expressed sequence tags (ESTs). A total of 4280 ESTs were differentially expressed in stressed bean nodules; of these, 2218 were repressed. Based on Gene Ontology analysis, these genes were grouped into 42 different biological process categories. Analysis with the PathExpress bioinformatic tool, adapted for bean, identified five significantly repressed metabolic pathways related to carbon/nitrogen metabolism, which is crucial for nodule function. Quantitative reverse transcription (qRT)-PCR analysis of transcription factor (TF) gene expression showed that 67 TF genes were differentially expressed in nodules exposed to oxidative stress. Putative cis-elements recognized by highly responsive TF were detected in promoter regions of oxidative stress regulated genes. The expression of oxidative stress responsive genes and of genes important for SNF in bacteroids analyzed in stressed nodules revealed that these conditions elicited a transcriptional response.

Changes in RACK1 expression induce defects in nodulation and development in Phaseolus vulgaris.

RACK1 is a scaffold protein with the ability to interact in a regulated manner with a diverse number of ligands from distinct signal-transduction pathways. This assessment allowed us to infer that it may be involved in different processes such as nodulation. In a recent study we showed by silencing, that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development in Phaseolus vulgaris. On the other hand, we have also observed that its over-expression provokes a dramatic phenotype in: (a) seedlings that have been exposed to heat, in which systemic necrosis is induced; and (b) in Agrobacterium rhizogenes-transformed roots, where nodulation is strongly inhibited and nodules show early senescent symptoms. These findings indicate that PvRACK1 may be an integrator of diverse signal-transduction pathways in processes as varied as nodulation, cell expansion, heat stress responses, and systemic activation of necrosis. 

A general method of protein purification for recombinant unstructured non-acidic proteins.

Typical late embryogenesis abundant (LEA) proteins accumulate in response to water deficit imposed by the environment or by plant developmental programs. Because of their physicochemical properties, they can be considered as hydrophilins and as a paradigm of intrinsically unstructured proteins (IUPs) in plants. To study their biophysical and biochemical characteristics large quantities of highly purified protein are required. In this work, we report a fast and simple purification method for non-acidic recombinant LEA proteins that does not need the addition of tags and that preserves their in vitro protective activity. The method is based on the enrichment of the protein of interest by boiling the bacterial protein extract, followed by a differential precipitation with trichloroacetic acid (TCA). Using this procedure we have obtained highly pure recombinant LEA proteins of groups 1, 3, and 4 and one recombinant bacterial hydrophilin. This protocol will facilitate the purification of this type of IUPs, and could be particularly useful in proteomic projects/analyses.

PvRACK1 loss-of-function impairs cell expansion and morphogenesis in Phaseolus vulgaris L. root nodules.

Receptor for activated C kinase (RACK1) is a highly conserved, eukaryotic protein of the WD-40 repeat family. Its peculiar ß-propeller structure allows its interaction with multiple proteins in various plant signal-transduction pathways, including those arising from hormone responses, development, and environmental stress. During Phaseolus vulgaris root development, RACK1 (PvRACK1) mRNA expression was induced by auxins, abscissic acid, cytokinin, and gibberellic acid. In addition, during P. vulgaris nodule development, PvRACK1 mRNA was highly accumulated at 12 to 15 days postinoculation, suggesting an important role after nodule meristem initiation and Rhizobium nodule infection. PvRACK1 transcript accumulation was downregulated by a specific RNA interference construct which was expressed in transgenic roots of composite plants of P. vulgaris inoculated with Rhizobium tropici. PvRACK1 downregulated transcript levels were monitored by quantitative reverse-transcription polymerase chain reaction analysis in individual transgenic roots and nodules. We observed a clear phenotype in PvRACK1-knockdown nodules, in which nodule number and nodule cell expansion were impaired, resulting in altered nodule size. Microscopic analysis indicated that, in PvRACK1-knockdown nodules, infected and uninfected cells were considerably smaller (80 and 60%, respectively) than in control nodules. In addition, noninfected cells and symbiosomes in silenced nodules showed significant defects in membrane structure under electron microscopy analysis. These findings indicate that PvRACK1 has a pivotal role in cell expansion and in symbiosome and bacteroid integrity during nodule development.

Plasma membrane integrity and survival of melanoma cells after nanosecond laser pulses.

Circulating tumor cells (CTCs) photoacoustic detection systems can aid clinical decision-making in the treatment of cancer. Interaction of melanin within melanoma cells with nanosecond laser pulses generates photoacoustic waves that make its detection possible. This study aims at: (1) determining melanoma cell survival after laser pulses of 6 ns at λ = 355 and 532 nm; (2) comparing the potential enhancement in the photoacoustic signal using λ = 355 nm in contrast with λ = 532 nm; (3) determining the critical laser fluence at which melanin begins to leak out from melanoma cells; and (4) developing a time-resolved imaging (TRI) system to study the intracellular interactions and their effect on the plasma membrane integrity. Monolayers of melanoma cells were grown on tissue culture-treated clusters and irradiated with up to 1.0 J/cm². Surviving cells were stained with trypan blue and counted using a hemacytometer. The phosphate buffered saline absorbance was measured with a nanodrop spectrophotometer to detect melanin leakage from the melanoma cells post-laser irradiation. Photoacoustic signal magnitude was studied at both wavelengths using piezoelectric sensors. TRI with 6 ns resolution was used to image plasma membrane damage. Cell survival decreased proportionally with increasing laser fluence for both wavelengths, although the decrease is more pronounced for 355 nm radiation than for 532 nm. It was found that melanin leaks from cells equally for both wavelengths. No significant difference in photoacoustic signal was found between wavelengths. TRI showed clear damage to plasma membrane due to laser-induced bubble formation.

Germination behavior, biochemical features and sequence analysis of the RACK1/arcA homolog from Phaseolus vulgaris.

Partial peptide sequence of a 36 kDa protein from common bean embryo axes showed 100% identity with a reported beta-subunit of a heterotrimeric G protein from soybean. Analysis of the full sequence showed 96.6% identity with the reported soybean G(beta)-subunit, 86% with RACK1B and C from Arabidopsis and 66% with human and mouse RACK1, at the amino acid level. In addition, it showed 85.5, 85 and 83% identities with arcA from Solanum lycopersicum, Arabidopsis (RACK1A) and Nicotiana tabacum, respectively. The amino acid sequence displayed seven WD40 domains and two sites for activated protein kinase C binding. The protein showed a constant expression level but the mRNA had a maximum at 32 h post-imbibition. Western immunoblotting showed the protein in vegetative plant tissues, and in both microsomal and soluble fractions from embryo axes. Synthetic auxin treatment during germination delayed the peak of RACK1 mRNA expression to 48 h but did not affect the protein expression level while the polar auxin transport inhibitor, naphtylphtalamic acid had no effect on either mRNA or protein expression levels. Southern blot and genomic DNA amplification revealed a small gene family with at least one member without introns in the genome. Thus, the RACK1/arcA homolog from common bean has the following features: (1) it is highly conserved; (2) it is both soluble and insoluble within the embryo axis; (3) it is encoded by a small gene family; (4) its mRNA has a peak of expression at the time point of germination stop and (5) its expression is only slightly affected by auxin but unaffected by an auxin transport blocker.

Effect of hyperosmotic conditions on flavin-containing monooxygenase activity, protein and mRNA expression in rat kidney.

Flavin-containing monooxigenases (FMOs) are a polymorphic family of drug and pesticide metabolizing enzymes, found in the smooth endoplasmatic reticulum that catalyze the oxidation of soft nucleophilic heteroatom substances to their respective oxides. Previous studies in euryhaline fishes have indicated induction of FMO expression and activity in vivo under hyperosmotic conditions. In this study we evaluated the effect of hypersaline conditions in rat kidney. Male Sprague-Dawley rats were injected intraperitoneal with 3.5M NaCl at a doses ranging from 0.3cm(3)/100g to 0.6cm(3)/100g in two separate treatments. Three hours after injection, FMO activities and FMO1 protein was examined in the first experiment, and the expression of FMO1 mRNA was measured in the second experiment from kidneys after treatment with NaCl. A positive significant correlation was found between FMO1 protein expression and plasma osmolarity (p<0.05, r=0.6193). Methyl-p-tolyl sulfide oxidase showed a statistically significant increase in FMO activity, and a positive correlation was observed between plasma osmolarity and production of FMO1-derived (R)-methyl-p-tolyl sulfoxide (p<0.05, r=0.6736). Expression of FMO1 mRNA was also positively correlated with plasma osmolality (p<0.05, r=0.8428). Similar to studies in fish, these results suggest that expression and activities of FMOs may be influenced by hyperosmotic conditions in the kidney of rats.

Fast, efficient and reproducible genetic transformation of Phaseolus spp. by Agrobacterium rhizogenes.

This transformation procedure generates, with high efficiency (70-90%), hairy roots in cultivars, landraces and accessions of Phaseolus vulgaris (common bean) and other Phaseolus spp. Hairy roots rapidly develop after wounding young plantlets with Agrobacterium rhizogenes, at the cotyledon node, and keeping the plants in high-humidity conditions. Callogenesis always precedes hairy-root formation, and after 15 days, when roots develop at wounded sites, the stem with the normal root is cleaved below the hairy root zone. Transgenic roots and nodules co-transformed with a binary vector can be easily identified using a reporter gene. This procedure, in addition to inducing robust transgenic hairy roots that are susceptible to being nodulated by rhizobia and to fixing nitrogen efficiently, sets the foundation for a high-throughput functional genomics approach on the study of root biology and root-microbe interactions. This protocol can be completed within 30 days.

Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics.

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.

Profilin tyrosine phosphorylation in poly-L-proline-binding regions inhibits binding to phosphoinositide 3-kinase in Phaseolus vulgaris.

The profilin family consists of a group of ubiquitous highly conserved 12-15 kDa eukaryotic proteins that bind actin, phosphoinositides, poly-l-proline (PLP) and proteins with proline-rich motifs. Some proteins with proline-rich motifs form complexes that have been implicated in the dynamics of the actin cytoskeleton and processes such as vesicular trafficking. A major unanswered question in the field is how profilin achieves the required specificity to bind such an array of proteins. It is now becoming clear that profilin isoforms are subject to differential regulation and that they may play distinct roles within the cell. Considerable evidence suggests that these isoforms have different functional roles in the sorting of diverse proteins with proline-rich motifs. All profilins contain highly conserved aromatic residues involved in PLP binding which are presumably implicated in the interaction with proline-rich motif proteins. We have previously shown that profilin is phosphorylated on tyrosine residues. Here, we show that profilin can bind directly to Phaseolus vulgaris phosphoinositide 3-kinase (PI3K) type III. We demonstrate that a new region around Y72 of profilin, as well as the N- and C-terminal PLP-binding domain, recognizes and binds PLP and PI3K. In vitro binding assays indicate that PI3K type III forms a complex with profilin in a manner that depends on the tyrosine phosphorylation status within the proline-rich-binding domain in profilin. Profilin-PI3K type III interaction suggests that profilin may be involved in membrane trafficking and in linking the endocytic pathway with actin reorganization dynamics.