An effective virus-based gene silencing method for functional genomics studies in common bean

BACKGROUND: Common bean (Phaseolus vulgaris L.) is a crop of economic and nutritious importance in many parts of the world. The lack of genomic resources have impeded the advancement of common bean genomics and thereby crop improvement. Although concerted efforts from the "Phaseomics" consortium have resulted in the development of several genomic resources, functional studies have continued to lag due to the recalcitrance of this crop for genetic transformation. RESULTS: Here we describe the use of a bean pod mottle virus (BPMV)-based vector for silencing of endogenous genes in common bean as well as for protein expression. This BPMV-based vector was originally developed for use in soybean. It has been successfully employed for both protein expression and gene silencing in this species. We tested this vector for applications in common bean by targeting common bean genes encoding nodulin 22 and stearoyl-acyl carrier protein desaturase for silencing. Our results indicate that the BPMV vector can indeed be employed for reverse genetics studies of diverse biological processes in common bean. We also used the BPMV-based vector for expressing the green fluorescent protein (GFP) in common bean and demonstrate stable GFP expression in all common bean tissues where BPMV was detected. CONCLUSIONS: The availability of this vector is an important advance for the common bean research community not only because it provides a rapid means for functional studies in common bean, but also because it does so without generating genetically modified plants. Here we describe the detailed methodology and provide essential guidelines for the use of this vector for both gene silencing and protein expression in common bean. The entire VIGS procedure can be completed in 4-5 weeks

Packaging of brome mosaic virus subgenomic RNA is functionally coupled to replication-dependent transcription and translation of coat protein.

In Brome mosaic virus (BMV), genomic RNA1 (gB1) and RNA2 (gB2), encoding the replication factors, are packaged into two separate virions, whereas genomic RNA3 (gB3) and its subgenomic coat protein (CP) mRNA (sgB4) are copackaged into a third virion. In vitro assembly assays performed between a series of deletion variants of sgB4 and wild-type (wt) CP subunits demonstrated that packaging of sgB4 is independent of sequences encoding the CP open reading frame. To confirm these observations in vivo and to unravel the mechanism of sgB4 copackaging, an Agrobacterium-mediated transient in vivo expression system (P. Annamalai and A. L. N. Rao, Virology 338:96-111, 2005) that effectively uncouples replication from packaging was used. Cultures of agrotransformants, engineered to express sgB4 and CP subunits either transiently (sgB4(Trans) and Cp-Trans) or in replication-dependent transcription and translation when complemented with gB1 and gB2 (sgB4(Rep) and Cp-Rep), were mixed in all four pair-wise combinations and infiltrated to Nicotiana benthamiana leaves to systematically evaluate requirements regulating sgB4 packaging. The data revealed that (i) in the absence of replication, packaging was nonspecific, since transiently expressed CP subunits efficiently packaged ubiquitous cellular RNA as well as transiently expressed sgB4 and its deletion variants; (ii) induction of viral replication increased specificity of RNA packaging; and most importantly, (iii) efficient packaging of sgB4, reminiscent of the wt scenario, is functionally coupled not only to its transcription via replication but also to translation of CP from replication-derived mRNA, a mechanism that appears to be conserved among positive-strand RNA viruses of plants (this study), animals (flock house virus), and humans (poliovirus)

Deletion of Highly Conserved Arginine-Rich RNA Binding Motif in Cowpea Chlorotic Mottle Virus Capsid Protein Results in Virion Structural Alterations and RNA Packaging Constraints

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of Bromoviridae and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Δ919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Δ919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Δ919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus

An effective virus-based gene silencing method for functional genomics studies in common bean

Abstract Background Common bean (Phaseolus vulgaris L.) is a crop of economic and nutritious importance in many parts of the world. The lack of genomic resources have impeded the advancement of common bean genomics and thereby crop improvement. Although concerted efforts from the "Phaseomics" consortium have resulted in the development of several genomic resources, functional studies have continued to lag due to the recalcitrance of this crop for genetic transformation. Results Here we describe the use of a bean pod mottle virus (BPMV)-based vector for silencing of endogenous genes in common bean as well as for protein expression. This BPMV-based vector was originally developed for use in soybean. It has been successfully employed for both protein expression and gene silencing in this species. We tested this vector for applications in common bean by targeting common bean genes encoding nodulin 22 and stearoyl-acyl carrier protein desaturase for silencing. Our results indicate that the BPMV vector can indeed be employed for reverse genetics studies of diverse biological processes in common bean. We also used the BPMV-based vector for expressing the green fluorescent protein (GFP) in common bean and demonstrate stable GFP expression in all common bean tissues where BPMV was detected. Conclusions The availability of this vector is an important advance for the common bean research community not only because it provides a rapid means for functional studies in common bean, but also because it does so without generating genetically modified plants. Here we describe the detailed methodology and provide essential guidelines for the use of this vector for both gene silencing and protein expression in common bean. The entire VIGS procedure can be completed in 4-5 weeks.</p

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

<div><p>Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme <i>de novo</i>, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of <i>Plasmodium berghei</i> (<i>Pb</i>). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out <i>in vitro</i> radiolabeling of heme in <i>Pb</i>-infected mouse reticulocytes and <i>Plasmodium falciparum</i>-infected human RBCs using [4-¹⁴C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the <i>de novo</i> pathway is absolutely essential for parasite development in the mosquito and liver stages. <i>Pb</i>KO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in <i>Pb</i>ALASKO parasites recovered when mosquitoes received an ALA supplement. <i>Pb</i>ALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.</p></div

Growth curves for intraerythrocytic stages of <i>P. berghei</i> WT and KO parasites in mice.

<p>Mice were injected intraperitoneally with 10⁵<i>P. berghei</i> infected-RBCs/reticulocytes and the parasite growth was routinely monitored as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003522#s4" target="_blank">Materials and Methods</a>. Multiple fields were used to quantify the parasite infected cells. The data provided represent the mean ± S.D. obtained from 6 animals.</p

Ability of <i>P.berghei</i> sporozoites (WT and KOs) to infect mice with and without ALA supplement to the animals.

<p>Mosquitoes were allowed to feed on mice (30 mosquitoes/mouse) and parasitemia in blood and mortality of the animals were assessed. The data represent 9 mice each from three different batches. Mq, mosquito; Mi, mice; <i>Pb</i>ALASKO(Mq^+ALAMi^+ALA), <i>Pb</i>ALASKO supplemented with ALA in mosquitoes and mice; <i>Pb</i>ALASKO(Mq^+ALAMi^−ALA), <i>Pb</i>ALASKO supplemented with ALA in mosquitoes but not in mice; <i>Pb</i>FCKO(Mq^+Blood), <i>Pb</i>FCKO supplemented with blood feeding in mosquitoes.</p

Oocyst and sporozoite formation in <i>P.berghei</i>-infected (WT and KOs) mosquitoes.

<p>(A) Mercurochrome staining of oocysts in the midgut preparations. Arrows indicate oocysts and the magnified images of oocysts are provided in insets. Scale bar: 100 µm. (B) Sporozoites in the salivary glands. Magnified images of sporozoites are provided in insets. Scale bar: 50 µm. (C) Quantification of oocysts. P values for <i>Pb</i>ALASKO and <i>Pb</i>FCKO with respect to WT are <0.02. P value for <i>Pb</i>ALASKO(Mq^+ALA) with respect to <i>Pb</i>ALASKO is <0.01 and <i>Pb</i>FCKO(Mq^+Blood) with respect to <i>Pb</i>FCKO is >0.05. The data represent 90 mosquitoes from 3 different batches. (D) Quantification of sporozoites. P values for <i>Pb</i>ALASKO, <i>Pb</i>FCKO, <i>Pb</i>ALASKO(Mq^+ALA) and <i>Pb</i>FCKO(Mq^+Blood) with respect to WT are <0.01. The data represent 90 mosquitoes from 3 different batches. UI, uninfected; Mq, mosquitoes; <i>Pb</i>ALASKO(Mq^+ALA) and <i>Pb</i>FCKO(Mq^+Blood), <i>P. berghei</i> KO parasites from mosquitoes supplemented with ALA and blood feeding, respectively.</p