An effective virus-based gene silencing method for functional genomics studies in common bean
© Díaz-Camino et al; licensee BioMed Central Ltd. 2011
Received: 5 May 2011
Accepted: 13 June 2011
Published: 13 June 2011
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.
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.
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.
Common bean (Phaseolus vulgaris) is the world's most important food legume for direct human consumption in developing countries such as Latin America and Eastern Africa. Although common bean production occurs in a wide range of cropping systems and environments, Latin America with its 8 million hectares in common bean production is the most important region as it accounts for nearly half of the global output .
Thus far, common bean crop improvement through biotechnological approaches has been limited due to its recalcitrance for genetic transformation. Genetic transformation of common bean has been achieved by using biolistic or Agrobacterium tumefaciens-based methods, but with low efficiency [2–8] and thus is not amenable for high throughput molecular and genetic techniques for analysis of gene function. An efficient common bean root transformation protocol for different cultivars and landraces of Phaseolus spp. with A. rhizogenes K599 was recently reported . This method, even though is useful to study genes involved in microbial symbiosis or root architecture, is not applicable to genes involved in other traits, such as those controlling shoot development and flowering.
Virus-induced gene silencing (VIGS) is an appealing reverse-genetic strategy that allows gene functional analysis in species not amenable to stable genetic transformation. However, its success relies on the ability of viral vectors to infect the plant species of interest. There are few examples of viral vectors that are suitable for use as VIGS vectors for legumes; these include pea early browning virus (PEBV), bean pod mottle virus (BPMV), a pseudorecombinant strain of cucumber mosaic virus (CMV) and apple latent spherical virus (ALSV). PEBV (genus Tobravirus) was successfully used in Pissum sativum, Medicago truncatula and Lathyrus od orata . BPMV (genus Comovirus) has been widely used to silence genes or to express foreign proteins in Glycine max[12–16]. Vectors based on a pseudorecombinant strain of CMV (genus Cucumovirus) and ALSV (genus Cheravirus) have been used for VIGS in soybean [17–19].
As far as we know all commercially available cultivars of soybean are susceptible to BPMV infection. In contrast, only a few common bean varieties including the cultivar Black Valentine are susceptible to BPMV. To demonstrate gene silencing mediated by BPMV vectors in common bean, we targeted the genes (PvNod22 and PvSACPDs) that encode, respectively, nodulin 22 (Nod22) and stearoyl-acyl carrier protein desaturases (SACPDs) in the cultivar Black Valentine. Furthermore, we used the BPMV vector for expressing the green fluorescent protein (GFP) in common bean and shown stable GFP expression in all common bean tissues where BPMV was detected thus establishing that the BPMV vector is also suitable for expression of heterologous proteins in common bean.
Construction of viral vectors and in vitro transcription
For generating a Nod22 silencing vector, a 360-bp DNA fragment was amplified from a Nod22 cDNA clone  using sequence-specific primers with Bam HI and Msc I sites (printed in bold): Nod22-FWD-5'GAGGCGGGATCC CAGGCGCTGT-TG'3; Nod22-REV-5' GTCTTCTGGCCA CTCTCCGTGCCC3'. The GFP gene was amplified by PCR from plasmid pSITE-2CA  using the primer pair GFP-FWD 5'ATCCGGATCCGTGAGCAAGGGCGAGGAGCTGTT3' and GFP-REV-5' ACCTTGGCCACTTGTACAGCTCGTCCATGCCGAGAG3'. PCR amplification was performed at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min for 30 cycles. The GFP and Nod22 amplified PCR products were gel purified and digested with Bam HI and Msc I and sub-cloned into pGG7R2-V previously digested with the same restriction enzymes and further verified by sequencing . Construction of BPMV-VIGS for silencing GmSACPDs was previously described . The recombinant BPMV-SACPD RNA2 transcript plus RNA1 from BPMV Kentucky strain Hopkins (K-Hop) or Hancock (K-Han) strains, were used for inoculation. To use plasmid pGG7R2-V as an empty vector control, the plasmid was first digested by Msc I and then re-ligated, giving rise to pGG7R2-M. This action removes the GFP fragment in pGG7R2-V . Transcripts from pGG7R2-M, but not from pGG7R2-V, are infectious. Transcript from pGG7R2-M, plus transcript from RNA1 (subgroup I) cDNA from strain K-Hop or from RNA1 (subgroup II) cDNA from strain K-Han were used for inoculation. The wild type strain K-Hop, which induces very severe symptoms in soybean, is a naturally occurring partial diploid that is diploid for RNA 1 (subgroups I and II) and monoploid for RNA 2 (subgroup II; ). Monoploid RNA1 reassortants containing RNA1 (subgroup I) induces significantly less severe symptoms than the naturally occurring partial diploid strain K-Hop . Plasmid constructs were used for in vitro transcription as previously described . Briefly, capped RNA transcripts were synthesized by incubating 1 to 5 μg of linearized plasmids in a 100-μl reaction mixture containing 40 mM Tris-HCl, pH 7.5; 6 mM MgCl2; 2 mM spermidine; 10 mM dithiothreitol; 50 units of RNasin (Promega Corp., Madison, WI, U.S.A.); 0.5 mM each ATP, CTP, and UTP; 0.1 mM GTP; 0.5 mM cap-analogue (m7G[5γ]G) (New England Biolabs, Ipswich, MA, U.S.A.]; and 50 units of T7 RNA polymerase (New England Biolabs) at 37°C for 2 h. Yield and integrity of the transcripts were analyzed by electrophoresis on a 1.0% agarose gel. RNA1 transcript, derived from RNA1 cDNA, and recombinant RNA2 transcript were used to rub inoculate fully expanded unifoliate leaves of common bean or soybean.
Plant inoculation and growth conditions
RNA transcripts were used to rub inoculate fully expanded unifoliate leaves of common bean (Phaseolus vulgaris cv Black Valentine) or soybean (Glycine max cv Essex). Mock, vector-inoculated, PvNod22-, PvSACPD-silenced or GFP expressing plants were analyzed 15 and 25 dpi. Four-six plants were included per treatment in each biological replicate. Plants were grown in a growth chamber at 24-26°C and 16 h photoperiod.
Sample preparation, RNA extraction and soybean Nod22 RT-PCR analysis
Sequences of primers used for RT-PCR and qRT-PCR.
Primer sequence (5→3, forward, reverse)
Elongation factor-1 alpha
Primer design and qRT-PCR analysis
Gene-specific primers to generate 140-150 bp PCR products were designed using the OligoPerfect™ (Invitrogen) software. Gene specific primers to amplify PvSACPD-C like (PvSACPD-C') and PvEf1-α were designed based on EST records available in the database (Table 1). Two pairs of non-overlapping PvNod22 specific primers were used to determine the real abundance of the Nod22 transcript (nod22-int. and nod22-ext.). By using a complex cDNA mixture, primers were preliminarily tested in semi-quantitative RT-PCR assays to verify the production of a unique PCR fragment of the expected length. Real-time RT-PCR reactions were performed in optical reaction tubes using an iCycler iQ5 apparatus (BioRad, Hercules, CA, USA). Pvnod22 and PvSACPD-C' transcript levels were determined with the SuperScript III SYBR Green One-Step qRT-PCR System (Invitrogen) according to the manufacturer's protocol in a final volume of 25 μL including 0.4 mM of each primer, 12.5 μL of the SYBR Premix (2X), 100 ng of total RNA, and sterile distilled DEPC-treated water. The cycling conditions were: 3 min at 50°C for cDNA synthesis, preheating for 5 min at 95°C followed by 40 cycles (denaturing for 15 s at 95°C, annealing and elongation for 15 s at 55.8°C and data acquisition at 81°C). A negative control reaction without template was also included for each primer combination. The melt curve protocol began immediately after amplification and consisted of 1 min at 55°C followed by 80-10 s steps with a 0.5°C increase in temperature at each step, to ensure the absence of primer-dimers and a single PCR product. Threshold values for threshold cycle (Ct) determination were generated automatically by the iCycler iQ5 software. Transcript levels for each of the target genes (PvNod22 and PvSACPD-C') were normalized to the endogenous elongation factor PvEf1-α transcript level. Three technical replicates were analyzed for each biological replicate. Each biological replicate resulted in similar trends and we report a mean value for each experiment.
Visualization of GFP expression in leaves and roots of HopV and HopGFP treated plants
GFP was observed under a Zeiss LSM 510 Meta confocal microscope attached to an Axiovert 200 M. GFP excitation was obtained at 488 nm using an Argon laser, an HFT UV 488/543/633 main dichroic excitation mirror and a BP 500-550 IR emission filter for detection. Simultaneously, autofluorescence was observed by exciting at 543 nm with a He/Ne laser; with the same main dichroic excitation mirror, a LP 560 emission filter and a BG 39 dichroic beam splitter. Images were processed using Adobe Photoshop 7.0 software (Adobe Systems Inc., Mountain View, CA, U.S.A.).
BPMV-based VIGS vector effectively silences Nod22 expression in common bean
The level of the Nod22 mRNA expressed 15 days postinoculation (15 dpi) was determined by using nod22-int. and nod22-ext. primer sets and the coding sequence of PvEf1-α as a reference transcript in common bean (Table 1; see also additional file 2: Annealing positions of Nod22 primers). The results indicated that Nod22 was not induced during viral infection (Figure 2, mock versus vector). Although Nod22 expression levels varied in Nod22-silenced plants (Figure 2, lower panel; VIGSNod22 P1-P3), these were diminished considerably (up to 50%) compared to mock and vector-treated plants. Similar results were also obtained in Nod22-silenced soybean plants (data not shown).
Silencing of SACPDs in common bean causes similar morphological changes to those induced in soybean plants
In addition to Nod22, we silenced PvSACPDs in common bean. Expressed sequence tags (ESTs from libraries prepared from pod, nodule or leaf tissues) encoding putative SACPD-like proteins were identified from the database (The DFCI Phaseolus vulgaris Gene Index [PhvGI], CV534591, TC14891, TC12707; ). Nucleotide sequences of PvSACPDs were compared with the soybean genes GmSACPD-A, B and C. Our analysis indicated that common bean SACPDs are related to the soybean isoforms GmSACPD-A/B (PvSACPD-A/B-like or A'/B') and C (PvSACPD-C-like or C'). Due to the high sequence similarity of SACPDs between common bean and soybean, we used a recombinant BPMV-based vector previously used to silence all three GmSACPDs in soybean . The recombinant RNA2-SACPD construct includes a highly conserved 258 bp fragment from GmSACPD-A. This sequence is highly conserved among all three GmSACPD isoforms as well as among the PvSACPDs isoforms (See additional file 3: Alignment of SACPD sequences).
Expression levels of foreign genes from BPMV vectors in common bean
We used the BPMV-based vector, previously described for applications in soybean , for gene silencing studies in common bean. To our knowledge, this is the first report that documents efficient and stable silencing of endogenous genes in common bean by VIGS. We also demonstrated stable and efficient systemic expression of the marker protein GFP, thus establishing that the BPMV vector is suitable for expression of heterologous proteins in common bean. The BPMV-GFP vector was previously shown to be stable after four serial passages in soybean cv Essex  and three passages in common bean cv. Black Valentine [; reported as data not shown]. Here we extend this finding by using confocal microscopy to demonstrate efficient GFP expression in all common bean tissues where BPMV is detected.
The BPMV-RNA2 vector is the only available plant-virus-based vector that is appropriate for stable expression of foreign proteins in common bean. Although the cowpea mosaic virus (CPMV)-RNA2 vector  could potentially be used for these applications, there is concern about its genetic stability due to homologous recombination as a consequence of the duplication of the cleavage sites. In engineering the BPMV-RNA2 vector, we altered the nucleotide sequence of the duplicated regions without affecting its amino acid sequence in order to minimize the chances of homologous recombination . Recently, Zhang et al  reported the development of a modified BPMV-RNA2 expression vector similar to that previously reported for the CPMV-RNA2 vector , in which they replaced the duplicated Q/M cleavage site with a foot and mouth virus autocatalytic 2A peptide. Incorporation of a 2A sequence between GFP (or protein of interest) and L-CP precludes the need to duplicate viral sequences and therefore is advantageous in terms of the genetic stability of constructs [26, 28]. Cleavage at a 2A site occurs between the last two amino acids of the sequence, resulting in that GFP (or the protein of interest) having all but one of the 2A amino acids attached to its C terminus, whereas the L-CP has only an additional proline at its N-terminus. Thus, the expressed protein (GFP) using such a vector is still not authentic in that it contains ~20 or more amino acids derived from the 2A sequence. Furthermore, due to the incompleteness of the cleavage mediated by 2A peptide, a significant portion of the L-CP subunits could carry GFP fused to their N termini, and these fusion proteins can be incorporated into virus particles. Such particles are predicted to be empty (noninfectious) since the N-terminus of the L-CP, now fused to GFP, is not available to interact with the viral RNA . Because of these reasons, the stability of the recombinant vector could be difficult to maintain through serial passage, a matter that was not tested by Zhang et al. .
In addition to protein expression in common bean, we used the BPMV-RNA2 vector to demonstrate efficient and stable silencing of endogenous genes coding for Nod22 and SACPDs. Nod22 is a small-heat shock protein (sHSP). sHSPs constitute a diverse family of molecular chaperones, crucial components of plant stress tolerance and normal development. Indeed, Nod22 transcripts are accumulated in response to pathogens  and root nodule development . Remarkably, when this protein is over-expressed in E. coli, it confers protection against oxidative stress . Phenotypic results on functional analysis of Nod22 in common bean and soybean, achieved in this work, characterized by the appearance of systemic necrotic lesions and interveinal necrosis (Figure 2) further supported the hypothesis that Nod22 is involved in plant defense responses.
Likewise SACPDs are also well known to be involved in plant defense . SACPD is the archetype member of a family of soluble fatty acid (FA) desaturases; these enzymes play an important role in regulating the overall levels of desaturated FAs in the cell. A mutation in the SACPD encoding SSI2 gene in Arabidopsis results in constitutive expression of pathogenesis-related (PR) genes, spontaneous lesion formation, increased accumulation of the phytohormone salicylic acid, and enhanced resistance to bacterial and oomycete pathogens . Analysis of suppressor mutations has shown that the altered defense signaling in the ssi2 plants is a result of their altered FA profile [32–35]. Silencing SACPD genes in soybean and rice also induce similar phenotypes as in Arabidopsis [13, 36]. Our work shows that this FA-derived defense-signaling pathway is also conserved in common bean (Figure 5).
The choice of BPMV strain(s) to use for VIGS studies in common bean is not an issue since the symptoms induced by infection involving RNA1s from both strain K-Han (mild symptoms on soybean) and K-Hop (moderate/relatively severe symptoms on soybean) are relatively mild on common bean and do not appear to interfere with the VIGS phenotypes (Figures 4 and 5). Provided efficient silencing can be achieved, it is obviously preferable to use symptomless or very mild virus strains in VIGS studies, mainly because of easier detection of silencing phenotypes. However, to use such symptomless virus strains, the inoculation protocol becomes cumbersome since the inoculum has to be increased in an alternative symptomatic host. For example, ALSV inoculum has first to be increased in Chenopdium quinoa and following two passages in C. quinoa, total RNA was prepared from the infected leaves and used to inoculate soybean leaves via particle bombardment . Furthermore, It should be emphasized that the virus-induced symptoms become evident long after the onset of virus infection and replication. Using genomics and proteomics technologies it was possible to identify at least 250 host factors that affect virus replication . Thus it is much more complicated than perceived in previous reports that commented on the rationale for using symptomless virus strains for VIGS work [17, 19, 27, 38].
Agroinfection is the most cost effective and efficient method for inoculating plants with virus-based vectors  and thus ideal for high-throughput applications including screening of cDNA libraries or EST collections. Although agroinfection has been used successfully for several different plant species, it is not yet amenable for use in common bean or soybean. We routinely and successfully use the transcript rub-inoculation method to introduce the BPMV vector into soybean and common bean. The efficiency of the transcript inoculation method depends on the quantity and quality of transcripts, regardless of the source of RNA1. In our experience, we obtain better RNA1 transcript yield with plasmid pGHoR1 than with plasmids pGG7R1 or pCRHaR1 . Considering that the VIGS phenotype is independent of the source strain of RNA 1, we routinely use pGHoR1 in our studies. We generally obtain 40-80% infection rate (average 60%), and this is very satisfactory for our purpose since we get 100% infection with first passage plants. Use of biolistics to introduce the vector into individual plants with 35S promoter-driven DNA constructs using a gene gun [27, 38] seems unnecessary considering the stability of our vector and the need to use transcripts only once for inoculation. We and others have successfully silenced or over-expressed a large number of soybean and common bean genes using the transcript rub-inoculation method [[13–16, 40] and Ghabrial, unpublished]. Until Agrobacterium-mediated inoculation methods become available for soybean and common bean, the transcript rub inoculation method has proven efficient for our laboratory.
The development of BPMV-VIGS vector is an important advance for the common bean research community since rapid functional studies in common bean have been greatly restrained due to the lack of appropriate tools. In this work, we have used the BPMV-based vector, previously described for applications in soybean , for gene silencing studies in common bean. As far as we know this is the first report that documents efficient silencing of endogenous genes in common bean by VIGS. Furthermore, we demonstrated efficient systemic expression of the marker protein GFP thus establishing that the BPMV vector is suitable for expression of heterologous proteins in common bean.
Acknowledgements and Funding
This work was funded in part by grants from United Soybean Board and Kentucky soybean Promotion Board (to SAG and AK) and from DGAPA UNAM IN203807 and IN202009. We thank Xochitl Alvarado-Affantranger and Wendy Havens for their technical support and Amy Crume for greenhouse care.
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