A rapid and efficient method for uniform gene expression using the barley stripe mosaic virus
© The Author(s) 2017
Received: 28 October 2016
Accepted: 30 March 2017
Published: 11 April 2017
The barley stripe mosaic virus (BSMV) has become a popular vector to study gene function in cereals. However, studies have been limited to gene silencing in leaves of barley or wheat. In addition, the method produces high variability between different leaves and plants. To overcome these limitations, we explored the potential of modifying the inoculation protocol for BSMV gene overexpression. An improved light, oxygen or voltage-sensing (iLOV) domain-based fluorescent protein was used as a reporter of gene expression to monitor the infection and spread of BSMV. Tobacco (Nicotiana benthamiana) leaves were infected via agroinfiltration and the leaves were homogenized to extract the BSMV particles and inoculate wheat tissues using the traditional leaf abrasion method or by incubation during seed imbibition in a Petri dish.
Compared to the leaf abrasion method, the seed imbibition method resulted in a high and uniform detection of iLOV in both roots and leaves of different wheat cultivars and other monocot and dicot species within 7 days after germination. The progression of viral infection via the imbibition method as measured by the expression of iLOV was more stable in different organs and tissues and is transmissible to the next generation.
Our results show that BSMV can be used as a vector for the expression of small genes such as iLOV in wheat roots and leaves. The inoculation by seed imbibition allows genes to be expressed rapidly and uniformly in wheat and different monocot and dicot species compared to the traditional leaf abrasion method. It also produces high successful transformation as early as 7 days post infection allowing gene function studies during the first generation of infected plants. Furthermore, the method is simple, rapid, and inexpensive compared to the production of transgenic plants.
As a result of rapid advances in genomics, the availability of powerful tools for gene function analysis has become a necessity, especially for important crops. In recent years, barley stripe mosaic virus (BSMV) vectors have been modified and showed important applications for high throughput assays due to its simplicity and ease of use . The BSMV virus is a tripartite (α, β, γ) positive strand RNA virus with RNAs capped at the 5′ end . These RNAs encodes all the genes necessary for viral RNA replication and viral propagation . Recently, a BSMV vector system coupling a ligation independent cloning strategy with an Agrobacterium tumefaciens-mediated delivery system has been engineered and provides substantial advantages in expense, cloning efficiency and ability to apply virus-induced gene silencing (VIGS) for high throughput genomic studies . However, the potential of BSMV vector for gene overexpression in wheat and the optimal inoculation protocol has been less studied [1, 4]. Virus-mediated overexpression (VOX) has been demonstrated for the first time in studies involving the green fluorescent protein (GFP) as reporter [1, 5, 6]. While GFP expression from BSMV was shown to be strong, studies reported that its overall level of expression in monocotyledonous plants is patchy [1, 4, 6]. It was suggested that this could be due to the relatively large size of GFP (720 bp), limiting local and systemic spread of BSMV, and causing instability in the viral genome . Insert sizes lesser than 500 bp were shown to be relatively more stable than larger inserts . An alternative strategy to verify the potential of BSMV vector for a more uniform and stable gene overexpression would be to use a smaller fluorescent protein to reduce gene size and increase stability. An improved light, oxygen or voltage-sensing (iLOV) domain of the plant blue light receptor, phototropin fluorescent protein has previously been demonstrated to be more stable compared to GFP in wheat leaves using BSMV . However, several cells containing chloroplasts do not express iLOV, showing that the expression is not uniform. This is probably due to the use of the leaf abrasion method of virus inoculation .
Furthermore, most protocols of VIGS involving the use of BSMV in plants are so far established for gene silencing in vegetative tissues with very limited success in infection at an early stage resulting in difficulties to evaluate the impact of gene silencing since results vary from different tissue samples [2, 7–9]. Transmission of BSMV through seeds allows a more uniform gene expression in different tissues without showing any viral symptoms . Based on this observation, we postulated that inoculation with BSMV particles at the earliest time possible may result in a more uniform gene expression. We have thus developed a new procedure of BSMV inoculation at the seed imbibition stage. This modification allows rapid and efficient gene overexpression in different wheat tissues, genotypes, and in different monocot and dicot species.
Plant material and growth conditions
Nicotiana benthamiana used for agroinfiltration was grown in a controlled environment chamber at 24 °C under 14 h photoperiod, 100 μmol m−2 s−1 (fluorescent and incandescent lighting) and 70% relative humidity. Different plant species and cultivars were used for BSMV inoculation: Triticum aestivum (wheat) cv. Atlas66, Norstar, Hordeum vulgare (barley) cv. Sophie, Zea mays (maize) cv. Pioneer Hybrid 3921, Brachypodium distachyon inbred line Bd21, N. benthamiana (tobacco) and Arabidopsis thaliana ecotype Columbia (Col-0).
Construction of Agrobacterium-mediated BSMV-iLOV vector
Agroinfiltration of N. benthamiana and viral inoculation
The pCaBS-α, pCaBS-β and pCaBS-γb:iLOV were introduced into A. tumefaciens strain EHA105 as described previously by Yuan et al. . Equal amounts of Agrobacterium harboring pCaBS-α, pCaBS-β and pCaBS-γb:iLOV were mixed (ratio 1:1:1) and incubated for 3–5 h at 28 °C. Infiltration of N. benthamiana leaves was performed using a 1-ml needleless syringe. The bacterial mixture was infiltrated into 3–6 spots (100 µl of resulting in approximately 3–4 cm2 infiltrated area) on each N. benthamiana leaf. After agro-infiltration, N. benthamiana plants were maintained in a controlled environment chamber. At 7 days post-infiltration (dpi), 0.5 g of spot-agroinfiltrated leaves were harvested and ground in 1 ml of 20 mM Na-phosphate buffer (pH 7.2) using a mortar and pestle. Homogenates can be directly used for virus inoculation or aliquoted in small volumes and stored at −20 °C for later use. Two methods of inoculation were tested in this study. Mechanical inoculation was performed using the traditional leaf abrasion method [2, 10]. Diatomaceous earth (1%, w/v) was added to the homogenate for mechanical inoculation onto 7-days old wheat leaves (first leaf). For the seed imbibition method, seeds of T. aestivum, H. vulgare, Z. mays, B. distachyon were germinated in a Petri dish containing N. benthamiana homogenate and distilled water. Different ratios of homogenate: distilled water were tested and a ratio of 1:100 (v/v) was used in this study, since this ratio did not lead to any alterations of seed germination and seedling development. Two different eudicot species (A. thaliana and N. benthamiana) were grown from seeds on 0.8% agarose and the diluted homogenate was directly spread on agarose after 3 days of seed stratification at 4 °C.
Quantitative RT-PCR analysis
Total RNA (including viral RNA) was isolated from different plant tissues using the RNeasy plant mini kit (Qiagen) and treated with on-column RNase free DNAase (Qiagen) before the reverse transcription step. Real-time quantitative RT-PCR was performed on a CFX96 Touch™ Thermal cycler (Bio-Rad) using the SsoFast EvaGreen Supermix (Bio-Rad). The iLOV RNA region was amplified by RT-PCR using the following primers ACAGATCAAGCGACTGTCCA (forward) and CACAGGTTGCAGGTGGAGTA (reverse). Amplification was performed with the following thermal cycling conditions: 5 min at 95 °C followed by 35 cycles of 95 °C for 15 s, 58 °C for 30 s. The copy number of BSMV:iLOV in infected tissue was normalized to the 18S rRNA and determined from a calibration curve using known amounts of iLOV cDNA as described previously .
iLOV fluorescence images were obtained using a confocal laser-scanning microscope (Nikon inverted microscope Eclipse Ti-E) or a NightOWL camera (Berthold LB 983 NC100) with an excitation wavelength of 488 and 450 nm, respectively. Fluorescence emission was detected at 550 nm. Z-stack images were generated through confocal microscopy to remove the out of focus signal collected within each individual image. In some experiments, bright field images were taken using transmitted light detection (TD) to show leaf and root structures.
Determination of growth parameters
Plants were harvested at 14, 21 and 31 days post inoculation (dpi). Entire plants were weighed after drying in a 70 °C oven for 3 days. Physiological variation in growth parameters were determined by total fresh and dry weight and by plant height.
qRT-PCR data are expressed as the mean of absolute quantification (copy number) ± standard deviation (SD) of 3 biological replicates. Comparisons of mean RNA abundance of BSMV:iLOV were conducted using one-way analysis of variance (ANOVA). Differences among means were analyzed using Tukey’s post hoc test at p values <.05. The significance test between treatments for physiological variation in growth parameters was determined by a Student’s t test. Significance was set at p values <0.05. Statistical analysis was performed using InStat 3.0. Graphs were made using GraphPad Prism 7.0.
Results and discussion
The efficiency of BSMV inoculation to silence gene expression in cereals has been documented and demonstrated in several studies [2, 6, 10, 12, 13]. Despite its substantial advantages in time, expense and efficiency, the system still has several limitations including an uneven distribution of gene silencing as shown using phytoene desaturase as reporter gene [2, 14]. To explore the potential use of BSMV for gene overexpression in different wheat tissues, a small gene (330 bp) encoding iLOV was cloned into a BSMV vector to inoculate N. benthamiana leaves via agro-infiltration.
To compare the level of expression in the different tissues, the iLOV RNA region was analyzed by RT-PCR to confirm the specificity of iLOV amplification products (Fig. 2c) and quantified by qRT-PCR (Fig. 2d). While inoculation of BSMV:iLOV into wheat plants either via seed imbibition or leaf abrasion allows the overexpression of iLOV (Fig. 2d), quantification analysis revealed a higher abundance of BSMV:iLOV copies in the first leaf of plants infected via seed imbibition at 7 dpi (Fig. 2d, upper panel). The level of BSMV:iLOV RNA at 21 days in leaf 1 is similar to the one at 7 days. In leaf 2, the level of BSMV:iLOV RNA is similar at 21 dpi between the two methods. However, the large standard error in the leaf treated with the abrasion method reflects a large variation in RNA copy number suggesting that viral propagation is uneven between the three different leaves used for analysis. This result is in agreement with the confocal analysis (Fig. 2a). The BSMV:iLOV abundance in the emerging third leaf was also determined by qRT-PCR. Although the quantification of BSMV:iLOV copies in the third leaf shows less variability, the seed imbibition method allows for a higher abundance of BSMV:iLOV in the growing leaf at 25 dpi suggesting that viral propagation is more efficient with the imbibition method. Root tissues were also harvested to verify the infection and spread of BSMV:iLOV in plants infected either via leaf abrasion or seed imbibition. Analysis by qRT-PCR reveals that BSMV:iLOV RNA is significantly more abundant in roots infected via seed imbibition, in contrast to leaf abrasion as soon as 4 dpi. This suggests that the imbibition method could be particularly useful to study the physiological impact of gene overexpression or RNAi during the early stages of plant growth and as early as 4 dpi.
In previous studies performing virus-induced gene silencing (VIGS) with the scratching method, the peak effect of gene silencing was observed at 20 dpi indicating that the imbibition method allows gene expression and function studies at a much earlier growth development stage. Together, these results confirm the presence of iLOV in root and leaf tissues, and suggest that the seed imbibition method greatly improves the distribution of BSMV and expression of iLOV.
In this study, we have developed a low-cost high-throughput inoculation method allowing rapid and uniform gene expression in different plants based on the barley stripe mosaic virus. Using this new approach, we demonstrated that inoculation greatly improves transfection compared to the traditional leaf abrasion method, and allows efficient and stable viral propagation in different tissues during the first generation as evidence with the iLOV fluorescent reporter. Hence, this protocol could help researchers to take full advantage of the BSMV system which is a powerful functional genomics tool for gene function characterization using gene overexpression or gene silencing in different plant species, particularly during different stages of plant growth.
AC performed the experiments; AC and MH designed the experiments, and interpreted the data and wrote the manuscript. Both authors read and approved the final manuscript.
The authors thank Dawei Li (State Key Laboratory of Agro-biotechnology at China Agriculture University) and Shawn Clark (National Research Council Canada) for providing the BSMV pCaBS-α, pCaBS-β and pCaBS-γ. They also thank Dr. F. Ouellet for sharing the CFX96 TouchTM Thermal cycler (Bio-Rad), Mélanie Grondin and Denis Flipo for their technical assistance in performing the confocal microscopy.
The authors declare that they have no competing interests.
Availability of data and materials
There is no data sets for this manuscript. The material is available from Dawei Li (see acknowledgements). We will gladly provide the BSMV material in Canada upon written consent from Dawei Li allowing our laboratory to distribute the material.
A.C. acknowledges support from the Faculty of Science at UQAM for a Ph.D. scholarship and for a scholarship exempting international student tuition fees. This work was supported by a Natural Sciences and Engineering Research Council of Canada Grant (OGP0138557) to M.H.
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