An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.
Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22) or phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.
Infectious clones of many plant viruses have been engineered for virus-induced gene silencing (VIGS), gene overexpression (VOX), and most recently, virus-enabled gene editing (VEdGE)1,2,3,4,5,6,7,8,9,10,11. As new viral constructs are developed, methods to successfully infect plant tissues with these modified viruses must also be considered. Current methods to launch virus infections in plants include particle bombardment, rub-inoculation of in vitro RNA transcripts or DNA clones, vascular puncture inoculation, or Agrobacterium tumefaciens inoculation (agroinoculation)5,12,13,14,15,16,17. Each of these inoculation methods has inherent advantages and disadvantages, which include cost, need for specialized equipment, and feasibility within a given plant-virus system. Methods that utilize infiltration or injection of Agrobacterium strains containing binary T-DNA constructs designed to deliver recombinant viruses are preferred, because they are simple and inexpensive. However, detailed agroinoculation methods for monocotyledonous species such as Zea mays (maize) are lacking.
One of the first reports of agroinoculation for virus delivery was published in 1986, when the genome of cauliflower mosaic virus (CaMV) was inserted into a T-DNA construct, and the resulting Agrobacterium carrying this construct was rub-inoculated onto turnip plants18. Additional methods for agroinoculation have since been developed. For example, in the case of foxtail mosaic virus (FoMV), Nicotiana benthamiana can be used as an intermediate host to generate virus particles in leaves that provide an inoculum source6. Rub inoculation of maize using infected N. benthamiana leaves is efficient, rapid, and simple, but the use of an intermediate host does not work for all maize-infecting viruses. Sugarcane mosaic virus (SCMV), for example, cannot infect N. benthamiana, requiring the use of alternative inoculum sources for vectors derived from this virus. In 1988, Agrobacterium containing maize streak virus (MSV), a DNA virus, was introduced into maize seedlings by injection (agroinjection), demonstrating Agrobacterium-based inoculation methods are also useful for monocots19. Despite this early success with agroinjection, few studies utilizing this technique in maize have been published, leaving open questions about the applicability of this method for RNA viruses and VIGS, VOX, and VEdGE vectors20,21,22. However, broad use of agroinjection in monocot species is promising, because this general approach has been utilized in orchid, rice, and wheat23,24,25,26,27,28.
This protocol was optimized for FoMV and Agrobacterium strain GV3101 and has also been applied to an SCMV vector. FoMV is a potexvirus with a wide host range that includes 56 monocot and dicot species29. FoMV has a 6.2 kilobase (kb) positive sense, single-stranded RNA genome that encodes five different proteins from five open reading frames (ORFs)30,31,32,33,34,35. FoMV was previously developed into both a VIGS and VOX vector for maize by incorporating an infectious clone onto a T-DNA plasmid backbone6,36,37. The viral genome was modified for VIGS applications by adding a cloning site (MCS1*) immediately downstream of the coat protein (CP) (Figure 1A)36. For VOX and VEdGE applications, the CP promoter was duplicated and a second cloning site (MCS2) was added to enable insertion of sequences of interest between ORF 4 and the CP (Figure 1B)6. The FoMV vector containing both MCS1 and MCS2 with no inserts is FoMV empty vector (FoMV-EV) (Figure 1).
SCMV is an unrelated virus that has been developed for VOX in maize38. It is a member of the Potyviridae family, of which several members have been engineered to express foreign proteins in planta39,40,41,42,43,44. The host range of SCMV includes maize, sorghum, and sugarcane45,46, making it valuable for gene functional studies in these major crop plants36,38. SCMV has a positive sense, single-stranded RNA genome of approximately 10 kb in length47,48. To create the SCMV VOX vector, the well-established P1/HCPro junction was utilized as an insertion site for heterologous sequences38. This cloning site is followed by sequence encoding a NIa-Pro protease cleavage site, leading to the production of proteins independent from the SCMV polyprotein (Figure 1C).
T-DNA plasmids carrying infectious cDNA of these recombinant viruses have been transformed into Agrobacterium strain GV3101. GV3101 is a nopaline type strain, which are well-known to be able to transfer T-DNA to monocotyledonous species, including maize26,28,49. Additionally, previous agroinjection studies have used the strains C58 or its derivative GV3101, as well19,20,22,27.
Three marker genes were used in the development of this protocol: two for gene silencing and one for gene expression. A 329 base-pair (bp) fragment from the maize gene lesion mimic 22 (les22, GRMZM2G044074) was used to construct the silencing vector FoMV-LES22. When les22 is silenced in maize, small, round patches of necrotic cells appear along the vasculature of leaves that expand and coalesce into large areas of necrotic leaf tissue50. FoMV-PDS, containing a 313 bp fragment from the sorghum gene phytoene desaturase (pds, LOC110436156, 96% sequence identity to maize pds, GRMZM2G410515), induces silencing of pds in maize, resulting in small streaks of photobleached cells along the vasculature of the leaves that lengthen over time51. The intact coding sequence for green fluorescent protein (GFP) was used to demonstrate protein expression for both FoMV (FoMV-GFP) and SCMV (SCMV-GFP). GFP expression in the leaves is typically most detectable at 14 days post inoculation (DPI)6. Although there have been previous studies utilizing agroinjection of viral vectors in maize, these experiments have only shown that agroinjection can facilitate viral infection from an infectious clone in maize seedlings and do not expand to recombinant viruses designed for VIGS or VOX applications19,20,21,22. The protocol presented here builds upon previous agroinjection methods, particularly Grismley et al.19. Overall, this agroinjection method is compatible with VIGS and VOX vectors, does not require specialized equipment or alternative hosts as inoculum sources, and decreases the overall time and cost required to set up and perform inoculations relative to other common methods that require biolistics or in vitro transcription. This protocol will facilitate functional genomics studies in maize with applications involving VIGS, VOX, and VEdGE.
Agrobacterium is an essential tool that facilitates numerous molecular biology techniques in plant-related research. This study provides an agroinjection protocol for inoculating FoMV and SCMV viral vectors directly into maize tissues for VIGS and VOX applications. The main goal is to increase the ease and utility of virus-based technologies for research in monocot crop plants. Although direct agroinoculation of maize has been reported for a few viruses, the authors are not aware of a detailed protocol, and there are no examples of VIGS and VOX applications in those studies19,22.
It has been reported, and was confirmed while developing this protocol, that the injection location is a key factor for successfully launching a systemic viral infection via agroinjection19. Consistently injecting the recommended location on the plant is assumed to be the largest variable, because the exact position of the meristem in maize seedlings is virtually undetectable by eye. To minimize interpersonal variation, dissecting a few maize seedlings down to the meristem is recommended to better visualize its location (Figure 3C). The meristem's position in relation to the coleoptilar node should be roughly the same for plants aged 4-7 days old. Additionally, practicing injection with a dyed liquid provides an easily visible demonstration of how the "inoculum" fills the leaf whorl, and because the injection site is marked with dye, the accuracy of the injection site can be corroborated (Figure 3G,H). Meristematic tissues are the most susceptible to agroinjection, but injecting Agrobacterium suspensions directly into this tissue results in undesirable morphological effects (Figure 6)19. Plants with damaged meristems survive, but the resulting defects are undesirable, and thus, direct injection of this tissue should be avoided.
There are several variables that may impact the successful launch of a systemic viral infection via agroinjection because three complex biological systems (plant, virus, and Agrobacterium strain) must interact in coordination. This complex interplay may be aided by the rapidly-dividing cells of the meristematic region, making it an ideal location for agroinoculation19. The Agrobacterium strain must be able to infect cells of the plant tissues to deliver the T-DNA carrying the viral genome, and the plant must be susceptible to the virus in order to initiate viral replication and systemic infection. Maize genotypes differ in their susceptibility to viruses (e.g., Mo17 is resistant to FoMV) or Agrobacterium strains, but the majority that were tested appear to be susceptible to both FoMV and SCMV (Table 1 and Table 2)53. For example, the inbred line FR1064 and the sweet corn variety Golden Bantam may be particularly susceptible to both GV3101 Agrobacterium and FoMV-based vectors.
The leaf number sampled and the timing of sampling for RT-PCR is critical for accurate assessment of viral infection. In the examples shown here, leaf number was determined by starting at the first rounded leaf (commonly known as the "thumb leaf") and counting upward. Leaves were sampled once they were expanded and the next leaf had begun emerging. However, which leaves are optimal for sampling might vary based on virus species used, growth conditions, and maize genotype. Therefore, an initial time course experiment is recommended when applying this protocol to a new virus system to optimize the sampling strategy with respect to leaves and timing.
The specific construct used significantly affects the efficiency of this protocol. For example, the empty vectors, FoMV-EV and SCMV-EV, and FoMV-PDS and FoMV-LES22,which both contain small inserts (313 bp and 329 bp, respectively), typically produce the highest percentages of plants with viral symptoms in these experiments (Tables 1 and Table 2). However, recombinant viruses carrying larger inserts of the GFP ORF (720 bp) in FoMV-GFP and SCMV-GFP, had much lower infection rates when compared to plants injected with the empty vector or gene silencing constructs. This trend may be due to the negative impacts on viral fitness caused by increasing amounts of exogenous genetic material in the viral genome. Several studies have shown that the insert stability of plant viral vectors is largely dependent on insert size and sequence36,54,55,56,57. Additionally, there was a notable difference in percentage of plants that become infected following inoculation with either the FoMV or SCMV empty vector, suggesting additional work is needed to optimize this protocol for SCMV (Table 1). These results indicate that some troubleshooting may be needed when developing a construct, because the sequence and length of the fragment can both affect efficiency.
Overall, this study has shown that agroinjection of maize seedlings is an effective inoculation method for two different RNA plant viruses, multiple vector configurations, and 11 genotypes of maize. This work with FoMV and SCMV, paired with previous works utilizing injection with maize chlorotic mottle virus (MCMV) or MSV, indicates that agroinjection is suitable for inoculating maize seedlings with infectious clones of both RNA and DNA viruses19,20,21,22. Additionally, this work further shows agroinjection is a viable method for VIGS and VOX vectors and can be applied to plants as young as four days old (Table 3). The protocol presented here is expected to be readily adapted by maize biologists to facilitate research in functional genomics studies involving transient gene silencing (VIGS) and overexpression (VOX). Agroinjection also has the capacity to facilitate virus-based gene editing approaches (VEdGE) that would otherwise be limited by reliance on plant transformation, potentially improving editing efficiency as well as accessibility58,59,60. Given the appropriate Agrobacterium strain, maize genotypes, and viral vectors are thoughtfully combined, inoculation by agroinjection is expected to become a valuable tool for transient gene function analyses in maize.
The authors have nothing to disclose.
Iowa State University is part of a team supporting DARPA's Insect Allies program HR0011-17-2-0053. This work was also supported by the Iowa State University Plant Sciences Institute, Iowa State University Crop Bioengineering Center, USDA NIFA Hatch project number 3808, and State of Iowa Funds. K.L.H. was also partially supported by the Iowa State University Predictive Plant Phenomics graduate training program funded by the National Science Foundation (DGE #1545453) and by Agricultural and Food Research Initiative grant no. 2019-07318 from the USDA National Institute of Food and Agriculture. The funders had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the funders.
We thank Nick Lauter (USDA-ARS, Ames, IA) for seed of maize inbred lines, Christian F. Montes-Serey (Iowa State University) for making the FoMV-GFP clone, and Tyler Austin (Iowa State University) for technical assistance.
1 mL syringes | Fisher Scientific | 14955450 | alternatively, BD 309659 |
15 mL Falcon Tubes | Corning Science | 352059 | |
1kb+ Ladder | ThermoFisher Scientific | 10787018 | For assessing sizes of PCR products |
25G x 5/8" PrecisionGlide Needles | Becton, Dickinson and Company (BD) | 305122 | |
28°C Incubator | For Agrobacterium | ||
37°C Incubator | For E. coli | ||
Acetosyringone | MilliporeSigma | D134406 | Optional |
Agar | MilliporeSigma | A4800 | |
Agarose | GeneMate | E-3120 | For making gels to check for virus/insert stability |
Agrobacterium tumefaciens Strain GV3101 | Carries vir plasmid encoding T-DNA transfer machinery, RifR, GmR, from lab stock | ||
Bsu36I | New England Biolabs | R0524 | |
cDNA Kit | ThermoFisher Scientific | K1672 | Maxima First Strand cDNA Synthesis Kit with Dnase |
Chloroform | Fisher Scientific | C298 | For RNA extraction |
Cuvettes | Fisher Scientific | 14955127 | 1.5 mL |
D-(+)-Glucose | MilliporeSigma | G7528 | Alkaline Lysis |
DH5alpha Competent E. coli Cells | New England Biolabs | C2987 | |
DNA Ligase | ThermoFisher Scientific | K1422 | Rapid DNA Ligation Kit |
EDTA (Ethylenediamine Tetraacetic Acid, Disodium Salt Dihydrate) | Fisher Scientific | S311 | Alkaline Lysis |
Ethanol | For RNA extraction | ||
Fertilizer | Peters Fertilizer 15-15-15 Concentrate | ||
Flat Inserts | T.O. Plastics | 715357C | For germinating seeds in trays |
Flats | T.O. Plastics | 710245C | For germinating seeds in trays |
FluorCam | Photon Systems Instruments | To assess maize plants for GFP expression before microscope | |
Fluorescence Microscope | |||
Gel Electrophoresis Box | |||
Gentamycin Sulfate | Fisher Scientific | BP918 | |
Glacial Acetate | Fisher Scientific | A38 | Alkaline Lysis |
Glycerol | Fisher Scientific | G33-500 | For saving frozen stocks of bacteria |
Go-Taq, 2X | Promega | M7123 | |
Hydrochloric Acid | Fisher Scientific | A144 | for pHing solutions |
Isopropanol | Sigma-Aldrich | 109827 | For RNA extraction |
Kanamycin, Monosulfate | Fisher Scientific | BP906 | |
Large Pots | Kordlok | SQL0550 | 5x5x4" or bigger. For transplanting seedlings. |
Luria Bertani (LB) Broth, Miller | Himedia | M1245 | |
Magnesium Sulfate Heptahydrate | Amresco | 662 | |
Maize Golden Bantam Sweet Corn Seed | American Meadows, West Coast Seeds | ||
Maize Inbred Seed | Our seed comes from our institution, but we are not able to provide this for other researchers. | ||
Maxima H Minus Reverse Transcriptase | ThermoFisher Scientific | EP0753 | |
MilliQ | Elga | Purelab Ultra | |
Monarch PCR & DNA Cleanup Kit | New England Biolabs | T1030 | |
PacI | New England Biolabs | R0547 | |
Peters Excel 15-5-15 Fertilizer | ICL Specialty Fertilizers | G99140 | |
Petri Dish, 95 mm x 15 mm | Fisher Scientific | FB0875714G | |
pH Meter | |||
Potassium Acetate | Fisher Scientific | P171 | Alkaline Lysis |
Primers | Our primers were synthesized through our institutional DNA facility or through IDT | ||
PspOMI | New England Biolabs | R0653 | |
Q5 High-Fidelity DNA Polymerase | New England Biolabs | M0491 | |
Rifampicin | EMD Millipore Corp | 557303 | |
Rnase A | ThermoFisher Scientific | 12091021 | Alkaline Lysis |
SbfI | New England Biolabs | R0642 | |
Scale | For weighing chemicals for media or buffers | ||
SDS (Sodium Dodecyl Sulfate) | Fisher Scientific | BP166 | Alkaline Lysis |
Sodium Hydroxide | Fisher Scientific | S318 | Alkaline Lysis |
Soil Substrate | SunGro Horticulture | SS#1-F1P | Sunshine Mix #1/Fafard-1P, any soil mix that maize grows well in is sufficient |
Spectrophotometer | For measuring OD600 | ||
Sybr Safe, 10,000X | Invitrogen | S33102 | For making gels to check for virus/insert stability |
Thermocycler | For PCR | ||
Tris Base | Fisher Scientific | BP154 | Alkaline Lysis |
Trizol | Ambion | 15596018 | For RNA extraction |
Weigh Paper | For weighing chemicals for media or buffers | ||
XbaI | New England Biolabs | R0145 |