Plant materials and inoculation
MDMV-OH5 was obtained from a Johnsongrass plant with mosaic symptoms collected from a field in Chillicothe, Ohio in October 2017 (public property collections and private property collections by permission of individual landowners were made, samples were not distinguished to original land ownership), and maintained in a greenhouse at Ohio State University (OSU) Wooster campus under OSU Institutional Biosafety Committee regulations. Plants were destructively sampled and not formally identified by taxonomic experts. Virus was transmitted from the source plant via rub inoculation by grinding leaf tissue in five volumes of 10 mM potassium phosphate, pH 7. Extract was rubbed onto leaves of 10 to 11 day old ‘Oh28’, ‘Early Sunglow’, and ‘Silver Queen’ between thumb and forefinger with inoculum mixed with 600-mesh silicon carbide (carborundum). All plant materials infected with modified virus constructs were grown in controlled growth chamber at 25 °C, 16 hr light and 8 hr dark conditions with light intensity of 13000 lumens.
Sequencing Mdmv Oh5
Total RNA was isolated from four Johnsongrass samples (named MDMV OH3 to MDMV OH6) using Directzol RNA Miniprep Kit (Zymo Research, USA). Complementary DNA (cDNA) was synthesized and used for reverse transcription-PCR (RT-PCR). Primers specific to MDMV OH1 (MDMV-7065F, MDMV-4272F, MDMV genR1) were used for MDMV genome sequence amplification (Additional file 1: Table S1). Amplified DNA fragments were sequenced using the same primers above and MDMV-6241R. MDMV OH5 was used for subsequent studies.
Terminal sequences of MDMV were determined by 5’- and 3’-RACE. For 5’-RACE, 1µ g total nucleic acid extracted from MDMV-infected plants were first annealed with primer WX24 (Additional file 1: Table S1), then used for first strand cDNA synthesis using Superscript III reverse transcriptase (ThermoFisher Scientific, USA), followed by RNaseH treatment as described by the manufacturer (ThermoFisher Scientific, USA). The cDNA was then passed through a Monarch® PCR and DNA cleanup column (New England Biolabs, USA), quantified and G-tailed with 0.25 mM dGTP by terminal transferase (New England Biolabs, USA). The G-tailed cDNA was used as template for PCR with primers WX25 and WX27, followed by a second amplification PCR with primers WX26 and WX29 (Additional file 1: Table S1). PCR amplified DNA was either sequenced directly using primer WX27 or cloned into pMINIT 2.0 vector (New England Biolabs, USA), then subject to sequencing. For 3’-RACE, 1µ g total nucleic acid extracted from MDMV-infected plants were first annealed with primer WX292 (Additional file 1: Table S1), then used for first strand cDNA synthesis using Superscript III reverse transcriptase (ThermoFisher Scientific, USA), followed by RNaseH treatment as described by the manufacturer (ThermoFisher Scientific, USA). The cDNA was then passed through a Monarch® PCR and DNA cleanup column (New England Biolabs, USA). The cDNA was used as template for PCR with primers WX236 and WX293, followed by a second PCR with primers WX29 and WX237. Amplified DNA was either sequenced directly using primer WX27 or cloned into pMINIT 2.0 vector (New England Biolabs, USA) and then subject to sequencing.
Creation Of Infectious Cdna Clone Of Mdmv Oh5
Full-length cDNA from MDMV OH5 was cloned into a binary vector pJL89 [51]. Several infectious MDMV OH5 clones in pJL89 were obtained, one of which, named pWX6, was selected for sequencing and further analysis.
Insertion of GFP at the N-terminal region of CP (pWX27)
Green fluorescent protein (GFP) gene sequence [54] was inserted using NEBuilder HiFi Assembly Master Mix (New England Biolabs Inc., USA) in-frame between pWX6 NIb and CP coding sequences with a duplicated cleavage sites inserted between nt 8386/8387: 15 nt (5’-CagGCcGGcGAgacc-3’, lower case indicating nucleotides changed to alter codons while retaining translated sequence) encoding amino acid sequence Q/AGET, cleaved by NIa-Pro at the beginning of the GFP gene; and 24 nt (5’-GAgGTtATcGAcGTgAAgCAcCAA-3’) encoding NIb cleavage site amino acid sequence of EVIDVKHQ/) at the end of GFP. Primers WX123/WX124 and WX126/WX127 were used to amplify the full-length GFP sequence, primers WX125/LRS764 and WX128/LRS765 for MDMV OH5 sequence, and LRS766/LRS769 were used to amplify the pJL89 vector (Additional file 1: Table S1). The GFP-encoding gene fragment was assembled into pWX6 using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs, USA), and subsequent clones were tested for infectivity.
Insertion of GFP at the N-terminal region of HCPro (pWX68)
GFP sequence was also cloned using NEBuilder HiFi Assembly Master Mix (New England Biolabs Inc., USA) in-frame between MDMV OH5 P1 and HCPro coding sequences with inserted NIb cleavage site sequence in pWX6 (nt 838/839), adding 12 nt 5’ (5’-GCcGAtCCtacc-3’), encoding amino acid sequence ADPT 5’, and 33 nt 3’ (5’- GAgGTAATcGAcGTgAAgCAcCAAGCcGGcGag-3’), encoding amino acid sequence EVIDVKHQ/AGE, cleaved by NIa-Pro) at the end of GFP. Nested primers WX36/WX37 and WX63/WX64 were used to amplify the full-length GFP gene sequence, and primers WX247 and WX250 were used to amplify pJL89 with MDMV sequence (Additional file 1: Table S1). The GFP gene fragment was assembled into pWX6 and recovered clones were tested for infectivity.
Creation of a triple gene insertion in the N-terminal region of HCPro (pWX56)
Infectious clone pWX27 with GFP inserted between NIb and CP was used as backbone vector for a triple partial gene sequence cloning between P1 and HCPro. Three maize genes, magnesium chelatase (ZmChlI, GenBank accession no. DQ084025, target region: 946–1193), lemon white 1 (ZmIspH, GenBank accession no. NM_001175829, target region: 740–988) and phytoene desaturase (ZmPDS, GenBank accession no. L39266, target region: 538–786) were selected for VIGS analysis. The triple gene fragment, 249 nt of each gene with total length of 747 nt, was synthesized (Eurofins Genomics, USA), cloned into pMINIT2.0 vector and verified by sequencing. The triple VIGS gene fragment was then amplified by PCR with primers WX251 and WX252, and the pWX27 backbone was amplified using primers WX247 and WX250, assembled using NEBuilder HiFi Assembly Master Mix (New England Biolabs Inc., USA) in-frame between P1 and HCPro to create pWX56. The triple VIGS DNA fragment contained nine additional 5’ nt (5’-GCcGAtCCt-3’) encoding amino acid sequence ADP at the beginning of the VIGS insertion, and 33 nt 3’ (5’- GAgGTAATcGAcGTgAAgCAcCAAGCcGGcGag-3’), encoding NIb cleavage site amino acid sequence of EVIDVKHQ/AGE, cleaved by NIa-Pro), between nt 838/839 of pWX27. Recovered clones were tested for infectivity and one infectious clone, pWX56, was selected for further analysis.
Infectivity testing by vascular puncture inoculation of in vitro transcripts
All full-length virus constructs were amplified by polymerase chain reaction (PCR) from plasmid DNA templates using primers containing 5’ T7 promoter sequences, WX3 (Table S1) and reverse primer MDMV GenR1 [47] complementary to the 3’-most 17 nt of the MDMV 3-terminal sequence and adding a 21 nt of poly(A) to the virus-sense strand of the amplicon. PCR was performed using PrimeSTAR GXL DNA Polymerase from Takara Bio USA (Mountain View, CA) according to manufacturer’s instructions.
In vitro RNA transcripts were synthesized using T7 ARCA RNA transcription kit (New England Biolabs, USA) rather than the transcription kit used for previous work reporting MDMV-OH1 infectious clone [47] and cleaned using with 2 M lithium chloride or the Monarch RNA cleanup kit (New England Biolabs, USA). Transcript quantity was estimated by NanoDrop (ThermoFisher Scientific, USA) and quality was assessed on non-denaturing 1% agarose 1X TBE (0.089 M Tris, 0.089M boric acid, 0.002M EDTA) gels.). Vascular puncture inoculation (VPI) was used to inoculate ‘Silver Queen’ maize seeds with 2.0 µg RNA transcript per seed as previously described [50, 45]. Inoculated seeds were germinated for two days at 30ºC, sown into sterilized soil, and grown in a growth chamber at 25ºC, 16 hr light and 8 hr dark conditions with light intensity of 13000 lumens. Infectivity of constructs was tested by reverse transcription polymerase chain reaction (RT-PCR) on leaves from individual plants with primers testing for MDMV (WX111 and WX112). RT-PCR was performed on fresh samples at each time point by grinding samples 1:20 (g/mL) in grape extraction buffer (GEB: 0.05M sodium carbonate buffer pH 9.6, 2% polyvinylpyrrolidone-40, 0.2% bovine serum albumin, 0.05% Tween-20), then boiling the samples diluted 4 µl into 50 µl of at 95°C for 10 min in GES buffer (0.1M glycine-NaOH pH 9.0, 50 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, and .01% beta-mercaptoethanol. One-step RT-PCR was performed with SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) and GoTaq polymerase (Promega Corp., Madison, WI) at 52°C for 40 min. followed by 2 min at 94°C and 32 cycles of 94°C for 15 seconds, 55°C for 20 seconds, and 72°C for 1 min, ending with a 7 min. 72°C extension.
Rub Inoculation Scale-up From Transcript-infected Material
To scale up infection with constructs after VPI inoculation with transcripts, VPI-infected leaves were harvested as early as systemic symptoms could be robustly confirmed (7–10 dpi), to maximize virus harvest while minimizing replication cycles in which insert can be lost, and stored at -80˚C for up to 3 months in 0.5 g aliquots. Frozen tissue was ground in five volumes of 10 mM pH 7 potassium phosphate buffer with 600-mesh silicon carbide (carborundum) added as abrasive. Extract was then rubbed onto leaves of 8 to 10-day old corn plants using thumb and forefinger, with 0.5 g frozen tissue providing enough inoculum for 20 plants and resulting in infection rates near 100%. Plants were symptomatic 5–7 days post rub inoculation.
Visualization Of Gfp Expression
GFP expression was visualized on leaves of Z. mays ‘Silver Queen’ at 7, and 21 days post rub inoculation. Images were taken with a Leica DFC460C (Leica Microsystems, USA) camera using fluorescence imaging with NIGHTSEA Green-only bandpass filter (NIGHTSEA, USA) at 3-sec exposure to separate green fluorescence from maize autofluorescence. Brightfield images were taken at 1-sec exposure.
Western Blotting
Western blotting
GFP protein expression was assessed by Western blotting. Plant tissue that tested positive by RT-PCR with primers WX358 and WX367 was saved at -80ºC, for each time point of 7, 14, and 21 days post rub inoculation, and later used for Western blotting. Thawed tissue was ground in 1 ml of radioimmunoprecipitation assay (RIPA) buffer amended with one tablet cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail (Millipore-Sigma, USA) and 300 µl 1M dithiothreitol (DTT) per 10 ml of buffer. Ground tissue was centrifuged at 16,000 g at 4°C for 20 min. Supernatant was transferred to new Eppendorf tubes and placed on ice for remainder of experiment. Total protein was determined using Pierce 660 nm Protein Assay kit and Pierce BCA Protein standards (ThermoFisher Scientific, USA) in a 96 well plate with 3 reps per sample.
Equal parts of leaf supernatant and 2X Laemmli sample buffer (Bio-Rad, USA) were mixed, boiled for 3 min and allowed to cool. Samples were loaded onto 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad, USA) at 25 µg total protein per lane. Recombinant E. coli GFP Protein (Abcam, USA) was loaded at 5 ng as a positive control. Lysates were electrophoresed in Tris/Glycine/SDS buffer (25 mM Tris; pH 8.8, 200 mM glycine, 0.1% sodium dodecyl sulfate) at 200V for 30 min. Proteins were transferred using Trans-Blot Turbo Transfer System on Trans-Blot Turbo Nitrocellulose Transfer Pack (Bio-Rad, USA) paper at 25V for 7 min. Membranes were blocked in 5% nonfat dry milk (NFDM) in TBS buffer (50 mM Tris and 150 mM NaCl, pH 7.5) for 1hr. Membranes were incubated on a shaker at room temperature for 1 hr with 1:3000 Anti-GFP antibody (Abcam, USA) in 1% TBS-T (TBS buffer with 0.1% Tween-20). Membranes were washed for 10 min 3 times in TBS-T. Membranes were incubated with 1:2,500 Goat Anti-Rabbit IgG H&L (Abcam, USA) for 1 hr and then washed for 5 min 3 times in TBS-T. Proteins were visualized by chemiluminescence using Clarity Western ECL Substrate (Bio-Rad, USA) on a ChemiDoc XRS System (Bio-Rad, USA).
Rt-pcr Insertion Stability Assays
Twenty plants were rub-inoculated with wild type and modified MDMV with GFP insert using verified VPI-sourced plant sap. Samples were collected at 7, 14, and 21 days post-rub inoculation from the youngest fully emerged leaf. Plant samples were screened by RT-PCR using the same method described above. Primers spanning whole GFP and GFP internal primers with MDMV-OH5 either upstream or downstream GFP insertion were used for RT-PCR analysis. Amplified DNA was electrophoresed on 1% agarose gels. Leaf samples were collected as described above and analyzed for insertion stability and quantified using either semi-quantification RT-PCR.
Individual target genes of triple VIGS insertion were confirmed using one of MDMV-OH5 specific primers either upstream (WX317) or downstream (WX315) of the triple VIGS insertion and specific primers (WX321 for ZmChlI, WX325 for ZmIspH, and WX327 for ZmPDS) to each of three target genes of magnesium chelatase, lemon white1 and phytoene desaturase (ZmChlI-IspH-PDS; triple VIGS). Photobleaching symptoms were observed and photographed, and chlorophyll content was measured with a MC-100 Chlorophyll Concentration Meter following manufacturer’s instructions (Apogee Instruments, USA). Three measurements were done for each leaf.
Rt-qpcr To Quantify Gene Silencing
Leaf samples were collected 7, 14, and 21 days post-rub inoculation, total RNA was extracted as described above and quantified using either semi-quantitative RT-PCR or RT-qPCR. One microgram total RNA from each sample was used for cDNA synthesis in a 20 µl reaction by using iScript™ Reverse Transcription Supermix (Bio-Rad, USA). Primer efficiency was determined using 1 µl cDNA at 1/5, 1/10, 1/20, 1/40 and 1/160 dilutions (Additional file 1: Table S6). qPCR was carried out by using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, USA) in a Bio-Rad’s CFX96 real-time C1000 touch thermal cycler under conditions of 95ºC for 30 s, 39 cycles of: 95ºC for 10 s and 60ºC for 30 s, then 95ºC for 10 s, melt curve 65ºC to 95ºC with an increment of 0.5ºC, 5 s. All samples were run with cDNA dilution of 1:5, cDNA derived from 10 ng total RNA. Gene quantification and analysis was done on target genes of ZmChlI, ZmIspH, ZmPDS as well as GFP, MDMV, and along with reference genes of membrane protein PB1A10.07c (MEP) and folypolyglutamate synthase (FPGS) [55]. Primers used for RT-qPCR analysis are shown in (Additional file 1: Table S1, S6). Ct was determined using E = 10^[-1/slope] (Additional file 1: Table S6).
Aphid Transmission Assays
Wild type MDMV-OH5 and modified MDMV-OH5 derived VIGS were transmitted by Rhopalosiphum padi. R. padi were maintained on virus-free ‘Early Sunglow’ maize plants in the cages at 25°C with a photoperiod of 15 hr light/ 9 hr dark. ‘Silver Queen’ maize plants were inoculated by VPI with in vitro RNA transcript. The infected tissues were collected and kept at -80ºC, which were used for rub inoculation. The virus source plants were prepared by rub inoculating seven-day-old seedlings ‘Early Sunglow’ with the VPI tissue and the plants were kept at 20°C with a photoperiod of 15 hr light/ 9 hr dark for 14 days. The leaves with strong MDMV symptoms (usually the 1/3 from tip in the 2nd leaf of plant) were sprayed with 10% sucrose, and were left to dry out before collection. The collected leaf was then cut into smaller pieces (around 1 cm2) and were placed in a small box (around 10 cm3) lined with wet tissue paper for maintaining the moisture. R. padi were then fed on source plants in the box for 10 min (acquisition access period), and after acquisition period, 10 aphids were moved onto the corn whorl per plant with 20 healthy ‘Early Sunglow’ plants in total for each treatment. The plants were covered with a plastic tube for 3 days, and then aphids were removed by NUVAN PROSTRIPS (AMVAC Chemical Corporation, USA) for 2–4 hours. Symptoms was scored at 7, and 14 days post-inoculation and infection was further confirmed by RT-PCR using primers WX317/WX315 for VIGS, and WX368/WX357 for GFP insertion.
Statistical analysis
Insert stability in MDMV-OH5 constructs at 7, 14, and 21 dpi was analyzed statistically. Constructs of pWX27, pWX68, and pWX56, three replicates of 20 plants inoculated per construct, were examined for integrity of insertion sequences using RT-PCR with primers flanking the insertion sites. Linear mixed model was used to analyze the effects of constructs, time, and their interactions on band types. F-statistics and probability values from the fit of linear mixed models to arcsine-square root transformed bands data. Data were arcsine-square-root transformed prior to analysis to stabilize variance. Since data were collected as temporal repeated measures on the same experimental units and as such were correlated in time, the random _residual_ statement and type option in GLIMMIX were used to account for, and model, the covariance structure (compound symmetry) of the within-subject data. Models were fitted using the GLIMMIX procedure of SAS.