Artificial miRNA mediated resistance in tobacco against Jatropha leaf curl Gujarat virus by targeting RNA silencing suppressors

The leaf curl disease of Jatropha caused by geminiviruses results in heavy economic losses. In the present study, we report the identification of a new strain of a Jatropha leaf curl Gujarat virus (JLCuGV), which encodes six ORFs with each one having RNA silencing suppressor activity. Therefore, three artificial microRNAs (amiRNAs; C1/C4, C2/C3 and V1/V2) were designed employing overlapping regions, each targeting two ORFs of JLCuGV genomic DNA and transformed in tobacco. The C1/C4 and C2/C3 amiRNA transgenics were resistant while V1/V2 amiRNA transgenics were tolerant against JLCuGV. The relative level of amiRNA inversely related to viral load indicating a correlation with disease resistance. The assessment of photosynthetic parameters suggests that the transgenics perform significantly better in response to JLCuGV infiltration as compared to wild type (WT). The metabolite contents were not altered remarkably in amiRNA transgenics, but sugar metabolism and tricarboxylic acid (TCA) cycle showed noticeable changes in WT on virus infiltration. The overall higher methylation and demethylation observed in amiRNA transgenics correlated with decreased JLCuGV accumulation. This study demonstrates that amiRNA transgenics showed enhanced resistance to JLCuGV while efficiently maintaining normalcy in their photosynthesis and metabolic pathways as well as homeostasis in the methylation patterns.

Viruses have emerged as major plant pathogens causing severe losses to agricultural production worldwide 1 . Geminiviridae is the second largest family of plant viruses 2 , possessing the potential to exploit a broad range of plants as its host. The geminiviruses are characterized by geminate virus particles that are categorized into nine genera based on their genome organization, insect vector, host range and phylogeny reconstruction, viz., Becurtovirus, Begomovirus, Curtovirus, Eragrovirus, Mastrevirus, Topocuvirus, Turncurtovirus, Capulavirus and Grablovirus. Based on the number of DNA molecules in the viral genome, they can be: monopartite (one DNA molecule) or bipartite (two DNA molecules: DNA-A and DNA-B) 3 . The viral genome in monopartite viruses and the DNA-A component of bipartite viruses are homologous and have a similar organization that includes the V1/AV1, C1/AC1, C2/AC2, C3/AC3, and C4/AC4 ORFs 3 .
The geminiviruses infect a wide range of economically important crops like cassava, cotton, grain legumes and tomato, causing significant financial losses across the world. These economic losses have been estimated to about US$ 1300-2300 million for cassava in Africa 4 , US$ 5 billion for cotton in Pakistan, US$ 300 million for grain legumes in India 5 and the US$ 140 million in Florida for tomato alone 6 . The DNA viral mutation rates roughly range between 10 -8 and 10 -6 substitutions per nucleotide per cell infection 7 . This high rate of recombination and mutation leads to their great diversity worldwide 8 , thereby increasing their host range and geographical distribution. Thus, geminiviruses are emerging as widely spread and diverse plant DNA viruses posing severe threat to crop production.
JLCuGV genes function as RNA silencing suppressors. For identification of potential RSS in the JLCuGV, the six ORFs of the genome (AV1, AV2, AC1, AC2, AC3 and AC4) were individually cloned in pCAM-BIA-1301 (Fig. 1a) and were independently agroinfiltrated in the in-house-developed stable green fluorescent protein (GFP)-silenced tobacco lines. The presence of strong green fluorescence indicated a reversal of GFP silencing. The assay was performed using the well-characterized RSS, FHVB2 (Flock house virus B2 gene), as a positive control 24 and empty vector and mock (only buffer) infiltration as a negative control [ Fig. 1b(iv)]. The infiltrated leaf patches were observed under UV at regular intervals of 7, 11 and 15 dpi (days post-inoculation; Fig. 1b(i-iii)). The GFP fluorescence was obtained with all six JLCuGV genes indicating that all the ORFs contained inherent RSS activity. This was confirmed by checking for the presence of GFP transcripts in the corresponding infiltrated regions (Fig. 1c). The cDNA generated from the leaves infiltrated with FHVB2 or individual JLCuGV ORFs showed a ~ 300 bp band for GFP transcript, but no band amplified from the mock control. However, detailed molecular analysis is required to characterize the strength, duration and complementation of the RSS activity exhibited by individual ORFs.
Transgenics show resistance against JLCuGV. To analyze the tobacco plants producing amiRNAs for resistance to virus infection, the agroinfectious clone of JLCuGV ( Supplementary Fig. S4) was used for infiltration. The agroinfiltration in WT and VA (vector alone) plants served as control. All the WT (total 3 out of 3 inoculated plants) and VA (total 3 out of 3 inoculated plants) plants showed curl-mosaic symptoms after 21 dpi, and the symptoms increased with an increase in time (Fig. 2a,b). Visible symptoms were not observed in the C1/C4 (L61,   Fig. 2c,d), but 3 out of 9 plants expressing amiRNA V1/V2 (L13, L14, L15) showed weak symptoms on leaves (Fig. 2e). At 28 dpi, the genomic DNAs from all these plants were checked for the presence of viral DNA by PCR (450 bp) using specific primers (Fig. 2f) and reconfirmed by checking for genomic DNA amplicon (2.7 kb) obtained by HindIII digestion of RCA product (Fig. 2g). The actin gene was amplified as loading control (Fig. 2f,g). The DNA bands were observed in virus-infected WT and VA plants, but no amplification was seen in case of C2/C3 and C1/C4 amiRNA transgenics. In virus-infected V1/V2 amiRNA transgenic plants, a faint band was obtained.
The WT, VA and V1/V2 amiRNA transgenics, which were found positive for JLCuGV infection, were further checked for the viral load using qRT-PCR for the quantitative determination of infection severity. The viral load of WT and VA was 4959 and 4999 copies/ng, respectively at 14 dpi, which increased by 1.93-and 1.89-fold respectively at 35 dpi (Fig. 2h). However, the viral load of V1/V2 amiRNA transgenics (5364 copies/ng at 14 dpi) decreased by 0.88-fold at 35 dpi (Fig. 2h).
For analyzing the changes in methylation patterns of transgenics in comparison to WT, the total bands were further classified as "A to P" (16 combinations; Table 3) based on their banding pattern. The banding pattern was classified into three types: no change (A to D), methylation (E to J) and demethylation (K to P; Table 3). The pairwise comparison between transgenics and WT revealed maximum "no change" and minimum "no change" in VA (65.03%) and C1/C4 amiRNA transgenics L67 (28.76%), respectively ( Table 3). The L43 of C2/ C3 amiRNA transgenics showed maximum change in methylation (14.51%) pattern compared to WT, whereas the lowest change in methylation pattern (9.59%) was observed in VA. The demethylation percentage was found to be maximum in L67 (45.08%) of C1/C4 amiRNA transgenics, while the lowest demethylation in VA (13.47%) in comparison to WT (Table 3).

Improved photosynthesis parameters in amiRNA expressing transgenics on JLCuGV infiltration.
For understanding the effect of virus infection on the photosynthesis process, the WT, VA and amiRNA transgenics (C1/C4, C2/C3 and V1/V2) were infected (in 3 replicates) with JLCuGV and analyzed for various photosynthetic parameters.

Metabolite profiling of amiRNA expressing transgenics. Metabolite analysis was performed in both
WT and amiRNA transgenics with and without JLCuGV agroinfiltration. Twenty-one common metabolites were detected in both healthy and virus infiltrated tissue (Supplementary Table S2). The identified metabolites were categorized into eight different groups like alkenes, amino acids, carboxylic acids, fatty acids, fatty alcohols, polyols, polyphenols and sugars. Sugars formed the largest group, followed by carboxylic acids and polyols. In WT, the concentration of sugar was more on geminivirus infiltration (68%) than the healthy tissue (54%), while the carboxylic acids were more in healthy (19%) than infected tissue (6%; Fig. 5a). In C1/C4 transgenics, healthy and virus infiltrated tissue showed less change in concentration of sugars (21% and 28%, respectively), carboxylic acids (36% and 34%, respectively) and polyols (38% and 33%, respectively; Fig. 5b). Also, in C2/C3 transgenics, healthy and virus infiltrated tissue showed less change in concentration of sugars (39% and 41%, respectively), carboxylic acids (27% and 21%, respectively) and polyols (32% and 30%, respectively; Fig. 5c). www.nature.com/scientificreports/ While in V1/V2 transgenics, the concentration of sugar was more on geminivirus infiltration (56%) than the healthy tissue (40%), while the carboxylic acids were higher in healthy (25%) than infected tissue (11%; Fig. 5d). The other compounds like polyphenols, fatty acids, fatty alcohols, alkenes and amino acids were less than 6% in both WT and transgenics with healthy and infected tissue. In amiRNA transgenics, except V1/V2 transgenics, no significant changes were observed in the concentration of metabolites in healthy (control) and virus infiltrated tissues. In WT, C2/C3 and V1/V2 transgenics, fructose and glucose content increased, with a maximum increase of 8.76-and 8.10-fold, respectively, in WT on infiltration. The C1/C4, however, showed a decrease in fructose and glucose content on virus infiltration. The sucrose content increased in transgenics (1.19-1.26-fold), whereas decreased by 1.95-fold in WT with virus infiltration ( Table 4). The malic and quinic acid content increased in C1/C4 and C2/C3 transgenics, whereas decreased in WT and V1/V2 transgenics on virus infiltration. The quinic acid content showed a noticeable increase of 3.15-fold and a decrease of 4.29-fold in C2/C3 and V1/V2 transgenics, respectively. The pyruvic acid showed a reduction in both WT and transgenics, with a remarkable decrease of 17.7-fold in WT on virus infiltration (Table 4). In WT, C2/C3 and V1/V2 transgenics, an increase in myo-inositol content (1.23-2.22-fold) was observed. The glycerol content decreased in both WT and transgenics, with a maximum decrease of 4.77-fold in C1/C4 transgenics on virus infiltration ( Table 4).

Discussion
This study shows that each of the 6 ORFs of the newly isolated JLCuGV genomic DNA (AC1, AC2, AC3, AC4, AV1 and AV2), independently show strong RSS activity. The reversal of GFP expression in GFP silenced tobacco lines on agroinfiltration of AC1, AC2, AC3, AC4, AV1 and AV2 ORFs, independently, confirmed the strong RSS activity in all the six ORFs. The positive amplification of GFP gene from the cDNA generated from infiltrated leaves, gave the molecular confirmation of RSS activity exhibited by JLCuGV ORFs. The RSS activity acquired by individual JLCuGV ORFs seems to have increased its virulence. In the case of geminiviruses, ORFs like AV2, AC1, AC2, and AC4 were reported as RSSs 26,27 . In Bhendi yellow vein mosaic virus, strong RSS activity detected in AC4 and βC1, while low RSS activity is detected for AC2 28 . In the Tomato leaf curl New Delhi virus, the RSS activity was identified in AC2, AV2 and AC4 proteins 22 . It was shown that the RSS activity of AC4 and AV2 appeared during early infection and supplemented the RSS activity of AC2 to sustain the response for a longer duration 22 . www.nature.com/scientificreports/ Here, we report for the first time that all six ORFs of JLCuGV genomic DNA have acquired a strong RSS activity, although detailed molecular analysis is required to characterize the strength, duration and complementation of the RSS activity exhibited by individual ORFs. As a strategy to boost plant resistance to virus infection, we designed amiRNAs targeting overlapping regions between ORFs C1/C4, C2/C3 and V1/V2 to silence their transcripts and hence inhibit their RSS activity. The N. benthamiana transgenics independently expressing C1/C4 and C2/C3 amiRNAs showed resistance against JLCuGV while transgenics expressing V1/V2 amiRNAs showed tolerance to JLCuGV. The WT and VA plants showed high viral load with severe disease symptoms, whereas no viral load and no symptoms were detected in C1/C4 and C2/C3 transgenics. This indicated that JLCuGV resistance in C1/C4 and C2/C3 transgenics could be due to the expression of amiRNA. Several other reports are available that have shown that amiRNAs are effective in bringing out the degradation or silencing of the target viral genes 23,29 .
Interestingly, amiRNA levels were lower in V1/V2 amiRNA transgenics as compared to the C1/C4 and C2/ C3 transgenics. The reason needs to be investigated though, in all probability, it may be due to the allelic states of amiRNA precursor, based on the integration of T-DNA into the plant genome, which may have altered the level of amiRNA expression 29 . The lower amiRNA expression level in V1/V2 amiRNA transgenics correlates with the development of mild symptoms and limited tolerance to virus infection. It was observed that the viral load in V1/V2 amiRNA transgenics was much lower than that observed in WT or VA plants. This shows the important role of miRNAs in providing protection from virus infection and the manifestation of pathogen symptoms 30 . Earlier, Vu et al. 29 have shown an inverse correlation between the expression levels of amiRNA and the development of disease symptoms.
DNA methylation plays a major role in protecting plants under both biotic and abiotic stresses. Several reports state that plants can methylate the invading virus genome to suppress their growth 30 . The changes in methylation in Nicotiana tabacum genome were observed after the Cucumber mosaic virus (CMV) infection 31. The predominance of CHH hypomethylation has emerged as a major defence response against viral infection 31 . The transgenics C1/C4 and C2/C3 amiRNA exhibited higher methylation across the genome after virus infection as compared to WT, VA and V1/V2 amiRNA transgenics. The higher methylation and demethylation in amiRNA transgenics, corelated with decreased JLCuGV accumulation. This suggested that the homeostasis in methylation pattern in C1/C4 and C2/C3 amiRNA transgenics helped to block the JLCuGV accumulation, leading to resistance against JLCuGV. It has been reported that geminiviruses counteract plant defence strategy by inhibiting DNA methylation. The geminiviral AC2 genes act as RSS by interacting and inhibiting adenosine kinase (ADK), an essential gene of the methylation pathway 32 . ADK plays a role in the production of the methyl group donor S-adenosyl methionine (SAM) and defects in ADK lead to a loss in methylation in plants 33 . As C2/ C3 amiRNA transgenics target the AC2 gene, the viral counter defence to suppress ADK was not functional, and the plants could successfully follow the methylation pathway to inhibit the virus growth.
Virus infection usually affects the process of photosynthesis 34,35 , such that decreased photosynthesis is correlated with increased disease severity and increased virus multiplication 36,37 . JLCuGV infiltrated WT showed severe symptoms and significantly decreased photosynthesis as compared to amiRNA transgenics (C1/C4 and C2/ C3). The V1/V2 transgenics showed mild symptoms and relatively less reduction of photosynthesis parameters as compared to WT. The decrease in photosynthesis rate has been previously reported in geminivirus-infected leaves of Eupatorium makinoi 35 and mustard after turnip mosaic virus infection 37 . Naidu et al. 38 suggested that a reduced level of chlorophyll-a may be responsible for the reduced rate of photosynthesis.
The transpiration rate, WUE and stomatal conductance were significantly higher in virus-infected transgenics compared to WT. The reduced transpiration rate and stomatal conductance were also observed in mustard after geminivirus infection 37 . The Ci and Ci/Ca were significantly higher in WT, suggesting that the CO 2 taken by the plants was not utilized during photosynthesis and got accumulated in the cells, increasing the Ci and Ci/Ca ratio. It also correlates with the reduced rate of photosynthesis observed in WT. Similar results were reported with tobamovirus infection for reduced stomatal conductance, transpiration rate and Ci in pepper 39 . The ɸCO 2 was significantly higher in transgenics, suggesting efficient CO 2 assimilation, which correlated with both increased photosynthesis rate and reduced Ci of transgenics. The reduced ɸCO 2 is reported for pea on Pea enation mosaic virus (family Luteoviridae) infection 40 . The higher ETR seen in amiRNA transgenics suggests better redox homeostasis. The high ETR was found associated with improved reactive oxygen species (ROS) homeostasis during salinity 41 . Although Guo et al. 37 report no change for ETR during geminivirus infection, a significant reduction in ETR was observed in grapevine on virus infection 42 .
Fv/Fm was maintained at a healthy 0.8 level 31 in both WT and transgenics even after virus infiltration. Similarly, no change in Fv/Fm was reported for geminivirus infected mustard 37 , and Pepper mild mottle virus (PMMoV) infected pepper 39 . ɸPSII was significantly higher in transgenics, suggesting proper functioning of PSII as it is a measure of PSII performance. In WT, ɸPSII was significantly reduced, and reduced ɸPSII is reported to cause the accumulation of reduced bound quinone, which causes damage to the primary electron acceptor plastoquinone of PSII 43 . Therefore, reduced ɸPSII can be correlated to the reduction of ETR observed in WT during infection. The qP indicates the efficient conversion of light into chemical energy 37 and significantly improved qP in transgenics can be attributed to the maintenance of photochemical quenching followed by increased photosynthesis rate in transgenics. The overexcitation of the photochemical system after turnip mosaic virus infection caused reduced qP in mustard 37 .
In WT, the sugar content was significantly increased and intermediates of the tricarboxylic acid (TCA) cycle (malic acid, pyruvic acid and citric acid) reduced after virus infiltration. This suggests that sugar metabolism is affected and the normal functioning of the TCA cycle is disturbed in WT after JLCuGV infection. In transgenics, no remarkable changes are observed, indicating better maintenance of metabolic pathways on virus infiltration. The increase in sugar content of WT is correlated with the virus infection in Tobacco mosaic virus-infected tobacco 44 47 . The decrease in sucrose concentration is reported in Potato virus Y (PVY)inoculated potato leaves, while the increase in sucrose content reported in Jatropha infected by geminivirus 48 .
In WT, a decrease in sucrose and an increase in fructose and glucose content after virus infiltration might be due to activation of invertase enzyme which converts sucrose into fructose and glucose 49,50 . An increase in glucose concentration is also reported in tomato infected with Tomato mosaic virus 50 and PVY-inoculated potato leaves 51 .
In conclusion, the study reports that a new isolate of JLCuGV was responsible for the leaf curl disease in Jatropha CP9 accession. All six ORFs of JLCuGV genomic DNA exhibited RSS activity in-planta. Three amiRNAs were designed against C1/C4, C2/C3 and V1/V2 transcripts by targeting the overlapping regions. Transgenics expressing these amiRNAs showed a resistance (C1/C4 and C2/C3) and tolerance (V1/V2) response to viral infection. A direct correlation of the viral load and disease symptoms with relative levels of amiRNA was observed. The photosynthetic parameters were significantly better in amiRNA transgenics as compared to WT on virus infiltration, suggesting that both light and dark centres of photosynthesis are maintained due to reduced viral load. Likewise, the metabolite profiles were not much altered in amiRNA transgenics, while sugar metabolism is disturbed in WT on virus infiltration, again suggesting resistance of the amiRNA transgenics to the virus. MSAP analysis revealed an increase in methylation and demethylation profiling in amiRNA transgenics as compared to VA and WT, suggesting that methylation pathways get activated in amiRNA transgenics, and increased level of methylation profiling is correlated with a degree of virus resistance in amiRNA transgenics. All the results indicated that the amiRNA transgenics were better adapted to resist/tolerate JLCuGV infection. Importantly, as amiRNAs designed in this study share homology to related geminiviruses that infect other crops like tomato, pepper and papaya, so these have the potential to be employed for developing geminiviral resistance in other susceptible economically important crops. Agroinfiltration of JLCuGV ORF constructs. The Agrobacterium cultures (OD 600 = 1; 1 ml per plant) of individual JLCuGV ORF constructs were injected into leaves of stable N. tabacum cv. Xanthi GFP silenced lines into separate patches in each leaf for identification of RSS activity. Agroinfiltration was performed by a method described by Hamilton et al. 52 . The Agrobacterium cultures (OD 600 = 1; 1 ml per plant) were injected into leaves of the N. benthamiana (WT and transgenics) at 4-6 leaf stage using a 2 ml syringe. The plants were kept in an insect-free chamber at a constant temperature (25-28 °C) under 14-16 h lighting.

Materials and methods
Detection and confirmation of RSS activity. The agroinfiltrated leaves of the GFP silenced lines were observed under UV light for visualization of green fluorescence at 7, 11 and 15 dpi. The ORFs having inherent RSS activity were expected to restore the GFP expression by suppressing the RNA silencing machinery.
Total RNA was isolated from the individual regions of leaves 52 agroinfiltrated independently with single JLCuGV ORF (AV1, AV2, AC1, AC2, AC3 and AC4) and FHVB2 construct, a well-known RSS used as control. The cDNA was prepared using 5 μg of total RNA using SuperScript IV First-Strand synthesis system (Invitrogen), as per manufacturer's instructions. cDNA (1 μl), from each sample was used for amplification of gfp gene (~ 300 bp) with specific primers (Supplementary Table S3). The cDNA from mock (only buffer infiltrated) leaves was used as a negative control as it lacked the GFP expression. The samples showing amplification of the gfp transcript confirmed the restoration of GFP expression in the agroinfiltrated region of the leaf, thereby confirming the inherent RSS activity of the particular ORF.
In silico designing and construction of amiRNA plasmids for tobacco transformation. The common overlapping sequences between the two genes (AV1-AV2, AC1-AC4 and AC2-AC3) were used for designing amiRNA (Table 1, Supplementary Fig. S5a). The amiRNA design was carried out by using the WMD3 (Web MicroRNA Designer 3) online tool (http://wmd3.weige lworl d.org). While designing of amiRNA, we also tried to take the sequences, which can be used to target the begomovirus infecting tomato, papaya, pepper,  Fig. S5b-d), so that same amiRNA construct can also be used against other begomoviruses. The three designed amiRNAs (Table 1) were amplified by four sets of primers (Supplementary Table S3) via overlapping PCR on the pNW55 vector backbone for obtaining pre-miRNA as given in Sharma et al. 53 . The pre-amiRNA fragment was then cloned in pRT101, using XhoI and BamHI sites for directional cloning, under CaMV-35S promoter. Then the entire cassettes of C1/C4, C2/C3 and V1/V2 amiRNA with CaMV-35S promoter were cloned in pCAMBIA-1301 using HindIII site. The schematic representation of the amiRNA gene construct in pCAMBIA-1301 showed in Supplementary Fig. S2. Tobacco transformation. Three amiRNA constructs (C1/C4, C2/C3 and V1/V2 amiRNA) cloned in pCAMBIA-1301 vector and vector backbone alone (VA) were mobilized into the A. tumefaciens strain LBA4404. The transformed agrobacterium was used for the transformation of N. benthamiana leaf discs, according to Clemente 54 . Molecular validation of transgenic plants expressing the amiRNA. The transgenic plants (T 1 ) were checked by GUS assay to confirm the transgene expression. These were later confirmed for gene integration by PCR. The PCR was carried out using the primers specific for amiRNA gene, gus gene and hygromycin (hptII) gene (Supplementary Table S3). The positive lines were then checked for copy number by quantitative Real-Time PCR (qRT-PCR).
Construction of infectious clone for agroinfiltration. The construction of infectious clone was performed in two steps. In the first step, the RCA product was double digested with BamHI and HindIII, then the digested product (0.65 kb) was cloned in pCAMBIA-1301 to get a recombinant vector pCAM-JLCuGV-Partial. In the second step, the full-length genome of RCA product generated by HindIII was ligated with pCAM-JLCuGV-Partial, to get a complete infectious clone pCAM-JLCuGV-Inf. The orientation of the inserts was confirmed with sequencing. The positive clones that contain tandemly repeated viral genomes having two ori (origin of replication) sites were selected. The infectious clone was transformed in A. tumefaciens LBA4404 strain. For causing infection, the agroinfectious clone of JLCuGV ( Supplementary Fig. S4) was infiltrated in leaves of WT and transgenic lines of each amiRNA (L61, L64, L67 of C1/C4 amiRNA; L41, L42, L44 of C2/C3 amiRNA and L13, L14, L15 of V1/V2 amiRNA) in three replicates.

Molecular confirmation of JLCuGV replicating in agroinfiltrated tobacco plants. The genomic
DNA was isolated from the tobacco plants 55 agroinfiltrated with JLCuGV infectious clone. The presence of an infectious clone of JLCuGV was checked by PCR with specific degenerate primers (Supplementary Table S3) using 100 ng of each genomic DNA as a template. The actin gene was amplified as a loading control using NtActF and NtActR primers (Supplementary Table S3). The complete begomoviral genome was amplified by RCA using a TempliPhi Amplification Kit (GE Life Sciences, UK). The amplified RCA product was digested with HindIII to get 2.7 kb genomic DNA.
Determination of viral load. Geminivirus copy number in the agroinfiltrated plant was determined as reported in Legarrea et al. 56 . The pCAMBIA-1301 containing JLCuGV genomic DNA plasmid was linearized with BglII restriction enzyme. The quantified plasmid DNA was used to calculate the number of copies based on the formula: where 650 denotes weight (Da) of a base pair.
The standard curve was prepared by serial dilution of 10 9 to 10 1 plasmid of pCAMBIA-1301 containing JLCuGV genomic DNA. The qRT-PCR reaction performed using the AV2 gene primers (Supplementary Table S3). The copy number was quantified using the Ct (cycle threshold) value plotted against the standard curve formula. Three lines of each transgenic event were used for the analysis.
Small RNA isolation. The total RNA was isolated from transgenic lines by Tris-SDS buffer 57 and then the small RNA was isolated by using different concentrations of polyethylene glycol (PEG) according to Singh and Jha 58 . The quality of the purified RNA was examined by 1% agarose gel electrophoresis and the absorbance ratio (A 260 /A 280 and A 260 /A 230 ) using Epoch Microplate Spectrophotometer (BioTek, USA).
The quantitative estimation of amiRNA. The cDNA was synthesized using small RNA as template and specifically designed stem-loop primers (Supplementary Table S3) by SuperScript IV First-Strand Synthesis System (Invitrogen). The quantitative estimation of amiRNA was carried out using cDNA with stem-loop primers based qRT-PCR as given in Czimmerer et al. 59 and actin gene was used as an internal control. www.nature.com/scientificreports/ cence measurement were kept the same as reported in Shukla et al. 60 . Photosynthetic gas exchange parameters and chlorophyll fluorescence parameters were recorded in WT and amiRNA transgenics (L61, L64, L67 of C1/ C4, L41, L42, L44 of C2/C3 amiRNA and L13, L14, L15 of V1/V2) at 21, 28 and 35 dpi.
Gas chromatography-mass spectrometry (GC-MS) analysis. The sample preparation and derivatization by N-methyl-N-trimethylsilyl trifluoroacetamide (MSFTA) was carried out as given in Lisec et al. 61 . The derivatized samples were then transferred into glass vials suitable for GC-MS analysis. The GC-MS analysis was carried out in a SHIMADZU prominent instrument (SHIMADZU Corporation, Kyoto, Japan) equipped with EI mass spectra and autosampler. The injection volume for each sample was 1.0 μl throughout the analysis. Gas chromatographic separation was performed using the silica capillary SH-Rtx-5 column (30 m × 0.25 mm) 62 . Injection temperature was 230 °C, the interface was set to 250 °C and the ion source was adjusted to 200 °C. Helium flow was 1 mL min −1 . After a 5-min solvent delay time at 70 °C, the oven temperature was increased at 5 °C min −1 to 310 °C, 1 min isocratic, cool-down to 70 °C, followed by an additional 5-min delay. The ion source temperature was 200 °C and mass ions in the range of 45-600 m/z were scanned at a rate of 1.0 scans s −1 .
MSAP analysis. MSAP analysis was performed as reported earlier 25