Wheat Myo-inositol phosphate synthase influences plant growth and stress responses via ethylene mediated signaling

L-myo-inositol phosphate synthase (MIPS; EC 5.5.1.4) is involved in abiotic stress tolerance, however its disruption and overexpression has also been associated with enhanced tolerance to pathogens. The molecular mechanism underlying the role of MIPS in growth, immunity and abiotic stress tolerance remains uncharacterized. We explore the molecular mechanism of MIPS action during growth and heat stress conditions. We raised and characterized the TaMIPS over-expressing rice transgenics which showed a reduced reproductive potential. Transcriptome analysis of overexpression transgenics revealed the activation of ET/JA dependent immune response. Pull-down analysis revealed the interaction of TaMIPS-B with ethylene related proteins. Our results suggest an essential requirement of MIPS for mediating the ethylene response and regulate the growth. A model is proposed outlining how fine tuning of MIPS regulate growth and stress tolerance of the plant.

Scientific RepoRtS | (2020) 10:10766 | https://doi.org/10.1038/s41598-020-67627-w www.nature.com/scientificreports/ teins sequences in Arabidopsis and Oryza 20 . Out of all the members from the gene family of respective organisms, primary enzyme coding sequence was used for multiple sequence alignment by ClustalOmega and phylogenetic tree was created using Neighbor-joining method (Fig. 1a). We found an extensive sequence conservation with presence of identical amino-acids in MIPS genes with two most conserved motifs i.e. (NGSPQN) and (SKSNV) in all the organism (Fig. 1b) (Fig. S1). We also performed a domain search in these sequences, identified the NAD_5_Binding (PF07994) and Inos-1-P_synth domain (PF01658). Overall this investigation suggests the significant evolutionary conservation of (NGSPQN) and (SKSNV) motifs in the NAD_5_binding and Inos-1-P_synth domain, respectively.
TaMIPS gene family, organization and expression analysis. We identified and isolated three MIPS transcript in wheat. All three TaMIPS gene are on wheat chromosome 4 of the A, B and D genome, so they were named TaMIPS-A, TaMIPS-B, TaMIPS-D according to their location. It was interesting to observe that MIPS gene family in Triticum aestivum consist of three genes only. Gene structure of MIPS homologues consist of 10,  S2). We further analysed the 2 Kb upstream sequence TaMIPS-B using PLACE database and found the regulatory elements related to light, SA and ET/JA responsive (Table S2). We also checked the homology between the TaMIPS transcripts and observed more than 99% homology between them. To investigate the differential expression of TaMIPSs homologs under various abiotic stresses, we carried out the quantitative RT-PCR in different stresses. We found elevated expression of TaMIPSs during heat stress which was prominent in TaMIPS-D (sixfold change) whereas TaMIPS-B and TaMIPS-D showed same expression compared to control condition (Fig. 1c). During cold stress, TaMIPS-D was found to have highest expression (fourfold change) among other members and followed by TaMIPS-B compared to control condition (Fig. 1c). We also investigated the expression of TaMIPS during salt stress and found that TaMIPS-A had highest expression amongst the three homologues followed by TaMIPS-B and TaMIPS-D. From this analysis, it appears that TaMIPS-A, TaMIPS-B and TaMIPS-D are stress inducible and TaMIPS-B was further characterized.

TaMIPS-B overexpression imparts thermotolerance in rice.
To characterize the TaMIPS-B, we raised the TaMIPS-B over-expressing rice transgenics. Of the various regenerants; at least independent 46 transgenic plants were identified, and five lines were chosen for detailed characterization (Fig. 2a). Southern blot analysis was carried out which revealed that transgenic line L5, L9 and L2 had a single copy insertion, while transgenic line L4 and L3 possess 2 and 3 transgene copies, respectively (Fig. 2b). Subsequent, expression analysis of TaMIPS-B transgenic lines was carried out and expression was observed in L4, L9, L2 whereas no signal was observed in L3 and L5 (Fig. 2c). This study suggests the expression of transgene in transgenic line L4, L9, L2 and no expression in L3 and L5. Transgene expressing Lines (L4, L9, L2) and silenced line (L3, L5) were used for further analysis and thermotolerance assay. 40-days old seeding were subjected to 45 °C ± 1 for 6 h consecutively for three days to assess the possible role of TaMIPS-B in heat stress. Control and treated plant samples of wild type and transgenics were collected to carry out the myo-inositol, MSI and MDA quantification. As expected, we found a significant increase in myoinositol levels in the transgenic lines L9, L2, L4 as compared to the wild type with maximum levels in line no 2. Further, upon heat stress a similar profile was observed however, a decrease in levels of myo-inositol was seen in lines L9, L2, L4 during heat stress whereas an increase in line L3, L5 (Fig. 3a). Membrane stability index (MSI) was also measured and MSI was high in transgenic lines L3 (78%), L9 (77%), L2 (77%), L4 (78%) as compared to wild type (72%) under control condition (Fig. 3b). During heat stress, transgenics line L3 and L2 showed highest membrane stability followed by L4, L5. Wild type plants had least membrane stability upon heat stress.
To determine the effect of heat stress, we checked the lipid peroxidation by quantifying the levels of MDA and found higher level of MDA in wild type plants upon heat stress as compared to the transgenic lines (Fig. 3c).
Photosynthetic efficiency of transgenics was also investigated under control and heat stress conditions. For heat stress treatment, 40-days old plants were subjected to 3-days heat treatment at 45 °C ± 1 for 6 h and transferred to green house after every treatment. Under control condition, F V /F M values were higher in transgenics as    Fig. 3d,f). After heat treatment, plants were shifted to green house and observed that transgenics recovered much faster than the wild type as F V /F M value of L2 reverted to 0.80 after three days followed by L4 (0.79) and L9 (0.78) (Fig. 3d,g). After 15 days of recovery, transgenic lines L9, L2, L4 recovered to their initial F V /F M ratio but transgenic lines L2 and L4 after recovery showed increase in F V /F M ratio compared to their control condition values.

Reduced grain yield upon overexpression of TaMIPS-B in rice.
When the effect of TaMIPS-B overexpression on reproduction was analysed. The seed setting rate in panicle of TaMIPS-B transgenics and wild type ( Fig. 4) was calculated. We observed a decrease in seed setting rate in all transgenic lines i.e. L9, L3, L4, L2 and a lowest percentage in L5 as compared to the wild type plant (Table 1). Reduced seed-setting rate suggest the negative effect of MIPS overexpression.

Gene expression analysis revealed enrichment defense pathway in TaMIPS-B transgenic.
To understand the role of MIPS in reduced reproductive potential, we carried out the whole transcriptome analysis of TaMIPS-B over-expressing transgenic under control and heat stress condition using Illumina HiSeq 2,500 sequencer with PE100 which resulted in generation of 57 to 70 million reads per sample. Paired end reads were assembled into 178,134 transcripts and 66,106 genes in TaMIPS-B over-expressing transgenics with N50 of 2,249 by Trinity. Finally, a total of 5,879 transcripts were found to be differentially expressed in the TaMIPS-B transgenics. Interestingly, we observed that the up regulation of defense genes like chitinase3, WRKY transcription factor 6, Pathogenesis-related protein PRMS along with ERF1B in TaMIPS-B over-expressing transgenics (Fig. 5). We carried out the gene ontology (GO) term enrichment analysis using upregulating genes in the TaMIPS-B over-expressing rice transgenic. To reduce the GO terms, we used REduced VIsualize Gene Ontology (REVIGO) 21 . We found multiple GO term like "defense response", "defense response to insect" and "regulation of salicylic acid metabolic process" (Fig. 6).

Identification of MIPS interacting protein in wheat.
To gain insight into the mode of action of MIPS, we investigated the presence of interacting proteins of TaMIPS-B as previous reports suggest its dual nature. We therefore performed the far-western analysis using purified His-TaMIPS-B protein (Fig. 7a) and crude plant extract and found two interacting signals in the range of 35-48 kD (Fig. 7b). Further, we performed in-vitro pull down using recombinant His-tagged TaMIPS-B expressed in bacteria E. coli. The protein obtained were separated by SDS page and Coomassie stained. We found three more protein bands along with the recombinant   We observed the presence of MIPS sequence in the first two bands which range in 48 kDa to 35 kDa (Table 2). This result suggests TaMIPS-B interacts with its homologs as molecular weight of TaMIPS-B and TaMIPS-D were in this range (Table S1). Further pull-down extract was subjected to LC-MS analysis to identity the interacting proteins. We found ethylene related proteins (Table 3).

Discussion
Myo-inositol has been associated with numerous roles i.e. cell wall biogenesis 1 , auxin storage 2 , phytic acid synthesis 3 , and oligosaccharides synthesis 4 . In this study, we have proposed that MIPS acts as a molecular switch to control growth and immunity.
MIPS gene family analysis in organism included in this study indicates their less diversification due to the presence of a relatively small gene family which usually consist of 1-5 genes. We identified previously reported conserved motifs (NGSPQN) and (SKSNV) in all the major MIPS coding gene from all the organism studied. MIPS is a phosphoprotein and its activity is regulated by phosphorylation at serine residue. Phosphorylation of www.nature.com/scientificreports/ serine residue of (NGSPQN) is predicted via GSK-3 which increases the MIPS activity whereas phosphorylation of serine residue of (SKSNV) via PKC decreases the activity 6 . Conservation of above said motifs across species including Homo sapiens, suggests a common mechanism of MIPS enzyme regulation at post-translational level. Expression analysis of TaMIPS in whole seedlings suggests differential expression of TaMIPS-B, TaMIPS-A, TaMIPS-D in wheat during induced stress conditions. TaMIPSs expression during stress revealed the stress inducible behavior of TaMIPS-A, TaMIPS-B and TaMIPS-D with highest expression of TaMIPS-D in thermal stress (heat and cold stress) whereas TaMIPS-A showed highest expression under salt stress. Similarly, high expression of AtMIPS2 in heat and AtMIPS3 in heat and drought has been seen in previous report where the AtMIPS1 showed less expression compared to AtMIPS2 and AtMIPS3 20 . Differential expression of CaMIPS1 and CaMIPS2 also demonstrated the similar situation in which CaMIPS2 was induced by environmental stress with no effect on CaMIPS1 22 .
We next carried out generation and physiological analysis of TaMIPS-B over-expressing rice transgenics. We observed two and three copy number of transgenes in L4 and L3, respectively. However, transgene expression was observed in transgenic lines L9, L2, L4 but no expression in L3 and L5. The probable reason could be the high copy number in case of L3 which led to the silencing whereas in L5 DNA methylation, position effect,   www.nature.com/scientificreports/ trans-inactivation could be the reason of gene silencing. Previous report correlates high transgene copy number with transgene silencing in tobacco 23 . Moreover, a recent study also demonstrated the overexpression of AtMIPS2 resulting in co-suppression of all the three AtMIPS genes which leads to reduced root length, delayed or absence of flowering along with reduced fertility 24 . Further these transgenics were used for thermotolerance assay. As expected, we observed increase in myo-inositol in transgenic line L9, L2, L4 with maximum level in L2 in control condition. A similar pattern was observed during heat stress but with slight decrease as compared to control conditions. When MDA (malondialdehyde) levels were checked in transgenic and wild type, we found low MDA levels in all transgenic line except L5. During heat stress, MDA levels increased in all the lines but the level of MDA in transgenics was lower as compared to wild type. MSI too has been correlated with heat tolerance under heat stress regime in wheat 25 . We observed higher MSI in transgenic line L9, L2, L4 along with L3 during heat stress and control condition compared to wild type. Heat stress also affects respiration and photosynthesis which results in decrease in plant productivity 26 . During heat stress regime, transgenic lines performed better especially transgenic line L2 which had the highest myo-inositol level at heat treatment day 3. High level of myo-inositol could be correlated to high photosynthetic efficiency of PSII. During recovery period after heat stress, transgenic lines recovered completely after 7 days whereas wildtype along with transgene silenced line L3 and L5 could not recover to control F v /F m ratio. Phenotypic analysis also showed a similar pattern; transgene silenced lines and wild type could not recover from the heat stress, even after 15 days of recovery whereas transgene expressing lines L9, L2, L4 showed growth and emergence of new leaves as early as 7 days of recovery in L2. Over-expression of SaINO1 showed significantly enhanced tolerance during salt stress in Arabidopsis 9 , whereas Ectopic expression of RINO1 in rice and overexpression of IbMIPS in sweet potato results in enhanced tolerance to salt and drought stress 10,15 . Previous report from our lab also demonstrated that overexpression of TaMIPS-B in Arabidopsis confers heat tolerance 27 and the present data also supplements our assumption that over-expression of TaMIPS-B results in enhanced heat tolerance in this instance in rice.
AtMIPS1 has been reported as metabolic enzyme and a transcriptional regulator 28 . Previous study has also revealed the existence of human MIPS as an enzyme complex with its isoforms and these complexes are tailored according to the tissue specific needs 8 . Our Far western and pull-down data suggests TaMIPS-B interaction with its homologues as well as 1-aminocyclopropane-1-carboxylate oxidase 3, serine/threonine-protein kinase CTR1 and might affect their activity as well. Therefore, we conclude that TaMIPS-B interacts with these proteins and forms an enzyme complex to carry out its function in the cell. Moreover, our results are substantiated by Arabidopsis interactome study which has predicted the interaction of AtMIPS1 with AtMIPS2 along with peroxidase and AtMIPS2 with ACO3 29 .
We also performed whole transcriptome analysis of TaMIPS-B transgenics and further gene ontology analysis which revealed activation of defense response. We observed the up-regulation of ERB1B in transgenics. ERF1B is considered as an integrating point for ethylene and jasmonate signals and activates the defense related gene like chitinase 3, 23 KDa jasmonate-induced protein, ß-glucosidase, peroxidase 2, WRKY transcription factor 6, glutathione S-transferase T3 and thaumatin-like protein which were reported to be upregulated in overexpression Col-0;35S:ERF1 transgenics 30 . Up-regulation of these genes in TaMIPS-B transgenic suggests the activation of ET/JA signaling. Overexpression of IbMIPS1 provides resistance to stem nematode 15 , presence of elements related in ET/JA in MIPS promoter and GO term "defense response to insect" supports the activation of ET/JA pathway. From this data, we can conclude that the up-regulation of MIPS results in activation of ET/JA-dependent defense pathway in TAMIPS over-expressing rice transgenics.
When we investigated TaMIPS-B over-expressing rice transgenic at reproductive stage and we observed a reduced in seed setting rate with maximum reduction in L2 which had highest level of MI. During heat stress, rate of evolution of ethylene in heat sensitive variety is higher than tolerant one, which is correlated to higher male sterility and results in reduced grain yield 31,32 . Decrease in seed setting rate in over-expressing TaMIPS-B transgenic might be because of over-production of ethylene which results in reduced fertility.
In conclusion, activation of SA defense response in Atmips1 and overexpression of IbMIPS1 provides resistance to stem nematode and salt stress 15,17 suggests us that plants perceive altered levels of myo-inositol as a stress effector molecule and result in activation of defense response. We proposed the hypothesis that receptors like kinases might be phosphorylating the serine residue of (SKSNV) motif which result in rapid increase or decrease in activity of myo-inositol phosphate synthase 6 and subsequent increase and decrease in level of MI is perceived as the stress condition. We can thus conclude that during normal conditions optimal level of MI are maintained and when the host encounters abiotic or biotic stress conditions, it increases or decreases MI level to activate ethylene response or SA-mediated defense responses depending upon the stress, type of stress and causative agents. It is therefore one of the important central regulators in balancing plant growth and immunity (Fig. 8).

Materials and methods
TaMIPS gene mining. Protein  Plant sample, growth and stress conditions. Wheat (Triticum aestivum) seeds (PBW 343) were surface sterilized by sodium hypochlorite for 20 min and grown at 22 ± 1 with 16:8 light and dark cycle for 10 days. 10-day-old seedlings were used for expression analysis under different stress conditions. For temperature stress, seedlings were exposed to 42 °C and 4 °C for 4 and 24 h respectively. For salt stress, seedlings were transferred to 200 mM NaCl solution for 24 h. Rice transgenics were raised using Oryza sativa ssp. Indica var. PB1. Sterilized seeds were used for callusing and transgenic generation was done according to protocol described by Nishimura et al. 34 . Regenerated plantlets were then transferred to a rooting media and subsequently to pots and raised to maturity. RNA isolation and cDNA synthesis. RNA was isolated from the different plant tissues (TaMIPS-B over-expressing rice transgenic seedling, control and stressed sample) using RNeasy plant mini kit (Qiagen, Germany). Samples were ground with liquid nitrogen and further proceeded according to the kit manual. Incolumn DNase treatment was done to remove the genomic DNA contamination. Quality and quantity of RNA samples were done by gel electrophoresis and nanodrop. 2 μg of RNA was used to make the cDNA using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific) and SuperScrip III First-Strand Synthesis System for Full length cDNA synthesis.

Cloning of TaMIPS-B CDS.
TaMIPS-B full length cDNA was amplified from cDNA (forward primer AAG GAT CCA TGT TCA TCG AGA GCT T, reverse primer AAG GTA CCT CAC TTG TAC TCC AGG AT) was cloned in pB4NU vector via restriction digestion by BamH1 and Kpn1. Clone was confirmed by restriction digestion and DNA sequencing and the confirmed clone was used to transform rice calli using EHA 105 strain of Agrobacterium tumefaciens harboring pB4NU-TaMIPS-B.
Promoter sequence analysis. Promoter sequence of TaMIPS-B was fetched from IWGSC chromosome assemblies using CLC genomic work bench. A 2 kb putative promoter sequence was searched against PLACE cis elements database 35 using CLC genomic workbench. www.nature.com/scientificreports/ Genomic DNA isolation and southern analysis. 100 mg fresh leaves transgenics were homogenized in liquid nitrogen and mixed with 500 μl CTAB buffer (2% w/v CTAB, 0.3 M NaCl, 20 mM EDTA, 100 mM Tris-Cl (pH-8) 0.2% BME). Genomic DNA was isolated according to Murray and Thompson 36 . Quality and quantity of genomic DNA was checked by gel electrophoresis and nanodrop (GE, US), respectively. Transgene copy number was checked using 10 μg of genomic DNA. DNA was digested with BamHI and separated by 0.8% agarose gel electrophoresis. DNA was blotted on Hybond-N Nylon membrane (Pharmacia) and fixed using UV cross linker (UVP HybriLinker, Analytik Jena, US). Blot was probed with hpt gene probe which was synthesized using PCR DIG probe synthesis kit (SIGMA-ALDRICH). Hybridization and detection were done using the non-radioactive method by DIG Luminescent Detection Kit as per the manufacturer guidelines (SIGMA-ALDRICH) with hybridization temperature 42 °C and washing 65 °C. Detection was carried out using CSPD solution and visualized by Fujifilm LAS4000 luminescence imager.
Northern analysis. Transgene TaMIPS-B expression was checked by Northern blot analysis in rice transgenics. Total RNA from leaves of selected transgenic lines was isolated. 15 μg of RNA was denatured at 65 °C for 5 min, separated on 1% formaldehyde agarose gel 37 and probed with TaMIPS-B gene probe. Blotting, hybridization and detection was done as described above.
Total plant protein extraction. Total plant protein was isolated from five wheat samples (PBW343) named spike zadok 65, heat stressed spike zadok 65, drought stressed spike zadok65, grain zadok 71, and grain zadok 75. 100 mg of leaves were homogenized in liquid nitrogen and mixed with 1 ml extraction buffer 50 mM NaHPO 4 , 150 mM NaCl, 0.5% Triton-X-100, 10% glycerol, 0.5 mM PMSF and 25 mM imidazole (IDZ). Samples were vortexed thoroughly followed by centrifugation at 10,000 g at 4 °C for 5 min. Total protein was collected from Supernatant and estimation of protein was done by plotting standard curve of BSA.
Membrane stability index. The Membrane Stability Index (MSI) was determined as explained by Singh and Khurana 38 . TaMIPS-B over-expressing rice transgenics leaf tissue (control and stressed) were placed in MQ water and initial conductivity (C1) was measured after incubating for 30 min at RT. Samples were then autoclaved for 20 min under 120 psi pressure and C2 was measured. MSI was calculated according to MSI = (1-(C1/ C2)).
Photosynthetic yield. Photosynthetic efficiency of TaMIPS-B over-expressing rice transgenics was measured using a PAM-210 (H.Waltz, Germany) under control, heat stress and recovery period. To measure the Fv/ Fm, plants were dark adapted for 30 min and measuring beam was applied to leaf to measure F 0 followed by application of saturating pulse using the F v /F m function to measure the F m (maximum fluorescence). F v /F m value calculated by instrument (PAM-210) using the formula as following:

MDA quantification.
Lipid peroxidation level in wild type and TaMIPS-B over-expressing rice transgenic plants was checked under control and stress conditions by quantifying the MDA content. 100 mg of tissue was homogenized by adding 500 μl of 0.1% (w/v) Trichloro acetic acid and centrifuged at max speed for 10 min and 500 μl of supernatant was mixed with 0.5% TBA (thiobarbituric acid) followed by incubated at 95 °C for 25 min. Reaction was stopped at incubating in ice and absorbance was measured at 532 and 600 nm.
Myo-inositol quantification. myo-inositol levels were quantified using myo-inositol assay kit (Megazyme, USA). TaMIPS-B over-expressing rice transgenics leaf tissue were homogenized in MQ water and centrifuged at max speed for 5 min and reaction was set as per the kit instructions. Absorbance of samples were measured at 492 nm and myo-inositol content was calculated by the formula: Myo-inositol = 0.2046 × ∆A Inositol where ∆A Inositol= Absorbance 2 -Absorbance1. Transcriptome analysis. Whole transcriptome comparison of TaMIPS-B over-expressing rice transgenics was done against wild type. Paired-end cDNA library preparation was prepared from pooled RNA sample of three gene expressing chosen transgenic lines. RNA sequencing was carried out by SciGenom, India. Denovo assembly of Reads from each library was carried out using software package Trinity 2.0.6 with default parameters 39,40 . Generated transcripts were used for differential expression analysis. Transcript abundance estimation was done using the software RSEM 1.2.7 which outputs the expression-normalizing value FPKM. Differential expression was carried out using abundance matrix files generated by RSEM, by software EdgeR 2.14 41 with the default parameters and TMM (Trimmed Mean of M-values) comparison was done across all the samples which resulted into a new matrix with normalized expression values across samples measured in FPKM. The expression patterns of the transcripts in the samples were restricted to the transcripts with significant differential expression (P-value 0.01, Fold change log2 scale).
Functional annotation and gene ontology (GO) enrichment analysis. Assembled transcripts were annotated using Trinotate pipeline (https ://trino tate.githu b.io/). Assembled transcripts were compared against Swissprot-Uniprot database, using blastn with an E-value 10 -5 . Transdecoder v.2.0.1 (https ://trans decod er.sourc eforg e.net/) was used to predict the longest ORF in assembled transcripts. Predicted ORFs were then annotated using blastp against the Swissprot-uniprot database with an E-value cut-off of 10 −6 . Further annotation was carried out using Trinotate as per the manual. REVIGO scatter plot was used to visualization of GO enrichment analysis (https ://revig o.irb.hr).
Western and far-western assay. To check the TaMIPS-B protein interaction, Far-western analysis was carried out using purified TaMIPS-B-His recombinant protein as bait and plant crude extract (Zadok 65 spike stage) as prey according to Wu et al. 42 . 500 μg prey protein along with BSA (Bovine serum albumin) and purified His-TaMIPS-B as negative and positive control, respectively, were blotted on membrane in two sets and one of it was proceeded with western blot analysis and the other blot used for far-western analysis. Western blot was probed and detected by anti-His antibody whereas other was proceeded to denaturing/renaturing of blotted prey protein for 12-16 h. Blot was then blocked with 5% milk and washed with PBST (Phosphate buffered saline with Tween-20) buffer three times. Membrane was then incubated with bait protein (Purified His-TaMIPS-B protein) at 4 °C for 12 h. Bait protein bound to prey protein was detected on blot after stringent washing and incubating it with primary (anti-His) antibody followed by secondary antibody. Visualization was done by chemiluminescence using Immobilon Western Chemiluminescent HRP Substrate (MERCK) according to manufactural instructions using Fujifilm LAS4000 luminescence imager.
Pull-down assays. Pull-down assay was preformed using the 500 μg of purified TaMIPS-B-His recombinant protein with 2 mg of crude wheat extract. Wheat extracts were prepared by homogenizing control samples of spike at Zadok 65 (spike_Z65) stage along with treatments i.e. heat and drought and grain sample (grain_Z71, grain_Z75) total plant protein extraction buffer (50 mM NaH 2 PO 4 , 50 mM NaCl, 1 mM PMSF, 0.1% Triton X-100). Homogenate was centrifuge at 5,000 rpm for 5 min and supernatant transferred to fresh MCT. Purified recombinant protein, Ni-NTA beads and plant crude extract were incubated at 4 °C with gentle shaking for 12 h. Beads were then centrifuged and washed extensively with washing buffer (same as above) and bound protein eluted with equal bed volume of elution buffer (same as above). The eluted protein was then subjected to SDS-PAGE electrophoresis and protein bands isolated and given for MALDI-TOF Mass Spectrometry (CIF, Biotech Centre, UDSC). Pull-down extract was also given for Liquid Chromatography Mass Spectrometry (LC-MS) analysis to Sandor, India.

Statistical analyses.
All values reported in this work are the average of at least two independent biological replicates having at least 15 seedlings. Error bars represent SE.