miR166h Directed Cleavage of Target Upon PGPR Inoculation Under Drought Stress and Tissue- Speci c Expression Analysis Under Abiotic Stresses in Chickpea

Ankita Yadav National Botanical Research Institute Sanoj Kumar National Botanical Research Institute Rita Verma National Botanical Research Institute Shashi Pandey Rai Banaras Hindu University Faculty of Science Charu Lata NISCAIR: Council of Scienti c and Industrial Research National Institute for Science Communication and Information Resources Indraneel Sanyal (  i_sanyal@rediffmail.com ) National Botanical Research Institute CSIR https://orcid.org/0000-0002-2592-773X


Introduction
Legumes are important crops known for their nitrogen supplementation in soil as well as nitrogen-xing capacity in association with soil bacteria. Chickpea (Cicer arietinum L.) is one of the food legumes and an imperative diet portion rich in proteins with essential amino acids. It lines second amongst all leguminous crops and is grown in dry/semi-dry regions of tropics as a rain fed crop ( (Kim et al. 2005). Hence, the present study is an attempt to delineate ATHB15 as the target of car-miR166 in chickpea using 5´RLM-RACE.
Further, PGPR's are investigated to have roles in host plant growth promotion by attuning morphophysiological, biochemical and molecular responses, increasing availability of soil micronutrients and production of phytohormones. Plants inoculated with PGPR have shown to be better survivors under drought stress in arid and semi-arid regions (Tiwari et al. 2016;Khan et al. 2018). Pseudomonas putida, a PGPR strain MTCC5279 (RA) has been reported to have miRNA-mediated drought stress mitigating roles revealed by a genome-wide analysis of stress responsive miRNAs upon inoculation with or without RA (Jatan et al. 2019). The study also highlighted the role of PGPR in modulating the expression of conserved miR166h and its potential target ATHB15.Hence, mapping of selected target cleavage by miR166h in chickpea through 5´RLM-RACE would provide better analysis of its role in chickpea and its relationship with its cognate target ATHB15. The tissue-speci c expression patterns of both miR166 and ATHB15 would also help to better understand the mechanism of action which is yet not fully deciphered.

Plant material, stress treatment and library construction
The tolerant desi chickpea cultivar BG-362 was selected for the experiment. The seeds were initially washed with RO water and further with 0.1% (w/v) mercuric chloride solution for surface sterilization and after washing three to four times with sterile Milli-Q water, the seeds were washed with 70% ethanol and repeatedly washed with sterile Milli-Q and soaked overnight. The overnight water imbibed seeds were placed in Petridishes on wet autoclaved Whatman No. 1 lter paper and incubated in growth chamber for 3-4 days. The germinated seeds were transferred to Hewitt media containing hydroponic trays under standard growth conditions for two weeks. After two weeks the experiment was designed for library construction involving 1% RA suspension inoculation in two trays for 24h and the rest two trays were kept uninoculated. The bacterial culture of RA available in our institute was grown with conditions at 250 rpm and 28°C. After 24h, the uninoculated and inoculated seedlings were subjected to drought stress using 20% (w/v) polyethylene glycol (PEG) 6000. The untreated non-stressed condition was maintained for control. The treated and untreated plants were harvested at 0, 72 and 168h time intervals. The time intervals for physiological and biochemical studies involve 0, 24, 72 and 168h. Hence, four set of plants included control, drought, drought+RA and RA. All the tissues were harvested in three independent biological replicates and were immediately in liquid nitrogen and preserved carefully after labelling in −80°C for further experimental analysis.

Morphological analysis
All four set of plants were evaluated for various morphological parameters in terms of drought stress response evaluation. The number of biological replicates of plants used for experimental analysis at each time interval was ve. The samples were wiped carefully after removing from hydroponics medium and different parameters were recorded thereafter. After the fresh weight analysis, the samples were kept in blotting sheets and were dried in hot air oven at 60°C for 4 days and then dry weight was recorded. The morphological parameters were studied and noted as described elsewhere (Tiwari et al. 2016).

Electrolyte Leakage (EL)
The electrolyte leakage (EL) of treated and untreated roots of chickpea plants was measured according to method explained by Lata et al (2011) and Tiwari et al (2016) with minor modi cations. About 100 mg of fresh root samples were taken from all sets and further incubated in 20 ml sterile deionized Milli-Q water in 50 ml sterile Falcon tubes for 40 mins at 120rpm at room temperature. After 1h, the initial electrolyte conductivity (E1) was measured using Orion 5star conductivity meter (Thermo Scienti c, USA). The tubes were further kept in boiling water with temperature 90°C in a water bath for 40 min and after cooling for some time, the nal electrolyte conductivity (E2) was recorded. EL was calculated as by the formula mentioned in previous reports (Tiwari et al. 2016).

Relative water content (RWC)
For the assessment of relative water content in both control and treated samples, the leaf samples were taken in triplicate (Lata et al. 2011;Tiwari et al. 2016). The leaf samples of similar size were collected and their fresh weight (FW) was recorded immediately. Afterwards, these samples were soaked in 30 ml sterile Milli-Q water for 4h in Petridish after which their turgid weight (TW) was recorded. These samples were then kept for drying in hot air oven at 60°C for 48h after which their dry weight (DW) was recorded.

Lipid peroxidation assay
The modi ed protocol of Heath and Packer (1968) was used for estimation of lipid peroxidation. The aldehyde product, malondialdehyde (MDA) was measured using 2-thiobarbituric acid (TBA) reaction. 0.1% (w/v) TCA solution was prepared from which 500 µl was used for homogenization of ~100 mg leaf tissue samples from all sample sets in triplicates. The mixture was centrifuged at 13,000 g at 4°C for 10 min. The 500 µl supernatant was mixed with 1.5 ml of 0.5% TBA and incubated for 25 min at 95°C. The reaction was inhibited after 5 min incubation on ice and the absorbance was measured at 532 nm and 600 nm in a microplate reader.

Proline estimation assay
The amino acid proline estimation was measured using the protocol of Carillo and Gibbon (2011). 1ml of 70% ethanol was used to homogenize ~100 mg of leaves. 50 µl ethanolic extract was mixed with reaction mixture prepared by mixing 1% w/v ninhydrin in 60% (v/ v) acetic acid and 20% (v/v) ethanol. The reaction was incubated at 95°C for 20 min followed by 5 min on ice and then absorbance was recorded in microplate recorder at 520 nm.

Total RNA isolation for control and treated samples
Total RNA was isolated using mirPremier® microRNA Isolation Kit (Sigma-Aldrich, USA) according to the manufacturer's instructions. The root samples were taken for quantitative real-time PCR analysis of all four sample sets for miR166h and its target ATHB15expression analysis. For tissue-speci c expression analysis, total RNA was isolated from different tissues as well as root, shoot and leaves with 15 days old seedlings harvested at time intervals 0 h, 1 h, 24 h and 72 h with different abiotic stress treatments like 20% polyethylene glycol (PEG) (6000), 100mM NaCl and 10µM ABA. Purity and quantity of isolated RNA samples was checked using Nano-Drop (Nanodrop 1000, Thermo Scienti c, USA) and 1.2% formaldehyde-agarose gel electrophoresis. Turbo DNA-free™ kit (Ambion, Life Technology) was then used for DNase treatment of 5 µg RNA.

cDNA synthesis of miR166h and ATHB15
The puri ed RNA samples were used for cDNA preparation using Taqman® MicroRNA Reverse Transcription Kit (Applied Biosystems, USA) according to the manufacturer's protocol. Stem-loop reverse transcription primer of miR166h and mature miR166h forward primer were designed according to the previous study (Jatanet al. 2019). Universal reverse primer was designed as described (Kramer et al. 2011). The target cDNA was prepared using Verso cDNA synthesis kit (Thermo Scienti c, USA) according to the manufacturer's protocol. The information regarding target gene prediction and its functions was gathered as per Jatan et al (2019).

Stem-loop quantitative real-time PCR (SL-qRT) for car-miR166 expression analysis
The SL-qRT-PCR analysis was performed to validate the expression levels of selected conserved car-

Phylogenetic analysis of target
The target was subjected to evolutionary phylogenetic analysis in different leguminous crops in comparison to chickpea using Molecular Evolutionary Genetics Analysis (MEGA X) (Kumar et al. 2018). The sequences were retrieved from NCBI using BLAST and were aligned further using CLUSTAL W and sequentially the data was analysed by neighbour-joining tree method using bootstrap test.

Phylogenetic analysis of pre-miR166h
Sequences of miR166 precursors (pre-miR166h) of different crops plants were collected from NCBI using BLAST application and then aligned using Clustal W. Subsequently, phylogenetic tree was constructed with the bootstrap value calculated with 1000 replicates using maximum likelihood method on MEGA-X.
2.14 Detection of miR166 cleaved target ATHB15 mRNA by 5´RLM RACE For the validation of predicted target of miR166h, modi ed 5´ RACE was performed. For this validation, the FirstChoice RLM-RACE Kit (Ambion, USA) was used with slight modi cations. 1 µg of total RNA from RA inoculated drought treated root samples were isolated, checked for purity and quantity and further subjected to adapter ligation. This was followed by cDNA synthesis in which ligated product was used as a template. Speci c gene reverse primers were designed for 5′ RLM-RACE and checked by OligoEvaluator™ (Sigma) (Supplementary Table 1). PCR reactions for cDNA ampli cations using primer combinations with PCR cycling conditions set according to manufacturer's instructions were performed.
The annealing temperature was optimized and the single PCR fragments were cloned into the pGEM-T Easy Vector (Promega, USA) and sequenced to identify the 5´end of the ampli ed target gene.

2.15
Relative expression analysis of miR166 in different chickpea tissues cDNA for miR166h, U6 and target were synthesised from different tissue samples of chickpea and further studied for relative expression analysis. Stem-loop primers for miR166h and U6 were used for reverse transcription. For transcript normalization, GAPDH was used (Garg et al. 2010). Expression analysis of miR166h and its target was performed in triplicates. The relative expression of the miR166h and target gene in different samples was calculated using 2 −ΔΔCt method (Livak and Schmittgen 2001).

Tissue-speci c expression analysis in abiotic stresses
The leaves, shoot and root samples were harvested from control and different abiotic stress treatments including drought, salinity and ABA treatments according to the manufacturer's instructions. The relative expression of the miR166h and target gene in different treated samples compared to the control was calculated using 2 −ΔΔCt method (Livak and Schmittgen 2001).

Statistical analysis
Relative expression data for miR166 and its target gene, ATHB15 from three independent biological replicates were calculated as the mean with standard error (mean ± SEM). One-way analysis of variance (ANOVA) using Duncan's multiple range tests (DMRT) was used for signi cant differences in variance between average values of control and treated plants with the analysis of signi cant difference between the means (p < 0.05). Standard deviation (SD) values were calculated using the mean of the replicates.

Results
The expression patterns of miR166h and ATHB15 were studied in the present study upon RA inoculation in chickpea tolerant cultivar subjected to drought stress. Control samples with standard conditions and RA inoculated and uninoculated samples with drought stress treatment were brought under study where differential expression patterns were observed. This miRNA and its target have been found to have inverse correlation expression patterns in chickpea. When comparing all four libraries as explained by (Jatan et al. 2018; Jatan et al. 2019) and we also researched that RA in relation to miR166h has roles in drought tolerance and response. Transcripts with ≥ 1.0 were considered as upregulated and that of ≤ −1.0 fold-change values as down-regulated.

Effect of drought stress on morphological parameters with and without RA-inoculation
To determine the effect of RA on drought stress treated plants, the morphological study was done. The RA-inoculated samples showed better growth with and without drought stress ( Figure. 1a and 1b). The primary root length was found to be increased by 42% and 50% in drought treated RA-inoculated plants as compared to drought treated uninoculated plants at late time intervals (72h and 168h) respectively. The shoot length was also comparatively higher with 14% increase in drought treated RA inoculated plants at 168h in comparison to drought treated control plants. The fresh weight and dry weight also indicated towards stress adaptive e cacy. Huge differences were observed in the numbers of lateral branches with almost (~3 fold) increase in number of lateral branches in RA-inoculated drought stressed plants after 168h of stress treatment. Signi cant difference in lateral roots number was observed in drought induced RA inoculated plants at 168h with 66% increase as compared to drought treated plants indicating the role of RA in drought stress endurance in chickpea.
3.2 Effect of drought stress on physiological parameters with or without RA inoculation Electrolyte leakage was progressively increased in drought treated plants as compared to control whereas RA inoculated drought stressed plants showed comparative decline in EL at 72h (50%) and 168h (80%). The RA inoculated plants showed better results as compared to drought at 72h (72%) and 168h (75%) (Fig. 2a). The RWC was progressively higher with increase in time to about 63% and 115% at 72 and 168h in drought treated RA inoculated samples respectively as compared to drought treated samples. The RA inoculated plants had relatively higher water content (69%) at 72h as compared to drought (Fig. 2b).

Effect of drought stress on biochemical aspects with or without RA inoculation
The MDA content was highest in 72h of drought treated plants and decreased with increase in stress exposure with lowest MDA content recorded in RA inoculated non stressed plants at 168h (0.04 nmols MDA mg −1 FW). The lipid peroxidation levels were less in RA inoculated drought treated plants at all time intervals involving 75% and 54% at 72h and 168h time intervals as compared to drought treated plants at the same time points (Fig. 2c). Further accumulation of compatible osmolyte, proline showed signi cant increase with progression of drought stress whereas signi cant decrease in its content was observed in RA inoculated plants in comparison to stress treatment. The RA inoculated drought treated plants showed comparatively less proline content as compared to drought treated plants by 20% (72h) and 40% (168h) (Fig. 2d).

Expression analysis of miR166h and ATHB15 under drought stress in different samples
The expression analysis of chickpea root samples for miR166h and ATHB15 in all four sample sets at two time points of drought stress (72h and 168h) was studied. The quantitative expression analysis indicated inverse correlation between the selected miRNA and its target. MiR166 was highly upregulated under drought with (~2.8 fold expression) at 7h and 168h with its target was found to be downregulated (-1.3 fold). In case of drought stressed RA-inoculated plants, the fold change in expression levels of miR166h was found to be increased as compared to drought stress at 72h (3.3fold) and 168h (3.2 fold) and the target was downregulated by -1.6 and -1.5 fold, respectively at respective stress durations. Further, RA inoculated non stressed plants resulted in downregulation of miR166h with upregulation of its target. MiR166h expression levels were found to be -1.1 and -1.5 fold whereas ATHB15 showed an upregulation with 1.9 and 1.6 fold change in expression at 72h and 168 h, respectively (Fig. 3). Hence, these quantitative expression levels indicate the role of miR166 in RA-mediated mechanism of mitigating drought stress.

Phylogenetic status of ATHB15 and pre-miR166h
We examined the phylogenetic status of miR166 target ATHB15 of chickpea with other leguminous plant species. A simple nucleotide BLAST of ATHB15 in NCBI was performed that identi ed sequence similarity ranging from 77 to 100% and query coverage of 81 to 100 %. Phylogenetic analysis revealed that the target ATHB15 of miR166in chickpea was closely related to ATHB15of Medicago truncatula (Fig. 4a) regulating vascular development and cambium formation (Li et al. 2020). Whereas, reduces the formation of lateral roots and nodulation when suppressed by its miRNA (Boualem et al. 2008). This target has not yet been characterized in many crop plants and hence need more interest. Similarly pre-miR166h was directed for phylogenetic relationship using same procedure. The sequence similarity was ranging from 36 to 100% and query coverage of 97 to 100 %. MiR166h of chickpea is most similar in Hevea brasiliensis with 98% query cover (Fig. 4b). Kuruvilla et al. (2016) also studied the roles of pre-miR166 in Hevea brasiliensisand hence more plants should be studied for roles in pre-miR166 in different plants.

Target validation for conserved car-miR166h
For experimental validation of regulatory target of car-miR166h was done using modi ed 5´end rapid ampli cation of cDNA ends (5' RLM-RACE) approach. Based on the sequencing results of mRNA cleavage of predicted target gene, ATHB15cleavage site was found opposite to the 10th position from the 5'end of the miRNA. Based on the predicted target for the conserved miR166h,11 RACE experiments were done using root samples of RA inoculated drought stress treated chickpea. The results presented in Fig. 5 indicate that our prediction approach correctly identi ed target ATHB15 for conserved miR166h in chickpea.

Analysis of cis-regulatory motifs in promoters of miR166
The promoter analysis was carried out for better understanding of miR166 regulation in chickpea in terms of abiotic stress response. To identify and study predicted putative cis-regulatory elements localised in the promoter region that was located according to the analysis based on the assumed position of transcription start site (TSS) was done using in silico approach. The observed promoter elements included light response, hormone response, plant development, metabolism, defence and stress responses and others. The different cis-regulatory elements analysed in promoter of pre-miR166h in chickpea with their putative positions have been annotated (Table.1). The cis-regulatory elements were not distributed uniformly in miR166 promoters. The light-responsive elements were most abundantly found in the promoters. Preferably ve types of stress-responsive elements were observed namely, ABRE (ACGTG, the abscisic acid (ABA) response element), CGTCA-motif (methyl-jasmonate-responsive cisregulatory element), TC-rich repeats (GTTTTCTTAC, defence and stress responsive cis-regulatory) elements and TCA-elements (CCATCTTTTT, salicylic acid responsive cis-regulatory elements) (Fig. 6). For better understanding of the roles of these cis-regulatory motifs, quantitative real-time PCR analysis of different tissues harvested from drought, salinity and ABA treatments was performed.

Tissue-speci c expression of miR166h and ATHB15
The relatively highest expression levels of miR166h was observed in pods (1.9 fold) followed by owers (1.8 fold), whereas the expression of ATHB15 was found to be highest in mature shoot (1.7 fold) followed by immature leaf (1.2 fold) (Fig. 7a). Further expression analysis of miR166 and its target was done in different tissues harvested after drought, salinity and ABA stress treatments. Expression patterns in different tissues and different stresses suggested inverse correlation between miR166h and its target. On exposure to drought stress, the expression of miR166h in leaf tissue was found to be downregulated under control conditions as well as during 20% PEG6000 treatment with -0.5 and -1.3 fold relative expression levels at 24h and 72h of stress exposure, respectively. The drought stress treated shoot at 24h showed 2.6 fold whereas at 72h the expression levels were declined to basal levels with 1.2 fold change. Further, root tissue analysis revealed high upregulation on drought exposure with 2.4 (1h), 6.4 (24h) and 6.6 fold expression at 72h of stress. In accordance to miR166h expression, ATHB15 showed decline in expression in leaf tissue at 24h and 72h with 1.6 and 2.0 fold expression, respectively while drought treated shoots showed -0.3 fold-change expression at other time intervals. Relative expression levels were found to be comparatively declined (-3.6 and -4.4 fold) in roots at later time points (Fig. 7b).
The expression analysis of conserved miR166h and ATHB15 was also studied under100mM NaCl treatment that revealed differential expression patterns at different time intervals. ATHB15 was found to be increasingly upregulated by the increase in time interval with 2.1-foldat 24h and 1.9 fold at 72h fold expression in control along with signi cant upregulation (3.4 and 5.3 fold) values when treated samples were subjected to relative fold expression study. The inverse expression patterns of miR166h were observed with that of ATHB15that showed highly downregulated expression with -0.4, -1.7 and -2.4 fold expression at 1h, 24h and at 72h. The salinity stress treated shoot samples showed downregulation of miR166h with signi cant fold change (-1.6 and -1.8) at 24h and 72h, respectively whereas ATHB15 showed highest expression at 72h with 2.8 fold expression. The target gene showed upregulation (0.6, 1.7 and 5.7 fold) in roots with increasing stress duration (1h, 24h and 72h). The miR166h showed a signi cant decline (0.8, -1.3 and -3.2 fold) with increasing time intervals indicating the important roles of miR166h in salinity endurance (Fig. 7c).
Variations in expression patterns were also observed upon treatment with 10µM ABA observed. MiR166h exhibited upregulation (2.1 fold) at 1 h in leaves but showed a decline (-3.2 fold) at 24 h and again an increase at 72h (2.9 fold) whereas ATHB15showed an upregulation (1.6 fold) at 1h but downregulation (-1.0 and -3.0 fold) at 24h and 72h, respectively. ABA treated shoots showed high upregulation of miR166h only at 24h (4.7fold) and no signi cant expression at 1h and 72h whereas basal expression levels were noted in ATHB15 upon ABA stress treatment at all time intervals. Roots treated with ABA showed highly downregulated expression (-4.0 and -3.5 fold) of ATHB15 at 24h and 72h, respectively with gradual increase in regulation in control samples whereas the miR166h expression was higher at 1h but declined with increase in time interval resulting in expression levels as 4.8 fold at 1h, 3.2 fold at 24h and 2.3 fold at 72h of stress (Fig. 7d).

Discussion
With the discovery of plethora of miRNAs in response to drought stress has led to initiation of in-depth research to decipher their mechanism of action ( . Drought stress is the most common and detrimental environmental stress factor that reduces crop production and yield throughout the world. Drought stress adaptation of crop plants can be achieved through breeding, genetic engineering or gene editing for enhanced agricultural productivity. However, microbial techniques involving microbes for improving stress tolerance of crop plants is gaining importance due to the constraints of labour, cost, time and ethical issues (Nautiyal et al. 2013). The present study discusses about the positive modulatory aspects of RA as well as miR166hmediated response to drought stress in desi chickpea genotypein stress alleviation. Plants subjected to drought stress perceive the simulations primarily through roots. Hence, root growth is one of the major indicators of stress management. In this study, the phylogenetic analyses of pre-miR166 and ATHB15 have been performed to understand their evolutionarily conserved nature. ATHB15 has been previously characterized to a lesser extent as the target of miR166 in Zinnia (Ohashi and Fukuda 2003) and Arabidopsis (Kim et al. 2005). Further, RLM-RACE is used for analysing miRNA-guided sequence-speci c mRNA target endo-nucleolytic cleavage (Llave et al. 2002;Donaire et al. 2011). This procedure has been successfully utilized for validation of miRNA targets (Llave et al. 2002; Jones-Rhoades and Bartel 2004). In this study, the RLM-RACE con rmed that ATHB15 is indeed the target of miR166hin desi chickpea genotype. The validated target mRNA transcript showed perfect cleavage site that mapped at the 10th from the 5′-end of the binding site for miR166hjustifying the previous report in A.thaliana (Kim et al. 2005). The earlier study reported the role of ATHB15 in vascular development while no such validation has been reported till date suggesting ATHB15 to be the mRNA target of miR166 in chickpea under drought stress or PGPR inoculation.
The cis-regulatory ACGT-containing ABREs have been functionally demonstrated in ABA-modulated transcription roles in various stress responsive genes subjected to ABA and water stress in maize by Pla et al. (1993). It has been demonstrated that bZIP proteins bind with more a nity to these elements and further regulate ABA signalling and abiotic stress responses (Liao et al. 2008). Such bZIP proteins bind to the cis-elements in the promoter region and modulate the stress responses by either directly binding or indirectly by activating various other stress responsive genes. Liao et al. (2008) reported negative regulatory ABA signalling involving ACGT elements leading to salinity and freezing tolerance in transgenic Arabidopsis. Promoter analysis of Oryza sativa lectin receptor-like kinases (LecRLKs) indicated the involvement of various cis-regulatory genes including TC-rich repeats under biotic stress (Passricha et al. 2017). ZmMYB30 of Zea mays is also enriched with TC-rich repeats in its promoter and has roles in terms of salinity stress (Luo et al. 2020). PDI gene family in Solanum lycopersicumalso has TC-rich repeats in promoter pointing their role towards the abiotic stress mitigation (Wai et al. 2021). The ESTs under salinity stress in Artemesia annua were also studied for the presence of cis-regulatory elements and showed the role of TCA-elements in salicylic acid response and stress modulation (Alam and Balawi 2020).
Tissue-speci c expression analysis is a useful tool to examine the transcriptional networks involving miRNAs and their targets in various plant tissues (Celik and Akdas 2019). miR166 has been studied in different plants for its relative expression and abundance in different plant tissues. In this study, contrasting expression patterns are noted between miR166 and its target gene in desi chickpea genotype upon different abiotic stress treatments including salinity, drought and ABA on different plant tissues. ).Phytohormonal treatment resulted in variation in expression patterns of both miR166h and ATHB15 where ABA stressed leaf showed miR166h upregulation at 1h and 72h and basal expression at 24h while ATHB15 was found to be highly downregulated at 72h. The ABA treated shoots however, drew no such signi cant expression relations for both miR166h and ATHB15except high expression levels in shoot samples at 24h for miR166h and basal expression of ATHB15in response to ABA. The stressed root tissues showed miR166h upregulation at 1h but gradual decline with increasing time interval while the target was also downregulated at 24h and 72h. Similar observation was reported earlier in Arabidopsis thaliana ). However, no signi cant difference in expression patterns of miR166h and its target was observed upon ABA treatment in Arabidopsis and rice ).

Conclusion And Future Perspectives
The tripartite plant-soil-microbe interaction is becoming an important area of research for crop improvement in terms of abiotic stress including drought. P. putida RA has been found to be bene cial in ameliorating drought stress in desi chickpea cultivar. Drought stress negatively regulated the crop by affecting the growth and development, including root length, shoot length, number of lateral roots, branches and internodes, increased EL and MDA content with reduced RWC, enhanced compatible solutes. While RA played an important role in positively modulating the stress response with improved plant growth, membrane integrity, water status with restored antioxidants and osmolytes. At molecular levels the regulation of various stress responsive genes with inverse correlation to miRNAs have also been found to be modulated by RA. A working hypothesis for the mechanism of RA-mediated drought stress mitigation in desi chickpea cultivar based on the plant responses to RA from this study and literature survey in different crops has been illustrated (Fig. 8).
miR166 known to be an important drought stress-responsive miRNA targeting the HD-ZIP III TF family. In our study, miR166h was found to have positive roles under drought stress. In RA-inoculated plants, both miR166h and ATHB15 showed inverse expression patterns suggesting the role of RA in drought stress alleviation and in growth promotion in RA inoculated plants. Based on our results as well as previous literature, we hypothesised the mechanism of RA and miR166h-mediated regulation of drought stress by targeting ATHB15 for enhanced drought endurance in chickpea (Fig. 9). RLM-RACE con rmed that ATHB15 is the target of miR166h in chickpea. This is the rst report on the validation ofATHB15 as the target of miR166hin chickpea upon PGPR inoculation under drought stress. The inverse correlation between miR166h and its target ATHB15 in different tissues of chickpea under different abiotic stresses indicated the crucial role of miR166h in stress mitigation by expression modulation of its target.
Hence, this study highlighted the bene cial roles of RA-inoculation in the modulation of miR166h and its target gene expression in drought stress mitigation and its utilization in various crop improvement strategies.    a. Evolutionary relationships of ATHB15: The evolutionary analyses were conducted in MEGA X and the evolutionary history was inferred using the Neighbor-Joining method. The associated taxa were clustered together in the bootstrap test (1000 replicates) with evolutionary distances computed using the pdistance method. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 2536 positions in the nal dataset. b. Evolutionary analysis by Maximum Likelihood method of pre-miR166h: The evolutionary analysis was conducted using MEGA X including Maximum Likelihood method and Tamura-Nei model. The tree with the highest log likelihood (-985.96) is shown. The initial tree(s) for the heuristic search were obtained automatically using Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura-Nei model, and then selecting the topology with superior log likelihood value.  The sequence of miR166h promoter indicated different cis-acting elements that regulate genes. The elements involved in different stress responses are marked with speci c color. Grey: cis-acting regulatory element involved in the MeJA-responsiveness (TGACG-motif), pink: cis-acting regulatory element involved in the MeJA-responsiveness (CGTCA motif), blue: cis-acting element involved in defense and stress responsiveness (TC-rich repeats), green: cis-acting element involved in salicylic acid responsiveness (TCA element) and yellow: cis-acting element involved in the abscisic acid responsiveness (ABRE).