Fine particular miRNA is extremely useful for studying

Fine tuning of gene expression remain as a focalproblem from long ago in genomics as it can determine the fate of many cellularand metabolic processes. The successful survival of plants, compared to otherorganisms strongly depends on the efficient gene regulatory mechanisms whichhelps to overcome their deterrents like being sessile, hostile environment andinvasion of a vast variety of pathogens. Though there are different ways forgene regulation, the recently recognized layer with the aid of microRNAs (miRNAs)piqued the central attraction and it provides new dimensions to the complexeukaryotic gene regulatory mechanisms in this genomic era. The precise andtargeted gene regulation makes miRNAs distinct and very efficient genemodulators. The attractiveness of miRNAs also lies in its myriads ofapplications where it can be exploited as a powerful tool in basic research aswell as for genetic modifications. Potentiality of miRNA research includes identificationof a miRNA and its target gene which is silenced by that particular miRNA isextremely useful for studying the function of the target gene (since majorityof the plant genes are yet to be characterized), genetic manipulation such asover-expression or knocking down of the characterized miRNAs will allowspecific regulation of its target genes for better adaptation of plants towardsthe stressful environment as well as for improving the crop yield, designingartificial miRNAs based on endogenous miRNAs to suppress the target geneexpression and enhancing the quality and quantity of agricultural productivity. MicroRNAs represent the class of small non-codingRNAs, which are impotent to code for proteins, and that made their functionsentirely different from just protein coding to a plethora of versatilefunctions in different aspects of growth, development, chromatin remodeling,genome stability and stress responses (Chen.

2009, Sun.2012). MicroRNAs aresingle stranded RNAs, endogenous in origin with ~18-24nt in length and bind toits complementary sequence present in the target mRNA. This leads to thepost-transcriptional silencing of the target mRNA either through mRNA cleavageor by translational repression based on the complementarity between the two.  Plant miRNAs usually have the property of nearperfect complementarity with the target mRNAs which directs the cleavage oftarget mRNAs whereas animal miRNAs having partial complementarity with theirtargets results in translational repression of target mRNAs (Rhoades et al.

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2002). miRNAs significantly differ from other members of the small non-codingRNA family such as small interfering RNAs (siRNAs) and piwi interacting RNAs(piRNAs) in their biogenesis as well as in mode of action (Bologna et al.2014). miRNAs are highly conserved across the plant kingdom based on strongsequence similarity and that is extremely useful for identifying plant miRNAswith computational approach which can be further validated through traditionalexperimental techniques ( Rhoades et al. 2002, Xie et al.

2005, Zhang et al. 2006a).The miRNAs were initially known as short temporalRNAs (stRNAs) and foremost reported in Caenorhabditiselegans by early nineties (Lee et al. 1993) but various plant miRNAs werediscovered latterly (Llave et al.

2002, Park et al. 2002, Reinhart et al. 2002).The biogenesis of miRNAs in plants are well studied and are depicted infigure1. miRNAs are produced from long single stranded RNAs with stem-loopstructure and known as primary miRNAs (pri-miRNA), which are transcribed frommiRNA genes by RNA polymerase-II like that of protein coding genes (Lee et al.

2002, Lee et al. 2004). These pri-miRNAs undergo two-step processes and bothare mediated by DICER/ Dicer Like (DCL) endonuclease, the former step resultsin ~60-70 nt long precursor miRNA (pre-miRNA) and the second step generates themiRNA-miRNA* duplex with the help of HYPONASTIC LEAVES 1 (HYL1),ds-RNA binding protein 1 (DRB1) and serrate (SE) (Schauer et al. 2002, Lee etal.

2003, Denli et al. 2004). The miRNA duplex produced in the nucleus ismethylated by HUA ENHANCER 1 (HEN1) and transported to the cytoplasm assistedby HASTY proteins (Park et al. 2005). Once it is exported to cytoplasm, withthe help of Hsc70/Hsp90 and ATP, one of the miRNA strand from the duplex get insertedinto the RNA-induced silencing complex (RISC) in which the ARGONAUTE (AGO)proteins guide the miRNA strand to its target mRNA and the other strandundergoes degradation (Olsen et al. 1999, Hammond et al. 2001, Vionnet. 2009, Iwasakiet al.

, 2010; Nakanishi, 2016). Great efforts have been done in the pastfew years for the identification of a large number of miRNA-mRNA module inplant-microbe interaction, but very few have functionally characterized andvalidated for agronomic importance. Elucidating the role of species specificmiRNAs in individual cases will deepen the possibility of their application intargeted gene regulation to enhance plant stress tolerance in connection withplant-microbe interaction and thereby improving the crop yield. Though manymiRNAs are conserved across the plant kingdom, they are functionally divergentacross species, and in different conditions (find another appropriate word!!).So the meticulous identification of rightful miRNAs as gene modulators withefficacy and reliability is very essential before using it for geneticmanipulation of crops.

A cumulative approach using bioinformatic tools andbiological experiments will open the doors to identify the rightful candidates.In this review, we tried to summarize the recent advances in    miRNA mediated regulation of genes relatedto plant-microbe interaction with the emphasis on role of plant miRNAs, whichcan be exploited for making improved crop varieties.  ReviewMicroRNAs as cardinalregulators in symbiotic plant-microbe interactionSymbiosis always has its place innature, in bridging two different species which live in close proximity andinteract to each other for the benefits of either one or both the candidates.Root nodule symbiosis and arbuscular mycorrhizal symbiosis are the most commonand well-studied symbiotic plant microbe systems. Though the mechanism andmolecular interplay driving the symbiotic plant microbe interaction is highlyestablished, the regulatory view especially via miRNA mode is still in infancy.

The root nodule (RN) symbiosis isspecific to legumes in which nitrogen fixation occurs with the aid of nitrogenfixing bacteria in specialized organs known as root nodules (Patriarca et al. 2004).In the context of RN symbiosis, miRNAs are outlined as the molecular signaturesin the regulatory network of nodule development and nutrient homeostasis (Simonet al.

2009). miR169 is a well-known player in modulating root noduledevelopment and meristematic activity. In Medicagotruncatula, miR169a has identified to target a CCAAT-binding family oftranscription factor known as HemeActivator Protein 2-1 (MtHAP2-1)and confines the protein expression in nodule meristematic zone (Combier et al.2006).

The authors proposed that miR169a mediated regulation of MtHAP2-1 may be essential for nodulecell differentiation since the gene is required for meristematic activity andmaintenance.  Likewise miR166 of M. truncatula targets a class -III homeodomain-leucine zipper(HD-ZIP III) family of transcription factors, which are associated with thenodule development. The study revealed that miR166 co-expressed with its targettranscription factors in vascular bundles, apical regions of roots and nodulescausing reduction in nodule number and lateral roots (Boualem et al.

2008). Thework by Lelandais-Briere et al. (2009) showed the differential expression ofmiRNAs from M. truncatula root tipsand mature nodules and described about their role in spatial development ofnodules. In soybean, Subramanian et al. (2008)reported the temporal differential expression of miRNAs in response toinoculation with Bradyrhizobium japonicum.In an attempt to find out the regulatory roles played by the differentiallyregulated miRNAs, they observed that miR169, has two putative targets insoybean and are identical to HAP2-1of M. truncatula which has alreadyreported for nodule development.

In addition, they have identified miR172,miR166, and miR396 regulate a putative Apetala2-like transcription factor,HD-ZIPIII-like transcription factor, and a cysteine protease respectively. Theyhave also suggested that few of the reported miRNAs are linked to the planthormone auxin signaling or its homeostasis which might be an important bridgefor nodule development. This category includes miR167 targeting auxin responseactivator ARF8, miR160 regulatesauxin response repressors ARF10, 16 and 17, miR393 targets an auxin receptor TIR1 and miR164 regulates a NAC1transcription factor (Rhoades et al. 2002, Kasschau et al.

2003, Mallory et al.2005). Further study on miR172 of soybean disclosed that ectopic expression ofmiR172 increased the number of nodules, expression of symbiotic leghemoglobinand non-symbiotic hemoglobin by limiting the level of an AP2 transcription factor. And another miRNA, miR156 negativelyregulates the expression of miR172. So the authors postulated that theantagonistic effects of miR156 and miR172 controls the nodule development insoybean (Yan et al. 2013). Similarly, ectopic expression of miR160 ended up inauxin hypersensitivity and cytokinin hyposensitivity which causes moderatelevel inhibition of primordium formation and inhibition of further developmentsof nodule (Turner et al.

2013). In addition, recently miR393j-3p was identifiedas potential regulator of nodule formation in soybean, by targeting a nodulingene known as Early Nodulin 93 (ENOD93) and the ectopic expression ofmiR393j-3p resulted in significant reduction in the number of nodules (Yan etal. 2015).

Many more miRNA repertoires have reported in association with therhizobia-legume symbiosis, but very few focused on the roles played by specificmiRNAs.The symbiotic association between mostof the flowering plants and glomeromycotian fungi is commonly known asarbuscular mycorrhizal (AM) symbiosis, which enhances the phosphateavailability to the plant partner and also improves plant resistance towardsabiotic and biotic stresses. The crucial role of miRNAs in the modulation of AMsymbiosis has identified in recent times. miR399 is a well-studied candidate inAM symbiosis in M. truncatula as wellas in tobacco plants. Studies have revealed that AM symbiosis leads toincreased expression of miR399, but that is not a remarkable signal forimproving the mycorrhizal colonization. Instead it act as a modulator forsufficient Pi uptake during AM symbiosis by targeting transcript of aubiquitin-conjugating enzyme PHO-2,which is a negative regulator of Pi starvation inducible genes (Branscheid etal.

2010). Another important finding was that both miRNA as well as miRNA*are differentially expressed during AM symbiosis and it indicates theirinvolvement in AM symbiosis. Several of these miRNAs and miRNA*starget disease resistance genes for promoting fungal growth (Devers et al.2011). Interestingly, studies revealed that miRNAs can prevent the over-colonizationof AM symbiotic fungi around plant roots. miR171h of M. truncatula negatively regulates NSP2, a transcription factor involved in both nodulation andmycorrhization. NSP2 is important inroot colonization by the fungal partner, so miR171h mediated transcriptionalregulation of NSP2 is considered as amechanism to prevent over-colonization or to limit the fungal growth spatially(Lauressergues et al.

2012). A very recent study has reported six AM symbiosismiRNAs in tomato and two out of six of these miRNAs belongs to the miR171family (miR171 and miR171g) and are capable of targeting the NSP2 genes (Wu et al. 2016). Thesestudies are suggesting that regulation of NSP2genes mediated by miR171 family serve as a general strategy in the regulationof AM symbiosis.

Unlike other members of miR171 family, miR171b promotes the establishmentof AM symbiosis. The miR171b has a mismatched cleavage site among the plantsthat establish AM symbiosis, so that it is unable to target LOM1 (LOST MERISTEMS1) gene, which isused to be down-regulated by miR171 family during AM symbiosis (Couzigou et al. 2016).Regulation of symbiosis via miRNAs is awide area of research with immense potential but less utilized and majority ofthe works focused on RN symbiosis part. Though miRNAs are identified as apromising signature for improving the effectiveness of symbiotic relations interms of plant mineral nutrition, we can hardly see any research work focusingmiRNA mediated improvement of association efficacy. Functional analysis ofmodified miRNA expression can assure producing new insights into enhancingplant mineral nutrition through the symbiotic plant-microbe interaction.MicroRNAsand plant disease susceptibilityDisease susceptibility is also animportant component as disease resistance, though they have opposite functionsand many factors contribute to make the plant infected.

Many scientific studieshave reported that alteration in miRNA profiles occur during pathogen infectionand many of the miRNA pathways got inactivated by the ingenious act of pathogenas a part of their counter defense programing. An engrossing discovery in this field isthe overexpression of Sp-miR396a-5p in tobacco plants resulted in increasedsusceptibility towards Phytophthora nicotianae infection. And the interestingpart is same transgenic plants showed increased resistance towards differentabiotic stress like salt, cold and drought stress though it showed increasedsusceptibility towards the infection. The dual role of Sp-miR396a-5p as apositive regulator of abiotic stress and negative regulator of biotic stress isobtained by tight regulation of NtGRF7 mediated down regulation of osmoticstress-responsive genes and by targeting NtGRF1 and NtGRF3 which in turninduces cytokinin mediated defense response pathways respectively (Chen et al.2015). Additionally the coordinated act of miR472 and RNA-dependent RNApolymerase 6 (RDR6) of Arabidopsis leads to the post-transcriptional silencingof resistance genes of coiled-coil nucleotide-binding leucine rich-repeats(CC-NB-LRRs/ CNLs) family.

The mutant lines, for both miR472 and rdr6 were moreresistant towards Pseudomonas syringae pv.tomato DC3000 (Pto DC3000) strainswith the bacterial effector AvrPphB. And the same time, transgenic plantsoverexpressing miR472 and RDR6 exhibited an enhanced susceptibility towards thebacterial strains. Thus miR472/RDR6 mediated silencing pathways execute thecontrol over plant’s basal defense, PAMP-triggered immunity (PTI) and also toeffector-triggered immunity (ETI) (Boccara et al. 2014).

Similar to this,miR400 of Arabidopsis targets peptatricopeptide repeat (PPR) genes, which inturn is a contributor of defense responses. The negative regulation of PPRgenes by miR400 turned the host plant susceptible for both bacterium PtoDC3000and the fungus Botrytis cinerea (Park et al. 2014). Moreover, miR825 andmiR825* of Arabidopsis mediate susceptibility towards Pto DC3000. miR825targets two ubiquitin-protein ligases whereas miR825* regulatestoll-interleukin-like receptor (TIR)-nucleotide binding site (NBS) andleucine-rich repeat (LRR) type resistance (R) genes, which are the majorcontributors to the resistance response. However another plant growth promotingrhizobacterium known as Bacillus cereus AR156 suppress the activity of miR825and miR825*, and primes induced systemic resistance (ISR) (Niu et al. 2016).Very recently, it has been reported that, there are wheat specific miRNAsinvolved in susceptible interaction with Puccinia striiformis f.

sp. tritici,causing stripe rust of wheat. These miRNAs and their down regulated targetswere implicated to have a role in causing the susceptibility interaction betweenthe host plant and the pathogen (Feng et al. 2016). In another study, it hasreported that miR408 of wheat post-transcriptionally regulates achemocyanin-like protein (TaCLP1) gene, that in turn is essential for mediatingresistance response to stresses like salinity, high cupric content and striperust.

So the induction miR408 increases wheat susceptibility towards striperust (Feng et al. 2013).  Anotherembraced part of miRNAs is the conversion of host to susceptible for parasiticinfection. The miR827 from Arabidopsis mediates susceptibility towards the beetcyst nematode Heterodera schachtii, which is a sedentary endoparasite.

HeremiR827 targets nitrogen limitation adaptation (NLA) genes that encode ubiquitinE3 ligase enzyme required to limit the activity of parasites. Inactivation ofmiR827 reduces plant susceptibility and over expression of miR827 leads tohyper susceptibility which strengthen the model (Hewezi et al. 2016).Inactivation of miRNAs and thereby miRNA mediated post transcriptionalregulation is another strategy related to disease susceptibility. For example,in the poplar plant susceptible to the infection of virulent Melampsoralarici-populina, the miRNAs which regulates defense signaling pathways gotinactivated in the stage of effector triggered susceptibility and that wasmarked as the major reason for its susceptibility (Li et al.

2016).