Fine particular miRNA is extremely useful for studying

Fine tuning of gene expression remain as a focal
problem from long ago in genomics as it can determine the fate of many cellular
and metabolic processes. The successful survival of plants, compared to other
organisms strongly depends on the efficient gene regulatory mechanisms which
helps to overcome their deterrents like being sessile, hostile environment and
invasion of a vast variety of pathogens. Though there are different ways for
gene regulation, the recently recognized layer with the aid of microRNAs (miRNAs)
piqued the central attraction and it provides new dimensions to the complex
eukaryotic gene regulatory mechanisms in this genomic era. The precise and
targeted gene regulation makes miRNAs distinct and very efficient gene
modulators. The attractiveness of miRNAs also lies in its myriads of
applications where it can be exploited as a powerful tool in basic research as
well as for genetic modifications. Potentiality of miRNA research includes identification
of a miRNA and its target gene which is silenced by that particular miRNA is
extremely useful for studying the function of the target gene (since majority
of the plant genes are yet to be characterized), genetic manipulation such as
over-expression or knocking down of the characterized miRNAs will allow
specific regulation of its target genes for better adaptation of plants towards
the stressful environment as well as for improving the crop yield, designing
artificial miRNAs based on endogenous miRNAs to suppress the target gene
expression and enhancing the quality and quantity of agricultural productivity. 

MicroRNAs represent the class of small non-coding
RNAs, which are impotent to code for proteins, and that made their functions
entirely different from just protein coding to a plethora of versatile
functions in different aspects of growth, development, chromatin remodeling,
genome stability and stress responses (Chen.2009, Sun.2012). MicroRNAs are
single stranded RNAs, endogenous in origin with ~18-24nt in length and bind to
its complementary sequence present in the target mRNA. This leads to the
post-transcriptional silencing of the target mRNA either through mRNA cleavage
or by translational repression based on the complementarity between the two.  Plant miRNAs usually have the property of near
perfect complementarity with the target mRNAs which directs the cleavage of
target mRNAs whereas animal miRNAs having partial complementarity with their
targets results in translational repression of target mRNAs (Rhoades et al.
2002). miRNAs significantly differ from other members of the small non-coding
RNA 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 strong
sequence similarity and that is extremely useful for identifying plant miRNAs
with computational approach which can be further validated through traditional
experimental techniques ( Rhoades et al. 2002, Xie et al. 2005, Zhang et al. 2006a).

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The miRNAs were initially known as short temporal
RNAs (stRNAs) and foremost reported in Caenorhabditis
elegans by early nineties (Lee et al. 1993) but various plant miRNAs were
discovered 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 in
figure1. miRNAs are produced from long single stranded RNAs with stem-loop
structure and known as primary miRNAs (pri-miRNA), which are transcribed from
miRNA 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 both
are mediated by DICER/ Dicer Like (DCL) endonuclease, the former step results
in ~60-70 nt long precursor miRNA (pre-miRNA) and the second step generates the
miRNA-miRNA* duplex with the help of HYPONASTIC LEAVES 1 (HYL1),
ds-RNA binding protein 1 (DRB1) and serrate (SE) (Schauer et al. 2002, Lee et
al. 2003, Denli et al. 2004). The miRNA duplex produced in the nucleus is
methylated by HUA ENHANCER 1 (HEN1) and transported to the cytoplasm assisted
by HASTY proteins (Park et al. 2005). Once it is exported to cytoplasm, with
the help of Hsc70/Hsp90 and ATP, one of the miRNA strand from the duplex get inserted
into the RNA-induced silencing complex (RISC) in which the ARGONAUTE (AGO)
proteins guide the miRNA strand to its target mRNA and the other strand
undergoes degradation (Olsen et al. 1999, Hammond et al. 2001, Vionnet. 2009, Iwasaki
et al., 2010; Nakanishi, 2016). 

Great efforts have been done in the past
few years for the identification of a large number of miRNA-mRNA module in
plant-microbe interaction, but very few have functionally characterized and
validated for agronomic importance. Elucidating the role of species specific
miRNAs in individual cases will deepen the possibility of their application in
targeted gene regulation to enhance plant stress tolerance in connection with
plant-microbe interaction and thereby improving the crop yield. Though many
miRNAs are conserved across the plant kingdom, they are functionally divergent
across species, and in different conditions (find another appropriate word!!).
So the meticulous identification of rightful miRNAs as gene modulators with
efficacy and reliability is very essential before using it for genetic
manipulation of crops. A cumulative approach using bioinformatic tools and
biological 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 related
to plant-microbe interaction with the emphasis on role of plant miRNAs, which
can be exploited for making improved crop varieties.  

Review

MicroRNAs as cardinal
regulators in symbiotic plant-microbe interaction

Symbiosis always has its place in
nature, in bridging two different species which live in close proximity and
interact to each other for the benefits of either one or both the candidates.
Root nodule symbiosis and arbuscular mycorrhizal symbiosis are the most common
and well-studied symbiotic plant microbe systems. Though the mechanism and
molecular interplay driving the symbiotic plant microbe interaction is highly
established, the regulatory view especially via miRNA mode is still in infancy.

The root nodule (RN) symbiosis is
specific to legumes in which nitrogen fixation occurs with the aid of nitrogen
fixing bacteria in specialized organs known as root nodules (Patriarca et al. 2004).
In the context of RN symbiosis, miRNAs are outlined as the molecular signatures
in the regulatory network of nodule development and nutrient homeostasis (Simon
et al. 2009). miR169 is a well-known player in modulating root nodule
development and meristematic activity. In Medicago
truncatula, miR169a has identified to target a CCAAT-binding family of
transcription factor known as Heme
Activator 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 nodule
cell differentiation since the gene is required for meristematic activity and
maintenance.  Likewise miR166 of M. truncatula targets a class -III homeodomain-leucine zipper
(HD-ZIP III) family of transcription factors, which are associated with the
nodule development. The study revealed that miR166 co-expressed with its target
transcription factors in vascular bundles, apical regions of roots and nodules
causing reduction in nodule number and lateral roots (Boualem et al. 2008). The
work by Lelandais-Briere et al. (2009) showed the differential expression of
miRNAs from M. truncatula root tips
and mature nodules and described about their role in spatial development of
nodules.

In soybean, Subramanian et al. (2008)
reported the temporal differential expression of miRNAs in response to
inoculation with Bradyrhizobium japonicum.
In an attempt to find out the regulatory roles played by the differentially
regulated miRNAs, they observed that miR169, has two putative targets in
soybean and are identical to HAP2-1
of M. truncatula which has already
reported 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. They
have also suggested that few of the reported miRNAs are linked to the plant
hormone auxin signaling or its homeostasis which might be an important bridge
for nodule development. This category includes miR167 targeting auxin response
activator ARF8, miR160 regulates
auxin response repressors ARF10, 16 and 17, miR393 targets an auxin receptor TIR1 and miR164 regulates a NAC1
transcription factor (Rhoades et al. 2002, Kasschau et al. 2003, Mallory et al.
2005). Further study on miR172 of soybean disclosed that ectopic expression of
miR172 increased the number of nodules, expression of symbiotic leghemoglobin
and non-symbiotic hemoglobin by limiting the level of an AP2 transcription factor. And another miRNA, miR156 negatively
regulates the expression of miR172. So the authors postulated that the
antagonistic effects of miR156 and miR172 controls the nodule development in
soybean (Yan et al. 2013). Similarly, ectopic expression of miR160 ended up in
auxin hypersensitivity and cytokinin hyposensitivity which causes moderate
level inhibition of primordium formation and inhibition of further developments
of nodule (Turner et al. 2013). In addition, recently miR393j-3p was identified
as potential regulator of nodule formation in soybean, by targeting a nodulin
gene known as Early Nodulin 93 (ENOD93) and the ectopic expression of
miR393j-3p resulted in significant reduction in the number of nodules (Yan et
al. 2015). Many more miRNA repertoires have reported in association with the
rhizobia-legume symbiosis, but very few focused on the roles played by specific
miRNAs.

The symbiotic association between most
of the flowering plants and glomeromycotian fungi is commonly known as
arbuscular mycorrhizal (AM) symbiosis, which enhances the phosphate
availability to the plant partner and also improves plant resistance towards
abiotic and biotic stresses. The crucial role of miRNAs in the modulation of AM
symbiosis has identified in recent times. miR399 is a well-studied candidate in
AM symbiosis in M. truncatula as well
as in tobacco plants. Studies have revealed that AM symbiosis leads to
increased expression of miR399, but that is not a remarkable signal for
improving the mycorrhizal colonization. Instead it act as a modulator for
sufficient Pi uptake during AM symbiosis by targeting transcript of a
ubiquitin-conjugating enzyme PHO-2,
which is a negative regulator of Pi starvation inducible genes (Branscheid et
al. 2010). Another important finding was that both miRNA as well as miRNA*
are differentially expressed during AM symbiosis and it indicates their
involvement in AM symbiosis. Several of these miRNAs and miRNA*s
target disease resistance genes for promoting fungal growth (Devers et al.
2011). Interestingly, studies revealed that miRNAs can prevent the over-colonization
of AM symbiotic fungi around plant roots. miR171h of M. truncatula negatively regulates NSP2, a transcription factor involved in both nodulation and
mycorrhization. NSP2 is important in
root colonization by the fungal partner, so miR171h mediated transcriptional
regulation of NSP2 is considered as a
mechanism to prevent over-colonization or to limit the fungal growth spatially
(Lauressergues et al. 2012). A very recent study has reported six AM symbiosis
miRNAs in tomato and two out of six of these miRNAs belongs to the miR171
family (miR171 and miR171g) and are capable of targeting the NSP2 genes (Wu et al. 2016). These
studies are suggesting that regulation of NSP2
genes mediated by miR171 family serve as a general strategy in the regulation
of AM symbiosis. Unlike other members of miR171 family, miR171b promotes the establishment
of AM symbiosis. The miR171b has a mismatched cleavage site among the plants
that establish AM symbiosis, so that it is unable to target LOM1 (LOST MERISTEMS1) gene, which is
used to be down-regulated by miR171 family during AM symbiosis (Couzigou et al. 2016).

Regulation of symbiosis via miRNAs is a
wide area of research with immense potential but less utilized and majority of
the works focused on RN symbiosis part. Though miRNAs are identified as a
promising signature for improving the effectiveness of symbiotic relations in
terms of plant mineral nutrition, we can hardly see any research work focusing
miRNA mediated improvement of association efficacy. Functional analysis of
modified miRNA expression can assure producing new insights into enhancing
plant mineral nutrition through the symbiotic plant-microbe interaction.

MicroRNAs
and plant disease susceptibility

Disease susceptibility is also an
important component as disease resistance, though they have opposite functions
and many factors contribute to make the plant infected. Many scientific studies
have reported that alteration in miRNA profiles occur during pathogen infection
and many of the miRNA pathways got inactivated by the ingenious act of pathogen
as a part of their counter defense programing.

An engrossing discovery in this field is
the overexpression of Sp-miR396a-5p in tobacco plants resulted in increased
susceptibility towards Phytophthora nicotianae infection. And the interesting
part is same transgenic plants showed increased resistance towards different
abiotic stress like salt, cold and drought stress though it showed increased
susceptibility towards the infection. The dual role of Sp-miR396a-5p as a
positive regulator of abiotic stress and negative regulator of biotic stress is
obtained by tight regulation of NtGRF7 mediated down regulation of osmotic
stress-responsive genes and by targeting NtGRF1 and NtGRF3 which in turn
induces cytokinin mediated defense response pathways respectively (Chen et al.
2015). Additionally the coordinated act of miR472 and RNA-dependent RNA
polymerase 6 (RDR6) of Arabidopsis leads to the post-transcriptional silencing
of resistance genes of coiled-coil nucleotide-binding leucine rich-repeats
(CC-NB-LRRs/ CNLs) family. The mutant lines, for both miR472 and rdr6 were more
resistant towards Pseudomonas syringae pv.tomato DC3000 (Pto DC3000) strains
with the bacterial effector AvrPphB. And the same time, transgenic plants
overexpressing miR472 and RDR6 exhibited an enhanced susceptibility towards the
bacterial strains. Thus miR472/RDR6 mediated silencing pathways execute the
control over plant’s basal defense, PAMP-triggered immunity (PTI) and also to
effector-triggered immunity (ETI) (Boccara et al. 2014). Similar to this,
miR400 of Arabidopsis targets peptatricopeptide repeat (PPR) genes, which in
turn is a contributor of defense responses. The negative regulation of PPR
genes by miR400 turned the host plant susceptible for both bacterium Pto
DC3000and the fungus Botrytis cinerea (Park et al. 2014). Moreover, miR825 and
miR825* of Arabidopsis mediate susceptibility towards Pto DC3000. miR825
targets two ubiquitin-protein ligases whereas miR825* regulates
toll-interleukin-like receptor (TIR)-nucleotide binding site (NBS) and
leucine-rich repeat (LRR) type resistance (R) genes, which are the major
contributors to the resistance response. However another plant growth promoting
rhizobacterium known as Bacillus cereus AR156 suppress the activity of miR825
and miR825*, and primes induced systemic resistance (ISR) (Niu et al. 2016).
Very recently, it has been reported that, there are wheat specific miRNAs
involved in susceptible interaction with Puccinia striiformis f. sp. tritici,
causing stripe rust of wheat. These miRNAs and their down regulated targets
were implicated to have a role in causing the susceptibility interaction between
the host plant and the pathogen (Feng et al. 2016). In another study, it has
reported that miR408 of wheat post-transcriptionally regulates a
chemocyanin-like protein (TaCLP1) gene, that in turn is essential for mediating
resistance response to stresses like salinity, high cupric content and stripe
rust. So the induction miR408 increases wheat susceptibility towards stripe
rust (Feng et al. 2013).  Another
embraced part of miRNAs is the conversion of host to susceptible for parasitic
infection. The miR827 from Arabidopsis mediates susceptibility towards the beet
cyst nematode Heterodera schachtii, which is a sedentary endoparasite. Here
miR827 targets nitrogen limitation adaptation (NLA) genes that encode ubiquitin
E3 ligase enzyme required to limit the activity of parasites. Inactivation of
miR827 reduces plant susceptibility and over expression of miR827 leads to
hyper susceptibility which strengthen the model (Hewezi et al. 2016).
Inactivation of miRNAs and thereby miRNA mediated post transcriptional
regulation is another strategy related to disease susceptibility. For example,
in the poplar plant susceptible to the infection of virulent Melampsora
larici-populina, the miRNAs which regulates defense signaling pathways got
inactivated in the stage of effector triggered susceptibility and that was
marked as the major reason for its susceptibility (Li et al. 2016). 

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