given a detailed description about the miRNA generation and role in crop development along with some articles as an example

2. Topic: Role of miRNAs in gene regulation and Crop
improvement
PRESENTED BY
A. Govardhani
TAD/2020-028
PRESENTED TO
Dr. M. Reddi Sekhar,
Professor and Head
Department of Genetics and Plant
Breeding
COURSE NO : GP-691
COURSE TITLE : Doctoral Seminar I

3. Contents
Introduction
Discovery of miRNA
Biogenesis of miRNA
• miRNA transcription
• miRNA processing
• Argonaute loading and sorting
Mode of action/gene silencing mechanism
• PTGS
• Synthesis of secondary siRNAs
• DNA methylation
Role of miRNA in crop improvement

4. Introduction
• RNA- Ribonucleic acid
RNA is one of the four major macromolecules of life and is
essential in the regulation and expression of genes
RNA

5. miRNA
• MicroRNAs (miRNAs) are 20-24 nucleotide long,
endogenous, non-coding RNAs.
• miRNAs are partially complementary to at least one or
additional messenger RNA
• miRNAs key principle is to downregulate/repress gene
expression through sequence complementarity
• Represses gene expression post transcriptionally by binding to
the 3′ untranslated region of target mRNAs
• miRNAs were first identified from nematode- Caenorhabditis
elegans at Victor Ambros and Gary Ruvkun's Laboratories in
1993
Dong et al., 2022

6. Discovery of miRNA
• The first miRNA family, lin-4, was identified
through a genetic screen for defects in the
temporal control of post-embryonic development
• In C. elegans, larvae has 4 distinct larval stages
(L1-L4)
• Mutations in lin-4 disrupt the temporal regulation
of larval development, causing L1 specific cell-
division patterns to reiterate at later
developmental stages
He and Hannon, 2004

7. • lin-4 encodes a 22-nucleotide non-coding RNA that is partially complementary to 7
conserved sites located in the 3′-untranslated region (UTR) of the lin-14 gene.
• lin-14 encodes a nuclear protein, downregulation of which at the end of the first
larval stage initiates the developmental progression into the second larval stage
He and Hannon, 2004

8. Biogenesis of miRNA
 miRNA biogenesis can be categorized into 3
steps
 miRNA transcription
 miRNA Processing
 Argonaute loading and sorting
Zhang et al., 2022

9. miRNA gene transcription
Phosphorylates
CTD, Facilitates
miRNA
transcription, 5’
end capping, co-
transcriptional
RNA processing, 3’
end
polyadenylation
Occupancy of Pol II at
miRNA loci and pri-
miRNA transcription
Recruiting Pol II to
promoters of miRNA
Interact and
colocalize Pol
II to promoter
Acetylation
of H3K14
Regulates miRNA
transcription positively
and negatively by
recruiting different
transcription machinery
Facilitates
transcriptional
elongation
Repress
transcription of
MIR genes
Promotes
transcription of MIR
genes
• Pri-miRNA formed in the nucleus have typical stem loop structure, having base
pairs of several hundred to thousand, along with 5’ cap and a 3’- poly A tail.
• miRNA genes are transcribed by DNA-dependent RNA Polymerase II (Pol II) and
produces the primary transcripts of miRNAs (known as pri-miRNAs)
Zhang et al., 2022
positively regulates the
occupancy of Pol II at
MIR promoters

10. miRNA processing
• Pri-miRNAs undergoes two constitutive cuts to
produce pre-miRNAs and to release the
miRNA/miRNA∗ duplex. This process is called
processing.
• Processing occurs in two distinct subnuclear bodies,
namely, Dicing bodies (D-bodies)
• DCL1, an RNAse III endoribonuclease, is responsible
for the cleavage of pri-/pre-miRNAs
• HYL1 and SE are the two cofactors of DCL1, which
interact with each other in D-bodies to ensure the
accuracy and efficiency of processing
Zhang et al., 2022

11. Dicer
• Dicer family is a unique class of RNase III enzymes
• Dicer consists of ATPase/DExD/H-box helicase domain at the
N-terminus, DUF283 domain,a Piwi/Argonaute/Zwille (PAZ)
domain in the middle and dual RNase III domains followed by
one or two dsRNA-binding domains in the C-terminal half
He and Hannon, 2004

12. Helicase domain:
Helicase domain serves as a protein–protein interaction surface recruiting co-
factor regulatory proteins
It also utilizes ATP hydrolysis to achieve processive cleavage of the long
dsRNA substrate
PAZ domain:
PAZ domain contains a conserved pocket for recognizing the terminus of the
dsRNA substrate, and the distance between PAZ and the RNase III catalytic
center determines the product sizes
Rnase III domain:
Each of the two RNase III domains cuts one of the dsRNA strands, leaving a
characteristic 2-nt overhang at 3′-end of the product
dsRNA binding domain:
The C-terminal dsRNA-binding domains (dsRBDs) serve as a protein–protein
interaction interface and nuclear localization signals, in addition to having
dsRNA-binding function
Fukudome and Fukuhara, 2016

13. • Plant genome consists of four distinct types of Dicer (DCL 1-4)
• DCL1 is the only Dicer protein that produces most 21-nt miRNA
• DCL2-4 are involved in siRNA-production
• DCL4 is the major producer of trans-acting siRNA (tasiRNA) and
phased siRNAs (phasiRNA)
• DCL2 can compensate for the loss of DCL4 and is essential for
secondary siRNA mediated transitive silencing
• DCL3 mainly produces 24-nt repeat-associated siRNAs derived
from transposons and DNA repetitive elements and participates in
transcriptional gene silencing (TGS) through RNA-dependent DNA
methylation
• Monocots have another distinct class of Dicer, DCL5 (also known as
DCL3b)
• DCL5 is specifically expressed in developing panicles and is
responsible for 24-nt reproductive phasiRNAs
Fukudome and Fukuhara, 2016

14. HYL1(Hyponastic Leaves 1)
• HYL1, an important chaperone of DCL1
• Plays an critical role in miRNA processing accuracy and
efficiency
• HYL1 comprises of three representative domains
N-terminal : two double stranded RNA-binding domain
C-terminal : one protein-protein interaction domain
• HYL1 antagonizes nuclear exosome to protect pri-miRNAs
from degradation
• HYL1 locates both in cytoplasm and nucleus
• In dark conditions HYL1 suffers from degradation
• Phosphorylation of HYL1 plays an important role in regulating
the activity and stability of HYL1
Zhang et al., 2022

15. HYL1
KETCH1 – Transport of HYL1 from cytoplasm to
nucleus to take part in miRNA biogenesis
COP1 – Translocates into the cytoplasm from the nucleus
and protects the degradation of HYL1
snRK2 - phosphorylate HYL1 and waken the position of
HYL1 during miRNA processing
CPL1 - dephosphorylates HYL1, thereby leading to
accurate processing and strand selection
RCF3 - interacts with phosphatases CPL1 and CPL2,
mediates the dephosphorylation of HYL1
PP4/SMEK1 - dephosphorylate and stabilize HYL1
MAPK3
Regulatory Factors influencing the HYL1 activity
Zhang et al., 2022

16. SE (Serrate)
• SE plays a critical role in pri-miRNA processing
• Involved in miRNA biogenesis and regulating the
expression of mRNA
• SE promotes pre-mRNA splicing by interacting
with Cap Binding Complex (CBC)
• SE plays an role in promoting the expression of
intronless genes via promoting the association of
Pol II with direct chromatin binding
Zhang et al., 2022

17. SE
Regulator Factors influencing the SE activity
CBP 20, CBP 80 - are involved in mRNA and pri-
miRNAs splicing
CHR2 - interact with SE to remodel pri-miRNA’s
conformation and impair its processing
SEAP1 - positively promotes miRNA biogenesis by
modulating pri-miRNA splicing, processing, and/or
stability
HEN2, RBM7, ZCCHC8A/B - members of the Nuclear
Exosome Targeting (NEXT) complex. SE and NEXT
complex promote the degradation of pre-miRNA
MAC5- interacts with SE and protects pri-miRNAs from
nuclease degradation
RH6/8/12- interact with SE and promote the phase
separation and the formation of D-bodies
miR863-3P - transcription level of SE is regulated by a
feedback loop manner
Zhang et al., 2022

18. Other regulatory factors influencing miRNA
biogenesis
miRNA
biogenesis
TGH - positively regulates
miRNA and siRNA biogenesis
PRL1 - promotes pri-miRNAs
accumulation by stabilizing pri-
miRNAs and enhancing the DCL1
activity
DDL - stabilize pri-miRNAs
GRP7, STA1, ILR1, NTR1 -
interacts with pri-miRNA and
promotes pri-miRNA splicing
CDC5 – miRNA biogenesis
RACK1 - a partner protein
of SE, promotes miRNA
biogenesis
STV1 - recruitment of pri-
miRNAs to HYL1 to promote
miRNA biogenesis
THP1 - formation of D-bodies,
thereby promoting miRNA
processing
MTA - introduces N6-
methyladenosine (m6A) into pri-
miRNAs and promotes miRNA
biogenesis by interacting with Pol
II and TOUGH
FHA2 promotes HYL1
binding
RH27- associates with pri-
miRNAs and interacts with
miRNA-biogenesis Zhang et al., 2022

19. targets a number of MIR
genes, and it is
responsible for the
acetylation of H3K14 of
these loci
• SMA1- affects MIR
transcription but also plays an
important role in the correct
splicing of DCL1
• XCT, STA1 – Positive
regulators
• Biogenesis of miR162 is dependent on DCL1, but the
increase of miR162 suppresses the abundance of
DCL1 mRNA by target cleavage
• Generation of miR838 depends on the processing of
DCL1 pre-mRNA
Zhang et al., 2022
Factors involved in pri-miRNA processing

20. Co-factor proteins and structural determinants that fine-tune two-step processing of
primary miRNAs by DCL1 in plants. a. base to loop b. loop to base
Fukudome and Fukuhara, 2016
70-350nt

21. • The mature miRNAs have 2 bases protruding at the 3′ end
(miRNA double-stranded complex).
• This miRNA/miRNA* complex is methylated at the 2′-OH
position of its 3′ end under the action of HEN1 (HUA
ENHANCER 1 ) protein to prevent degradation from SMALL
RNA DEGRADING NUCLEASE (SDN) exonucleases
proteins
• HEN1-mediated methylation is considered as the core
mechanism of miRNA stability regulation.
• HEN1 protein is present in both the nucleus and cytoplasm
• HEN1 induces the event of methylation at dual sites
• The miRNA/miRNA∗ duplexes are thought to be translocated
from the nucleus to the cytoplasm by HASTY (HST), a
homologous gene of animal Exportin 5 (EXPO5)
• miRNA turns out to be uridylated by uridyl-transferase for the
direct degradation process
Dong et al., 2022; Choudhary et al., 2021

22. Argonaute loading and sorting
• The miRNA duplex gets disconnect in the cytoplasm and a
binding protein, known as ARGONAUTE (AGO) assists the
loading of the guide strand into the RISC apparatus
• The basis for selecting the 5p or 3p strand is based on
thermodynamic stabilities at 5ʹ ends of the miRNA duplex or
5ʹ U at 1ST nucleotide
• The strand with relatively low 5ʹ stability or 5ʹ uracil is loaded
into AGO selectively and is considered to be the guide strand
• The unloaded strand is referred to as the passage strand
• AGO1 protein binds to a good number of plant miRNAs that
carry 5′ Uridine.
• The passenger strand (miRNA∗) is removed and degraded
• This process is called Argonaute loading and sorting
Zhang et al., 2022; Choudhary et al., 2021

23. Argonaute
• 10 different types of AGO proteins have been found in
Arabidopsis thaliana
• They can be grouped into three clades: AGO1/5/10,
AGO2/3/7 and AGO4/6/8/9
• AGO2, AGO4, AGO7 and AGO10 have been demonstrated
in the gene silencing pathway of target RNAs
• AGO1 protein is involved in PTGS as the main component
of RISC that binds to a short guide RNA (miRNA or
siRNA)
• AGO4 and AGO6 : involved in the repeat-associated siRNA
pathway
• AGO7 plays a role in the formation of ta-siRNA
• AGO protein not only serves as an effector, but also plays a
critical role in maintaining the stability of miRNA
Dong et al., 2022; Zhang et al., 2022

24. Factors in regulating miRNA stability control
Zhang et al., 2022

25. RISC
• ARGONAUTE (AGO) assists the loading of the guide
strand into the RISC apparatus
• RISC can be assembled in the nucleus and exported
to the cytosol by EXPO1.
• It contains four domains: the N-terminal domain (N),
PIWI/ Argonaute/Zwille (PAZ) domain, MID domain,
and the P-element-induced wimpy tested (PIWI)
domain.
• The PAZ domain can bind to RNA and the PIWI
domain has RNase H activity.
• MID and PAZ domains bind to the 5′ phosphate and 3′
end of small RNAs
• The PIWI domain cuts target RNAs through its
endonuclease activity
• The assembly of RISC requires the molecular
chaperone HSP90, and this process is facilitated by
Cyclophilin 40/Squint (CYP40/SQN) and inhibited by
Protein Phosphatase 5 (PP5) Dong et al., 2022; Wang et al., 2019

26. Mode of action
Plant miRNAs exploit two leading
mechanisms to regulate the gene expression of
targets
PTGS
Transcript cleavage
Translation repression
Synthesis of secondary siRNA
DNA methylation
Zhang et al., 2022

27. Zhang et al., 2022

28. Transcript/Target cleavage
• Involves the cleavage of target mRNA
at the accurate position under the
miRNA guidance at 5′ monophosphate
• AGO proteins having PIWI domain
help to smoothen the process of
cleavage forming an RNaseH-like fold
• The fold has endonuclease activity
• After cleavage, 5′ and 3′ ends are
available for exonucleases activity for
degradation
• In Arabidopsis, 3′ end undergoes
degradation by EXORIBONUCLEASE
4 (XRN4)
Choudhary et al., 2021

29. Translation repression
• miRNAs directed translation inhibition is less chronic probably because of
the universal presence of miRNAs intended for cleavage
• Diverse factors rope in to facilitate the process are KATANIN1
(microtubule severing enzyme), GW repeat protein SUO, processing body
component VARICOSE, and endoplasmic reticulum membrane protein
ALTERED MERISTEMPROGRAM1 (AMP1)
• The mechanism of translational repression in plants is still unknown
Rogers et al., 2013; Choudhary et al., 2021

30. Synthesis of secondary siRNAs
• miRNAs involves in the initiation
of phase secondary siRNAs
(phasi-RNAs)
• AGO prefers cleavage to generate
5′ or 3′ fragment become stable
by binding of SUPPRESSOR OF
GENE SILENCING 3 (SGS3).
• The recruitment of RNA
DEPENDENT RNA
POLYMERASE 6 (RDR6) allows
the cleavage fragment to convert
into dsRNA followed by dicing
into phasi-RNAs at a 21
nucleotide gap
Choudhary et al., 2021

31. DNA methylation
• miRNAs both in plants and in animals
are capable of transcriptional gene
silencing
• In rice, DCL3-dependent long miRNAs
of 24 nucleotides are sorted to AGO4 and
trigger cytosine DNA methylation at both
MIR and target loci
• DCL3-dependent small RNAs,
heterochromatic small interfering RNA
are generated in the nucleus and loaded
into AGO4 in the cytoplasm prior to
import into the nucleus where they act in
RNA-directed DNA methylation
• The partial redundancy of AGO6 and
AGO4 in RNA-directed DNA
methylation suggests a functional
specialization of the
AGO4/AGO6/AGO9 clade.
Rogers et al., 2013

32. Role of miRNA in Crop improvement
miRNA
Shoot
meristem
Leaf
Vascular
and Root
Inflorescence
meristem
Biomass
yield
Grain yield
Shelf life of
fruit
Flowerin
g pattern
Sex
determinati
on
Parthenocar
py

33. Shoot meristem
• The STM (shoot meristemless)-WUS
(Wuschel)-CLV (Clavata) pathway plays a
key role in the maintenance of meristem
activity
• miR394 is generated in the L1 layer on the
surface of the SAM and diffuses down to
the Organizing Center
• In the OC, expression of Leaf Curling
Responsiveness (LCR) is inhibited
• LCR further regulates
CLAVATAWUSCHEL (CLV-WUS)
negative feedback loop for proper SAM
development and specification
• ARGONAUTE10 (AGO10) specifically
sequesters miR166/165 to upregulate Class
III homeodomain leucine zipper
transcription factors (HD-ZIP III TFs) to
maintain SAM development
Dong et al., 2022

34. TAS3 ta-siRNA
determines the
adaxial side by
inhibiting the
expression of ARF3
and ARF4 on the
abaxial side of
leaves
Leaf
polarization
Leaf number and
regulates the size
of meristem
Leaf senescence
Involved in evolution of
composite leaves
Dong et al., 2022
Model for the role of miRNAs in shoot apex
Leaf Development
synergisticall
y regulate
trichome
initiation

35. Role of miRNAs in Vascular and Root Development
• The vascular bundle consists of three neatly arranged
tissues: xylem, procambium/cambium and phloem
• HD-ZIP III gene family is strongly expressed in
vascular bundles of roots, stems and leaves
• Overexpression of miR165 reduce the transcription
level of all members of the HD-ZIP III family, thus
regulating the polar differentiation of vascular tissue
cells and affecting plant morphogenesis
• miR166 controls the development of vascular cells and
phloem cells by regulating the Homeobox 15 protein
Dong et al., 2022

36. Dong et al., 2022
Role of miRNAs in the development of vascular and root

37. Function of miRNA in inflorescence meristem
• As plants grows from juveniles to
adults, downregulation of miR156
dampens the inhibition of SPL
expression, which in turn promotes
miR172 transcription. miR172
triggers the development of
inflorescence meristem by reducing
the mRNA level of AP2-like genes.
• Spatiotemporal functions of
miR165/166 and their targets HD-ZIP
III genes, together with miR164,
restrict the functions of CUCs in
specific regions of the boundary to
maintain the inflorescence meristem.
Dong et al., 2022

38. • overexpression of miR156 in
Arabidopsis increased plant branching
and further helped in increase of
biomass by more than 300%
Biomass yield
improvement
• Os-miR156 target gene OsSPL-14 is
associated with yield. Overexpression of
OsSPL14 is known to promote panicle
branching, decrease tiller number and
increase grain yield
Grain yield
improvement
• miR156 targets CNR(color-less never
ripe) gene associated with the tomato
fruit development and ripening
Shelf life of
fruit
Mandal et al., 2021

39. • miR164 is involved in the boundary
formation between organs or in
between whorls, miR172 is involved in
the inner whorl organ differentiation
Flowering
pattern
• Feminized tassels of maize mop1 and
ts1 (tasselseed1) exhibits low level of
miR156 which indicate the link
between miR156 and sex
determination genes
Sex
determination
• Aberrant expression miR167 targets
auxin response factor 8 (ARF8) that
produce parthenocarpic fruit in
Arabidopsis and tomato
Parthenocarpy
Mandal et al., 2021

40. Role of miRNAs in abiotic stress
• Plant miRNAs target the transcriptional factors that brilliantly
furnish the various developmental aspects even under stress
conditions.
Choudhary et al., 2021

41. Role of miRNAs in biotic stress
• miRNA168 elevation has taken place by
infection of Soybean mosaic virus G7
• Tomato leaf curl virus-infected plant with
accumulated miR156 did not reveal any sign
of disease in tomato resistant cultivars
• The expression of miR2911 and miR1030 is
induced after the Xanthomonas axonopodis on
Populas species
Choudhary et al., 2021

42. CASE STUDY
• Rice blast is threatening the global food security which causes a yield loss
of 10-30%, and under favourable conditions for Magnaporthe oryzae, the
losses could be up to 80% of potential yield
• Pi54 gene confers resistance to many predominant strains of blast pathogen
from across India
Arora et al., 2021

43. Materials
• japonica rice line Taipei 309 (susceptible) and its
near monogenic blast resistant rice line having
Pi54; TP-Pi54-2 (Taipei309Pi54) in T6 generation
were grown and maintained in net house
• Tetep and HR12 lines were used as resistant and
susceptible control
• Magnaporthe oryzae strain PB-1 was subcultured
on potato dextrose agar medium
• Spore suspension was inoculated on 21 days old
seedlings
Arora et al., 2021

44. Methodology for small RNA library preparation
Total RNA was isolated
RNA integrity number was also assessed on Bioanalyzer RNA 6000 Pico chip
Enrichment of siRNA from the total RNA -Ambion flash page
Deep sequencing was performed via Applied Biosystems SOLiD
Four small RNA libraries were prepared using Total RNA seq kit
.
Arora et al., 2021

45. Data processing and Bioinformatics analysis
Raw reads were generated from all the four rice libraries
Reads mapped to tRNAs, rRNAs were filtered out
Reads mapped against precursor miRNAs- miRBase sequence database release 16
FPKM analysis was performed for differential expression profiling of mature miRNAs
FPKM values were used to calculate the fold change as ln (up>2, down <2)
Remaining reads were mapped to rice genome using miRanalyzer (minimum read count > 10),
filtered using Xcelris proprietary script and miRcheck script
Novel miRNAs predicted
All the miRNAs available were BLAST searched against miRBase
Arora et al., 2021

46. Target prediction
Target prediction for selected rice miRNAs was done using psRNA Target
miRNA target sequences were searched with the O. sativa cDNA set available
at The Institute for Genomic Research
Predicted target genes were evaluated by scoring
Those sequences with a total score of less than 3.0 points were considered as
miRNA targets
Arora et al., 2021

47. Resistant and Susceptible rice plants along with their disease phenotypes after 7 dpi. a
Taipei309Pi54 rice plant (resistant). b Taipei309- rice plant (susceptible). c Disease reaction on
HR12 rice plant (positive control). d Disease reaction on Taipei309- rice plant. e Disease reaction
on Taipei309Pi54 rice plan
Arora et al., 2021

48. Venn diagram showing overall differential
regulation in Taipei309Pi54 (resistant) and
Taipei309- (susceptible) rice lines
miRNAs are unique to
Taipei309Pi54 UR
miRNAs are
unique to
Taipei309Pi54
DR
miRNAs are
unique to
Taipei309-
UR
Taipei309-
DR
miRNAs are
common between
Taipei309Pi54 DR
& Taipei309- UR
miRNAs are
common to
Taipei309Pi
54 UR &
Taipei309-
DR
miRNAs are common to
Taipei309Pi54 UR &
Taipei309- UR
central cluster, no miRNA is
common to all the 4 categories
Arora et al., 2021
Arora et al., 2021

49. Heat Map showing overall differential regulation of miRNAs in rice line. a Up-regulated miRNAs
in resistant line. b Down-regulated miRNAs in resistant line. c Common miRNAs in resistant and
susceptible line
Arora et al., 2021

50. Expression profiling of various blast responsive positive and negative regulators with respect to
their profiling in resistant as well as susceptible rice lines. a) Positive regulators of rice blast,
b) Negative regulators of blast resistance, c) Positive and negative regulators present exclusively in
resistant rice line, d) Expression profiling of osa-miR531b in resistance and susceptible rice lines

51. Analysis of Target genes. Functional annotation & Target Ratio of predicted targets of differentially expressing
microRNAs in Resistant rice line (Taipei309Pi54). a Upregulated microRNA targets b Down regulated microRNA
targets. c Targeting ratio analysis of resistant and susceptible rice lines (Abbreviations: SBP Squamosa promoter-
binding domain, ZnFP- Zinc finger proteins, ATPBP- ATP binding protein, DRP- Disease resistance proteins like
RPP13, F-box- F-box domain containing protein, HD-Zip- Class III HD-Zip protein 4, NTF- Nuclear transcription
factor Y, RK- Receptor kinases, STPK’s- Serine/threonine-protein kinase, UCE- Ubiquitin conjugating enzyme
In total 142 miRNAs in resistant line showed a
total of 2424 targets giving targeting ratio of 17.07
and 140 miRNAs in susceptible line showed 2099
targets with a targeting ratio of 14.99
Arora et al., 2021

52. • Target genes with E- value ranging from 2.5 to 3.0 and genes responsible for disease
resistance were selected for validation by semi qRT-PCR and qRT-PCR.
• Majority of the target genes for up-regulated miRNAs showed down-regulation,
demonstrating their inverse relation with miRNAs in terms of expressions
• Highest down-regulation was observed in NBSDS target gene with log2 fold change of -1.59
• The OsWAK129b gene (LOC_Os12g42070) targeted by miR815c showed less down-
regulation in resistant rice line - 0.08 log2 fold) as compared to susceptible rice line (- 0.86
log2 fold)
Real time expressions of target genes in transgenic lines. a Targets of Upregulated
miRNAs, b Targets of Downregulated miRNAs
Arora et al., 2021

53. Conclusion
• miRNA candidates identified are miR159c, miR167c,
miR2100, miR21180, miR2118l, miR319a, miR393, miR395l,
miR397a, miR397b, miR398, miR439g, miR531b, miR812f,
and miR815c, and they manifest their role in balancing the
interplay between various DR genes during Pi54 mediated
resistance
Arora et al., 2021

54. Nutrient uptake
• The obligatory activity of the plant is the uptake of nutrients
and their homeostasis
• It is crucial for the normal growth and development of plant
• Sulphur is one of the vital element in the plant life cycle,
translocated as sulphate and primarily required for the
structural integrity
• miR395 targets ATP sulphurilases & its transporter
• miR399 is responsible for the homeostasis of phosphate.
Under the deficiency condition, miR399 is activated and
targets the PHOSPHATE 2 GENE (PHO2) due to Pi
responsive gene
• miR398, miR408 and miR857 are the intrinsic components of
signaling network operate in the maintenance of copper in
cells
Choudhary et al., 2021

55. Nutrient status and functional responses of miRNAs in
various plants
Choudhary et al., 2021

56. CASE STUDY
• Nitrogen (N) and phosphorous (P) are the most limiting factors
reducing wheat production
• 30-40% of these applied fertilizers are utilized by crop plants,
remaining is lost through volatilization, leaching and surface
run off
• It increases the cost of the farmers along with harming the
water bodies through eutrophication.
Sagwal et al., 2022

57. Materials
• Chosen 10 genotypes based on published literature on
N/PUE
• Experiment was carried out in earthen pots following a
completely randomized block design (CRD) in triplicates
• Nitrogen was applied in the form of CaNO3.4H2O in two
doses - 0.18 gm (low) and 0.37 gm (optimum)
• Phosphorous was given in the form of KH2PO4 as 0.07 gm
(low) and 0.15 gm (optimum)
• Macro and Micro nutrients are supplied in the form of
Hoagland’s solution
• N and P was done by Kjeldahl’s and Olsen’s methods
• Agronomic data like grain yield per plant, harvest index,
biomass per plant were calculated
Sagwal et al., 2022

58. Methodology
Sagwal et al., 2022

59. Frequency and distribution of nitrogen and phosphorous
responsive miRNA and gene-specific SSRs
In various crops In subgenome of wheat
In wheat chromosomes On the basis of their role in different cellular activities
Sagwal et al., 2022

61. miR171a marker screened over 10 wheat genotypes.
Green and red arrow represent genotypes with low and
high NUE respectively
miR167a marker screened over 10 wheat genotypes. Green and red arrow
represent genotypes with low and high PUE respectively Sagwal et al., 2022

62. Dendrogram showing
clustering of 10 wheat
genotypes on the basis of
polymorphic N
responsive miRNA and
genic SSRs. Red and
blue color indicates the
N deficient and efficient
wheat genotypes.
Dendrogram showing
clustering of 10 wheat
genotypes on the
basis of polymorphic
P responsive miRNA
and genic SSRs. Red
and blue color
indicates the P
deficient and efficient
wheat genotypes.
Sagwal et al., 2022

63. 2-D plot of principal
component analysis (PCA)
for 10 wheat genotypes in
response to N. Red and blue
color indicates deficient and
efficient N wheat genotypes
2-D plot of principal
component analysis
(PCA) for 10 wheat
genotypes in response to
P. Red and blue color
indicates deficient and
efficient P wheat
genotypes
Sagwal et al., 2022

64. Sagwal et al., 2022
Allelic data of N responsive SSRs have a total of 138 alleles in two
populations. Among them, 78 alleles were found in population 1 and
60 alleles were found in population 2. Number of polymorphic loci
in population 1 (93.75%), was observed to be higher than in
population 2 (84.38%). Allelic data obtained from P responsive
SSRs identified a total of 112 alleles in both populations. Among
them, 58 alleles were found in population 1 and 54 were identified
from population 2. Population 1 was found to be more polymorphic
than population 2. Number of polymorphic loci for population 1 was
observed to be 85.19%, whereas for population 2 it was 81.48%

65. WEGO output for GO of the N and P responsive miRNAs targeted genes in wheat. The
x-axis of plot showed three GO categories, whereas left and right side of y-axis
represent the percentage of target genes and number of miRNA target genes
Sagwal et al., 2022

66. Expression profiling of P responsive miRNAs targeted genes in wheat and their
hierarchical clustering on the basis of their expression in anatomical stages
• Expression potential percentage was higher in leaf followed by flag leaf and shoot.
• Ser/thr protein kinase and purple acid phosphatase were found to be highly expressed
followed by phosphate transport protein,
Sagwal et al., 2022

67. Expression profiling of P responsive miRNAs targeted genes in wheat and their
hierarchical clustering on the basis of their expression in developmental stages
Target gene glutamate receptor and phosphate transport protein were upregulated at
inflorescence emergence, whereas SOD up-regulated at milk development stage
Sagwal et al., 2022

68. Expression profiling of P responsive miRNAs targeted genes in wheat and their
hierarchical clustering on the basis of their expression under different P starvation
conditions and abiotic stresses
Under P starvation conditions, NAC transcription factor, zinc finger protein, and
glycosyltransferase were found highly expressed
The expression profiling of these genes under low P implies their role in senescence
and nutrient remobilization Sagwal et al., 2022

69. Conclusion:
• Existed a positive correlation between the phenotype and genotype data
• miR171a can discriminate between N efficient and deficient wheat genotypes
• miR167a can distinguish the P efficient and deficient wheat genotypes
Sagwal et al., 2022

70. Role in phenylpropanoid biosynthesis
• Several miRNAs have been identifed, such as
miR1438, miR5532, miR172i, miR1873, and
miR829.1 targeting phenylpropanoid
biosynthesis genes in Podophyllum hexandrum
• In Arabidopsis, miR156 regulates anthocyanin
accumulation by targeting the SPL9 gene
Jeena et al., 2022

71. Artificial miRNA (amiRNA) strategy
• The amiRNA method produces miRNAs that silence target
genes while not interfering with other genes' function
• The gene sequence can be used to construct mature
amiRNA with the original preconserved miRNA's stem‐loop
structure and complementary sequence to target mRNA
• The amiRNA:miRNA duplex can be inserted directly into
the stem‐loop structure of the transgenic plant to target
specific mRNA
• The Arabidopsis miR156 gene can be transferred to
eggplants with unknown genetic information to study the
function of this miRNA
Rani et al., 2022

72. CASE STUDY
• Tomatos appear to be an ideal haven for many viruses, and so far
more than 10 major viruses have been found
• Aphids are sap-sucking insects of the order Hemiptera
• Aphids are capable of efficiently transmitting over 100 types of
plant viruses
• Aphids transmit many single-stranded plant RNA viruses
Faisal et al., 2021

73. • Acetylcholinesterase is an enzyme in the insect’s
central nervous system that catalyzes the breakdown
of neurotransmitter acetylcholine into its acetyl-CoA
and acetate components
• Dysfunction in acetylcholine will result in muscle
atrophy, will cause the paralysis and even the death of
the insect
• Acetylcholinesterase is encoded by two
acetylcholinesterase (Ace 1 and Ace 2)
• Ace 1 would possibly encode a key catalytic enzyme,
Faisal et al., 2021

74. Materials
• Plant material : Jamila and Tomaland
• agrobacterium strain LB4404 was transfected with the
amiRNA vector by electroporation
• Vector- pFGC5941
Faisal et al., 2021

75. Methodology
Jamila and Tomaland seeds were sterilized with Naocl
Transferred to Ms media
Precultured explants were cocultivated with Agrobacterium LB4404 containing
amiRNA for 20 min
Transferred to MS media
After 48hrs, agro-cocultivated CL explants were transferred to antibiotic selective MS
media
After 14 days, the CL explants were transferred to a fresh media devoid of antibiotics
for further shoot regeneration and multiplication for six weeks
The regenerated shoots were rooted in 0.5 µM of indole-3-butyric acid supplied half-
MS medium and were successfully developed ex vitro
Faisal et al., 2021

76. PCR and northern blotting were used to analyze the putative transgenic plants
(T0)
T1 transgenic plants carrying the amiRNA vector were separately grown in
plastic pots
Five adult aphids were placed in clip cages (diameter 20 mm, height 15 mm
and length 70 mm) on T1 transgenic, empty vector nontransgenic and control
plants
All the adults were removed after seven days, and only five two-day-old
nymphs remained
qRT-PCR Analyses of Target Ace 1 Gene was done in Aphids
population of aphids was counted in each clip cage after 14 days, and their
numbers were recorded as the fecundity of five nymphs
Faisal et al., 2021

77. Faisal et al., 2021

78. • Transformed seedlings were screened through PCR
amplification and Northern blotting at T0 generation
Northern blot analysis of
amiRNA in tomato transgenic
plants. Lane 1 = Control plant;
Lane 2 = Plant with empty
vector, Lane 3 = Transgenic
plant.
Faisal et al., 2021

79. Aphid performance assay on T1
transgenic tomato plants. Inset is the
larger image of the aphids’ colony in a
clip cage
Faisal et al., 2021
Results -significant reduction in the aphid population.
Jamila genotypes expressed best results compared to Tomalnad
Effect of gene silencing using amiRNA on
aphid populations after 14 days of feeding on
transgenic tomato plants

80. miRNA prediction tools
Tool Method Prediction URL
MirScan simple comparative methods Similarity in
candidate gene
http://genes.mit.edu/mi
rscan/
MiRAlign comparative method, similarity
score is estimated for every
candidate gene using structural and
sequential features
prediction of miRNA
genes
http://bioinfo.au.tsingh
ua.edu.cn/miralign/
MIRcheck similarity score is based on six
features including structural,
sequential and conservative features
miRNA genes in plants http://web.wi.mit.edu/b
artel/pub/software.ht m
miPred machine learning classification
algorithm based on sequential,
structural and thermodynamical
features
classifies effectively
miRNA genes and
pseudo hairpins.
http://www.bioinf.seu.e
du.cn/miRNA/

81. Conclusion
• miRNAs are acutely conserved non translated RNAs and
are playing significant operating roles in the regulation of
developmental factors for cellular interaction, proliferation
and coordination by carrying out the promising events.
• The understanding of miRNAs creates realization for
independency at different levels
• miRNAs participate in broad range of biological alteration
either creating positive or negative feedback loop and
promote up and down regulation of genes
• Still, the current knowledge is insufficient to explore the
potential impact of miRNA. It is required to elucidate the
rooting of miRNAs and its unpredictable reach extending to
the major aspects in development

82. References
Arora, K., Rai, A.K., Devanna, B.N., Dubey, H., Narula, A and Sharma, T.R.
2021. Deciphering the role of microRNAs during Pi54 gene
mediated Magnaporthe oryzae resistance response in
rice. Physiology and Molecular Biology of Plants. 27(3): 633-647.
Choudhary, A., Kumar, A., Kaur, H and Kaur, N. 2021. MiRNA: the
taskmaster of plant world. Biologia. 76(5): 1551-1567.
Dong, Q., Hu, B and Zhang, C. 2022. microRNAs and Their Roles in Plant
Development. Frontiers in Plant Science. 13: 824240.
Faisal, M., Abdel-Salam, E.M., Alatar, A.A. 2021. Artificial microRNA-
Based RNA Interference and Specific Gene Silencing for
Developing Insect Resistance in Solanum lycopersicum.
Agronomy. 11: 136.
Fukudome, A and Fukuhara, T. 2017. Plant dicer-like proteins: double-
stranded RNA-cleaving enzymes for small RNA
biogenesis. Journal of Plant Research. 130(1): 33-44.
He, L and Hannon, G.J. 2004. MicroRNAs: small RNAs with a big role in
gene regulation. Nature reviews genetics. 5(7): 522-531.

83. Jeena, G.S., Singh, N., Shikha., Shukla, R.K. 2022. An insight into microRNA
biogenesis and its regulatory role in plant secondary metabolism.
Plant cell Reports. 1-21.
Mandal, K., Boro, P and Chattopadhyay, S. 2021. Micro-RNA based gene
regulation: A potential way for crop improvements. Plant Gene. 27:
100312.
Rani, V and Sengar, R.S. 2022. Biogenesis and mechanisms of
microRNA‐mediated gene regulation. Biotechnology and
Bioengineering. 119(3): 685-692.
Rogers, K and Chen, X. 2013. Biogenesis, turnover, and mode of action of
plant microRNAs. The Plant Cell. 25(7): 2383-2399.
Sagwal, V., Sihag, P., Singh, Y., Mehla, S., Kapoor, P., Balyan, P., Kumar, A.,
Mir, R.R., Dhankher, O.P and Kumar, U. 2022. Development and
characterization of nitrogen and phosphorus use efficiency responsive
genic and miRNA derived SSR markers in wheat. Heredity. 1-11.
Wang, J., Mei, J and Ren, G. 2019. Plant microRNAs: Biogenesis,
Homeostasis, and Degradation. Frontiers in Plant Science. 10: 360.
Zhang, L., Xiang, Y., Chen, S., Shi, M., Jiang, X., He, Z and Gao, S. 2022.
Mechanisms of MicroRNA Biogenesis and Stability Control in Plants.
Frontiers in Plant Science. 13: 844149.