miRNAs based disease resistance.pptx Primary mi RNAs Precursor mi RNAs Biogensis Case studies Disease resistance

1. miRNA based disease resistance
Harish J
PAMB1087
Department of Plant Pathology
UNIVERSITY OF AGRICULTURAL SCIENCES,GKVK
BANGALORE

2. miRNA
• microRNA (abbreviated miRNA) is a small single-stranded non-coding
RNA molecule (containing about 22 nucleotides) found in plants,
animals and some viruses.
• Found in simple bryophytes to advance angiosperms which target
mRNA and suppress expression either by direct cleavage or
translational repression.
• The discovery of the first microRNA (miRNA), lin-4, in 1993 by the
Ambros and Ruvkun groups in Caenorhabditis elegans

3. • Most miRNAs are transcribed from DNA sequences into primary
miRNAs (pri-miRNAs) and processed into precursor miRNAs (pre-
miRNAs) and mature miRNAs.
• miRNAs interact with the 3′ UTR of target mRNAs to suppress
expression
• miRNAs have been shown to activate gene expression under certain
conditions

7. miRNA- mediated regulation is involved in various biological
processes viz.
* Growth and developmental regulation,
* Biomass yield,
* Grain quality regulation,
* Biotic and abiotic stress adaptations,
* Extracellular miRNAs have been widely reported as potential biomarkers for a variety
of diseases and they also serve as signaling,
* Aberrant expression of miRNAs is associated with many human diseases.
The technologies include gain/loss of expression in constitutive, tissue-specific manner
of miRNA or targets, artificial target mimic technology, artificial miRNA, RNA
interference etc.

8. Biogenesis of mature miRNA and technologies use to alter
miRNA target genes module expression
 Biosynthesis of miRNA from MIR gene
 RNA interference of Pri or Pre–miRNA
 TM- Target Mimic technology
 STTM- Short Tandem Target Mimic technology
 Spongy miRNA technology
 AMO- Anti-microRNA Oligonucleotides
 Modification of target gene by CRISPR/Cas9 technology
 Overexpression of cleavage resistance target genes
 Overexpression of miRNA gene.

9. Biogenesis of miRNAs
• miRNA biogenesis starts with the processing of RNA polymerase II/I
II transcripts post- or co-transcriptionally
• About half of all currently identified miRNAs are intragenic and
processed mostly from introns and relatively few exons of protein
coding genes
2 pathways of mi-RNAs Biogenesis
• The Canonical Pathway of miRNA Biogenesis
• Non-canonical miRNA Biogenesis Pathways

10. *This pathway is the dominant pathway
*Pri-miRNAs are transcribed from their genes and then processed into pre
miRNAs by the microprocessor complex, consisting of an RNA binding
protein DiGeorge Syndrome Critical Region 8 (DGCR8) and a ribonuclease
III enzyme, Drosha
*DGCR8 recognizes an N6-methyladenylated GGAC and other motifs within
the pri-miRNA.
*Drosha cleaves the pri-miRNA duplex at the base of the characteristic
hairpin structure of pri-miRNA
*Once pre-miRNAs are generated, they are exported to the cytoplasm by an
exportin 5 (XPO5)/RanGTP complex
*Then, processed by the RNase III endonuclease Dicer removes terminal
loop , resulting in a mature miRNA duplex

11. Non-canonical miRNA Biogenesis Pathways
• These pathways make use of different combinations of the proteins i
nvolved in the canonical pathway, mainly Drosha, Dicer, exportin 5,
and AGO2
• The non-canonical miRNA biogenesis can be grouped into Drosha/D
GCR8-independent and Dicer-independent pathways.
• Mirtrons and 7-methylguanosine (m7G)-capped pre-miRNA were Dic
er-dependent pathways
• Dicer-independent miRNAs are processed by Drosha from endogeno
us short hairpin RNA (shRNA) transcripts
• Dicer-independent miRNAs are processed by Drosha from endogeno
us short hairpin RNA (shRNA) transcripts

14. Mechanisms of miRNA-mediated gene regulation
• miRNAs bind to a specific sequence at the 3′ UTR of mRNAs induce
translational repression ,deadenylation and decapping
• In some conditions, mRNA regions including the 5′ UTR and coding
sequence were the target regions of miRNAs within promoter region
• The binding of miRNAs to 5′ UTR and coding regions have silencing
effects on gene expression
• while miRNA interaction with promoter region has been reported to
induce transcription

15. A. MicroRNA-Mediated Gene Silencing via miRISC
• miRNA-induced silencing complex (miRISC) consists of the guide
strand and AGO
• The target specificity of miRISC is due to its interaction with
complementary sequences on target mRNA, called miRNA response
elements (MREs).
• A fully complementary miRNA:MRE interaction induces AGO2
endonuclease activity and targets mRNA cleavage
• In animal cells, the majority of miRNA:MRE interactions are not fully
complementary
• Most MREs contain at least central mismatches to their guide
miRNA, preventing AGO2 endonuclease activity
• The formation of a silencing miRISC complex starts with the
recruitment of the GW182 family of proteins by miRISC

16. • Effector proteins , such as the poly(A)-deadenylase complexes
PAN2-PAN3 and CCR4-NOT
• Target mRNA poly(A)-deadenylation is initiated by PAN2/3 and
completed by the CCR4-NOT complex
• The interaction between the tryptophan (W)-repeats of GW182 and
poly(A)-binding protein C (PABPC) promotes efficient deadenylation
• decapping takes place facilitated by decapping protein 2 (DCP2) and
associated proteins (52), followed by 5′−3′ degradation by
exoribonuclease 1 (XRN1)

17. B. Translational inhibition
• MiRNAs inhibit translation at the initiation step
• (i) PABP displacement mediated by GW182
• (ii) recruitment of the translational repressors through GW182
• (iii) dissociation of eukaryotic initiation factor-4A (eIF4A) from the
cap-binding complex eIF4F

18. C. Gene activation by miRNAs
• MiRNAs also induce up-regulation of their targets.
• MiRNAs have been implicated in gene activation triggered by
promoter-targeted small RNAs, known as RNA activation (RNAa)

19. d. miRNA- mediated up regulation through 3’UTR
binding
• Up-regulation of certain transcripts can also be mediated by miRNA
binding to mRNA 3 un translated regions (3 UTRs). Resulting in
either translation activation or RNA stability enhancement.

20. Dynamics OF miRNA actions
• miRNA-mediated gene regulation is dynamic and helps to buffer
gene expression to a steady state.
• Factors that may contribute to the robustness of miRNA-mediated
gene regulation include the functionalized compartmentalization and
shuttling of miRISC within the cells.
Subcellular Compartmentalization of
miRNAs
• miRISC and target mRNA have been observed to localize in multiple
subcellular compartments including rough endoplasmic reticulum
(rER), processing (P)-bodies, stress granules (SG), the trans-Golgi
network (TGN), early/late endosomes , multivesicular bodies (MVB),
lysosomes, mitochondria and the nucleus.
• facilitate miRNA post-transcriptional gene regulation are enriched at
sites where miRISC:mRNA complexes localize.

22. Circulation of miRNAs
• miRNAs can be released into extracellular fluids
• Extracellular miRNAs can be used as biomarkers for a variety of
diseases
• Extracellular/circulating miRNAs in biological fluids, such as plasma
and serum, cerebrospinal fluid, saliva, breast milk, urine,tears,
colostrum, peritoneal fluid, bronchial lavage, seminal fluid and
ovarian follicular fluid
• found in vesicles such as exosomes, microvesicles, and apoptotic
bodies.

23. miRNA-mediated manoeuvring against biotic stresses
• Overexpressing target mimicry of miR396 to block its target Growth
Regulating Factor (GRF) exhibited enhanced resistance against
Magnaporthe oryzae in rice plants (Chandran et al., 2018; Liebsch and
Palatnik, 2020).
• STTM of miR482/2118 in tomato also showed resistance against bacteria
and oomycete (Canto-Pastor et al., 2019).
• In Brassica, miR1885 was induced during TuMV infection which further
down regulated its targets TIR-NBS-LRR class of disease resistance
proteins (He et al., 2008).

25.  identify a novel defence strategy of host plants by exporting specific
miRNAs to induce crosskingdom gene silencing in pathogenic fungi
and confer disease resistance.
Materials and methods
1. Fungal isolation
2. RNA extraction, RNA gel blotting, quantitative real-time PCR
3. Cloning and constructs

26. a, Cotton wilt disease symptoms caused by infection with V. dahliae strain
V592 and growth of hyphae recovered from infected cotton (Vda Cotton).
b, Cotton miRNAs were detected in three individual Vda Cotton colonies
using low molecular weight RNA gel blots.
c, miR166 and miR159 were induced in V592-infected cotton and
Arabidopsis roots (cotton/V592 and Col-0/V592). miR156 and miR167
were not induced after V592 infection.

27. 1. The Clp-1 and HiC-15 knockout mutants VdaΔclp-1 and VdaΔhic-15 exhibited reduced
virulence in cotton compared with wild-type V592 at 20-dpi
2. Disease grades for all pathogen infection assays at 15, 20 and 30 days post
inoculation (dpi)
3. V. dahliae VdaClp-1m and VdaHiC-15 carrying the miRNA-resistant Clp-1m or HiC-
15m displayed enhanced virulence in cotton.

28. Results
 Infection with Verticillium dahliae (a vascular fungal pathogen
responsible for devastating wilt diseases in many crops) cotton
plants increase production of microRNA 166 (miR166) and miR159.
 Found two V. dahliae genes encoding a Ca2+ dependent cysteine
protease (Clp-1) and an isotrichodermin C-15 hydroxylase (HiC-15),
and targeted by miR166 and miR159, respectively
 V. dahliae strains expressing either Clp-1 or HiC-15 rendered
resistant to the respective miRNA exhibited drastically enhanced
virulence in cotton plants

30. Conclusion
• miRNAs are powerful gene regulators, and that they not only help
control mRNA stability and translation but are also involved in
transcription.
• Our understanding of when and how miRNAs can exert regulatory
effects on transcription is limited
• investigations based on miRNA-mediated processes in plant-
pathogen interactions have considerable implications in devising
new strategies for disease control and ultimately improve crop
productivity.
• High-throughput sequencing has enabled the researchers to uncover
the role of plant miRNAs during pathogen invasion.
• miRNA can be very useful as biomarkers for disease resistance
characteristics during breeding programs.