This is a Ph.D. Thesis research presentation of Mr. Bhor Sachin Ashok (M.Sc.Biotechnology) for the fulfillment of the requirement of the Ph.D. degree by The United Graduate School of Agricultural Sciences, Ehime University, Matsuyama, Japan. This research work was supervised by Prof. Kappei Kobayashi (Assoc. Prof., Ehime Univ., Japan).

1. TheUnitedGraduateSchoolof AgriculturalSciences,
EhimeUniversity
Establishment of novel systems to explore the
molecular mechanisms underlying chlorosis induced
by virus and viroid pathogens
(ウイルスおよびウイロイドによって誘導される退緑症状の分
子機構を探る新規実験系の確立)
By:
Bhor Sachin Ashok
(Reg. No. 4741008C)
Supervisor:
Prof. Kappei KOBAYASHI,
Ehime University, Matsuyama, Japan

2. Background information
 Chlorosis – The most common feature that occurs during
development of plant viral diseases
 Mechanism underlying the virally induced chlorosis is still
poorly understood
 Understanding the mechanism underlying virus-induced
chlorosis would provide a new insight into crop protection.

3.  To address the mechanisms for chlorosis-
 Transgenic tobacco plants has previously generated
 It expresses Cauliflower mosaic virus (CaMV) Tav gene under
the control of chemical-inducible GVG promoter.
Cauliflower mosaic virus (CaMV)
 CaMV genomic DNA codes for at least
six functional proteins:
Mov, Atf, Vap, Gag, Pol and Tav
 Gag, Pol and Tav proteins are essential
for basic replication
(Kobayashi and Hohn, 2003)

4.  Transgenic tobacco plant constitutively expressing CaMV-Tav
gene produces chlorosis or symptom like phenotypes
(Baughman et al., 1987)
 Expression of defense-related genes in Tav transgenic tobacco
plants (Takahashi et al., 1989)
Historical landmarks in the development of Tav-
transgenic plants
Baughman et al., 1987 Takahashi et al., 1989

5. Dexamethasone (Dex) inducible GVG promoter
Tav coding sequence was introduced
downstream the dexamethasone (Dex)-
inducible GVG promoter
GVG fusion
transcription factor
Tav
Dex
Dex
＋

6. Dex inducible Tav expression system
iTav tobacco Chlorosis
Dex
Control Dex+
(Waliullah et al., 2014, 2015)

7. Hypersensitive response (HR)
Pathogen attack
R proteins
NDR1
NPR1
EDS1
Rar1
SGT1
Hsp90
NRG1
ADR1
Immune
chaperons
/ co-
chaperons
SA and JA
pathway
related genes
Role of plant immune system in disease resistance
mechanism

8. Peach latent mosaic viroid targets the chloroplast
Hsp90 through RNA silencing to produce albino
symptoms
(Navarro et al., 2012)

9. CMV-Y satellite RNA induces yellow symptoms in
tobacco through silencing of CHLI gene
(Shimura et al., 2011 and Smith et al., 2011)
 These reports suggest the involvement of different mechanisms in the
viral induction of chlorosis, but the detailed mechanisms underlying the
chlorosis development through these mechanisms remain to be studied.

10. 1) Establishment of virus induced gene silencing
(VIGS) for analysis of immunity responsive genes
in Tav induced chlorosis
2) Establishment of system for artificial induction of
chlorosis symptom through the silencing of
chloroplast genes in transgenic tobacco-
i) Chloroplast Hsp90(Hsp90C), and
ii) Chlorophyll biosynthetic gene (CHLI)
Objectives of the study

11. Objective- 1
Establishment of virus induced gene silencing
(VIGS) for analysis of immunity responsive genes
in Tav induced chlorosis

12. R proteins
NDR1
NPR1
EDS1
Rar1
SGT1
Hsp90
NRG1
ADR1
Immune
chaperons
/ co-chaperons
SA and JA
pathway
related genes
Analysis of role of plant immune system in virally
induced chlorosis
iTav-tobacco
Dex treatment
Chlorosis ???

13. Construction and confirmation of recombinant
ALSV-vectors
a)
Fig. 1. a) To construct VIGS vectors DNA fragments of immunity responsive genes
were introduced into XhoI and BamHI sites of pBICAL2 binary vector. b)
Confirmation of recombinant ALSV vectors by BglII digestion.
RB
P35S HEL Tnos
LB
pBICAL1 PRO-co C-PRO POL
XhoI-SmaI-BamHI
RB
42KP
P35S Vp25 Vp20 Vp24 Tnos
LB
VIGS target gene(s)
pBICAL2
b)

14. Standardization of methodology for silencing of
various immunity related genes
Agro- inoculation
(ALSV-immunity
responsive gene)
Silencing of
endogenous gene
N. benthamiana
Crude sap extraction
Virus concentration
Silenced plant
Mechanical sap
inoculation to iTav
plant
Photo bleaching symptoms in PDS
silenced plant
No symptoms in other constructs
Virus inoculum
3 6-7 8
iTav tobacco
(Weeks)
Fig. 2. Schematic representation of standardization of agro-inoculation methodology
for VIGS in N. benthamiana and iTav-tobacco.

16. Outline of an experiment
Fig. 6. Schematic representation of VIGS for silencing of immunity responsive genes in iTav
tobacco plants and Dex-treatment for inducible chlorosis in transgenic tobacco plants.

17. 50 µM
Dex
Mock 50 µM
Dex
Mock
50 µM
Dex
Mock
SR1 iTav-3 iTav-12
a) Spraying 50 µM
Dex solution
Dex
treatment
b) Infiltration of
different
concentrations of
Dex solution
Fig. 7. Standardization of Dex treatment to 7 week old iTav 3 and 12 line plants by a)
Spraying with 50 µM Dex solution daily for a week, and b) infiltration of different
concentrations (0, 1, 5, 20, 50 µM Dex solutions in the leaf. Photographs takes 1 week
after Dex treatment.
Older iTav tobacco plants does not responds to
Dex treatment

18. Objective- 2
Establishment of system for artificial induction of
chlorosis symptom through the silencing of chloroplast
genes in transgenic tobacco-
I. Chloroplast Hsp90 (Hsp90C), and
II. Chlorophyll biosynthetic gene (CHLI)

19. (Shimura et al., 2011 and Smith et al., 2011)
(Navarro et al., 2012)
Hsp90C mRNA
Peach latent mosaic viroid
(PLMVd)
CHLI mRNA
Symptom development through the anti-viral
siRNA-mediated silencing of chloroplast protein
genes
Hsp90C
CHLI

20.  The full length sequence of NtHsp90C was obtained by RACE,
cloning and sequencing
Hsp90 signature
ATP binding site
1. Chloroplast Hsp90 (Hsp90C)

21. Vector construction and agrobacterium mediated
transformation
RB 35S GVG E9 NOS-P HPT NOS-T 3A 6xUAS LB
Hsp90C
Hsp90C
GUS spacer
SpeI XhoI
Sse8387I PmeI
0.1 picomole
insert [each]
+
270 bp ►
►
22
kb
0.1 picomole
pGVG-vector
1025 bp ►
Agrobacterium mediated
transformation of
tobacco with pGVG-
Hsp90C-hpRNA
construct
Raising T0 plants
and harvesting T1
seeds
Fig. 8. Components of Hsp90C-hpRNA construct, schematic representation of pGVG-
Hsp90C-hpRNA vector used in this study and Agrobacterium mediated co-cultivation
of tobacco.

22. Induced silencing of Hsp90C produces chlorosis
and growth suppression phenotypes in transgenic
seedlings
Fig. 10. Chlorosis and growth suppression phenotypes of T2 generation of transgenic
lines grown on a medium containing Dex.

23. Expression analysis of Hsp90C in transgenic lines
upon Dex treatment
Fig. 11. Quantitative RT-PCR analysis of Hsp90C gene expression.

24. Induced silencing of Hsp90C produces chlorosis
and growth suppression phenotypes in transgenic
plants
Fig. 12. Chlorosis and growth suppression phenotypes observed in Dex-treated
transgenic plants (7 dpt).

25. Progression of chlorosis in iTav-tobacco and
Hsp90C-silenced transgenic tobacco lines
Fig. 13. Decrease in total chlorophyll content during the progression of induced
chlorosis.

26. Downregulation of CPRGs in transgenic tobacco
plants during the early phase of chlorosis
development
Fig. 14. Quantitative RT-PCR analysis of chloroplast and photosynthesis-related
gene expression.

27. Induction of plant defense genes in transgenic
plants during the early phase of chlorosis
development
Fig. 15. Quantitative RT-PCR analysis of plant defense gene expression (2 dpt).

28. Chloroplast
ROS
production?
SA
Dex-trigger
GVG fusion
transcription factor
Hsp90C-hpRNA transgene
hpRNA
siRNA
Degradation of Hsp90C mRNA
Nucleus
Cytoplasm
Activation of retrograde
signaling ?
Compromised
protein import
PR genes
Rapid upregulation of
plant defense genes
CPRGs
Downregulation of
CPRGs
Reduced Hsp90C
levels
A possible model explaining the molecular events
in chlorosis through the induced Hsp90C silencing
in transgenic tobacco
Fig. 16. Schematic representation of molecular events in chlorosis induced by
silencing of Hsp90C transgenic tobacco plants .

29. (Shimura et al., 2011 and Smith et al., 2011)
(Navarro et al., 2012)
Hsp90C mRNA
Peach latent mosaic viroid
(PLMVd)
CHLI mRNA
Symptom development through the anti-viral
siRNA-mediated silencing of chloroplast protein
genes
Hsp90C
CHLI

30.  For silencing of ChlI gene, we designed ChlI-amiRNAs using
WMD3 online tool (www.wmd3.weigelworld.org)
2. Chlorophyll biosynthetic gene (CHLI)
Fig. 17. Predicted mature amiRNAs sequences targeting Nicotiana tabacum CHLI-
mRNA.

31. Construction of pGVG-CHLI-ami vector and Agro-
transformation
Fig. 18. Schematics of construct for inducible expression of the CHLI-amiRNAs
used in this study.

32. Induced silencing of CHLI-amiRNAs produces
chlorosis and growth suppression phenotypes in
transgenic seedlings
Fig. 20. Development of chlorosis in transgenic seedlings grown on growth medium
containing Dex (15 DAS).

33. Expression analysis of CHLI mRNA upon Dex
treatment
Fig. 21. Expression analysis of CHLI gene by qRT-PCR (2 dpt).

34. Inducible silencing of CHLI gene expression in
transgenic tobacco plants produces
chlorosis symptoms
Fig. 22. Induced chlorosis observed in Dex-treated transgenic plants (7 dpt).

35. Time course assay to monitor the progression of
chlorosis
Fig. 23. Decrease in total chlorophyll content during the development of chlorosis in
transgenic plants.

36. CHLI-amiRNA induced chlorosis accompanies
downregulation of other CPRGs and
chaperone during early phase of chlorosis development
Fig. 24. Expression analysis of CPRGs and chloroplast chaperone by qRT-PCR
(2 dpt).

37. Induction of defense genes during the early phase
of chlorosis induced by CHLI silencing
Fig. 25. Expression analysis of plant defense genes by qRT-PCR (2 dpt).

38. A schematic model presenting molecular events upon
the onset of chlorosis symptoms through the silencing
of CHLI gene in CHLI-amiRNA transgenic plants
Chloroplast
ROS
production?
Dex-trigger
GVG fusion
transcription factor
CHLI-amiRNA transgene
Pre-miRNA
miRNA
Degradation of CHLI mRNA
and translational inhibition
Nucleus
Cytoplasm
Activation of retrograde
signaling ?
Less chlorophyll
biosynthesis
PR genes
Rapid upregulation of
plant defense genes
CPRGs
Reduced CHLI
levels
Downregulation of
CPRGs
Tetrapyrrole
intermediates
Fig. 26. The schematics of biochemical and molecular events upon induction of
chlorosis in CHLI-amiRNA transgenic tobacco plants.

39. iTav Hsp90C-
hpRNA
CHLI-
amiRNA
 Chlorosis with
mosaic pattern
 Growth
suppression
 Chlorosis with
altered morphology
 Growth
suppression
 Chlorosis with
green spots
 Growth
suppression
Summary / Conclusion
iTav-3 Hsp90C-4 CHLI-2-8

40. Trigger iTav Hsp90C-
hpRNA
CHLI-
amiRNA
Progression of
chlorosis
 0-2 days
 2-5 days
 5-7 days
CPRGs:
 CHLI
 RSSU
 LHCa/b
 HSP90C
Plant defense
genes:
 PR1a
 PR4
 SAMDC

41. Main papers:
1. SACHIN ASHOK BHOR, MD. SHAMIM AKHTER and KAPPEI KOBAYASHI.
Artificial induction of chlorosis symptom through the silencing of chloroplast
genes in transgenic tobacco. Indian Phytopathology, (In press).
2. Sachin Ashok Bhor・Chika Tateda・Tomofumi Mochizuki・Ken-Taro Sekine・
Takashi Yaeno・Naoto Yamaoka・Masamichi Nishiguchi・Kappei Kobayashi.
Inducible expression of magnesium protoporphyrin chelatase subunit I (CHLI)-
amiRNA provides insights into Cucumber mosaic virus Y satellite RNA-induced
chlorosis symptoms. VirusDisease, (DOI 10.1007/s13337-017-0360-1).
3. Sachin Ashok Bhor・Chika Tateda・Tomofumi Mochizuki・Ken-Taro Sekine・
Takashi Yaeno・Naoto Yamaoka・Masamichi Nishiguchi・Kappei Kobayashi.
Inducible transgenic tobacco system to study the mechanisms underlying
chlorosis mediated by the silencing of chloroplast heat shock protein 90.
VirusDisease, (DOI 10.1007/s13337-017-0361-0).
Additional papers:
1. Sachin Ashok Bhor, Md. Shamim Akhter, Takashi Yaeno, Naoto Yamaoka,
Masamichi Nishiguchi, Masanori Kaido, Kappei Kobayashi. Simple and
Quantitative Detection of Apple latent spherical virus Vector by a Spot
Hybridization. International Journal of Modern Botany, 6(2):31-36. (2016).
List of publications

42.  This study was supported partly by UGAS, EU and JSPS
KAKENHI grants 26292026 and 15K14664 to Prof. Kappei
Kobayashi
 I am grateful to Rotary Yoneyama Memorial Foundation for
providing scholarship for doctoral studies
 Supervisor Prof. Kappei Kobayashi, Co-supervisors,
Members of the dissertation committee, Co-authors and
PMBV lab members are greatly acknowledged
 Also, thankful to staff members of UGAS, EU.
Acknowledgements

43. Thank you ..!!!