What are all the bacterial symbionts involved in transmission and persistent of plant virus and their roles.

1. Bacterial symbionts in vector and its role
in plant virus transmission
1
B. Sangeetha, (Ph. D Scholar)
Dr. P. Renukadevi, Assistant Professor (Plant Pathology)
Dr. V. G. Malathi, Adjunct Professor (Plant Virology)
Department of Plant Pathology,
Tamil Nadu Agricultural University, Coimbatore, India.

2. Content
 Genesis of symbionts
Symbionts in Aphid
Symbionts in whitefly
 Symbionts in mealybug , leafhopper , thrips
2

3. Symbiosis:
 Symbiosis origin - Greek means together
 Permanent association between two or more
distinct individuals , atleast during a part of their life cycle.
Symbionts:
 An organism in a symbiotic relationship.
Examples: Fungi , Baceriophage , Lichens , mycorrhiza
Harpreet Singh Raina et al ., 2015
3

4. Nearly 100 years ago
Paul Buchner from
Germany documented
array of both
endosymbiotic fungi and
bacterial associates of
arthropods
The title of the book
“Endosymbiosis of
animals with plant
microorganisms”.
Genesis of bacterial symbionts
4

5.  1967- Lynn Margulis 'endosymbiont theory'.
 1994- Jonnes F.J -First report of endosymbiotic
bateria associated with PLRV by Myzus persicae
 1998 -A. E. Douglas -Nutritional Interactions in
Insect-Microbial Symbioses: Aphids and Their
Symbiotic Bacteria Buchnera
5
Jonnes F.J et al ., 1994

6. Animal Host Symbiont Symbiont
Contribution
Medicinal leech Enteric bacterium Blood digestion
Aphid Bacterium
(Buchneria aphidicola)
Amino acid
synthesis
Shipworm Gill cell bacterium
Cellulose digestion
and nitrogen
fixation
Sponges, jellyfish, sea
anemones, corals
Zooxanthellae
(spherical algae)
Increased
calcification of
corals; donation of
organic carbon;
Termites Enteric bacteria
Organic acid
production,
nitrogen fixation &
cellulose digestion
Humans
Enteric bacteria
(e.g. Escherichia coli)
Donation of
vitamins and
precursor
molecules there of
(= provitamins)
6

7. Examples of Bacterial Endosymbionts of
Insects
7

8. Ubiquitous - prevalent in arthropods.
Strictly transmitted vertically.
Types of symbionts
Primary symbionts
Secondary symbionts
Xiao-Li Bing, et al ., 2013
8
Bacterial symbionts

9. Bacterial Symbionts
Primary symbionts:
 Candidatus Portiera aleyrodidarum” (Oceanospirillales)
 Buchnera aphidicola
Secondary symbionts:
 “Candidatus Cardinium hertigii” (Bacteroidales)
 “Candidatus Fritschea bemisiae” (Chlamydiales)
 “Candidatus Hamiltonella defensa” (Enterobacteriales)
 Arsenophonus spp. (Enterobacteriales)
 Wolbachia spp. (Rickettsiales)
 Rickettsia spp. (Rickettsiales)
 Cardinium ( Bacteroidetes )
9
Harpreet Singh Raina et al ., 2015

10. Insect feeds on phloem sap which, although is rich in carbohydrates,
but lacks essential amino acids.
Lacking nutrients are expected to be compensated by the bacterial
community harboured by the insect.
Secondary endosymbionts -confer them :
Temperature tolerance
Increased resistance to parasitic wasps
Increased resistance to insecticides
Sex determination
Providing fitness benefits
Causing host plant specialization
Harpreet Singh Raina et al ., 2015
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Role and functions

11. • A bacteriocyte (Greek - bacteria cell) also called a
mycetocyte - insect groups such as aphids, german
cockroaches and weevils.
• Endosymbiotic bacteria such as Buchnera species,
which provide essential aminoacids and other
chemicals to their host.
• Bactriome
Baumann et al, 2000
TEM of aphid bacteriocyte
Larger - Buchnera
Smaller-Hamiltonella defensa
11

12. Aphids and its hosts
Aphids Hosts plants
Aphis fabae Bean
A. craccivora Cowpea
Acyrthosiphon pisum Pea
Rhopalosiphum padi Maize
Metopolophium dirhodum
Sitobion avenae
Oats
12Johannes F. J. M. van den Heuvel et al ., 1994

13. Bacterial symbionts in aphids
13
Caroline De Clerck et al ., 2015

14. 14
Bacteriocytes in aphid
Christian Braendle et al ., 2003

15. Aphid species having endosymbionts
Vector Virus Endosymbionts
Myzus persicae PLRV
BWYV
Buchnera aphidicola
Acyrthosiphon pisum PEMV
BLRV
BYDV
Buchnera aphidicola
Hamiltonella , Regiella ,
Serratia , Rickettsia ,
Spiroplasma
Pentalonia nigronervosa BBTV Buchnera aphidicola
Wolbachia
15
Johannes F. J. et al ., 1994

16. Charactization of Endosymbiont
Total DNA extraction
PCR by using 16S ribosomal DNA sequences
Clones and Sequencing
Confirmation
16Caroline De Clerck et al ., 2015

17. 17

18. GroEL
 GroEL belongs to class of proteins called Chaperonin.
 They are ring shaped protein complexes essential in the
cell mediating ATP dependent polypeptide folding.
 Costa Gourgopolas (1970) isolated a mutant of E.coli
that were unable to support the Lamda phage.
 E.coli gene responsible for this phenotype was given the
name GroEL- assembly aiding function.
18
Adi Kliot and Murad Ghanim 2013

19. Contd….
 Van den Henvel 1994 Myzus persicae protein extracts
 Out of five proteins one bind the CP antibody.
 The protein was present in high concentration.
 This protein was homologous to E.coli well described
protein called GroEL.
 Which was previously called symbionin- production
by endosymbionts of pea aphid Acyrthosiphon pisum
now referred to as (MpBsym) Myzus persicae -
Buchnera GroEL
19

20. Role of bacterial chaperones in virus
transmission
20
Virion assembly
stage within the
host
Ability to bind –
proteins of
different
structure
Adi Kliot and Murad Ghanim 2013

21. Role of symbionin of Buchnera spp in
aphid
 The aphid endosymbiont, Buchnera spp., is known to
produce copious amounts of the protein symbionin (a
homolog of Escherichia coli GroEL) which binds to
luteoviruses and other viruses that are transmitted in a
circulative, nonpropagative manner .
 Symbionin binds to virus in the hemolymph it protects
the virus from the aphid immune system.
Alberto Fereres and Benjamin Raccah.,2015
21

22.  Potato leafroll virus (PLRV) is a single-stranded RNA
virus that belongs to the luteovirus group.
 PLRV particles also bind to native symbionin ,these
virus particles when acquired into the haemocoel of
an aphid interact with symbionin.
22

23. Assays for the study
Monoclonal antibody raised for PLRV
Anti-idiotypic antibodies (AiAbs) were raised to
PLRV-specific MAbs (monoclonal antibodies) virus
(BNYVV)
23

24. Two-dimensional SDS PAGE
24
(a) Two-dimensional profile of whole-body proteins of M.
persicae stained with CBB. (b,e) Immunoblots of two-
dimensional Uv separated whole-body proteins of
M.persicae incubated with purified PLRV particles and
AiAb (c)

25. Bacterial symbionts in Myzus persicae
25
A B
Immunogold-labelling of the
primary endosymbiont (e) in the
mycetocyte (m) of M. persicae
using AiAb.
Cross-section through a 1-day-old
nymph
Endosymbiont in mycetocyte

26. ELISA to determine binding in vitro of vector-
borne plant viruses to native p63 of M. persicae
26
All viruses were applied at 10 µg/ml PBS-Tween.
1-PLRV
2-BWYV
3-BICMV
4-TSWV
5- CPMV
6-BNYVV

27. Effect of Tetracycline treatment
27
Western blots of haemolymph proteins probed with anti-p63 antibodies, and of
whole-body homogenates of aphids probed with anti-PLRV antibodies. Tc,
tetracycline-treatednymphs; Co, control nymphs, not treated with the antibiotic.

28.  Luteovirus particles are composed of two types of
capsomeres.
 The predominant one is CP .
 Another minor one, believed to be on the surface of
the virion, is the read-through (RT) protein.
Gray et al., 2014
Beet western yellows virus - Interaction
with Buchnera
28

29. Beet western yellows virus
 BWYV engineered to be encapsidated with CP alone (with no
RT protein subunits) did not bind to Buchnera GroEL.
 in vivo studies showed that BWYV virions lacking the RT
protein were significantly less persistent in the haemolymph
than were virions with the RT protein.
 This led to the hypothesis is that the interaction between
Buchnera GroEL and the RT protein protects the virus from
rapid degradation in the haemolymph.
 Comparison of the RT domain from different luteoviruses and
PEMV revealed several conserved amino acid residues that
may be important for the interaction with Buchnera GroEL
29

30. Bacterial symbionts in whitefly
Primary endosymbiont:
Portiera
Secondary endosymbiont:
 Wolbachia
 Rickettsia
 Arsenophonus
 Cardinium
 Hamiltonella
 Fritschea
30

31. Diverse population of secondary symbionts in
insects -(FISH)
A–B: FISH of Arsenophonus (yellow)
and Portiera (red) in T. vaporariorum under
bright field (A) and dark field (B).
C–D: Fritschea (blue) and Portiera (red) in
NW2
E–F: Hamiltonella (green) and Portiera(red) in
MEAM1 .
G–H: Rickettsia (blue) and Portiera (red) in
MEAM1
I–J: Cardinium (blue) and Portiera (red) in T.
acaciae .
K–L: Wolbachia (blue) in B. tuberculata .
31

32. Presence of secondary endosymbionts in
B. tabaci
P-pleomorphic endosymbionts
C-coccoid endo symbionts
immunogold labelling with
antibody to Buchnera GroEL
protein antibody.
In whiteflies fed on antibody to
protein TYLCV titre declined.
Morin et al ., 1999
32

33. Binding between GroEL and TYLCV CP in
Y2H system
Morin et al., 2000
33

34. Immunocapture PCR of TYLCV using
antibody to GroEL
Gottlieb et al ., 2010
34

35. B. tabaci cryptic species China 1 ( B. tabaci biotype ZHJ3) additional
bacterium which belongs to the Alphaproteobacteria subdivision of the
Proteobacteria and has a close relationship with human pathogens of the
genus Orientia was identified . Orientia like organism (OLO)
New endosymbiont identified in China-1 population
35

36.  Different Bemisia tabaci biotypes harbour different
secondary symbionts.
 Morin et al ., identified GroEl analogue of Buchnera in B
biotype of B. tabaci
 Whiteflies fed with antibodies to Buchnera protein . No
TYLCV DNA was detected .
 Physical interaction between GroEL and TYLCV –CP was
demonstrated by TEM studies showed (immunogold)
binding with Buchnera not with Portiera.
 Yeast hybrid test ,I –PCR ,
 Q and B biotype Spain
36

37. Involvement of GroEL in TYLCV
37
P- pholem, s – stylet , e- esophagus , fc- filter chamber , mg- midgut
, psg- primary salivary gland , bc- bacteriocyte , h-hindgut

38. Secondary symbiont Rickettsia in China 1
biotype of whitefly
Infection with Rickettsia spp., a facultative endosymbiont of
whiteflies, altered TYLCV-B. tabaci interactions.
 B. tabaci strain infected with Rickettsia acquired more
TYLCV from infected plants, retained the virus longer, and
exhibited nearly double the transmission efficiency compared to
an uninfected B. tabaci strain with the same genetic background.
Adi Kliot et al., 2014
38

39. Representative double FISH of TYLCV (red) and Rickettsia
(green) in B. tabaci midguts dissected from Rick+ female
whiteflies collected from TYLCV-infected tomatoes.
Adi Kliot et al., 2014
FISH of TYLCV (red) and Rickettsia(green) in B. tabaci midguts
dissected from Rick+ female whiteflies collected from TYLCV-infected
tomatoes.
39

40.  B biotype - Middle East-Minor Asia 1 (MEAM1)
 Q biotype -Mediterranean (MED)
40

41. Different Q types
Israeli Q lacks Hamiltonella - cannot effectively transmit
–TYLCV
Chinese Q contains Hamiltonella- can efficiently
transmit TYLCV to tomato plants.
41

42. 42
Percentage of H1 vs. H2 Q whiteflies (Bemisia tabaci) that acquired TYLCV
after 13 acquisition access periods (AAPs) as determined by conventional PCR.

43. 43
Relative amount of TYLCV (normalized to the host nuclear bactingene) in H1 (black
dots) and H2 (red dots) Q whiteflies (B. tabaci) after 10 acquisition access periods
(AAPs) as determined by q-PCR.

44. Polymerase chain reaction (PCR) and Flourescence in situ
Hybridisation (FISH) commonly used for identification and
localization of bacterial endosymbionts in B. tabaci

44
DNA isolation
16S rRNA sequences
Clone and sequence
Confirmation
Harpreet Singh Raina et al ., 2015

45. 45
Agarose gel electrophoresis of 16S rDNA PCR product of different bacterial
endosymbionts, amplified from total DNA of Bemisia tabaci samples of Delhi
population. (A) Represents PCR results for primary endosymbiont Portiera (1kb). (B)
Wolbachia (650bp). (C) Rickettsia (800bp). (D) Arsenophonus (630bp). (E) Cardinium
(440bp)
Harpreet Singh Raina et al ., 2015

46. 46
FISH staining of different bacterial endosymbionts in whole
mount of B. tabaci using bacteria specific LNA probes.
Localization of (A)Wolbachia
and Portiera.
(B) Localization of Rickettsia
and Portiera.
(C) Localization of
Arsenophonus and Portiera.
(D) Localization of Cardinium
and Portiera
(a) Merged image showing
overlap of Portiera and
respective secondary
endosymbiont.
(b) Presence of Portiera in
bacteriocytes.
(c) Presence of respective
secondary endosymbiont in
bacteriocytes
(d) Phase contrast.
Harpreet Singh Raina et al ., 2015

47. 47
Comparative frequency distribution of different bacterial endosymbionts by
diagnostic PCR and FISH in samples of B. tabaci from different locations.

48. 48
Western flower thrips, Frankliniella occidentalis
(Thysanoptera:Thripidae)
Erwinia - facultative symbiont De Vries et al., 2001
Bacterial symbionts of thrips

49. Cont..,
First instar larvae -take up bacteria from the leaves or
from the faeces or saliva deposited by other thrips.
Most very young first instar thrips larvae are not
infected with gut bacteria (prevalence of 20%)
 Second larval stage, are 100% infected.
Bacteria are not directly transmitted from mother to
offspring, but larvae acquire bacteria from the leaves
right after they hatch.
49
De Vries et al., 2001

50. 
50
Tomato spotted wilt virus (TSWV)
Close association of Erwinia sp

51. Western flower thrips preference for thrips damaged leaves
over fresh leaves enables uptake of symbiotic gut bacteria
51

52. (%)Percentage of adult western flower thrips that transmitted Tomato spotted
wilt virus.
Symbiotic thrips (Sym).
Bacteria-free thrips aposymbiotic (Apo). (Tetracycline)
The percentage of adults that transmitted the virus determined by recording the
incidence of local lesions that developed on a Petunia leaf disk fed on by an adult
for two days. Transmission – percentage of thrips that successfully transmitted
virus.
52

53. No effects of symbiotic gut bacteria of thrips on
tospovirus transmission efficiency
53

54. Bacterial symbionts in mealybugs
Tremblaya princeps
Plant sap-sucking insects with an obligate association with
prokaryotic endosymbionts .
Particular endosymbionts are acquired through vertical, maternal
transmission - stored within the mealybug’s body cavity in
aggregates of specialized cells called bacteriocytes.
Phylogenetic analyses are consistent with an infection of a mealybug
ancestor – precursor endosymbiont followed by the vertical
transmission of the endosymbiont to progeny.
Charles JG et al ., 2006
54

55. Diversity of Bacterial Endosymbionts Associated with
Leafhoppers (Macrosteles )
 Endosymbiotic microbiota of the Macrosteles leafhoppers Macrosteles striifrons
and Macrostele sexnotatus
 PCR, cloning, sequencing, and phylogenetic analyses of bacterial 16S rRNA
genes identified
 Two obligate endosymbionts:
 “Candidatus Sulcia muelleri”
 “Candidatus Nasuia deltocephalinicola”
Five facultative endosymbionts:
 Wolbachia
 Rickettsia
 Burkholderia
 Diplorickettsia
 A novel bacterium belonging to the Rickettsiaceae
55Charles JG et al ., 2006

56. 56
(A) Epifluorescence image of the whole abdomen.
(B) Confocal image of the bacteriome. Red, green, and blue signals
indicate “Ca. Sulcia muelleri,” “Ca. Nasuia deltocephalinicola,” and host nuclear
DNA, respectively
Fluorescence in situ hybridization of the endosymbionts “Ca. Sulcia
muelleri” and “Ca. Nasuia deltocephalinicola” within the bacteriomes of the
leafhopper Macrostele striifrons.
Charles JG et al ., 2006

57. Conclusion
 We can say that convincing evidence establishing
the role of endosymbionts in the transmission of
plant viruses have been obtained.
 So far only in two cases however, the exact
mechanism of how the symbionin helps a virus in
crossing the midgut and salivary gland barrier
leads to be understood.
57

58. Conclusion
 The convincing evidence establishing the role of
endosymbionts in the transmission of plant
viruses have been obtained so far only in two
cases .
 However the exact mechanism of how the
symbionin helps the virus in crossing the midgut
and salivary gland barrier needs to be
understood.
58