Nematode effector proteins play a key role in plant parasitism. Effectors are secretory proteins that alter host cells to suppress defenses and facilitate infection. They are synthesized in gland cells and injected into plants through the stylet. Effectors can have different targets, such as modifying the cell wall, altering metabolism or hormone signaling, and suppressing immunity. Characterizing effectors provides insights into plant responses and resistance mechanisms. Recent studies have identified effectors that interact with components of auxin signaling or the NADPH oxidase complex to promote parasitism. Going forward, RNA interference targeting important nematode genes holds promise for developing resistant crop varieties.

1. Nematode effector proteins
1
BY
C. MATHIVATHANI
2016801402

2. OutlineFlow of seminar
 Introduction
 Effectors
 Plant responses to effectors
 Effectors as probes
 Functional characterization
 Research findings
 Conclusion
 References
2

3. INTRODUCTION
Plant parasitic nematodes are unique - that
develop an intimate and sustained obligate parasitic
relationship with their host plants.
• Need of special strategy
 To infest the plants,
 To fight the plant innate immune system and
 To sustain their life on plants.
3

4. Are plants so week?
4

5. A. Pre –Infectional resistance/ Passive resistance
B. Post –Infectional resistance/Active resistance
PLANT DEFENSES
PAMP Signals
Organisms betray their presence
with recognizable molecular
signals on their surfaces - pathogen
associated molecular patterns
(PAMPs).
DAMP Signals
Signals that trigger PTI responses
resulting from damage caused by
the invasion – damage associated
molecular patterns (DAMPs).
First line of defense involves production of salicylic acid - signal to invoke defense
mechanisms
5
Mellato et al., 2006

6. JA = Jasmonic acid
SA = Salicylic acid
ISR = Induced Systemic Resistance
SAR = Systemic Acquired Resistance
PR = Pathogenesis-Related genes
> peroxidase and catalase
Induced Resistance 6
Mellato et al., 2006

7. How nematodes achieve
parasitism ?
7

8. Nematode secretions from different organs
Cuticle Excretory pore
Rectal glandsAmphids
Oesophageal
glands
8

9. Role of Nematode secretions
Involve in
• Hatching
• Self-defense
• Movement through plant tissue
• Establishment & maintenance of feeding structures.
• Parasitic strategies differ among species of
phytonematodes.
(Hussey & Grundler, 1998)
9

10. EFFECTORS
Effectors are secretory proteins that alter host
cells to suppress host defense mechanisms and facilitate
infection by the pathogen so it can derive nutrients from
the host.
(Presti et al., 2015)
10

11. Characteristics of effectors
 Effector proteins - modular proteins.
 Synthesized - response to specific signals.
 Regulated by nervous system.
 Expression - very specific to developmental stages.
 Different effectors are required to be produced in time and
space during the parasitic process.
 Gland cells - a mixture of secretory granules containing
individually packaged effector proteins, at any particular time.
Presti et al. (2015)
11

12. Types of effectors
 Apoplastic effectors  Cytoplasmic effectors
12
Humira et al. (2006)

13. Secretion from Oesophageal glands
• Key role in the parasitism of plants.
Primary source of secreted effectors
13

14. Structure of Oesophagus
• Muscular metacarpus
• Pump chamber
• Three active secretory
gland cells
• One dorsal & two sub
ventral.
14

15. • Valve of dorsal gland cell - near the base of the stylet.
• Valves of sub ventral gland cells - back of the meta- corporal
pump chamber and release secretions into the tube of the
oesophagus.
15

16. 16

17. Mechanism of Effectors
1. Delivery of effectors into host cells
2. Action of effectors in the apoplast
3. Suppression of plant immunity
4. Some effectors alter plant behavior and development
5. Molecular mimicry by effectors
(Hogenhout et al., 2008)
17

18. Parasitic cycle of sedentary endoparasites
J2 enter &
migrate through
cortex
Causes cellular
damage and
necrosis
Penetrate the
endodermis
Initial feeding
site
Inject the
secretions
through stylet
Formation of
nutrient sink
18

19. Secretory proteins are synthesized in the nuclear region of the
gland cell.
Stored in membrane-bounded granules that are transport to the
ampulla.
Inject secretory proteins through stylet in the root cell of
susceptible plant.
The stylet penetrates the cell wall but does not puncture the
plasma membrane.
Secretion deposit outside the plasma membrane and perforate in
the Cytoplasm through pores.
19

20. Effectors interact with receptor and other host protein.
Organize & maintain feeding cell & form feeding tube.
Directly regulate change in host gene expression.
Gland secretions alter morphological, physiological & molecular
changes in recipient cells.
Enable them to function as nutrient sink.
20
Quentin et al. 2013

21. Morphological changes in
Oesophageal glands
Migration
Initiation
of
Feeding
cells
Cell wall modifying proteins are
switched.
e.g. ß-1,4-endoglucanases and
pectate lyases.
Sub-ventral
glands
Dorsal gland
Increase in activity during the
initiation of feeding cells .
2 1
21
Hewezi et al. 2013

22. Plant responses to nematode secretions
• Plant response to nematode parasitism - susceptible or resistant
crop cultivars occurs in feeding sites surrounding the
nematode head.
(Williamson & Kumar, 2006)
• Dramatic changes in host cell morphology and gene
expression of host plant tissues – received much attention.
Eg., Root-knot, cyst and reniform nematodes.
(Jones, 1981)
22

23. Feeding cells by sedentary endo - parasites
23

24. Nematode effectors in establishment of feeding cell 24

25. Nematode effectors as probes
 Cell wall architecture
 Metabolism
 Modifications of the plant cell wall
 Effectors mimicking plant compounds
 Plant triggered immunity and susceptibility
 Alteration of hormonal signalling
 Suppression of defense signalling
(Mitchum et al., 2013)
25

26. 26Nematode effectors as probes

27. Cell wall architecture
• A host response pivotal to feeding cell formation is controlled
by cell wall modifications.
• The recent glycan-specific antibodies and fluorescence
imaging - beginning to reveal the distinct molecular
architecture of feeding cell walls.
(Davies et al., 2012).
• Contributing to these changes - numerous classes of plant
proteins involved in cell wall modification and biogenesis,
such as expansins, extensins, cellulose synthases and UDP-
glucose dehydrogenases.
(Siddique et al., 2012).
27

28. Metabolism
• As feeding cells develop, the large central vacuole is reduced
to several small vacuoles and is replaced by dense cytoplasm
reflecting one of the most impressive features of nematode
feeding cells – increased metabolism.
(Ithal et al., 2007)
• Altered metabolism under direct influence of the nematode.
(Lee et al., 2011)
28

29. Modifications of plant cell wall
• The structural complexity is reflected - wide array of
nematode-secreted enzymes & able to depolymerize various
structural polysaccharides of the cell wall, including cellulose,
hemicellulose and pectins.
(Davis et al., 2011)
• Sedentary parasitic nematodes also secrete other effector
proteins, such as CELLULOSE-BINDING PROTEINs
(CBPs), that function in plant cell wall modifications during
the sedentary phase of parasitism.
(Ding et al., 1998)
29

30. • Functional characterization of a CBP from the sugar beet cyst
nematode Heterodera schachtii revealed that CBP functions
through a direct association with (Arabidopsis thaliana) Pectin
methyl esterase protein3 (PME3).
• CBP overexpression increased PME3 activity in plants,
thereby reducing the level of methyl esterified pectin in cell
walls - facilitating the access of cell wall-modifying enzymes
to cell wall polymers.
(Hewezi et al., 2008)
30

31. 31

32. Mimicking plant compounds
• Effectors mimicks host proteins.
• Affect plant signalling, hormone balance, and cell morphogenesis.
• The CNs secrete active CLAVATA3/ESR (CLE)- like proteins in
plants, CLE-like peptides play an essential role in meristem
differentiation.
• Secreted through cytoplasm of host cells, from which they are
transported to the plant apoplasm
• where they mimic plant CLE signalling peptides and interact at the
plasma membrane with leucine - rich repeat (LRR) receptor kinase
family proteins, resulting in the formation and maintenance of
syncytia.
(Guo et al., 2011).
32

33. Effector - triggered Immunity and
Susceptibility
• Sources of specific ETIs are resistance genes.
• Activated ETI effectively disrupts the feeding and
development of sedentary endoparasitic nematode species.
The Mi gene of tomato codes for receptors to the
effector molecules introduced by root-knot nematodes
that would otherwise suppress plant defenses and
facilitate the development of feeding sites.
33

34. Examples
• RKN calreticulin (Mi-CRT) secreted by root knot nematode
sedentary stages during induction and maintenance of the giant
cells.
• The role of Mi-CRT as a suppressor of host innate immunity.
• The venom allergen-like effector Gr-VAP1 from
Globodera rostochiensis to interact with an allelic form of the
apoplastic papain-like cysteine protease Rcr3 of tomato -
increasing plant susceptibility to the nematode.
• In resistant tomato plants this modification of Rcr3 is
perceived by the trans membrane Cf2 receptor which triggers a
hypersensitive response.
34

35. Alteration of Hormonal signals
• Two cyst nematode effector proteins were recently found to
associate physically with key components of the auxin-
signalling pathway.
• The 19C07 effector from H. schachtii was found to interact
with the Arabidopsis auxin influx transporter LIKE AUXIN1 3
(LAX3) in the plasma membrane.
• Overexpression of 19C07 in Arabidopsis augmented the rate of
lateral root emergence, an indication of increasing auxin influx
thereby facilitating their incorporation into the developing
syncytium.
(Lee et al., 2011)
35

36. Suppression of defense signalling
• Parasitic nematodes use a variety of effectors to directly or
indirectly subvert host immune responses in order to mediate
susceptibility.
• The secreted SPRY domain-containing (SPRYSEC) effector
family identified in Globodera rostochiensis & Globodera
pallida - the largest effector family found in plant parasitic
nematodes to date
• Members of this family are involved in the modulation of plant
immunity.
(Cotton et al., 2014)
36

37. 37

38. 38

39. Nematode effectors as probes
• Recent studies - changes and differences in gene expression in
response to these different infection strategies of host
(Kyndt et al., 2012)
• Plant’s first line of defense against the invading nematode is
triggered in response to (DAMPs) or (PAMPS).
(Win et al., 2012)
39

40. • Many effectors secreted during migratory phase of infection
facilitate nematode penetration and migration by degrading
components of the plant cell wall, as well as enable nematodes to
dampen down the plant’s immune system.
(Smant & Jones, 2011).
• Effector proteins secreted during sedentary phase of parasitism –
co- ordinately regulate plant defense suppression with cellular
reprogramming to form a metabolically highly active feeding cell.
(Kaloshian et al., 2011).
40

41. Overview
Effectors/ Gene
Product
Species in which
identified
Possible function
β-1,4 endoglucanase
(cellulase)
G. rostochiensis
G. tabacum
H. glycines
H. Schachtii
M. incognita
Cell-wall degradation
Pectate lyase M. javanica
G. rostochiensis
H. glycines
Cell-wall degradation
Polygalacturonase M. incognita Cell-wall degradation
Chorismate mutase H. glycines
M. javanica
G. rostochiensis
Alter auxin balance
feeding cell formation
Thioredoxin
peroxidase
G. rostochiensis Breakdown of H2O2,
protect against host
defenses
41

42. Co - immunolocalization
• Localize multiple effectors simultaneously within the oesophageal gland
secretory granules for understanding effector packaging.
• Example, some effectors (e.g. Endoglucanases (ENGs)) are clearly secreted
into the apoplast during nematode intra - or intercellular migration in the
root .
(Wang et al., 1999).
• Others directly interact with specific cytoplasmic proteins or targeted to the
nucleus, must enter the host cell to function in parasitism
(Hewezi & Baum, 2013).
42

43. Functional characterization
RNA Interference/ Silencing
43

44. Silencing
• Silencing the H. glycines gene HgALD, encoding
fructose-1,6-diphosphate aldolase converts glucose into
energy during parasite invasion into its host.
• Transgenic soybean roots expressing an RNAi construct
targeted to silence HgALD revealed 58% reduction of
females formed by H. glycines.
• Application of RNAi for the enhancement of nematode
resistance by suppression of important effector proteins.
(Tripathi et al., 2017)
44

45. Research findings
45

46. • Hs-Tyr promoted plant growth and changed the root
architecture.
• In Arabidopsis caused changes in the homeostasis of
several plant hormones especially Auxin.

47. Subcellular localization of Hs-Tyr::GFP within the epidermal cell
(a) The green fluorescence - Hs-Tyr::GFP fusion protein localized in the cytoplasm of
the cells.
(b) Merged image shows the GFP signal in bright field.

48. 48

49. 49

50. • MiMsp40 is a novel Meloidogyne-specific effector that is injected
into plant cells by early parasitic stages of the nematode - that
plays a role in suppressing PTI and/or ETI signals to facilitate
RKN parasitism.
50

51. 51

52. • UGD2 and UGD3 are needed for the production of cell wall
ingrowths in syncytia and that their lack leads to a reduced host
suitability for H. schachtii
• Resulting in smaller syncytia, lower number of developing
nematodes, and smaller females.
52

54. This gene plays a key role in manipulating plant defense signal
responsive genes to maintain the GCs, thus promoting successful
parasitism in the host plants.
Misp12 protein has a potential through down-regulation of SA and
JA- dependent defense responses genes to promote the latter
parasitism of M. incognita during the mature stages.

55. Increased antioxidant protection and interruption of salicylic acid signaling are key aspects
of 10A06 function in addition to other physiological and morphological changes caused by
altered polyamines, which are potent plant signaling molecules.

56. GUS staining in H. schachtii - induced feeding sites
N, Nematodes;
S, syncytium.

57. • Pathogen-stimulated ROS are primarily catalyzed by the plasma membrane–resident
nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and cell wall–bound
peroxidases.
• NADPH oxidase is a multi subunit enzymatic complex in mammalian neutrophils
mediating microbial killing.
• Homologs of the NADPH oxidase catalytic subunit gp91phox are present in various
plants and known as respiratory burst oxidase homologs (RBOHs) with 10 family
members in Arabidopsis thaliana .
• NADPH oxidase–derived ROS function as a pathogenicity factor to facilitate infection
of Arabidopsis by nematode to establish a long-term feeding relationship with host
plants

58. • Illustration of the early processes during nematode infection of wild-type
(susceptible) and atrbohD/F mutant (resistant) roots.

59. • Host ferredoxin : thioredoxin system can be exploited
cunningly by M. javanica, revealing a novel mechanism
utilized by plant–parasitic nematodes to subjugate plant innate
immunity and thereby promoting parasitism.
59

60. Future prospects
• Bioengineering crops with dsRNA of phytonematode genes
can disrupt the developmental life cycle of parasitic nematodes
and therefore holds great promise to develop resistant crops
against plant-parasitic nematodes.
60

61. Conclusion
• The use of host plant resistance - not very effective - lack
of novel sources of resistance & race-specific resistance
even if found, can easily be overcome due to the
emergence of more virulent biotypes.
• Nematicides – Not ecofriendly.
• Identification of nematode genes involved in parasitism -
utilization of nematode inducible plant genes for creating
new forms of durable plant resistance.
61

62. References
• Habash, S. S., Radakovic, Z. S., Vankova, R., Siddique, S., Dobrev, P., Gleason, C.,
& Elashry, A. (2017). Heterodera schachtii Tyrosinase-like protein-a novel
nematode effector modulating plant hormone homeostasis. Scientific Reports, 7.
• Niu, J., Liu, P., Liu, Q., Chen, C., Guo, Q., Yin, J., ... & Jian, H. (2016). Msp40
effector of root-knot nematode manipulates plant immunity to facilitate parasitism.
Scientific reports, 6, 19443.
• Rehman, S., Gupta, V. K., & Goyal, A. K. (2016). Identification and functional
analysis of secreted effectors from phytoparasitic nematodes. BMC microbiology,
16(1), 48.
• Xie, J., Li, S., Mo, C., Wang, G., Xiao, X., & Xiao, Y. (2016). A novel Meloidogyne
incognita effector Misp12 suppresses plant defense response at latter stages of
nematode parasitism. Frontiers in plant science, 7.
• Hewezi, T. (2015). Cellular signaling pathways and posttranslational modifications
mediated by nematode effector proteins. Plant physiology, 169(2), 1018-1026.
62

63. • Hewezi, T., Juvale, P. S., Piya, S., Maier, T. R., Rambani, A., Rice, J. H., ... & Baum, T.
J. (2015). The cyst nematode effector protein 10A07 targets and recruits host
posttranslational machinery to mediate its nuclear trafficking and to promote parasitism
in Arabidopsis. The Plant Cell, 27(3), 891-907.
• Quentin, M., Abad, P., & Favery, B. (2013). Plant parasitic nematode effectors target
host defense and nuclear functions to establish feeding cells. Frontiers in plant science,
4.
• Mitchum, M. G., Hussey, R. S., Baum, T. J., Wang, X., Elling, A. A., Wubben, M., &
Davis, E. L. (2013). Nematode effector proteins: an emerging paradigm of parasitism.
New Phytologist, 199(4), 879-894.
• Jaouannet, M., & Rosso, M. N. (2013). Effectors of root sedentary nematodes target
diverse plant cell compartments to manipulate plant functions and promote infection.
Plant signaling & behavior, 8(9), e25507.
• Karczmarek, A., Overmars, H., Helder, J., & Goverse, A. (2004). Feeding cell
development by cyst and root‐knot nematodes involves a similar early, local and
transient activation of a specific auxin‐inducible promoter element. Molecular plant
pathology, 5(4), 343-346.
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64. I Thank everyone
for your valuable
presence….
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