Role of Autophagy in Plant-Pathogen interaction.pptx

Doctoral Seminar
(Course: PL.PATH-692)
Speaker: Boggala Vajramma
Roll Number: 12127
Ph.D. 1st year
Seminar leaders: Dr. Anirban Roy
Dr. Deeba Kamil
Role of Autophagy in Plant-Pathogen Interaction
INDIAN AGRICULTURAL RESEARCH INSTITUTE
Division of Plant Pathology
New Delhi.
Chairman: Dr. Deeba Kamil

1. Introduction 2. History
3. Types of
Autophagy
4. Autophagy-
related genes
(ATGs)
5. Molecular
mechanism of
Autophagy
6. Role in Plant-
Pathogen
interaction
6. Case studies 7. Conclusion
8. Future
prospects
Overview

Plant immune
response against
pathogens
Host plants fend off pathogens with several layers of defence
 The plant immune system is highly complex and involves PTI, ETI, and SAR.
 Protein degradation pathways are emerging as a central component of innate immunity.
 Special emphasis is on autophagy, whose manipulation can strongly affect immunity in Plant-Pathogen
interaction
Ubiquitination
Autophagy
Sumoylation

INTRODUCTION
 Autophagy (Greek word)= Self-eating
 Autophagy: It is an evolutionarily conserved degradative (Catabolic) process from yeasts to plants,
through which damaged organelles, non-functional proteins, and harmful microbial invaders are
delivered to vacuoles (in yeast and plants) or lysosomes (animals) for degradation.
 It not only maintains cellular homeostasis under starvation but also modulates the host cellular
response to abiotic and biotic stress conditions
 In recent years, it has emerged to play important roles in plant-pathogen interactions and acts as a
powerful tool to defend against pathogen infection in plants.
(Tang and Bassham, 2018; Shimamura et al., 2019)

Timeline of Notable Events in Autophagy Research
( Frake and Rubinsztein, 2016 )

Types of Autophagy in Plants
Micro-Autophagy Mega-Autophagy
Macro-Autophagy
 (Directly engulf cytosolic contents).
Cytoplasmic material congregates
on the vacuole surface and
becomes trapped by the
invagination of the tonoplast.
 The tonoplast undergoes fission to
release autophagic bodies.
 Double membranous autophagosomes
forms around cytosolic contents, then
it fuses with the vacuole for their
eventual degradation.
 Best characterized
 The Hydrolases released directly into
the cytoplasm by tonoplast rupture,
where they degrade cytoplasmic
material insitu.
 The most extreme form of autophagy,
often represents the final stage of
programmed cell death.
 Only present in plants during
developmental PCD, not in pathogen
infection
Chaperon-mediated Autophagy
(CMA): In Animals Independent of
vesicles.
Based on mechanism involved:
Wang et al., 2021

Selective Autophagy
 Unlike bulk autophagy, selective autophagy contributes to intra-cellular homeostasis under non-starved conditions by
selectively degrading the cargo material via specific autophagic receptors.
 Aggregated proteins (Aggrephagy), excess peroxisomes (pexophagy), damaged chloroplasts (Chlorophagy), mitochondria
(Mitophagy) and invading pathogens (Xenophagy)
Ran et al., 2020
 Xenophagy: During pathogen
attack, selective autophagic
receptor NEIGHBOR OF
BRCA1 (NBR1)/Joka 2/P62
binds to pathogen proteins
and mediates its Autophagic
degradation

AuTophaGy-related proteins (ATGs)
1). ATG 1/ ATG 13 Kinase complex ATG 1 Initiates & regulates autophagosome formation in response to
stress/pathogen infection
ATG 13
ATG 11
ATG 101
2). ATG 9 Recycling complex
(Transmembrane protein)
ATG 2 Recruits’ lipids to the expanding phagophore & promotes
phagophore expansion
ATG 18
3). Phosphotidylinositol 3 kinase (PI3K)
complex
Beclin 1/ ATG 6 Decorates the phagophore with (PI3P), which is essential for the
vesicle nucleation
VPS 14
VPS 15
VPS 34
4). Two Ubiquitin-like conjugation systems: ATG 3 (E2 like) Mediates the expansion of Phagophore & autophagosome
maturation
ATG 8-PE lipidation system & ATG 4
ATG 12- ATG 5 conjugation system (E3 like) ATG 5
ATG 7 (E1 like)
ATG 8*
ATG 10
ATG 12
ATG 16
 In Arabidopsis more than 40 ATGs have been identified. These are grouped into four complexes
Yang et al., 2020

Molecular mechanisms of Autophagy
2. Vesicle
nucleation
Su et al., 2020
1. Induction

Role of Autophagy during plant-pathogen interaction
 Does autophagy benefit the host as a defence
mechanism??..
 Does autophagy benefit the microbe by facilitating
survival and replication??..
 Or both??..
 Anti-death and Pro death roles of Autophagy
 Restricts Programmed Cell Death
(PCD): During necrotrophic
infection, autophagy serves an anti-
death role to limit the disease
lesion by suppressing ROS
accumulation and subsequent
disease development
 Promote PCD: In the
biotrophic type of host-
pathogen interactions, host-
regulated autophagy kills
surrounding uninfected
cells, limiting disease
spread
(Hofius et al., 2009; Lenz et al., 2011)
 Autophagy in plant pathogenic fungal infection
 The energy requirement for glycerol accumulation in
Appressorium development is transported from
autophagy-related cell death of neighboring conidia cells
to generate sufficient turgor
 Autophagy deficient mutants failed in fungal penetration
and disease development

(Liery et al., 2017
 ßc1 induces host Calmodulin-
like protein CaM, which
interacts with SGS3 to aid in
autophagic degradation
 In TuMV VPg interacts with SGS
3, resulting in its autophagic
degradation
 In polerovirus P0 induces ER-
derived autophagy for
degradation of membrane-
bound Argonaute 1 (AGO1).
Autophagy is highjacked by pathogen factors to promote infection
 P. infestans reprogram cellular
trafficking through secretion
of effector proteins through
haustoria.
 RXLR effector, PexRD54 has
evolved a canonical AIM to
bind the potato ATG8CL.
 Which depletes NBR1/Joka2
from ATG8CL complexes and
antagonizes the defense-
related autophagy
coordinated by NBR1/Joka2.
 In an ongoing evolutionary arms race, pathogens have acquired the potent ability to hijack and subvert
autophagy for their benefit.
SGS3: Suppressor of gene silencing
 R. solanacearum protein AWR5 inhibits directly the activation of TOR and
stimulates autophagy during infection.

(Fangfang et al.,2020)
In yeast, ATG39, an Autophagy receptor located at the perinuclear endoplasmic reticulum (ER), is dispensable for
yeast micronucleophagy, with this information they wanted to know the role of nuclear autophagy under Gemini
TLCYnV infection in degrading viral C1 protein
Case Study-1
Autophagy in plants
against pathogen
 Tomato leaf curl Yunnan virus (TLCYnV) is a monopartite begomovirus
that has six open reading frames (ORFs) – two in the virion sense (V1 and
V2) and four in the complementary sense (C1, C2, C3 and C4).
 Among these, the replication initiator protein (Rep), known as C1, is the
only viral protein necessary for viral DNA replication in the nucleus

Subcellular localization and degradation of the TLCYnV C1 protein
 The accumulation of the C1-YFP protein decreased after 24 hpi in
contrast to the change observed for its transcript
 Whereas the YFP protein increased after 24 hpi, correlating with
its transcript accumulation
The TLCYnV C1 protein forms granules in the cytoplasm
after 24 hpi.
 To examine the subcellular localization of the
TLCYnV C1 protein and its degradation
 With transiently expressing C1- yellow fluorescent
protein (YFP) and YFP in histone (H2B)-red
fluorescent protein (RFP) transgenic N. benthamiana
leaves at 24, 48, 72 and 96 h post-infiltration (hpi)
The TLCYnV C1 protein accumulation decreases after 24 hpi and is susceptible to
degradation

Autophagy inhibitors block the degradation of C1
 Both 3-MA & E64d treatments significantly inhibited the
degradation of C1 and enhanced its expression in the nucleus and
in the cytoplasm at 48 hpi, compared with DMSO or MG132
 C1-YFP protein accumulated to higher levels in 3-MA or
E64d-treated leaves compared with DMSO or MG132-
treated leaves.
 But no observable impact on the accumulation of the
C1 mRNA .
 The treatment with another autophagy inhibitor, Con
A, also increases C1-containing granules in the
nucleus and cytoplasm at 48hpi and the increased
accumulation of total C1 protein
The autophagy pathway is involved in the degradation of the nuclear protein C1
 The observed altered fluorescence signal and the
decreased protein levels of C1-YFP in the nucleus
prompted them to analyze whether the degradation
of C1-YFP occurs via autophagy or some other
protein degradation system

ATG8 interacts with C1 to form small granules in the cytoplasm
 None of the tested ATGs showed interaction with c1
other than ATG8h
 The C1–SlATG8h
interaction was
confirmed by BiFC in
H2B transgenic N.
benthamiana plants
 Also revealed that
interaction occurs in
the cytoplasm.
 Confirmed the C1-SlATG8h
associate complex in the cell.
 But no Myc-C1 or Myc-C4
was co-purified using GFP-
Trap
 Notably, the accumulation of
C1 significantly decreased
when this protein was co-
expressed with CFP-SlATG8h.
 SlATG8h was present in the
cytoplasm and nucleoplasm, but
not in the nucleolus, when
expressed alone or with C1
 whereas C1 localized in the
nucleus at 24 hpi
 However, When C1-YFP co
expressed with CFP-SlATG8h, it
was redistributed to the CFP-
SlATG8h-labeled punctuate
structures in the cytoplasm at 60
hpi.
 After getting that C1 is degraded via
the autophagy pathway , then
examined which ATG proteins are
involved in this process using Y2H
(SlBeclin1 and SlATG8a serving as
positive control)
SlATG8h interacts with C1 and probably directs it to autophagosomes for degradation

The AIM of C1 is responsible for the formation of the ATG8h-C1 interaction complexes
in the cytoplasm and the induction of C1 degradation
 The C1 protein with a mutated AIM (C1mAIM) was still able to
interact with SlATG8h in Y2H and BiFC assays, but it lost the
ability to translocate from the nucleus to the cytoplasm
 BiFC also revealed that C1 interacts with SATG8h in the
cytoplasm in H2B-RFP N. benthamiana leaves, but not C1mAIM
 C1mAIM-YFP was found not only in the nucleoplasm, but also
in the nucleolus.
 Compared with C1-YFP, C1mAIM-YFP showed stronger
fluorescence
 which also correlates with increased protein accumulation.
 Overexpression of Myc-SlATG8h (Autophagy) promoted
the degradation of C1-YFP, but not that of C1mAIM-YFP
The expression of C1 induces autophagy, that the core autophagy protein ATG8h binds C1 via
its AIM, and that this interaction probably tethers C1 to autophagosomes for degradation.
 The interaction between ATG8h- C1
prompted them to investigate whether
there is an AIM (ATG8 Interacting motif)
contained in C1
 Y2H and BiFC assays was performed for
C1 or C1- mutated AIM (C1mAIM) and
SlATG8h.

Autophagy defends against TLCYnV infection by recognizing the C1 AIM in N. benthamiana
and S. lycopersicum plants
 All three mutants showed severe symptoms with from TLCYnV in
N. benthamiana or S. lycopersicum plants compared to control
 Consistently, higher levels of TLCYnV
genomic DNA were found in ATG8h,
ATG5, or ATG7- silenced N. benthamiana
or S. lycopersicum plants
Suggest that ATG8h, ATG5, and ATG7 are
required for autophagy-mediated anti-
TLCYnV defense.
 Mutation in the AIM in TLCYnV led to more
severe symptoms and higher viral genomic
DNA levels compared with wild-type
 The high accumulation of the ATG8 protein
was observed the in the infection by
TLCYnVWt infectious clone, but not
TLCYnVmAIM infectious clone
The AIM is the key motif in C1 that is recognized and bound by ATG8, resulting in autophagic
degradation.
In planta assay in ATG5, ATG8h & ATG 7 silenced plants
against TLCYnV
In planta assay with TLCYnVmAIM infectious clone

The XPO1-dependent nuclear export pathway is involved in the ATG8h-
mediated degradation of C1
 Both LMB Treatment & VIGS knockdown of XPO 1 enhanced the
C1-YFP fluorescence signal in the nucleus
 Y2H assays revealed strong interactions between ATG8h
and XPO1
 No interaction was found between XPO1a and C1.
 BiFC assays showed the interaction between ATG8h and
XPO1a was distributed in the nucleus and in the
cytoplasm.
Consistently, higher C1-YFP protein accumulation
was also detected.
 The subcellular localization of C1 and C1mAIM
prompted them to investigate whether the autophagic
degradation of C1 was dependent on the nuclear export
pathway
 Tested with Leptomycin B (LMB): Nuclear export
inhibitor and other with downregulation of exportin 1
(EXPO 1) with VIGS vector
XPO1 is involved in the exportation of the C1-ATG8h interaction complex
via binding to ATG8h.

 Accumulation of the C1 protein in the nucleus of plant induces autophagy.
 The autophagy-related protein ATG8h interacts with C1 and translocate it from the nucleus to the cytoplasm;
this shuttling is dependent on the XPO1-mediated nuclear export pathway.
 Then, other ATGs in the cytoplasm, including ATG5 and ATG7, are recruited into autophagosomes.
 The autophagosomes formed would finally fuse with the vacuole to degrade its substrates, including C1, for
degradation.
Sum up:

Case Study-2:
Pathogen hijack plant
autophagy
 ARGONAUTE 1 (AGO1) appears in membrane-bound and membrane-free (soluble) forms, and that miRNAs
copurify with membrane-bound polysomes through their association with AGO1
 VSR protein P0 from Polerovirus encodes an F-box protein targets the PAZ motif and its adjacent upstream
sequence of AGO1 and hijacks the host SCF type ubiquitin-E3 ligase to promote proteasomal degradation of
AGO1. (SCF: S-phase kinase-associated protein1 (SKP1)- cullin 1 (CUL1)-F-box protein; VSR: Viral Suppressor Protein)
 However, inhibition of the proteasome was unable to block P0- mediated degradation of AGO1. Instead, it
was shown that AGO1 degradation was blocked by inhibition of trafficking pathways that lead to the
vacuole
With this information they wanted to know the viral f box protein P0 mediated ER derived autophagic degradation
of AGO1

P0 Encounters AGO1 on the ER and Both Proteins Are Co-delivered to the Vacuole
Most AGO1 protein was detected in
microsomes & its abundance decreased
after β-Es treatment.
 Again staining root cells that
express P0-mRFP with ER-Tracker
demonstrated its partial association
with the ER
 P0- mRFP were closely associated with
the ER, and some were also enriched
with the GFP-AGO1 signal.
 AGO1 protein level decreases
as the free-mRFP accumulates
(vacuolar degradation product
of P0-mRFP)
 suggesting that P0- mRFP
might be delivered to the
vacuole along with its target
AGO1.
 Accumulation of P0-mRFP−
and GFP-AGO1−containing
bodies within the
vacuoles.
 To examine microsomal & soluble
AGO1 pools degradation used
the (β-Es)-inducible promoter
XVE:P0-myc line of Arabidopsis
 To see whether both AG01 & P0
are associated with the ER with
transiently expressed GFP-AGO1
and P0-mRFP along with an ER
marker N. benthamiana
The ER as a major site from which P0 induces AGO1 degradation and from which it is co
delivered to the vacuole

Autophagy Deficiency Impairs P0 Delivery to the Vacuole, yet Is Unable to Block AGO1
Degradation
 In the cells of the
meristematic zone, both
autophagy mutants
displayed cytosolic
structures that contain
P0-mRFP
 That were absent from
wild-type cells
 None of the autophagy mutants was able to block AGO1
degradation.
 On the contrary, its transcript level was significantly
increased, ruling out the possibility that the decline of
protein level is due to reduced transcript level
 The mRFP-ATG8a protein was detected in the cytosol and the
nucleus, as well as in small bodies representing autophagosomes
 P0-GFP predominantly colocalized with mRFP-ATG8a in these
bodies.
 To examine whether P0 associates with
autophagy components and whether it
relies on autophagy for its function with
transiently expressing P0-GFP and mRFP-
ATG8a in N. benthamiana
 To determine whether the
degradation of P0 bodies is
regulated by autophagy,
introduced the XVE:P0-
mRFP into atg5-1 and
atg7-2
P0 can bypass autophagy deficiency to achieve a reduction in AGO1 protein level even when its delivery to the
vacuole appears to rely on canonical autophagy

P0 is engulfed by ATI1/2-decorated bodies on the ER and is co delivered to the Vacuole.
 At 30 hfa, detected the P0-GFP signal
colocalizing with ATI1- mCherry labeled
bodies
 Notably, at 120 hfa, the P0-GFP signal was
engulfed by a ring-shaped ATI1- mCherry
signal
 Vacuoles exhibited a large number of bodies, many of which
appeared to be labeled with both P0-mRFP and ATI1-GFP proteins
Quantification of the relative abundance of
each of the body types within vacuoles of P0-
mRFP−expressing cells revealed that
 80.5% of these bodies contained both
proteins (yellow bar)
 17% contained only ATI1-GFP (green bar),
and
 2.5% contained only P0-mRFP (red bar).
 Next Cells in the root elongation zone of Arabidopsis
seedlings harbouring pATI1:ATI1-GFP and XVE:P0-
mRFP, imaged following treatment with β-Es +
ConcA.
 To get insight into the P0 delivery pathway from ER
to-vacuole
 Examined the possible involvement of the ER and
autophagy-associated protein (ATI1/2) with
transiently expressing P0-GFP and ATI1-mCherry N.
benthamiana
This indicates that the majority of the P0 cargo is delivered to the vacuole via ATI-bodies

P0 Induces the Flux of ATI-Bodies to the Vacuole
 Further quantification
revealed a 5-fold increase
in the number of vacuolar
ATI1-GFP bodies in P0-
RFP induced plants
 Strikingly, this induction of ATI1-bodies was not
accompanied by either increased transcript nor
protein levels of ATI1-GFP
 The accumulation of P0- and ATI1-labeled autophagic
bodies in vacuoles suggests that whether P0 may induce
an ER-derived degradation pathway involving ATI1.
 To investigate this the pATI1:ATI1-GFP/XVE:P0-mRFP
transgenic line and its corresponding parental line that
does not contain P0- mRFP (pATI1:ATI1-GFP) were treated
with β-Es and ConcA
 Significantly more basal flux of ATI1-GFP bodies in the vacuoles of
the plants expressing P0-RFP than the parental line,
A posttranslational effect driven by P0 for the induction of the ATI degradation pathway

ATI1 Interacts with AGO1 on the ER, and ATI1/2 Deficiency Attenuates P0-Mediated
Decay of Membrane Associated AGO 1
 The accumulation of both GFP-AGO1 and ATI1-mCherry bodies within
the vacuole lumen
 Ring-like shapes of ATI1-mCherry signal engulfing GFP-AGO1 signal
(morphology that is preserved up to the vacuole).
 Both proteins colocalized on the ER, wherein
ATI1 engulfed the GFP-AGO1 signal, forming a
ring-like shape
 AGO1 interacted with ATI1 and ATI2, although
less strongly than with control (ATG8).
 Highest interaction of ATI1 with the AGO1
occurred at PolyQ-PAZ N-terminal half.
 AGO1 degradation was attenuated in the
ati1ati2KD mutant background, despite a
higher expression level of P0-myc.
 After confirming that P0 induces the ATI
degradation pathway, further examined
whether AT expressing GFP-AGO1 and ATI1-
mCherry I1/2 are involved in AGO1 regulation
independently of P0 using transiently
expressing GFP-AGO1 and ATI1-mCherry
N.benthamiana
ATI1/2 are required especially for membrane-bound AGO1 degradation via P0.
 To evaluate ATI1 and AGO1 mutual
dynamics transgenic lines harboring
pAGO1:GFP AGO1 and XVE:ATI1-mCherry
were used

Sum Up:
 The viral suppressor of RNA silencing P0 is known to target plant antiviral ARGONAUTE (AGO) proteins for degradation via
an autophagy-related process.
 P0 targets endoplasmic reticulum (ER)-associated AGO1 by inducing the formation of ER-related bodies then co delivered
to the vacuole as cargos.
 This process involves ATG8- interacting proteins 1 and 2 (ATI1 and ATI2) that interact with AGO1 and negatively regulate
its transgene-silencing activity.
 All together, reveal a layer of ER-bound AGO1 posttranslational regulation is manipulated by P0 to subvert plant antiviral
defense.

Conclusion
 Autophagy has emerged as a central part of the plant weaponry against invading pathogens, involved in
pathogen sensing, phagocytosis and then selectively removal of intra cellular pathogens
 Its significance for plant defense is supported by the evolution of microbial strategies to manipulate the host
autophagy machinery for their enhanced virulence and disease establishment.
 In addition, autophagy in eukaryotic phytopathogens has evolved as an essential process in the development of
functional infection structures.
 Expression of Autophagy Related Genes (ATGs) may be valuable in agricultural applications, as this can confer a
number of benefits like enhanced crop growth, yield, increased stress tolerance and defense response against
pathogen attack.
 The manipulation of autophagy pathways for the development of disease-resistant crops with increased yields
is now feasible and may play a significant role in sustaining agriculture in changing climatic scenario

Future prospects
The identification and characterization of new regulatory mechanisms in plant defence is a critical area for
research.
 A key direction of future research will be the identification and characterization of selective autophagy
receptors that drive plant defense responses and are still hidden in the gray shades of ‘bulk’ autophagy.
 There exist, largely unexplored crosstalk between autophagy and other cellular pathways that govern
proteostasis, hormone signaling, and programmed cell death in plant–microbe interaction.
 The transcriptional control of autophagy should be another fruitful area for further research
 Future investigations need to address whether and how autophagy can be re engineered to obtain disease
resistant plants

Role of Autophagy in Plant-Pathogen interaction.pptx

Role of Autophagy in Plant-Pathogen interaction.pptx

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