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Role of extracellular vesicles in plant defence

THESIS
SUBMITTED TO
MAHARSHI MARKANDESHWER (DEEMED TO BE UNIVERSITY) MULLANA –
AMBALA
FOR THE AWARD OF B.Tech. DEGREE

IN
BIOTECHNOLOGY
SUBMITTED BY
MD SOBAN ALI
(Roll No. 11204503)

SUPERVISOR
DR. YASHIKA WALIA DHIR
(Assistant Professor)

DEPARTMENT OF BIOSCIENCES &TECHNOLOGY MAHARSHI

MARKANDESHWER (DEEMED TO BE UNIVERSITY) MULLANA – AMBALA
(HARYANA)
2020-2024
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DEPARTMENT OF BIO-SCIENCES AND TECHNOLOGY

MAHARISHI MARKANDESHWAR (DEEMED TO BE UNIVERSITY)

MULLANA-AMBALA

CERTIFICATE

This is to certify that the review work included in the dissertation report entitled “Role of
extracellular vesicles in plant defence” submitted in partial fulfillment of requirement for the
award of degree of B.Tech Biotechnology, Maharishi Markandeshwar (Deemed to be
University), Mullana-Ambala is a bonafide work done by Md Soban Ali under my guidance.

Dr. Yashika Walia Dhir Dr. Adesh K. Saini

Assistant Professor (Professor and Head)
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DECLARATION

I, Md Soban Ali hereby declare that the review work entitled “Role of extracellular vesicles
in plant defence” done by me under the guidance of Dr. Mayank Chaudhary, Assistant
Professor, Maharishi Markandeshwar (Deemed to be University), Mullana-Ambala is
submitted in partial fulfillment for the award of degree of B. Tech Biotechnology.

I declare that this review work is purely original and is not submitted for the award of any other
degree or diploma to any other institute or university in India or abroad.

Date:

Place: Mullana Signature of the Candidate

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ACKNOWLEDGEMENT

It is my heartiest pleasure to express to my sincere gratitude to my supervisor Dr. Yashika

Walia Dhir , (Assistant Professor), Department of Biosciences & technology, Maharishi

Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India, for shorting out

the research problems, continuous inspiration and providing helpful advice.

I feel prickly conscience to remain with a single word of gratitude for the pivotal role played

by Dr. Adesh Shaini (Head) Department of Biosciences & technology for technical guidance,

sympathetic attitude and timely help at different stages of present investigation.

I wish to record my profound respect and grateful thanks to Dr. Yashika Walia Dhir (Associate

Professor) for their continuous encouragement, moral and emotional support and useful

suggestions all through the course of my research work.

My vocabulary fails to find appropriate words for the intense feeling of loving gratitude

towards my beloved parents Mr. Md Ali and Ms. Kausar Begam who has always stood beside

me like a tower in giving me the most inspiration, encouragement and illuminating my life path

with their eternal love and blessings. I cannot imagine if I would do anything without them.

Last but not least, I thank to almighty God without whom nothing is possible in this world.

Md Soban Ali

Roll No. 11204503

B.Tech biotechnlogy

Department of Biosciences & technology,

MMDU Mullana, Ambala Haryana

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Sr. No. PARTICULARS PAGE No.

1. Introduction 8

2. Both Plant And Plant‐Pathogenic MicrobesanAN 9-10

Release Evs

3. Roles Of Evs In Plant–Fungus Interactions 11-13

4. Roles Of Evs In Plant–Bacteria Interactions 14-17

5. Roles Of Evs In Plant–Virus Interactions 18-21

6. Roles Of Plants Evs In PTI, ETI, AND SAR 21-26

8. Conclusion 26-27

9 Refrence 27-30
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ABSTRACT

Extracellular vesicles (EVs) are tiny, bubble-like structures wrapped in a protective layer of fat

that are released by both eukaryotic cells and bacteria. They transport a variety of active

substances such as genetic material, proteins, and fats. In mammals, EVs play diverse roles like

facilitating communication between cells, influencing cancer development, and contributing to

disease processes. Conversely, research on plant EVs has been limited despite their

significance. Recent findings show that both plants and their invaders, like fungi and bacteria,

produce EVs, which are crucial in plant-pathogen interactions. Plant EVs contain small RNA

molecules and defense-related proteins, which can be absorbed by harmful fungi, reducing their

ability to cause harm. On the other hand, EVs from certain bacteria trigger plant immune

responses by releasing specific molecules, affecting defense mechanisms. This review

highlights the pivotal role of EVs in mediating interactions between plants and pathogens,

underscoring the importance of key molecules like small RNAs and proteins found in plant

EVs.

EVs serve as vital components in plant defense mechanisms, acting as messengers for

intercellular communication and carriers of defense-related substances. Here's a simplified

breakdown of their significance:

Communication Hub: EVs mediate communication between plants and microbes,

transporting essential molecules like proteins and RNAs. This facilitates effective responses to

pathogens

Defense Booster: Plant EVs actively engage in defense against diverse pathogens, delivering

defense-related molecules to target cells and bolstering plant resistance to threats

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Cargo Carriers: EVs harbor a diverse array of molecules, including proteins, lipids, and

nucleic acids. These cargo molecules encompass antimicrobial peptides, enzymes, and

signaling molecules crucial for defense responses

Systemic Defense: By inducing systemic resistance, plant EVs prime distant tissues to fend off

pathogens. They aid in disseminating defense signals across the plant, preemptively preparing

it for potential threats

Stress Adaptation: EVs facilitate plant adaptation to environmental stressors by transferring

stress-responsive molecules. This capability helps plants endure both biotic and abiotic

stresses, promoting overall survival and resilience

METHODOLOGY

The information on the immune reactions caused by viral and viroid stresses in plants was

gathered from a variety of sources, such as (PUBMED, SCOPUS, GOOGLE SCHOLAR and

Several internet sources), and was later compiled and further assembled in a simplified manner

with the single goal of advancing knowledge on this subject.

INTRODUCTION
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Plants face a constant barrage of pests and pathogens throughout their lives. Over time, they

have evolved various defense mechanisms to fend off these threats. When pathogens attack,

plants recognize specific molecular patterns called pathogen-associated molecular patterns

(PAMPs), triggering an immune response known as PAMP-triggered immunity (PTI)

In response, pathogens have developed effector proteins that undermine PTI. Plants, in turn,

detect these effectors through resistance (R) genes, which activate a stronger immune response

called effector-triggered immunity (ETI) [Naveed et al., 2020; Qi et al., 2011] . Additionally,

plants can produce signaling molecules like glycerol-3-phosphate and pipecolic acid in infected

tissues, which move to uninfected areas to trigger systemic acquired resistance (SAR) [Chanda

et al., 2011; Hartmann et al., 2018; Jung et al., 2009; Návarová et al., 2012].

Extracellular vesicles (EVs) play a crucial role in the exchange of molecules between plants

and pathogens. These small membranous vesicles are produced and secreted by cells under

various conditions. EVs can be categorized into apoptotic bodies, microvesicles, and exosomes,

each with distinct formation mechanisms [Akers et al., 2013; György et al., 2011; Liebana-

Jordan et al., 2021; van der Pol et al., 2012].

Initially thought to be waste disposal structures, exosomes are now recognized for their roles

in cell communication, development, tissue repair, fertilization, and immune responses. They

contain various bioactive substances, such as nucleic acids, proteins, and lipids, which can be

delivered to target cells [Théry et al., 2002; Doyle & Wang, 2019.]

While research on EVs in plants has been slower compared to animals, recent studies show that
both plants and pathogens can release EVs, which are crucial for their communication. This
review covers the latest findings on EV roles in plant-microbe interactions and the significant
molecules found in plant EVs, such as small RNAs and proteins [Cai et al., 2018; Cai, He &
Jin, 2019].

BOTH PLANT AND PLANT‐PATHOGENIC MICROBES CAN RELEASE EVS

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In 1967, Jensen's group utilized electron microscopy to observe extracellular vesicles (EVs) in

carrot cells (Halperin & Jensen, 1967). Despite this early observation, the presence of a cell

wall in plant cells was initially seen as a barrier to the production or detection of vesicles,

leading to a lack of research on plant EVs in subsequent decades. However, in 2009, de la

Canal's group isolated EVs from sunflower apoplastic fluids, demonstrating that plant cells,

similar to animal cells, can secrete exosomes into the intercellular.

Over the past decade, studies have revealed that plant EVs originate from multivesicular body

endosomes (MVBs) and exocyst-positive organelles (EXPO), containing distinct sets of RNAs,

proteins, lipids, and metabolites. These EVs are functionally involved in defensive responses

against pathogens (Cai et al., 2018; He et al., 2021; Liu et al., 2020; Regente et al., 2017;

Rutter & Innes, 2017).

Bacterial pathogens also release EVs, with Gram-negative bacteria producing outer membrane

vesicles (OMVs) by pinching off part of their outer membrane (Kulp & Kuehn, 2010). OMVs,

ranging from 20 to 400 nm, contain various macromolecules and play roles in bacterial

functions such as cell-to-cell communication and modulation of host immunity (Bose et al.,

2020; Manning & Kuehn, 2011).

Research on EVs produced by pathogens has mainly focused on human-pathogenic gram-

negative bacteria, with relatively fewer studies on fungal EVs. However, recent research has

shown that fungal EVs contain various cargo and elicit notable biological responses in immune

cells. Plant-pathogenic fungi and bacteria also release EVs, contributing to biofilm formation,

virulence, and modulation of plant immunity (Bose et al., 2020; Katsir & Bahar, 2017;

McMillan et al., 2021).

Fundamental questions remain regarding the passage of EVs through cell walls and their

uptake by plant cells. Demonstrated that plant EVs can penetrate the cell wall, potentially
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facilitated by cell wall-modifying proteins. Further research is needed to understand the

mechanisms of EV transport and uptake in plant-microbe interactions.

FIGURE 1

Cargo of plant extracellular vesicles (EVs). PEN1, plant‐specific penetration 1; sRNA, small

RNA; TET, tetraspanin; tyRNA, tiny RNA

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ROLES OF EVS IN PLANT–FUNGUS INTERACTIONS

Plant EVs inhibit fungal growth and virulence by transferring their cargo into fungal

cells.

A proteomic analysis of extracellular vesicles (EVs) from sunflower seedlings revealed the

presence of defense-related proteins (PR proteins, disease resistance dirigent protein, Gnk2

antifungal protein), GDSL lipase acyl hydrolases, lectins, and germin-like proteins. Similarly,

EVs released by tomato root cells contain proteins involved in plant-pathogen interactions and

inhibit fungal spore germination (Palma et al., 2020), suggesting a vital role for plant EVs in

communication with fungal pathogens.

Small RNAs (sRNAs), including microRNAs (miRNAs) and small interfering RNAs

(siRNAs), silence genes with complementary sequences. They regulate fungal transcript

expression during host-induced gene silencing, affecting pathogen development. Exosome-like

vesicles found in extrahaustorial matrix may mediate sRNA transfer between plants and fungi.

Plant EVs transfer sRNAs to fungal cells, reducing virulence. They selectively load sRNAs,

potentially mediated by RNA-binding proteins like AGO1. Arabidopsis EVs are enriched in

tiny RNAs (tyRNAs), possibly derived from various RNA sources (Baldrich et al., 2019). The

specific uptake of EVs by pathogens may involve ligand-receptor interactions (Regente et al.,

2017; Rutter & Innes, 2017).

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FIGURE 2

Function of plant extracellular vesicles (EVs) in plant–microbe interactions. (a) Plant EVs can

be taken up by pathogens and can inhibit the virulence of pathogens by releasing their cargos

(small RNAs [sRNA], proteins, tiny RNAs [tyRNA], and lipids). Meanwhile, pathogen‐

associated molecular patterns (PAMPs) from pathogens can be loaded into plant EVs in

pathogen‐infected cells. (b) Plant EVs may be functionally related to the signal transmission

of PAMP‐triggered immunity (PTI) and effector‐triggered immunity (ETI). First, PAMPs in

plant EVs released by infected cells are recognized by receptor‐like kinases (RLKs) located on

the membrane to activate PTI in adjacent uninfected cells. Second, plant EVs contain abundant

proteins involved in PTI and ETI. When plant cells perceive pathogens, the contents of plant

EVs secreted by these cells may change. These EVs can be taken up by adjacent pathogen‐free
 P a g e | 13

cells and induce them to produce PTI and ETI downstream signals. (c) Plant EVs may be

functionally related to systemic acquired resistance (SAR) signal transmission. Plant EVs

contain SAR‐related proteins and PAMPs. During SAR, EVs released by pathogen‐infected

cells may be transported to systemic tissues via the apoplast to transmit SAR signals when they

are taken up by target cells or induce an immune response by the recognition of PAMPs and

RLKs in systemic tissues. ER, endoplasmic reticulum; MVB, multivesicular bodies

endosomes; TGN/EE, trans‐Golgi network/early endosome.

Fungal EVs induce a phytotoxic response in plants

While research has extensively explored the functions of plant EVs in interactions with

pathogenic fungi, fewer studies have focused on fungal EVs in plant-fungus interactions.

Recent findings shed light on the significance of fungal EVs:

1. Phytotoxic Effects: EVs from F. oxysporum f. sp. vasinfectum induced a phytotoxic

response in cotton and Nicotiana benthamiana plants. Remarkably, these EVs exhibited

a deep purple color, suggesting a potential role in pigment and/or toxin biosynthesis.

2. Proteomic Analysis: EVs isolated from F. oxysporum f. sp. vasinfectum and Z. tritici

contained sets of putative virulence-associated proteins. This indicates that fungal EVs

may play crucial roles in host plant-fungus interactions, possibly contributing to

pathogenicity.

These findings underscore the importance of understanding fungal EVs' functions, which could

lead to insights into novel mechanisms of pathogenicity and potential targets for disease control

in agriculture.
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ROLES OF EVS IN PLANT–BACTERIA INTERACTIONS

Functions of plant EVs in plant–bacteria interactions

While research on plant extracellular vesicles (EVs) in plant-bacteria interactions is limited,

studies in mammalian systems suggest parallels in immune response modulation. EVs released

by bacteria-infected mammalian cells contain pathogen-associated molecular patterns

(PAMPs) and host proteins, promoting an immune response in adjacent cells. Similarly, EVs

from Pseudomonas syringae-infected Arabidopsis carry signal transduction proteins linked to

biotic stress, hinting at a role in immune signal transmission (Rutter & Innes, 2017).

Mammalian cells transmit sRNAs to bacterial cells via EVs, reducing antibiotic-resistant

biofilm formation (Koeppen et al., 2021). P. syringae-infected Arabidopsis shows doubled EV

secretion, suggesting EV involvement in bacterial resistance. Plant EVs, containing sRNAs

and pathogenesis-related proteins, likely inhibit bacterial growth and virulence by cargo

delivery to bacterial cells (Cai et al., 2018); (Regente et al., 2017).

These findings hint at the potential of plant EVs in modulating immune responses and

inhibiting bacterial pathogens, although further research is needed to elucidate their specific

mechanisms in plant-bacteria interactions.

OMVs activate plant immune responses

Proteomic analyses of outer membrane vesicles (OMVs) secreted by plant-pathogenic bacteria

have identified effectors and various virulence factors, indicating their role in modulating plant

immune responses (Bose et al., 2020; Chowdhury & Jagannadham, 2013; Kulkarni et al., 2015;

Sidhu et al., 2008). Ronald's group was the first to demonstrate this function, showing that

OMVs from Xanthomonas campestris pv. campestris and Xanthomonas oryzae pv. oryzae

induce a burst of reactive oxygen species (ROS) and the expression of defense-related genes

in Arabidopsis (Bahar et al., 2016).

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Western blot analysis detected the immune elicitor EF-Tu in OMVs. In plants, pattern-triggered

immunity (PTI) is mediated by pattern-recognition receptors (PRRs) like FLS2 and EFR, which

recognize flagellin and EF-Tu, respectively, to induce PTI (Gómez-Gómez & Boller, 2000;

Zipfel et al., 2006). Arabidopsis mutants lacking EFR are insensitive to OMVs, suggesting the

ROS burst is mediated by EFR (Bahar et al., 2016). Immune co-receptors BAK1 and SOBIR1

are also involved in OMV perception and downstream signaling.

Kuehn's group found that OMVs from Pseudomonas syringae and Pseudomonas fluorescens

activate immune responses against bacterial and oomycete pathogens. Effector-triggered

immunity (ETI) is induced by bacterial type III secretion system (T3SS) effectors (Jones &

Dangl, 2006; Spoel & Dong, 2012). While T3SS components are associated with OMVs,

OMVs from T3SS mutants still induced immune responses, indicating that T3SS effectors are

not essential for OMV-induced immunity (McMillan et al., 2021).

The resistance to bacterial infection mediated by OMVs was only slightly affected by

proteinase K treatment or boiling, suggesting that non-protein components of OMVs

(metabolites, lipids, small protein epitopes) are responsible for their protective effects. OMVs

induced both salicylic acid (SA)-dependent and -independent immune responses. For example,

P. syringae OMVs increased ICS1 expression and SA accumulation, unlike P. fluorescens

OMVs, indicating differences in immune responses triggered by OMVs from different bacteria.

Notably, P. syringae OMVs induced a more sustained MAPK phosphorylation compared to the

well-characterized PAMP signal flg22, suggesting that OMVs elicit more complex immune

responses than a single PAMP. Further research is needed to identify OMVcomponents that
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activate plant immunity, which could aid in developing novel biopesticides.

FIGURE 3

Bacterial outer membrane vesicles (OMVs), which contain protein effectors, can elicit plant

protective immune responses. The immune co‐receptors BAK1 and SOBIR1 mediate the

perception of, and the response to, OMVs. The OMVs induce immune responses via salicylic

acid (SA)‐dependent/‐independent processes and MAPK pathways

OMVs facilitate infection by pathogenic bacteria

Many studies have explored the functions of outer membrane vesicles (OMVs) in mammalian

systems, where they enhance the secretion efficiency of virulence factors & Kuehn, 2014;
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Lloubes et al., 2013). While the direct uptake of OMVs by plant cells has not been documented,

it is plausible that plant-pathogenic bacteria use extracellular vesicles (EVs) to deliver various

cargo to plant cells. Plant cell walls, primarily composed of pectin, cellulose, and

hemicellulose, act as barriers to the transport of factors from bacteria to plant cells. To

overcome this obstacle, bacteria have evolved a range of cell wall-degrading enzymes using

type II secretion systems (T2SSs), such as polygalacturonases, cellulases, amylases, and

xylanases. These enzymes degrade the plant cell wall, facilitating the transport of virulence

factors into the cell (Faulkner & Robatzek, 2012; Gibson et al., 2011; Hématy et al., 2009;

Rajeshwari et al., 2005; Szczesny et al., 2010).

Büttner's group demonstrated that T2SS substrates, such as xylanases, are secreted by

Xanthomonas campestris pv. vesicatoria via OMVs. T2SS mutants did not produce or secrete

xylanases, and immunoelectron microscopy confirmed the presence of xylanases in OMVs

from wild-type X. These findings suggest that OMVs provide an alternative route for

transporting virulence factors, including T2SS substrates, to the extracellular

microenvironment, playing a functional role in pathogen infection.

Lindow's group discovered that OMVs are involved in the migration ability and virulence of

the aerobic, gram-negative bacterium Xylella fastidiosa (Ionescu et al., 2014).OMV secretion

was suppressed by a diffusible signal factor-dependent quorum-sensing system. A ΔrpfF strain

with disrupted quorum signaling showed increased OMV production and greater virulence to

plants compared to the wild-type strain, but lower adhesion to glass or plant surfaces. OMV

treatment significantly suppressed the adhesion of X. fastidiosa cells to xylem vessels and

facilitated cell movement within the xylem, enhancing virulence. It remains to be seen whether

OMVs from other plant-pathogenic bacteria have similar functions.

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ROLES OF EVS IN PLANT–VIRUS INTERACTIONS

Extensive research has delved into the functions of extracellular vesicles (EVs) in mammals

during viral infections. For instance, exosomes from hepatitis C virus (HCV)-infected human

hepatoma Huh7.5.1 cells were found to carry full-length viral RNAs, proteins, and particles.

These exosomes successfully transmitted the infection to naive cells, establishing a productive

infection (Ramakrishnaiah et al., 2013). This suggests that exosome-mediated viral

transmission plays a significant role in the virus's ability to evade the immune system.

Conversely, exosomes can also inhibit viral transmission through various mechanisms.

Pathogen-associated molecular patterns (PAMPs), such as viral nucleic acids, are recognized

by pattern recognition receptors (PRRs) that trigger innate immune responses (Kawai & Akira,

2009). For example, exosomes from HCV-infected cells transferred viral PAMP (RNA) to

plasmacytoid dendritic cells (pDCs), activating host immune responses like the production of

antiviral type 1 interferons (Dreux et al., 2012). Thus, exosomes secreted by virus-infected

cells can both enhance and inhibit viral infections (Jia et al., 2021).

In contrast, the role of plant EVs in plant-virus interactions remains underexplored. Turnip

mosaic virus (TuMV), a positive-sense, single-stranded RNA virus from the order

Picornavirales, provides a useful model. Research by Zheng's group revealed that EVs in

TuMV-infected Nicotiana benthamiana leaves contain viral replication complexes (proteins

and RNA), as shown by immunogold labeling and confocal microscopy (Movahed et al., 2019).

Proteomic analyses of EVs from TuMV-infected plants identified numerous proteins involved

in TuMV replication, such as putative poly(A)-binding protein (PABP) and infection-related

proteins like S-adenosylhomocysteine hydrolase (AGO2), along with proteins involved in plant

immunity, such as the 14-3-3 protein.

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These findings suggest a dual role for EVs in plant-virus interactions. First, EVs containing

viral replication complexes may facilitate the spread of viruses from infected cells to

neighboring cells or systemic tissues via the phloem. Further studies should investigate whether

EVs isolated from phloem sap of virus-infected tissues contain viral replication complexes,

though obtaining large volumes of phloem sap is challenging. Second, EVs may act as carriers

of PAMPs or signal molecules, transmitting immune signals from infected cells to adjacent

cells. It would be intriguing to determine whether viral components in plant EVs function as

PAMPs to trigger plant immune responses (Figure 4).

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FIGURE 4

Plant extracellular vesicles (EVs) play a significant role in the immune response against

viruses. When plant cells are infected with viruses, they release EVs containing viral proteins

and RNA. These EVs serve multiple functions, including facilitating the transmission of
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viruses between neighboring cells and acting as carriers of pathogen-associated molecular

patterns (PAMPs) to trigger immune responses in plants.

ROLES OF PLANT EVS IN PTI, ETI, AND SAR

When plants encounter pathogens, they respond by proliferating multivesicular bodies, leading

to increased exosome secretion (An et al., 2006; Wang et al., 2014) . In particular, Arabidopsis

infected with Pseudomonas syringae shows higher exosome secretion compared to uninfected

plants, indicating that these exosomes might play a role in plant resistance to bacterial

pathogens . Salicylic acid (SA) is a crucial phytohormone involved in various defense

mechanisms, such as systemic acquired resistance (SAR), basal defense, and resistance gene-

mediated defense . SA treatment in Arabidopsis has been found to promote exosome secretion,

suggesting that these exosomes are key players in transmitting immune signals between cells

(Rutter & Innes, 2017). Proteomic analyses have identified numerous proteins linked to

immune signal transmission, reactive oxygen species (ROS) signaling, and other defense-

related proteins in these exosomes (Movahed et al., 2019; Regente et al., 2017; Rutter & Innes,

2017).

Plants have a two-tiered innate immune system: pattern-triggered immunity (PTI) and effector-

triggered immunity (ETI). PTI is the first line of defense, activated when pattern recognition

receptors (PRRs) on the cell membrane detect pathogen-associated molecular patterns

(PAMPs) from pathogens (Li et al., 2018). PRRs include receptor-like kinases (RLKs) and

receptor-like proteins (RLPs) (Wu et al., 2019). In Arabidopsis, exosomes from both infected

and control plants have been found to contain RLKs such as CARD1, KIN7, LRR-RLK 2, and

SIF3, suggesting their role in PTI (Movahed et al., 2019; Rutter & Innes, 2017). Plant exosomes

are primarily found in extracellular spaces, likely contributing to PTI (Figure 2b).
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Pathogens have evolved effectors to suppress PTI and facilitate infection. In response, plants

have developed resistance (R) proteins that recognize these effectors and trigger ETI. For

example, the RPM1-interacting protein 4 (RIN4) in Arabidopsis is phosphorylated by P.

syringae effectors, activating R protein signaling and initiating an immune response . RIN4 and

other interacting proteins are present in plant exosomes, indicating their involvement in ETI .

The exosome proteome also includes calcium-dependent protein kinases (CDPKs) such as

CPK3 and CPK21, which regulate various immune responses, including ROS production and

transcriptional reprogramming. Thus, plant exosomes can communicate with neighboring cells

to transmit or amplify immune signals (Figure 2b).

Inducers of SAR, including azelaic acid, glycerol-3-phosphate, pipecolic acid, and N-hydroxy-

pipecolic acid, are produced in pathogen-infected tissues and transferred via the phloem to

systemic tissues, establishing SAR (Chanda et al., 2011; Hartmann et al., 2018; Jung et al.,

2009; Návarová et al., 2012). SAR-related proteins such as DEFECTIVE IN INDUCED

RESISTANCE 1 (DIR1), THIOREDOXIN h3 (TRXm3), and PATHOGENESIS-RELATED

1 (PR1) increase in phloem exudates from SAR-induced leaves (Carella et al., 2016;

Champigny et al., 2013; Chanda et al., 2011). Azelaic acid and glycerol-3-phosphate are

transported through plasmodesmata (Lim et al., 2016). In mammals, exosomes are secreted

into bodily fluids under various conditions, suggesting that plant exosomes could also transport

via the phloem to deliver their cargo to systemic tissues. Carella et al. (2016) identified

numerous mobile proteins from phloem exudates during SAR induction, including TRXh3,

which increases in phloem exudates from leaves infected with P. syringae (Carella et al., 2016).

TRXh3 is crucial for SA-dependent local resistance and SAR by catalyzing an SA-induced

NPR1 oligomer-to-monomer reaction (Tada et al., 2008). These findings indicate that plant

exosomes mediate SAR signal transport via the phloem (Figure 2c). Further research should
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explore whether pathogen-induced plant exosomes can enhance disease resistance and induce

SAR.

In mammals, exosomes from bacteria-infected cells contain PAMPs and bacterial proteins,

triggering immune responses in target cells (Schorey et al., 2015; Wang et al., 2018) . For

example, exosomes from Mycobacterium tuberculosis-infected macrophages contain PAMPs

and can activate immune responses in target cells. Plant PTI is similarly activated by PAMPs

recognized by PRRs, mainly RLKs. Recent studies show that phosphorylation levels of 17

RLKs change significantly in systemic leaves of SAR-induced plants, suggesting their

involvement in SAR (Zhou et al., 2021). Thus, plant exosomes likely contain PAMPs that

contribute to local resistance and SAR (Figure 2). Further research is needed to determine if

plant exosomes contain PAMPs or other pathogen-derived components.

Additionally, exosomes from Helicobacter pylori-infected macrophages contain sRNAs that

induce inflammatory responses by promoting cytokine production. Plant exosomes also

contain sRNAs that inhibit fungal virulence by targeting virulence-related genes. Exploring the

role of these sRNAs in plant immunity could provide new insights into exosome function.

In mammals, exosome composition and function change under stress (Beninson & Fleshner,

2014) . Plant exosomes also contain diverse cargo, including nucleic acids, proteins, and lipids

(Cai et al., 2018; He et al., 2021; Liu et al., 2020; Regente et al., 2017; Rutter & Innes, 2017).

However, this cargo has mostly been analyzed qualitatively. Applying omics methods to study

differentially accumulated cargo (sRNAs, mRNAs, lncRNAs, proteins, and lipids) in pathogen-

induced plant exosomes will enhance our understanding of their role in local resistance and

SAR.

Plant exosome proteomes reveal various signaling proteins even in the absence of pathogen

infection. Investigating whether these proteins are activated or become active upon pathogen
 P a g e | 24

recognition will be crucial. Posttranslational modifications (e.g., phosphorylation,

glycosylation, acetylation) regulate protein activity and interactions (Li et al., 2015).

Proteomics can identify modified EV proteins after pathogen infection, providing deeper

insights into exosome functions in plant immunity.

TABLE 1

Summary of proteins in proteome of both phloem and extracellular vesicles

Locus Gene Description Accumulation level in

symbol PEXPsm versus PEXMock

AT3G16470 JAL35 Jacalin‐related lectin 35 Decreased

Cytosolic isocitrate
AT1G65930 CICDH Decreased
dehydrogenase (NADP)

AT4G09000 GF14 14‐3‐3‐like protein GF14 chi Decreased

Probable calcium‐binding
AT1G18210 CML27 Increased
protein

AT3G53420 PIP2‐1 Aquaporin PIP2‐1 Decreased

AT4G23600 CORI3 Cystine lyase Decreased

Late embryogenesis abundant

AT2G44060 – Increased
protein, group 2
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Locus Gene Description Accumulation level in

symbol PEXPsm versus PEXMock

14‐3‐3‐like protein GF14

AT1G22300 GRF10 Decreased
epsilon

Ribulose bisphosphate
AT1G67090 RBCS1A Decreased
carboxylase small chain 1A

AT1G70890 MLP43 MLP‐like protein 43 Increased

AT1G72150 PALT1 Patellin 1 Decreased

AT1G76180 – Dehydrin Decreased

AT1G22530 PATL2 Patellin 2 Decreased

AT1G35720 ANN1 Annexin D1 Decreased

Glycine‐rich RNA‐binding
AT2G21660 CCR2 Decreased
protein 7

Glyceraldehyde‐3‐phosphate
AT1G13440 GAPC2 Decreased
dehydrogenase

Fasciclin‐like arabinogalactan
AT2G45470 FLA8 Decreased
protein 8

AT2G44790 – Uclacyanin‐2 Increased

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[Chanda et al., 2011; Hartmann et al., 2018; Jung et al., 2009; Návarová et al., 2012].

Note

PEXPsm, petiole exudates from Arabidopsis leaves inoculated by virulent or avirulent

Pseudomonas syringae; PEXMock, petiole exudates from leaves inoculated by MgCl2. These

data come from comparative analysis of proteomics data generated by Carella et al. (2016) and

Rutter and Innes (2017).

CONCLUSION

Both plant cells and plant pathogens release EVs, just like animals do. Plant EVs contain

various defense-related cargoes, such as proteins, nucleic acids (sRNA, tyRNA), and lipids,

and play significant roles in interactions between plants and pathogens. These EVs can be taken

up by fungi, hindering their virulence by transferring sRNAs and defense-related proteins into

pathogens. Moreover, plant EVs carry a diverse array of proteins involved in sensing and

transmitting plant-pathogen interactions, contributing to signal transduction in PTI, ETI, and

SAR.

EVs from plant-pathogenic bacteria and fungi also facilitate communication across kingdoms.

Bacterial EVs harbor different immune stimulants, triggering plant immune responses through

PRR- and BAK1/SOBIR1-mediated signaling pathways. Additionally, bacterial EVs activate

plant immune responses via SA-dependent and independent processes, owing not to immune

stimulants but to the metabolites, lipids, and small protein epitopes they contain. Furthermore,

bacterial EVs assist in cell movement within host plants and augment virulence. Fungal EVs

induce phytotoxic responses in host plants and seem pivotal in certain infection processes.
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Our understanding of the involvement of EVs in plant-microbe interactions remains limited

and fragmented, especially when compared to mammalian counterparts. The precise functions

of EVs necessitate further elucidation. Research focusing on isolating plant EVs relevant to the

plant immune response, characterizing their composition, and delineating their function is

crucial. Such endeavors will be instrumental in comprehending the roles of EVs in plant-

microbe interactions and will greatly facilitate the development of sustainable agricultural

methods.

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