Plant Virus Transmission Advanced article

by Insects • Introduction
Article Contents

• The Importance of Insect Vectors

Alberto Fereres, Instituto de Ciencias Agrarias, Consejo Superior de Investiga-
• Taxonomy
ciones Cientificas, Madrid, Spain • The Major Transmission Modes: Persistent
Versus Non-persistent; Circulative Versus
Benjamin Raccah, Department of Plant Pathology, The Volcani Center, Bet Non-circulative
Dagan, Israel • The Mechanism of Non-persistent Transmission
• The Role of the Capsid Protein in the
Transmission of Non-persistent Viruses
• Virus-Encoded Proteins That Affect
Non-circulative Virus Transmission by Insects
• The Mechanism of Non-propagative, Circulative
Transmission
• Viral Proteins Involved in Transmission: the Coat
Protein and the Read-Through Protein
• Insect Proteins Involved in Virus–Vector
Interactions
• The Interaction between Bacterial
Endosymbinots Proteins and Circulative Viruses
• Analysis of Virus Transmission by Electrical
Penetration Graphs (EPGs)
• Effects of Virus Infection on Vector Behaviour
• Control of Virus Diseases by Interfering
with Vectors and Transmission
• Acknowledgements

Online posting date: 15th April 2015

Most plant viruses depend on insect vectors for Cultural, physical and novel biotechnological tools
their survival, transmission and spread. They trans- can provide virus control by interfering with vec-
mit plant viruses by two principal modes, circula- tor landing and the retention of viruses in their
tive (circulating through the insect’s haemocoel, vectors.
CV) and non-circulative (carried on the cuticle lin-
ing of mouthparts or foregut, NC). Transmissibil-
ity and specificity between NC viruses and their
vectors depends on the coat protein (CP) of the Introduction
virus in addition to virus-encoded helper proteins.
Circulative viruses cross the gut, circulate in the Insect vectors of plant viruses are found in 7 of the 32 orders
haemocoel and cross the salivary glands to ren- of the class Insecta. Hemipterans are by far the most impor-
der the insect infective. Circulative luteoviruses tant virus vectors, comprising more than 70% of all known
insect-borne viruses. Among these, aphids and whiteflies are the
depend on small CP and the read-through pro-
major vectors of plant viruses transmitting more than 500 virus
tein (RTD) for transmission. Electrical penetration
species. Two major classifications of viruses have been proposed:
graphs have provided evidence on insect feeding attending to the time the vector remains viruliferous [persistent,
behaviour and virus transmission. Recently, stud- semi-persistent (SP) or non-persistent (NP)] or the route of the
ies have shown that viruses can modify vector virus within its vector [non-circulative (NC) or circulative (CV)].
behaviour in a way that transmission is enhanced. More recently, a third classification was proposed based on the
localization of virus–vector retention sites: cuticula-borne or sali-
eLS subject area: Virology vary gland-borne. A number of viral and insect proteins have
How to cite: been found to control some virus–vector association, but many
Fereres, Alberto and Raccah, Benjamin (April 2015) Plant Virus remain unknown. Interference with vector landing by manipula-
Transmission by Insects. In: eLS. John Wiley & Sons, Ltd: tion of insect vision together with novel molecules that outcom-
Chichester. pete viruses from the retention sites in their vectors could help
DOI: 10.1002/9780470015902.a0000760.pub3 reducing plant virus epidemics.

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 Plant Virus Transmission by Insects

The Importance of Insect Vectors The Major Transmission Modes:

Most plant viruses depend on vectors for their survival for two
Persistent Versus Non-persistent;
principal reasons: Circulative Versus Non-circulative
Plant viruses demonstrate a high level of specificity for the group
1. An impermeable cuticle coats the plant epidermis, preventing of insects that may transmit them (a virus that is transmitted by
entry of virus particles (animal viruses enter readily through one type of vector will not be transmitted by another). CV viruses
natural openings). Most vectors are insects (non-insect vec- that propagate in their insect vectors are not considered in this
tors include mites, nematodes and fungi). Several plant article. The list of the major insect-borne virus groups and their
viruses may spread by contact or vegetative reproduction. vectors is summarised in Table 1.
Many insects such as hemipterans are well adapted to their
role as vectors by their capacity to pierce the epidermis and
Modes of transmission
delicately deposit the virus in the cytoplasm without risk-
ing the integrity of the plant cell. Recent findings propose In the 1930s, Watson and Roberts proposed modes of virus
that viruses have adapted to their vectors modifying their transmission by insects. The basis for their assigning viruses to
behaviour to maximise their own spread. these modes was the duration of virus retention in the vector.
2. Plants are rooted and lack independent mobility. Therefore, Originally, they proposed two modes: NP for short retention or
many viruses depend on insects for transport among hosts ‘less than the time the virus survives in leaf extracts’; and per-
(unlike animals that, by their own mobility, transport the sistent for extended retention, often for life. However, several
virus to new niches). viruses showed an intermediate retention in their vector. This led
Sylvester to designate the term SP viruses (cited in the study by
Raccah, 1986). In time, a different terminology was proposed for
Insect-borne plant viruses may cause severe or even crippling modes of transmission, based on the site at which the virus is
losses to many annual and perennial crops. On occasion, insects retained in the insect. Thus, NP viruses were termed stylet-borne,
are responsible for transition from a non-spreading form to the whereas persistent viruses were termed CV. In time, additional
epidemic form of diseases. Outbreaks of disease caused by insect attributes were attached to each of the modes of transmission
vectors are demonstrated in two examples. In perennials, the (Table 2). NP viruses are acquired and inoculated during brief
almost total destruction of the citrus industry in the 1930s in probing times, do not require a latent period in the vector and are
Argentina and Brazil is attributed to the aphid Toxoptera citricida. transmitted by many aphid species, mostly by those not colonis-
In annuals, outbreaks of Tomato spotted wilt virus (TSWV) or ing the crop. SP viruses need longer periods (hours) for acquisi-
begomoviruses in recent decades is attributed to the spread of the tion and transmission than do NP viruses. They have a narrower
thrips Frankliniella occidentalis and the whitefly cryptic species range of vector species. However, they do not require latent period
complex, Bemisia tabaci, respectively. and are lost when the vector moults. In persistent viruses, several
hours or even days are needed for efficient acquisition and inoc-
ulation. They have a narrow range of vectors, mostly those that
colonise the crop, pass through moult and need a latent period.
Many thorough biological, microscopical, immunological,
molecular techniques and electronic monitoring feeding devices
Taxonomy have subsequently been used to elucidate the mechanisms of
transmission. Two principal modes of transmission emerged: (1)
Insect vectors of plant viruses are found in 7 of the 32 orders CV or internal, where the virus crosses gut barriers and enters
of the class Insecta. The majority of vectors are found in the the circulatory system of the insect and accumulates inside the
two orders of insects with pierce-sucking mouthparts (number of salivary glands, and (2) NC or external, where the virus remains
species in parenthesis): Hemiptera (300) and Thysanoptera (6). attached to the cuticle of the insect mouthparts or foregut and
Other vector species are found in five orders of chewing insects: does not cross gut barriers.
Coleoptera (30), Orthoptera (10), Lepidoptera (4), Diptera (2) and
Dermaptera (1).

The Mechanism of Non-persistent

Mechanisms of transmission Transmission
Progress in the molecular biology of viruses and their vectors Virus particles, but not their naked nucleic acids, are the
has assisted greatly in the localization of virus retention sites in pathogenic units that are transmitted by insects to initiate infec-
their vectors and in identifying motifs in the viral genome and in tion (Ng and Falk, 2006). On the other hand, viral nucleic acids
viral and vector proteins, thus adding to the understanding of the (either DNA or RNA) are sufficient to cause infection when intro-
process of virus transmission by insects. duced to plant cells by artificial means (rubbing, bombardment,

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 Plant Virus Transmission by Insects

Table 1 Major groups of viruses and insect species that serve as vectors
Virus groups Mode Persistence Localization Insects involved
Alfamovirus NP Few hours Stylets Aphids
Badnavirus SP Days Unknown Mealybugs and leafhoppers
Begomovirus P Weeks Salivary glands Whiteflies
Crinivirus SP Days Foregut/Cibarium Whiteflies
Carlavirus NP Few Hours Stylets Aphids or whiteflies
Caulimovirus NP Many hours Acrostyle Aphids
Closterovirus SP Many hours Foregut Aphids or mealybugs
Comovirus SP Days Unknown Beetles
Cucumovirus NP Few hours Stylets Aphids
Curtovirus P Weeks Unknown Leafhoppers
Enamovirus P Weeks Salivary glands Aphids
Fabavirus NP Few hours Stylets Aphids
Ipomovirus SP Days Unknown Whiteflies
Ilarvirus P Days Unknown Thrips
Luteovirus P Weeks Salivary glands Aphids
Machlomovirus SP Many days Unknown Leafhoppers
Macluravirus NP Few hours Unknown Aphids
Mastrevirus P Weeks Unknown Leafhoppers
Nanovirus P Weeks Salivary glands Aphids
Potyvirus NP Few hours Stylets Aphids
Sequivirus SP Few hours Foregut Aphids
Sobemovirus SP Days Unknown Beetles
Torradovirus SP Days Stylets Whiteflies
Tymovirus SP Days Unknown Beetles
Waikavirus SP Few days Foregut Leafhoppers
NP, non-persistent; SP, semi-persistent; and P, persistent.

Table 2 Principal characteristics of the modes of virus transmission by insects

Feature External (non-circulative) Internal-circulative
Non-persistent Semi-persistent Persistenta
Duration of retention Brief (few hours) Intermedia (few days) Long
(days to months)
Duration of acquisition Brief Intermediate (hours) Long
and transmission
(seconds) (hours to days)
Latent period Not required Not required Required
Tissue where virus is Epidermis and parenchyma Epidermis, parenchyma Mostly phloem
acquired and inoculated and phloem
Pre-acquisition fasting Increase transmission No effects No effect
Passage through moult Negative Negative Positive
Insect species specificity Low Intermediate High
Sequential inoculation Poor Intermediate Good
a All circulative viruses except Pea enation mosaic virus (PEMV) are transmitted in a persistent manner. PEMV is assisted by an umbravirus that allows

the virus to invade tissues other than phloem and duration of the acquisition and inoculation periods is similar to viruses transmitted in a non-persistent
manner.

agro-infection, etc.). This suggests that protein molecules encap- continuous mechanical inoculation (see details in the following
sidating the nucleic acid are needed to interact with specific sites sections). Recently, the precise location and chemical nature of
present in the vector. Investigation of the role of the coat protein the first NC virus receptor within the vector mouthparts has been
(CP) in virus transmissibility was possible due to the occurrence identified. In addition, the specific probing behaviour activities
of virus strains that differ in their specificity for vector species of insect vectors linked to the transmission of plant viruses have
and the occurrence of strains that have lost transmissibility after also been elucidated with the help of electronic devices.

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 Plant Virus Transmission by Insects

The Role of the Capsid Protein from PAMV to PVX, resulted in its becoming aphid transmissible
(reviewed by Raccah et al., 2001).
in the Transmission
of Non-persistent Viruses
Cucumoviruses Virus-Encoded Proteins That Affect
Non-circulative Virus Transmission
For Cucumber mosaic virus (CMV), Gera and co-workers pro-
vided evidence that the genome of a poorly transmissible strain by Insects
became transmissible when encapsidated in vitro with the CP
of highly transmissible strain (Perry, 2001; Raccah et al., 2001; The vectors of potyviruses and caulimoviruses cannot transmit
Pirone and Perry, 2002). A follow-up of these studies was purified virus particles unless these are presented in mixture
achieved by Perry and co-workers who designed chimaeric RNA with a non-structural virus-encoded protein (Blanc et al., 2001;
3 cDNA constructs in order to introduce mutations in the CP Raccah et al., 2001).
(cited in the study by Perry, 2001). As a result of these studies,
three amino acid mutations in the CP were found to affect trans-
mission of CMV by Aphis gossypii. In a more recent study, these
Potyviruses
authors discovered that the transmissibility of CMV by Myzus The helper phenomenon was first reported by Kassanis and
persicae requires two mutations in the CP (in positions 25 and Govier (cited in the study by Raccah et al., 2001) showing
214) in addition to the mutations in positions 129, 162 and 168 transmission of the NAT virus PAMV became transmissible in
that were reported in their former study (Perry, 2001). Charge the presence of transmissible Potato virus Y (PVY). Later, they
alterations in the metal-ion-binding βH–βI loop exposed at the demonstrated that potyvirus transmission requires an HC in addi-
surface some non-transmissible CMV mutants are thought to be tion to the virus particles. In addition, they showed that trans-
responsible for the disrupted virus–vector interaction (Liu and mission occurs only if the virus is acquired in mixture or after
co-workers, cited in the study by Ng and Falk, 2006). the acquisition of the HC. This led to the formulation of the
‘bridge’ hypothesis, where the HC binds to aphid mouthparts on
Potyviruses one side and to virions on the other; thus, ensuring virus reten-
tion until release into the next host. Sequencing of the potyviral
In order to identify the determinants of potyvirus transmission by genome and identification of the resulting protein assisted in char-
aphids, the amino acid sequences of the CP of aphid-transmissible acterising it as a non-structural protein encoded by the HC-Pro
(AT) and non-aphid-transmissible (NAT) virus strains were com- region of the potyvirus genome. The helper function in transmis-
pared. The comparison revealed a conserved amino acid triplet, sion was assigned to the N-terminal and central regions of the
Asp-Ala-Gly (DAG) within the highly non-conserved and HC-Pro. The HC proteins have a predicted molecular mass rang-
exposed amino terminal end of the CP. The NAT strains were ing between 50 and 60 kDa. The proposed biologically active
found to have a mutated triplet. A mutation from Gly to Glu (DAG form is a dimer. The domains that are involved in vector trans-
to DAE) was introduced in the CP of an AT strain of Tobacco mission were traced by comparing strains with active and inactive
vein mottling virus (TVMV), rendering it non-transmissible. HC. For TVMV, the loss of HC activity was associated with a
The role of the DAG motif of the CP in aphid transmission was mutation in the highly conserved Lys-Ile-Thr-Cys (KITC) motif
then confirmed also for an NAT strain of Zucchini yellow mosaic where Lys was changed to Glu (E to K). This mutation was also
virus (ZYMV) by changing Thr to Ala (DTG to DAG), this time present in other potyvirues (mutants of PVY and ZYMV HCs).
restoring transmissibility. Effects on transmission of TVMV The KITC motif of the HC is not involved in binding to viri-
were noted not only for the DAG triplet but also for amino acids ons [transmission-defective ZYMV-Ct with K instead of E in the
in its immediate vicinity (several studies all cited in the study by KLSC motif was bound efficiently to virions in overlay blotting
Raccah et al., 2001). experiments (reviewed by Raccah et al., 2001)].
Electron microscopic studies provided evidence that the DAG Another conserved motif in the central region of the HC-Pro
motif in potyviruses is involved in retaining the virus in the gene, Pro-Thr-Lys (PTK), was found to be associated with HC
aphid’s mouthparts. The mechanism is apparently via an inter- assistance in transmission of ZYMV. A mutation from Pro to Ala
action of the DAG with a virus-encoded protein named helper in the PTK motif resulted in loss of helper activity. The PTK motif
component (HC), as shown by the protein-blotting overlay tech- was found to affect the HC binding to virions in overlay blotting
nique (reviewed by Raccah et al., 2001). experiments (reviewed by Raccah et al., 2001). A proposed model
summarising the interaction among the virions, the HC and the
Potexviruses aphid stylets is depicted in Figure 1.
The role of the HC in retaining the virus in the stylet was
Potato aucuba mosaic virus (PAMV) is not transmissible by shown by comparing aphids fed on mixtures of transmissible
aphids, but its transmission is possible when it is assisted by TEV or TVMV virions and functional PVY HC or TVMV HC
potyviruses. The DAG motif of the CP sequence of PAMV is not (motif KITC) with those fed on non-functional HC (motif EITC)
present in Potato virus X (PVX) but transfer of the DAG motif (reviewed by Raccah et al., 2001).

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 Plant Virus Transmission by Insects

Food canal

Potyvirus

Common canal
Salivary canal
Cuticular lining
CaMV

CMV
P2

HC-Pro

Figure 1 Model describing the different strategies for virus–vector interaction in non-circulative transmission by aphids. These strategies enable retention
of virus particles on the common canal of the maxillary stylets at the surface of the cuticular lining. In the capsid strategy, CMV, a motif of the coat protein
directly binds to the vector’s receptor. In the helper strategy used by potyviruses, virus–vector binding is mediated by the helper component (HC-Pro), which
creates a ‘molecular bridge’ between the two. HC-Pro can be acquired alone or together with the virion. Caulimoviruses (CaMV) also use the helper strategy,
but a different protein (P2) acts as a bridge between the virus and the vector.

Caulimoviruses led to the speculation that a helper is needed for transmission of

carlaviruses and closteroviruses (Raccah et al., 1990).
Caulimoviruses have also adopted a helper-dependent trans-
mission strategy, but in a rather more complex manner than
potyviruses. Cauliflower mosaic virus (CaMV) requires two Mode of transmission of viruses
viral-encoded non-structural proteins, P2 and P3. A P2-P3-virion by beetles
complex is formed, with P2 binding to the aphid, whereas P3
binding to the virions (Drucker et al., 2002). Furthermore, the HC Beetle vectors of plant viruses are known in four families
motif directly involved in specific vector recognition was iden- (Chrysomellidae, Coccinellidae, Curculionidae and Meloidae)
tified at position 6 of the N-terminus of P2. A single mutation (Gergerich, 2001). Beetle-borne viruses have a unique mode of
of one amino acid that may appear spontaneously changes the transmission. The viruses are transmitted in the beetle’s regur-
spectrum of vectors transmitting CaMV (Moreno et al., 2005). gitant and there is no latent period in the vector. The original
Recently, Martinière et al. (2013) found that the formation of assumption was that regurgitant components selectively inac-
transmission-specific inclusion bodies of CaMV are not acquired tivate particles of beetle non-transmissible viruses. However,
by their aphid vectors, but rather, they react immediately to intra- mixing preparations of a variety of viruses with beetle regur-
cellular stylet punctures and transiently dissociate, forming trans- gitant had insignificant effect on most viruses (beetle-borne or
missible P2-virion morphs throughout the cell that increase the not). Some beetle-borne viruses are CV, as they were found to
acquisition success of the virus. move into the insect haemolymph immediately after ingestion.
Indirect evidence suggests that helper is involved in several Beetles can also be rendered viruliferous by injecting virus into
other systems. The semi-persistently transmitted Parsnip yellow the haemolymph. However, Wang and co-workers found that
fleck virus is not transmissible by aphids unless acquired with the beetles may transmit viruses even if they are not carried in the
Anthriscus yellows virus. A dense material with virus-like parti- haemolymph. The retention of inoculativity of beetles differs
cles was seen in aphids’ mouthparts after acquisition of the virus. for different beetle vectors; thus, Epilachna varivestis retains
Rice tungro spherical virus (RTSV) is transmissible by several Cowpea severe mosaic virus for 1 day, whereas Cerotoma trifur-
Nephottetix leafhopper species. RTSV assists the transmission cata transmitted the same virus for several days. The virus does
of a second virus, the Rice tungro bacilliform virus. In addition, not propagate in the beetle as the virus titre declines with time.
Maize chlorotic dwarf virus is semi-persistently transmitted by Gergerich and co-workers demonstrated the unique role of the
leafhoppers and is considered to have HCs (cited in the study by regurgitant in the infection process. Viruses not transmissible by
Hull, 1994). Lack of vector transmissibility of purified virions beetles were mechanically infectious to wounded hosts, but when

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 Plant Virus Transmission by Insects

regurgitant was added to the inoculum mixture, only beetle-borne to the salivary glands, then, (6) via the salivary duct in the maxil-
viruses remained infectious. The inability of virus particles to lary stylets, the virus is transported by the saliva to internal plant
infect hosts was not due to inactivation since, when purified tissue (mostly phloem) (Figure 2).
away from the regurgitant virus particles regained infectivity. Virus particles are retained in the haemolymph for several
This finding suggests that an inhibitor in the regurgitant affected weeks. Survival in the haemolymph may depend on the presence
the host itself or the interaction between virus and host and that of symbionin (see the following discussion). In the Luteoviri-
viruses transmissible by beetles differ from other viruses in the dae, virus particles that are carried in the haemolymph need to
fast translocation to non-wounded cells through the xylem and cross the basal lamina of the accessory salivary gland (ASG) in
in the manner in which they initiate primary infection. order to be ejected by the salivary secretions to the plant tissues.
The basal lamina of ASG consists of collagen that may serve as
a selective filter, allowing differential binding and passage virus
The Mechanism of particles. On the way to exterior, virus particles must be trans-
Non-propagative, Circulative ported across a third preferential barrier, the plasmalemma of the
ASG, by receptor-mediated endocytosis. It is likely that the virus
Transmission movement across these barriers is involved with different viral
proteins or protein domains. Nanoviruses and begomoviruses,
CV (internal) viruses are carried in the interior of the vector however, are specifically retained in the principal salivary glands
body (Gray and Gildow, 2003; Hogenhout et al., 2008; Gray (PSGs) (Watanabe and co-workers, cited in the study by Gray
et al., 2014). Some of the CV viruses propagate in the insect et al., 2014). Circulative viruses are not transovarially transmit-
and are therefore termed CV-propagative. A list of CV and ted with the exception of the begomovirus Tomato yellow leaf curl
CV-propagative viruses is given in Table 1. The luteoviruses and virus-Is (TYLCV-Is) that can also be sexually transmitted.
the enamovirus PEMV are the best-studied CV viruses.

The transmission cycle The role of viral capsid protein for insect
transmission of circulative viruses
The transmission cycle of a CV virus includes six stages: (1) The
aphid stylets, while piercing and sucking are inserted intercellu- Protein subunits are important for the specificity of transmission
larly to reach the phloem sieve elements, (2) ingestion from the of CV viruses. Rochow showed that strains of Barley yellow
infected host plant reaches the alimentary system of the vector, dwarf virus (BYDV) that are transmitted by one aphid species
(3) passage of the virus through the vector’s gut, (4) retention in become transmitted by another aphid species if co-infected with
the haemocoel or other internal tissues, (5) passage of the virus another strain of BYDV (reviewed by Gray et al., 2014). This

Hemocoel

PSG ASG

Midgut

Hindgut

Food
Salivary canal
canal

Phloem

Figure 2 Schematic diagram of an aphid feeding and luteovirus transmission. Arrows indicate the circulative route for virus transmission through the insect’s
body. Virus particles circulate up through the food canal and cross the midgut and hindgut reaching the haemocoel. Then virus particles cross the accessory
salivary glands and return to the plant via the salivary canal (from Gray et al., 2014). Reproduced with permission form from Gray et al., 2014 © Elsevier.

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 Plant Virus Transmission by Insects

phenomenon was attributed by Rochow to heteroencaspsidation, Insect Proteins Involved

where the non-transmissible RNA is encapsidated with some
protein subunits of the transmissible strain. in Virus–Vector Interactions
Recently, the retention sites and specific proteins acting as
receptors of both NC and CV viruses have been identified. A
non-glycosylated protein deeply embedded in the chitin matrix of
Viral Proteins Involved in the aphid’s maxillary stylets is involved in the retention of CaMV.
Transmission: the Coat Protein This protein receptor present in three effective vector species but
absent in a non-vector is located exclusively at the stylet tips in
and the Read-Through Protein the bottom bed of the common duct where the food and salivary
canals fuse together (Uzest et al., 2007). The acrostyle, a specific
Pea enation mosaic virus (PEMV) and luteovirus particles are anatomical structure within the common duct of aphid’s maxil-
composed of two types of capsomeres (Gray et al., 2014). The lary stylets, was found to be the precise location where CaMV is
predominant one is CP (c. 22–24 kDa). Another minor one, retained by their vectors (Uzest et al., 2010). Using a proteomic
believed to be on the surface of the virion, is the read-through approach, four cuticular proteins that were extracted, separated
(RT) protein (c. 55–58 kDa). The RT protein results from a larger and identified from M. persicae were able to bind in vitro to active
protein translated via the weak stop codon of the CP. The open potyviral HC-Pro but not to the mutated HC-Pro of the same
reading frame encodes for a 72- to 74-kDa protein, of which virues (Dombrovsky et al., 2007).
the C-terminal half of the resulting protein is digested yielding A similar approach was taken to show that four proteins from
a 55- to 58-kDa proteins. This protein is also found when CP is Schyzaphis graminum are involved in the ability to bind to the CV
obtained from virus preparations. Virions encapsidated with the Cereal yellow dwarf virus-RPV polerovirus (Yang et al., 2008).
CP alone were not transmitted by aphids (but are found in the These proteins from S. graminum origin seem to play a key role
haemocoel following feeding). Moreover, these virions are infec- in the high level of vector specificity, possibly by facilitating
tive when agro-inoculated (cited in the study by Gray and Gildow, the passage of the virus through the gut and salivary gland
2003). These findings led to the conclusion that the RT protein tissues. Similarly, two proteins isolated from head tissues of the
is needed for aphid transmission. Mutants of Beet western yel- aphid vector, Sitobion avenae, have been identified as potential
low virus (BWYV) without the RT protein were not detectable in receptors for another CV virus (BYDV-MAV; Luteoviridae) (as
the ASGs and are non-transmissible by aphids. Mutants of RTD cited in the study by Yang et al., 2008).
in various domains at the C-terminus did not affect aphid trans- Specific retention of a crinivirusin in the anterior foregut and/or
missibility. Mutation at the N-terminus of the RTD resulted in a cibarium of its whitefly vector was mediated by the minor cap-
protein that did not incorporate in the virus particle, but ingested sid protein CPm (Chen et al., 2011). This was observed using a
particles are found in haemolymph. This suggests that the CP pro- unique immunofluorescent localization approach in which viri-
vides the signal for crossing the hindgut barrier, whereas the RT is ons or recombinant virus capsid components were ingested by
the protein that associates with the ASG. However, recent reports whiteflies using artificial membrane feeding.
show that particles encapsidated with the 22-kDa CP alone were Transcriptional response of B. tabaci to a begomovirus iden-
found not only in the haemolymph but also in the ASG cells and tified 1606 genes and 157 biochemical pathways that were dif-
in the salivary duct. This finding seems to be in contrast with the ferentially expressed in viruliferous whiteflies, explaining why a
hypothesis that the RT is needed for crossing the ASG barrier. negative impact of the virus on the longevity and fecundity of the
In addition, in the case of the nanovirus Faba bean necrotic B biotype of B. tabaci was found (reviewed by Gray et al., 2014).
yellows virus (FBNYV), a helper protein is required for trans- These findings may lead in the future to the use of viral genes
mission. However, the origin – virus or plant – of the helper encoding for proteins that are defective in the ability to assist
protein was yet not determined (Franz and co-workers as cited transmission in transgenic plants. This may prevent vector inocu-
in the study by Gray et al., 2014). Furthermore, proteins present lation. In addition, plants encoding for molecules (e.g. peptides)
in the phloem of cucurbits have been reported to enhance virus able to bind to cuticle protein receptors in the vector mouthparts
transmission of luteoviruses (Bencharki et al. and co-workers as may interfere with the process of virus retention. If successful,
cited in the study by Gray et al., 2014). this form of virus prevention will complement those based on
reduced multiplication and movement.

Geminiviruses
The Interaction between Bacterial
The role of the CP in Geminivirus transmission was determined
by exchanging the CP gene of two viruses differing in vector Endosymbinots Proteins
specificity. Thus, injection of the recombinant whitefly-borne and Circulative Viruses
African cassava mosaic virus (ACMV) with the Beet curly top
virus (BCTV) CP enabled transmission by leafhoppers of ACMV. Aphids are known to host primary endosymbiotic bacteria of the
This suggests that the CP is needed to pass from the haemocoel genus Buchnera in specialised cells located in the abdomen myce-
to the salivary glands (Hull, 1994). tome (Gray et al., 2014). These bacteria produce a protein called

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 Plant Virus Transmission by Insects

symbionin. The RT protein mentioned earlier was found to inter- to produce a GroEL protein that facilitates transmission of bego-
act with the GroEL (a bacterial protein showing homology with moviruses. Furthermore, some other whitefly endosymbionts of
symbionin). Mutational analysis of the RT protein of beet west- the Rickettsia genus contribute to the susceptibility to chemical
ern yellows luteovirus attributes the virus-binding capacity to a insecticides and heat tolerance (reviewed by Gray et al., 2014).
conserved region in the GroEL molecule. BWYV engineered to
be encapsidated with CP alone (with no RT protein subunits)
did not bind to Buchnera GroEL. In addition, in vivo studies Analysis of Virus Transmission
showed that BWYV virions lacking the RT protein were signifi-
cantly less persistent in the haemolymph than were virions with
by Electrical Penetration Graphs
the RT protein. This led to the hypothesis is that the interaction (EPGs)
between Buchnera GroEL and the RT protein protects the virus
from rapid degradation in the haemolymph. Comparison of the Electronic devices can distinguish between the intercellular and
RT domain from different luteoviruses and PEMV revealed sev- intracellular environments, which makes it possible to know
eral conserved amino acid residues that may be important for the when plant cell membranes are punctured by insect stylets
interaction with Buchnera GroEL. In a more recent study, Hogen- (Fereres and Moreno, 2009; Stafford et al., 2012). When a cell
membrane is punctured a very distinctive electrical penetration
hout and co-workers demonstrated by mutational analysis of the
graph (EPG), signal is recorded in the form of a potential drop
gene encoding for MpB GroEL that the PLRV binding site is
(pd), which is associated to NP virus transmission (Powell, 1991).
located in the equatorial domain and not in the apical domain of
Other distinct waveforms and activities that are relevant to virus
the symbionin (cited in the study by Gray and Gildow, 2003). The
transmission of phloem-restricted viruses by aphids and white-
exact function of the symbionin, however, is not known. Bou- flies are E1 (phloem salivation, linked to virus inoculation) and
vaine et al. (2011) reported that symbionin is restricted to the E2 (phloem ingestion, linked to virus acquisition) (Figure 3).
bacteriocytes and embryos, and not present in haemolymph or Acquisition of stylet-borne viruses occurs after very brief (<1
gut in Acyrthosiphon pisum and Rhopalosiphum padi, and there- min) probes and only when cell membranes are punctured by the
fore cannot participate in the protection of virus particles in the stylets as shown by electron microscopy and EPG (pd signals).
haemolymph. Therefore, it is not known if symbionin contributes Detailed analysis of direct current-EPG signals during intracellu-
in protecting luteoviruses as they travel through the haemolymph lar stylet punctures (pd) allows to differentiate three specific and
to the salivary gland or facilitate passage across the ASG barrier. distinct subphases: II-1, II-2 and II-3. Acquisition of stylet-borne
In addition to the primary endosymbiont Portiera, the whitefly viruses is associated to subphase II-3. Acquisition during the first
B. tabaci carries a secondary endosymbiont, Hamiltonella, able pd is not only restricts to typical NP viruses such as CMV or

Probe ‘C’

Aphid attached Potential drop

to a gold wire while
probing on a leaf

Epidermis
EPG waveform in the epidermis & I II III
mesophyll: Potential Drop or ‘pd’
associated to the transmission of Mesophyll
potyviruses, cucumoviruses pd sub-phase: II-1 II-2 II-3 8 sec
caulimoviruses & enamoviruses

Phloem

E1 E2
EPG waveforms in the phloem: E1 & E2
1s
linked to the transmission of
luteoviruses and poleroviruses

Figure 3 Electrical penetration graphs (EPGs) associated to the transmission of plant viruses by aphids.

8 eLS © 2015, John Wiley & Sons, Ltd. www.els.net

 Plant Virus Transmission by Insects

PVY but also occurs for SP viruses such as CaMV. The main Aphid attraction to volatiles emitted from plants infected with
difference is that CaMV is preferentially acquired after com- CV viruses was also reported (Eigenbrode et al., 2002). Further-
mitted phloem ingestion, whereas typical NP viruses are only more, attraction to infected plants may be reversed after the vector
acquired during brief superficial intracellular punctures. Work feeds on a virus source and becomes viruliferous. Rajabaskar
conducted by Fereres and co-workers showed that subphase II-1 et al. (2013) found that non-viruliferous M. persicae settled pref-
within the first intracellular puncture was associated to the inoc- erentially on potato plants infected with Potato leafroll virus,
ulation of NP viruses (PVY and CMV). On the basis of this whereas the reverse was the case for viruliferous aphids that pre-
finding and the fact that both salivary and alimentary canals fuse ferred mock-inoculated plants.
together in a common duct at the very tip of the maxillary stylets, Many other examples show how plant viruses can manipu-
the ingestion–salivation hypothesis was proposed. The results late vector behaviour to enhance their transmission and spread.
obtained also suggested that watery salivation was the mecha- TSWV-infected thrips changed their probing behaviour after
nism involved in flushing out virus particles from the common virus acquisition and made much more inoculative probes than
duct during cell penetration. Later work using PEMV as a marker non-infected thrips (Stafford et al., 2011). The settling and feed-
for intracellular salivation confirmed this hypothesis (Powell, ing behaviour of B. tabaci was modified after acquisition of
reviewed by Fereres and Moreno, 2009). Later, EPG-assisted TYLCV-Is in a way that whiteflies settled faster and increased
transmission studies showed that inoculation of the SP CaMV the duration of the salivation phase linked to the transmission of
was linked exclusively to subphase II-2 of the first pd suggest- the virus (Moreno-Delafuente et al., 2013).
ing that NP and SP viruses are inoculated in a different manner
(Moreno et al., 2012). Studies on the feeding behaviour activities
associated to the transmission of plant viruses by aphids, white- Control of Virus Diseases
flies, hoppers, mealybugs and thrips have been recently reviewed by Interfering with Vectors
by Stafford et al. (2012).
and Transmission
In this article, we discuss measures aimed against vector activity
Effects of Virus Infection on Vector and transmission (Antignus, 2012; Bragard et al., 2013). These
Behaviour measures are among the most successful approaches used to
suppress virus epidemics. Other control measures (e.g. breeding
As explained earlier, viruses need their vectors for survival and for resistance to the pathogen, sanitation, prevention and natural
spread (Mauck et al., 2012). Viruses have evolved and adapted and pathogen-derived resistance) will not be discussed in this
to their insect vectors to increase their chances of transmission article and reader should consult the Further Reading list. Control
from plant to plant. There are many cases in which both viruses measures against vectors and vector activities can be grouped into
and vectors benefit from their mutual association by changes four classes: (1) reducing vector populations, (2) reducing virus
induced in their shared host plant after virus infection. In several sources, (3) interference with vector landing and (4) interference
cases, the intrinsic rate of increase and the proportion of alate with the transmission process.
aphid morphs often increase in virus-infected plants (reviewed Reducing vector populations: Despite the wide range of
by Fereres and Moreno, 2009). However, a more striking hypoth- insecticides available, chemical control is not the preferred
esis (called vector manipulation) was proposed by Mauck and solution to prevent vector activity. Many viruses are introduced
co-workers to explain how viruses could change vector behaviour into crops by visiting insects that inoculate during their first
in a way that the transmission and spread of a NC virus was probing activities. Vectors for NP (and partly SP) viruses need
enhanced. In their studies, they found that CMV-infected plants relatively short inoculation times – much shorter than the time
emitted a deceptive signal of plant volatiles that attracted aphids, needed for insecticides to kill. In addition, insecticides can
which rejected the less suitable infected plant soon after probing. induce restlessness in insects, with the result that they make
This particular pull–push behaviour of aphids is known to opti- more inoculation attempts than do calm insects. Exceptions are
mise NP virus transmission. Their findings show that the trans- vectors that colonise the crop and transmit phloem-restricted
mission mechanism is a major factor shaping pathogen-induced viruses, for which insecticides may reduce virus spread. New
changes and how viruses have learned to manipulate vector biotechnology-based approaches such as genetically modified
behaviour to optimise their own spread. Similar results were aphid-resistant plants that express protease inhibitors, dsRNA,
found by Carmo-Sousa et al. (2014) that revealed a sharp change antimicrobial peptides or repellents can effectively reduce vector
in aphid settling and probing behaviour over time when exposed numbers (Will and Vilcinskas, 2013). In aphids, RNAi-mediated
to CMV-infected plants. Soon after, plant contact aphid vectors gene silencing can reduce the expression of salivary gland pro-
dramatically increased the number of short superficial probes and teins or kill the pea aphid, A. pisum when fed species-specific
intracellular punctures when exposed to CMV-infected plants. dsRNA targeting vATPase transcripts (Whyard et al., 2009).
At a later stage (second hour of recording), aphids diminished Reducing virus sources: Use of virus-free seeds and/or prop-
their feeding on CMV-infected plants as indicated by much less agative organs results in minimal primary infection. This can be
time spent in phloem salivation and ingestion (E1 and E2). Such complemented by removal of sources of infection in and around
changes in aphid behaviour on CMV-infected plants leads to opti- the crop, removal of plant remains from a former season and, if
mum transmission and spread of the virus. necessary, creation of a time gap between crops and/or space gap

eLS © 2015, John Wiley & Sons, Ltd. www.els.net 9

 Plant Virus Transmission by Insects

and retention by aphids. Mineral oil of an appropriate viscos-

ity and unsulfonated residues was found effective to reduce the
efficiency of transmission by vectors. This measure is still pop-
ular for protection against NP viruses, particularly in nurseries.
The mode of action seems to be by interference with virus
binding by probing aphids. The leaf surface must be fully cov-
ered: full coverage demands frequent applications (up to twice
a week) of large volume at high pressure. Combination of oil
with pyrethroids (insecticides that have insect-repelling qualities)
was tested successfully in Israel and in England (Raccah, 1986).
Novel molecules – for example, peptides – could be designed to
outcompete with virus CP or non-structural virus-encoded pro-
teins needed for virus attachment to insect receptors and thus
interfere with transmission (Blanc et al., 2014).

Acknowledgements
Figure 4 Non-woven agrotextiles are commonly used as a physical barrier
to protect vegetables from insect landing and preventing virus epidemics. In memorial of Prof. Michael Thresh, for his outstanding contri-
butions in the field of Plant Virus Epidemiology.

between plots. These operations will reduce the numbers of vir-

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