how plant virus survive in their host

1. Name: Mesele Tilahun ID: 02013035 Exam type: PhD qualification Dec.21/2021
Question.1) Plant virus have to make it's own protein in host cell. The translation system of host is
believed to be one mRNAto one protein. However, plant virus should express many different proteins
from a single mRNA.There are many different ways for plant virus to overcome this discrepancy in the
translation system. Describe various strategies (at least seven strategies) of translation for plant virus to
survive in plant host cell. (Dr. Moon).
Viruses have evolved a wide range of sophisticated mechanisms to optimize the ability to replicate or
at least to survive the host defences. Regulation of gene expression is a key aspect of such processes
and control of mRNAtranslation in particular represents an important focus for virus-host interactions.
Viral genomes do not encode for any components of the translation machinery. Hence viral protein
synthesis is wholly dependent on hosts. It is not so surprising that some viral RNAs have similar
structures to the 5’-cap and 3’-poly (A) tail mimicking eukaryotic mRNA to compete for translation
apparatus. However, most RNA viruses have been shown to evolve alternative translation initiation
mechanisms. In addition to translation initiation, RNA viruses also express their overlapping open
reading frames (ORFs) by recoding and read-through during the elongation and termination stages.
Some strategies of translation for plant virus to survive in plant host cell is reviewed below.
1. Multi genome: mono partite to more than octapartite genome
The genome of plant viruses is composed of one or several single-stranded (ss) or double-stranded (ds)
RNA or DNA molecules. Among DNA viruses the caulimoviruses, of which cauliflower mosaic virus
is the main representative, possess a dsDNA genome with ss interruptions, and geminiviruses with
mono- or bipartite morphology possess a circular ssDNA genome. The vast majority of plant viruses
possessssRNA genome of + polarity since their RNAsare useddirectly asmessengerRNA.There exist
only fewplant viruses whose genome is composed of dsRNA(plant reoviruses, e.g.,wound tumor virus)
or of ssRNA of (–) polarity (complementary to mRNA; plant rhabdoviruses, e.g. tomato spotted wilt
virus); in these cases,the RNA is first transcribed and only then translated. Among plant pathogens
there exist also viroids (free-living small circular ssRNAs that induce their own replication and are not
translated), satellite particles (whose small linear RNA requires a helper virus for replication, modifies
the symptoms induced by the associated virus and is often not translated: the helper virus is not
dependent on the satellite) and virusoids (containing viroid-like RNA in addition to virus-like, linear
RNA; the viroid-like RNA possesses properties in common with viroids and satellite RNAs, and in
some cases it seems to be required for virus infectivity).
Multipartite virus genomes are composed of severalsegments, eachpackagedin a distinct viral particle.
Multipartite viruses rapidly modify the copy number of each segment/gene from one host species to
another, a putative benefit if host switches are common. One multipartite virus functions in a
multicellular way: The segments do not all need to be present in the same cell and can functionally
complement across cells, maintaining genome integrity within hosts. Viruses show exceptional
variation in how they package their genetic information for transmission to future generations. Most
viruses have their genetic information carried by a single molecule of DNA or RNA packaged in a
transmission vehicle, the viral particle. Multipartite viruses are very common parasites of plants and
fungi.
The genome parts of multicomponent viruses are separately encapsidated. For infection of a cell, more
than one particle is thus required, and this reduces the efficiency of mechanical transmission. However,
in nature these viruses are often transmitted by seed or by invertebrate vectors allowing large numbers
of virus particles to enter per cell. Since RNA-dependent RNA polymerases lack error-correcting
mechanisms, the estimated error frequency for these enzymes lies between 10-3
and 10-4
. Clearly, for a
fixed error level, the longer the RNA,the greater the possible loss of information. This problem may be
resolved by the presence of multipartite genomes.

2. Since during encapsidation different genomic RNAs are withdrawn randomly from the intra cellular
pool of RNAs,a number of combinations exists and this strategy may ensure that the optimally adopted
combination of composite viruses arises at each round of replication. With multipartite genome viruses,
new pseudo recombinants can be obtained in vitro and in vivo. This increase in variability could
represent an important evolutionary advantage, especially since recombination at the RNA level takes
place only at low frequency. Division of the genome into more than one segment can result in
monocistronic RNAs. For example, the genome of brome mosaic virus (BMV) consists of three RNA
segments, of which RNA1 and 2 contain a single cistron. Other viruses such as cucumber mosaic virus
(CMV) and alfalfa mosaic virus (AlMV) are similar in this respect. Red clover necrotic mosaic
dianthovirus (RCNMV) has two RNAsegments, of which RNA2 is monocistronic.
Fig.1 show Segmented, tripartite linear ssRNA(+) genome composed of RNA1, RNA2, RNA3. Each genomic
segment has a 3' tRNA-like structure and a 5'cap.
2. Polyprotein
Polyprotein processing is a major strategy used by many viruses to generate many functional protein
products from a single open reading frame. In this strategy the viral genome contains a long ORF which
is translated and then cleaved into smaller, functional proteins by viral proteinases. Since these
proteinases form part of the polyprotein, initial cleavages should be autocatalytic.
The potyvirus group is one of the most important groups of plant viruses and members characteristically
express their genome through a polyprotein process. The potyviral genome is approximately 10 kb in
length and encodesa single polyprotein that is processedby three viral proteinases to yield nine or more
mature proteins. Two of the proteinases, P1 and helper component-proteinase (HC-Pro),each catalyse
cleavage only at their respective C termini. The remaining cleavage sites (at least six) are processed by
the NIa proteinase, a homologue of the picornaviral 3C proteinase. This enzyme possessesa serine-type
proteinase fold but contains a nucleophilic Cys residue ratherthan Serat the active site. The best known
members of the group are tobacco etch virus (TEV) and potato Y virus (PVY).
At present most is known about the gene products of TEV and their expression. Thus, the following sections deal
mainly with this virus. The main features of the TEV genome are: (1) a VPg is attached to the 5' end. (2) a 5' non-
coding region of 144 nt rich in A and U; (3) a single large ORF of 9161 nt initiating at residue 145-147, which
could encode for a polyprotein with about 3000 amino acids (about 340 K); and (4) a 3' untranslated region of 190
bases terminating in a poly(A) tract.
Fig.2 show polyprotein; Monopartite, linear, ssRNA(+) genome of 10 kb in size. 3' terminus has a poly
(A) tract. 5' terminus has a genome-linked protein (VPg)

3. 3. Subgenomic RNA
subgenomic RNAs which are usually 3’ co-terminal with one of the genomic RNAs and are packaged
when they possess the encapsidation site. The viral RNAs are encapsidated in the same and/or distinct
virus particles, the capsid being generally made up of a single protein species.The expression of internal
genessuch coat protein (CP)of the positive RNAviruses is frequently mediated via subgenomic RNAs.
These mRNAs are encapsidated in some viruses, but not in others. Among plant RNA viruses, the
mechanism of synthesis of the subgenomic RNA encoding the CP has been observed in several viruses,
i.e., TMV, CMV. Two mechanisms explained in the synthesis of subgenomic RNA species:
(1) During (-) RNA strand synthesis by the RdRp, premature termination could lead to the formation of
(-) RNAstrandsof subgenomic length that could serve as template to generate the subgenomic (+) RNA;
alternatively,
(2) the subgenomic (+) RNA could be synthesised via internal initiation on (-) RNA strands of genomic
length. The evidence from in vivo and in vitro experiments with various RNA viruses clearly tends to
favor the second mechanism. Since subgenomic RNAs contain at their 3' end the elements required for
the production of complementary subgenomic RNAchains, various explanations have been put forward
to account for the lack of autonomous replication of subgenomic RNA. These are that
(1) the sequence contained within the subgenomic RNA is insufficient for replication of the subgenomic RNA;
(2) the subgenomic RNA, which is frequently a highly efficient mRNA, may not be available for replication; and
(3) the subgenomic RNA would be produced late in infection or at time when negative-strand synthesis has ceased.
Synthesis of subgenomic (SG) messenger RNAs (mRNAs) by (+) strand RNA viruses allows the
differential expression of specific viral genes, both quantitatively and temporally. SG RNAs have the
following properties:
(i) they are made in infected cells but do not interfere with the normal course of viral
replication;
(ii) the SG RNAsequences are shorter than their cognate genomic RNAs;
(iii) their sequences are usually co-terminal with the 3′ genomic sequence but sometimes are
co-terminal with the 5′ sequences.Yet other viruses make SG RNAs which contain a 5′ co-
terminal leader joined to a 3′ co-terminal sequence;
(iv) typically, whether a messenger SG RNA contains only one ORF, or multiple ORFs, with
some rare exceptions, only the 5′ ORF is translated.
Although most SG RNAs function as messengers and are translated, other SG RNAs, generally those
with 5′ co-terminal sequences, have other functions. Among (+) strand RNA viruses of plants, only
viruses of the Potyviridae and Comoviridae as well as the Sequiviruses of the Sequiviridae family do
not use this strategy
Fig.3 subgenomic; monopartite, linear, ssRNA(+)
genome of 6.3-6.5 kb. The 3'-terminus has a tRNA-like
structure. The 5' terminus has a methylated nucleotide
cap (m7G5'pppG).
Many (+) strand RNAviruses use subgenomic (SG) RNAs as messengers for protein expression, or to
regulate their viral life cycle. Three different mechanisms described for the synthesis of SG RNAs. The
first mechanism involves internal initiation on a (−) strand RNAtemplate and requires an internal SGP
promoter. The second mechanism makes a prematurely terminated (−) strand RNA which is used as
template to make the SG RNA.The third mechanism uses discontinuous RNA synthesis while making
the (−) strand RNAtemplates. Most SG RNAs are translated into structural proteins or proteins related
to pathogenesis: however other SG RNAs regulate the transition between translation and replication,
function as riboregulators of replication or translation, or support RNA–RNArecombination. In this
review we discuss these functions of SG RNAsand how they influence viral replication, translation and
recombination.

4. The mechanism of BMV subgenomic RNA4 formation from genomic RNA3by using the in vitro RdRp
system provided, the first unequivocal evidence that the subgenomic RNA of a positive-strand RNA
virus is synthesized (at least in vitro) by internal initiation of positive-strand RNA synthesis on a
negative-strand template. The internal promoter involved in RNA4 synthesis was identified using an
altered negative-RNA3 strand as template. Deletions 3' from the RNA4-corresponding sequence were
performed on the RNA3negative-strand to identify the core sequence required for initiation of positive-
RNA4 synthesis. They are located, respectively, 20 nucleotides downstream from the RNA4 start site
and 17 nucleotides into the RNA4 sequence. An oligo (U) region 3' from this negative-strand core
promoter sequence seems to function as a spacer, ensuring accessibility of the promoter for the viral
RdRp, since its removal leads to an important decrease in RNA4 synthesis. Further studies have been
performed to investigate the nature and behaviour of sequences influencing RNA4 production in vivo.
The roles of additional downstream sequences and positional effects on promoter functions have also
been studied. Four regions can be identified as playing a role in RNA4 initiation in vivo. First, as
demonstrated in vitro, initiation of subgenomic RNA synthesis does not require more than 17
nucleotides of the RNA4 sequence (nucleotides +1 to +17). Second, sequences 3' from the start site of
RNA4 can be divided into three different domains. They correspond to the 20 nucleotides upstream
(nucleotide -1 to -20), an oligo(U) stretch (nucleotides -20 to -38) downstream from this region, and
finally, an (A+U)rich sequence (nucleotides -38 to -95) adjacent to the oligo (U).The region containing
the first two domains contributes favouring correct initiation of positive-RNA synthesis. The entire
promoter sequence is 112 nucleotides long. TMV has two separate subgenomic promoter sites in the
negative-strand, which control synthesis of the mRNAs for P30 (expressed relatively early) and CP.
4. Read-through
In some RNAs, the stop codon of the 5 gene may be “leaky”, which allows a proportion of ribosomes
to continue translation until the next stop codon. Such “read-through”, or “stop-codon suppression”,
results in a C-terminally extended version of the protein. The first cistron in the genomic viral RNA may
have a "leaky" termination codon (UAG or a UGA) that can be suppressed by a host transfer RNA (tRNA), thereby
permitting some of the ribosomes to read through into a downstream cistron as a result, giving rise a second longer
functional polypeptide. Therefore, the read-through process requires at least two elements.
- First, a suppressor tRNA; the nature of a possible candidate has been proposed for TMV and for tobacco
rattle tobravirus.
- Second, the nucleotide context surrounding the termination codon and in particular the two downstream
codons appear important for readth-rough of TMV RNA in vivo and in vitro.
The eukaryotic release factor complex, eRF1/eRF3/GTP, normally decodes stop signals efficiently.
However, depending on the context of the stop codon and the presence of the suppressor tRNA,
competition between the eukaryotic release factor complex and a suppressor tRNA for the stop signal
shifts in favor of the tRNA,resulting in a read-through rate of about 1-10%. This phenomenon was first
reported for TMV and later for at least 17 plant virus genera, including the Luteoviridae and
Tombusviridae. A large number of positive-sense ssRNA plant viruses use the read-through strategy to
produce components of RNA-dependent RNApolymerases (RdRp, e.g. tobamo and tombusviruses) or
elongated coat proteins (luteoviruses). For the tobamoviruses, tobraviruses, tombusviruses, carmoviruses and
for RNA1 of soil borne wheat mosaic furovirus (SBWMV), this read-through process allows synthesis of the
putative polymerase, and for RNA2 of SBWMV, RNA1 of pea enation mosaic virus and the luteoviruses, the
termination codon of the capsid protein gene is overcome to allow synthesis of a longer protein involved in
transmission, virus assembly or other functions
5. Leaky and scanning
The most widespread mechanism used by viruses to express polycistronic RNAs is leaky scanning, in
which a fraction of scanning ribosomes bypass the first start codon and initiate translation at
downstream start codons. Leaky scanning occurs when the first start codon resides in a suboptimal

5. context, lacking both a purine at position -3 and a guanosine at position +4, or when it is of a non-AUG
type (i.e. a triplet differing from AUG at one position). This can lead to two or more proteins with a
common C-terminal region being translated from one open reading phase,or to totally different proteins,
if the start codons are located in different phases. Leaky scanning can result in production of two or
more proteins with a common C-terminal region if translated from one open reading phase,or in totally
different proteins, if the start codons are located in different phases.
Cowpea mosaic virus M-RNAprovides an example of leaky scanning leading to translation of at least
two co-Cterminal polyproteins from one large ORF. In this case, two upstream AUGs in suboptimal
context (UGCAUGA at position 151 and ACAAUGU at 161) appear to be bypassed by scanning
ribosomes, which then initiate translation at the third start codon in an optimal context (GAAAUGGat
512). Initiation events in one reading phase at the second and the third AUGs give rise to two distinct
polypeptides essential for replication and cell to-cell movement, respectively. It should be noted that
cowpea mosaic virus belongs to the group of picorna-like plant RNAviruses that lack a cap structure
and very often use an IRES-dependent initiation mechanism. Besides leaky scanning,IRES-mediated
initiation has also been suggested to contribute to translation from the third AUG of cowpea mosaic
virus M-RNA.
According to the scanning model, when the first AUG resides in a very weak context, some ribosomes
start translation at that point but most continue scanning and initiate further downstream. This leaky
scanning enables the production of two separate proteins from one mRNA. Leaky scanning is the most
common phenomenon in eukaryotic mRNAs as well as viral mRNAs with overlapping ORFs allowing
the translation from downstream initiation codons. It is dependent on the context of the first startcodon.
When the first initiation codon is weak or in a poor context or too close to the 5’ end to be recognized
efficiently, majority of the ribosomes initiated translation from a downstream startsite. It is very striking
that leaky scanning occurs even when the two initiation codons are far apart.
Fig4. shows Leaky scanning is a phenomenon in which a weak initiation codon triplet on mRNA is sometimes
skipped by ribosome in translation initiation. The 40S ribosomal subunit continues scanning to further in itiation
codon. The weak initiation codon can be an ACG, or an ATG in a weak Kozak consensus context. This way a
mRNA can encode for several different proteins if the AUG are not in frame, or for proteins with different N-
terminus if the AUG are in the same frame. Upstream ORFs (uORF) are small coding regions preceding an ORF,
The latter must be translated by leaky scanning or termination-reinitiation.
6. Nested ORF
Anested gene is a gene whose entire coding sequence lies within the bounds (between the start
codon and the stop codon) of a larger external gene. The coding sequence for a nested gene differs
greatly from the coding sequence for its externalhost gene. Typically, nested genes and their host genes
encode functionally unrelated proteins, and have different expression patterns in an organism.
There are two categories of nested genes:
- genes nested within an intron of a larger gene
- genes which lie opposite the coding sequence of a larger gene
Barley yellow striate mosaic virus(BYSMV) is an example of characterizing nested ORF. BYSMV
contains three transcriptional units residing between the P and M genes compared with four units in the
corresponding region of NCMV. Unexpectedly, the middle mRNA in this region encodesgene5 nested

6. in an alternative frame within gene4 via a leaky scanning mechanism. The gene5 encodes a small
hydrophobic protein targeting to the endoplasmic reticulum (ER). The sequence of BYSMV and
NCMV have a similar genome organization and the high sequence identity of their major structural
proteins indicates that the two viruses are more closely related to each other than to other plant
rhabdoviruses. In the genome organization of the ORFs encoding the four ancillary proteins located in
the junctions between the P and M genes of BYSMV and the similar NCMV region. The four ORFs of
BYSMV are organized into three transcriptional units, in which gene5 is nested within gene4 in an
alternative reading frame suggesting that the two genes are expressed from the same mRNA and that
gene 5 might be expressed by a leaky ribosome scanning mechanism.
Fig.5 show nested ORF; Negative-stranded RNA linear genome, about 13 kb in size. Encodes for six (LNYV) to
nine (NCMV) proteins.The viral RNAdependent RNApolymerase binds the encapsidated genome at the
leader region, then sequentially transcribes each gene by recognizing start and stop signals flanking
viral genes. mRNAs are capped and polyadenylated by the L protein during synthesis.
7. Overlapping ORF
An overlapping gene a gene whose expressible nucleotide sequence partially overlaps with the
expressible nucleotide sequence of another gene. In this way, a nucleotide sequence may make a
contribution to the function of one or more gene products. Overlapping genes are present and a
fundamental feature of both cellular and viral genomes. viruses overlap must be between coding
sequences but not mRNA transcripts, and is defined when these coding sequences share a nucleotide on
either the same or opposite strands. Unidirectional overlaps are more frequent in genomes of viruses.
The topology of overlapping genes in viruses is determined both by the host cell type as well as by
constraints unique to viruses. Despite viruses having diverse genomes (RNAor DNA in single-stranded
or double-stranded form) and lifestyles, overlapping CDSs are found across all known virus groups.
The proportion of viruses with overlapping CDSs within their genomes varies from double-stranded
RNAviruses having fewer than a quarter to almost three-quarters of retroviridae (single-stranded RNA
using reverse transcriptase) and single-stranded DNA genomes containing overlapping CDSs.
Segmented viruses, those with the genome split into separate pieces and packaged either all in the same
capsid or in separate capsids, are more likely to contain an overlap than non-segmented viruses. he role
of overlapping genesin reducing the rate of viral evolution has been most intensively examined in RNA
viruses, which have higher mutation rates,smaller genomes and less CDS overlap than DNAviruses of
comparable length. Distribution of overlap length within virus’s points towards overlaps being favoured
for several different reasons, with short CDS overlaps enabling translational coupling, whereas long
overlaps being retained mainly when they generate genetic novelty that increases fitness. Virus capsid
size restrictions driving the evolution of gene overlaps has been a focal point of investigation due to
early observations of dramatic viability loss in viruses with genomes engineered to be longer than wild
type
Fig.6 Overlap ORF IN RNA2: Segmented, bipartite linear ssRNA(+) genome composed of RNA.1=7.8 kb and
RNA-2=5.4 kb. Each genomic segment has a VPg linked to its 5' end and a 3' poly(A) tract.
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7. Question.2) Describe the resistance genes to plant virus and compare to other genes conferring
resistance to bacteria and fungi. (Dr. SY. Kwon)
A. Resistance Genes (R Genes) in Plants
Plants have developed numerous approaches to resist infection by viruses. On the other hand, in many
instances viruses have evolved to overcome these various resistance responses and barriers. The extent
to which viruses can overcome some or all of these responses and barriers determines the extent to
which they are able to colonize plants of a given genotype or species. The activation of plant defence
to restrict pathogen invasion is often conferred by resistance (R) proteins. The most prevalent class of
R proteins contain
 leucine-rich repeats (LRRs),
 a central nucleotide binding site and
 a variable amino terminal domain.
Other classes possess an extracellular LRR domain, a transmembrane domain and sometimes an
intracellular serine/threonine kinase domain. R proteins function in pathogen perception and/or the
activation of conserved defence signaling networks. Upon infection, specific effectors produced by
pathogens and presumed to promote growth in host tissue, are either directly recognized by different R
proteins or are recognized by a targetedplant protein which is itself guarded by Rproteins. Subsequently,
various defence signaling networks are activated via R protein phosphorylation, oligomerization,
degradation, conformational changesand by the shuttling of R proteins betweenthe plant cell cytoplasm
and the nucleus. The overall outcome is dramatic cellular reprogramming and the activation of
coordinated defence responses both locally at the site of infection as well as systemically throughout
the plant. Many R gene loci appear to be under positive genetic selection, which rapidly diversifies
paralogous sequences. Some R genes are present in plant genomes at single loci as either a single
sequence or an allelic series whilst others reside within tight or loose clusters of related R sequences.
For a century, plant breeders have genetically characterized and used R genes to reduce the impact of
pathogens on crop production. More recently, various transgenic approacheshave been testedto provide
broader spectrum control and improved durability.
B. Innate Immunity and Resistance in Plants:
Resistance mechanisms in the host plant is governed by an incompatible gene for gene interaction
involving the resistance (R)gene product from the host and the corresponding Avr gene product from
the pathogen. Plants respond to an intruding pathogen either by their constitutive or pre-formed defenses
or via host resistance induction. Pre-formed defenses include the cuticle or thick waxy surfaces, cell
walls, antimicrobial compounds and enzyme inhibitors that block the entry of pathogens or prevent
vectors from transmitting viruses. Induced resistance is the ability of plant cells to activate and increase
their level of resistance to infection after perceiving or being stimulated by intruding pathogens.
In early years,two major categories of disease resistance were recognized as
 non-host resistance and
 host resistance.
Non-host resistance occurs when all genotypes within a plant species show resistance or fail to be
infected by a particular virus, which can be due to the host lacking susceptibility factors required by a
particular virus. Another manifestation of non-host resistance is the plant’s capability of recognizing
common pathogen (or microbe)-associated molecular patterns (PAMPs or MAMPs). MAMPs are
evolutionarily conserved molecules and are often indispensable for the pathogen’s life. They serve as
defense elicitors detected by pattern recognition receptors (PRRs) located in the plasma membrane of
plants. Examples of MAMPs include structural elements from within the fungal cell wall and the
bacterial flagellum. Such type of non-specific immune system, known as innate immunity, maintains
an inborn immunity of each cell and generates systemic signals emanating from the infection sites. The
innate immunity system is believed to be of common evolutionary origin in the defense systems of
pathogens in higher eukaryotes. While MAMPs/PAMPs and corresponding PRRs are yet to be fully
recognized in plant virus infections, the terms are often used in animal virology. For example, the

8. replicating intermediate dsRNAof +RNAviruses is recognized asa PAMPtriggering interferon induced
virus resistance.
In natural ecosystems plant disease epidemics are rare. This is because most plant species can
successfully defend themselves against the various infection strategies deployed by different types of
plant attackers, for example, fungi, oomycetes, bacteria, viruses, insect pests and nematodes. These
interactions are generally evolutionarily stable and the non-adopted plant species is referred to as a
‘non-host’. In these encounters, pathogens frequently reveal their presence to the plant’s ‘innate
immune system’through microbe-associated molecular patterns (MAMPs)formerly termed pathogen-
associated molecular patterns (PAMPS). MAMPs are not present in the plant and therefore signal
‘nonself’. Pathogenic species have evolved the ability to overcome preformed defences, to evade
MAMPsdetection and/or to suppressactively plant defence responsesand thus infect one to many plant
species and cause visible disease. Often the affected host tissues are restricted to either a specific organ
or cellular location, for example roots, leaves, flowers, the epidermis or vascular tissue. In these so
called ‘compatible interactions’,the plant activates various ‘basal defence’strategies to restrict the
colonization of ‘virulent’ pathogens.
Host resistance: is when the genetic polymorphism for susceptibility is observed in plant species. This
category of resistance is called specific resistance or cultivar resistance. Systemic acquired
resistance (SAR) has been depicted as a third category based on the studies involving tobacco mosaic
virus(TMV). SAR involves a spread of resistance resulting in a diminishing susceptibility to secondary
pathogen invasion of distal tissues. It is usually manifested by the formation of necrotic lesions either
as a form of hypersensitive response (HR) or as a disease symptom.
Resistance is often differentiated in accordance with the mode of interaction between the host and the
pathogen. One of the most common mechanisms of resistance is dominant resistance conferred by an
incompatible interaction between the host resistance(R) gene and the pathogen avirulence (avr) gene.
A majority of the R genes identified encode proteins containing nucleotide binding sitesand leucine-
rich repeats, and elicit a HR in response to the presence of a pathogen often conferred by dominant
alleles, this active resistance to virus invasion is manifested by cell death or necrosis at the infection
court preventing the spread of infection. Due to the high mutation rate of RNA viruses the interaction
betweenthe host resistance factorsand virus avirulance receptorsis frequently suppressed often making
dominant resistance less durable.
In contrast to dominant resistance, recessive resistance is thought to be more durable as the recessive
mutations render a host non permissive to viral infection due to the absence of specific host factors that
are required for a virus to complete its infection cycle. This loss of susceptibility does not require any
activity in plants thus is sometimes called passive resistance.This resistance is considered more durable
because the virus can defeat the host resistance only if they can adapt to the missing factors.
Resistance mechanisms may also vary according to the stage of viral infection cycle in the host plants.
In the early stage of viral infection, some cultivars resist the accumulation of virus particles that are
significant to effectinfection. Asa result, no virion canbe detectedin the Potato LeafRoll Virus (PLRV)
resistant cultivars following virus challenge. This resistance to virus accumulation precludes upward
movement and root infection as reported earlier for Soil-borne Cereal Mosaic Virus (SBCMV).
Different forms of resistance to virus movement have been observed in different potato cultivars. Some
of these include impaired movement of PLRV from sieve elements to the phloem bundles or restricted
movement of PLRV within leaves or from leaves to petioles and induction of phloem necrosis. Due to
the suppression of virus accumulation in the phloem, a low concentration of virus is present in the leaf
that contributes to the reduced acquisition of virus by insect vectors. Consequently, this results in
resistance for plant to plant transmission or secondary spread of infection within a field
In the early 1940s, Harold Flor formally investigated the genetics of the interaction between flax and
the fungal flax rust pathogen and proposed the ‘gene-for-gene model’. This model predicts that plant
resistance will occur only when a plant possesses a dominant ‘resistance gene’ (R) and the pathogen

9. expresses the complementary dominant ‘avirulence gene’ (Avr). Incompatibility is therefore
determined by complementary pairs of dominant genes. An alteration or loss of the plant resistance
gene (R changing to r) or of the pathogen avirulence gene (Avr changing to avr) leads to disease
(compatibility). Although the gene-for-gene model is not applicable to all host–pathogen interactions,
it holds true when the invading pathogen species colonizes primarily through living plant tissue and the
host induces a rapid and highly localized cell death response at the site of infection called the
‘hypersensitive response’ (HR). Once 20th
century plant breeders realized that plant resistance was
frequently inherited asa dominant trait, plant breeding programmes were developed to identify resistant
germplasm in wild relatives of crop plants and then introgress the corresponding resistance R genes for
agricultural benefits. Occasionally the outcome was stable disease control.
C. Pathogen Effectors andAvirulence Gene Products:
Why does a virulent pathogen make Avr gene products? Phytopathogenic bacteria were found to
produce a suite of molecules termed ‘effectors’which are delivered directly into the plant cells during
initial infection by a specialtype of secretion system,termed ‘type IIIsecretion’.These effectorseither
alter the plant’s physiological state to benefit pathogen colonization or are used to suppress the
activation of host plant defences. In response,the plant has evolved specific countermeasures to protect
against these pathogen-induced cellular reprogramming events. The protective countermeasures
included the evolution of R genes whose products lead to the direct or indirect recognition of a specific
subset of pathogen effectorgene products and the activation of defence responsesin the attackedtissues.
Only this recognized subset of effectors is identified as Avr genes by plant genotypes possessing the
corresponding R gene.
D. Resistance Gene Types, Mode ofInheritance and Patterns ofExpression
1. Dominant resistance genes
Since 1992 many disease resistance genes have been cloned from experimentally tractable species such
as maize, Arabidopsis, tomato and tobacco and subsequently from experimentally more challenging
species, for example, barley, flax, potato, pepper, rice and even more recently from hexaploid wheat.
The majority of known R proteins group into just a few main classes based primarily upon their
combination of a limited number of structural motifs (Table1). Most R proteins control only pathogen
races, which express the corresponding effector protein(s) and are therefore called race-specific R
proteins. Occasionally, effective resistance is conferred against multiple races and even different
pathogen species. These R proteins are called race-non-specific. The most prevalent class of
functionally defined R genes encode intracellular nucleotide-binding/leucine-rich repeat (NBLRR)
proteins, with either an N-terminal putative leucine zipper (LZ) or other coiled coil (CC) sequence.
Alternatively, at the N-terminus of these NB-LRR R proteins there is a region with high similarity to
the Toll and Interleukin 1 receptor protein, which are involved in innate immunity in drosophila and
mammals. This region is referred to as the TIR region. The only other intracellularly located R class
encode serine/threonine protein kinases, for example, tomato Pto.The tomato plasma membrane bound
(Cf) proteins which confer resistance to the fungal pathogen Cladosporium fulvum represent another
class of R proteins.
2. Resistance proteins domains: Each resistance protein provides the plant with up to two
unique capabilities;
 firstly, recognition of specific pathogen(s) and
 secondly activation of defence responses.
For a few R proteins both functions have been formally demonstrated to reside within a single protein
sequence,for example tomato Pto. However,for the majority only a single role is known or suspected.
For example, domain swap experiments between the flax rust L and P resistance proteins have so far
identified the LRR region to have a role in recognizing specific pathogen races.

10. Table 1. Resistance genes conferring resistance to viruses.
Resistance genes Plant species Virus targets
Dominant genes
N N. tabacum TMV
, ToMV
Rx1 S. tuberosum PVX
Rx2 S. tuberosum PVX
Sw-5 S. lycopersicum TSWV
, TCSV
, GRSV
HRT A. thaliana TCV
Y1 S. tuberosum PVY
RCY1 A. thaliana CMV
Tm-22 S. lycopersicum ToMV
, TMV
RTM1 A. thaliana TEV
RTM2 A. thaliana TEV
RT4-4 P. vulgaris CMV
Tm-1 S. lycopersicum TMV
, ToMV
PvVTT1 P. vulgaris BDMV
Ny-1 S. tuberosum PVY
JAX1 A. thaliana PlAMV
RTM3 A. thaliana TEV
RCY1 A. thaliana CMV
Tm-2 S. lycopersicum Tobamoviruses
L1, L2, L3, L4 C. annuum Tobamoviruses
Rsv1 G. max SMV
Ctv Poncirus trifoliata CTV
I P. vulgaris BCMV
Recessive genes
sbm2 P. sativum PSbMV
cyv1 and cyv2 P. sativum ClYVV
pvr1 S. lycopersicum TEV
pvr1/pvr2 C. annuum PVY
, TEV
Mutant eIF4E S. tuberosum PVY
pot-1 S. lycopersicum PVY
, TEV
mo11/mo12 L. sativa LMV
sbm1 P. sativum PSbMV
, BYMV
rym4/5 H. vulgare BaMMV
, BaYMV
rymv1 O. sativa RYMV
Nsv C. melo MNSV
pvr2 + pvr6 C. annuum PVY
, TEV
bc-3 P. vulgaris ClYVV
rym7 H. vulgare BaMMV
Virus names: Barley mild mosaic virus (BaMMV); Barley yellow mosaic virus (BaYMV); Bean
common mosaic virus (BCMV); Bean dwarf mosaic virus (BDMV); Bean yellow mosaic
virus (BYMV); Clover yellow vein virus (ClYVV);Cucumber mosaic virus (CMV); Groundnut
ringspot virus (GRSV); Lettuce mosaic virus (LMV); Melon necrotic spot virus (MNSV);Pea seed-
borne mosaic virus (PSbMV); Pepper veinal mottle virus (PVMV); Plantago asiatica mosaic
virus (PlAMV); Potato virus X (PVX); Potato virus Y(PVY); Rice yellow mottle
virus (RYMV); Soybean mosaic virus (SMV); Tobacco etch virus (TEV); Tobacco mosaic
virus (TMV); Tomato chlorotic spot virus (TCSV); Tomato mosaic virus (ToMV); Tomato spotted wilt
virus (TSWV); Turnip crinkle virus (TCV).

11. Table.2 Resistance genes conferring resistance to bacteria and fungus
Gene Plant Pathogen Infection type
Pto Tomato Pseudomonas syringae pv. tomato (avrPto) bacteria
PBS1 Arabidopsis Pseudomonas syringae pv. phaseolicola (avrPphB) bacteria
RPS2 Arabidopsis Pseudomonas syringae pv. maculicola (avrRpt2) bacteria
Bs2 Pepper Xanthomonas campestris pv. vesicatoria (avrBs2) bacteria
RRS-1 Arabidopsis Ralstonia solanacearum Bacteria
Pi-ta Rice Magnaporthe grisea (avrPita) fungus
Cf-9 Tomato Cladosporium fulvum (Avr9) fungus
Ve1, Ve2 Tomato Verticillium albo-atrum wilt fungus
Xa-21 Rice Xanthomonas oryzae pv. oryzae (all races) Bacteria
RPW8 Arabidopsis Multiple powdery mildew species fungus
Rpg1 Barley Puccinia graminis f.sp. tritici fungus
RFO1 Arabidopsis Fusarium oxysporum f.sp. matthiola
Fusarium oxysporum f.sp. raphani
wilt fungus
Xa27 Rice Xanthomonas oryzae pv. oryzae (avrXa27) Bacteria
Pi-d2 Rice Magnaporthe grisea fungus
The major classes of cloned plant disease resistance genesa
3. Recessive resistance genes: From approximately 40 cloned resistance genes, which confer
resistance to fungal and bacterial pathogens only four are recessive: barley melo, Arabidopsis
edr1, Arabidopsis rrs1-r and rice xa5. Whilst the functions of dominantly inherited R gene
products are beginning to emerge, the molecular mechanisms involved in recessive resistance
are less well understood.
4. Conserved recessive resistance: Naturally occurring or chemically induced mutations in the
barley and Arabidopsis mlo gene confer broad-spectrum resistance to all races of powdery
mildew fungi. Wild type Mlo encodes a novel class of plant-specific seven transmembrane
domains proteins. It is located in the plasma membrane and acts as a negative regulator of
defence and/ or programmed cell death. MLO protein co-localizes in the cell and physically
interacts with ROR2/PEN1which belongs to the syntaxin classof proteins known asmediators
of the fusion of vesicles with target membranes. Vesicle mediated exocytosis at the site of
attempted fungal infection leads to localized ‘papilla’ formation which inhibits penetration.
Therefore,it has been proposed that MLO is involved in some vesicle-associated processes at
the plant cell plasma membrane that the fungus has evolved to utilize for its own growth
advantage.
5. Recessive cereal resistance genes: The rice gene xa5 confers broad-spectrum resistance to
most races of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. Wild type Xa5
encodes a subunit of the general transcription factor TFIIArequired by RNApolymerase II. It
has been suggested that the mutant xa5 protein possesses an enhanced ability to interact with
the acidic transcription activation domain of the bacterial avirulence protein Avrxa5. This in
turn leads to retardation of host cell transcription and ultimately to rapid cell death and
resistance.
6. Recessive non cereal resistance genes: The Arabidopsis gene rrs1-r confers broad-spectrum
resistance to multiple strains of the causal agent of bacterial wilt, Ralstonia solanacearum. It
encodes a novel protein that structurally resembles the prevalent TNLsub-class of R proteins.
However, in contrast to ‘classical’ R proteins RRS1 also contains at its C-terminus a WRKY
motif characteristic of some plant transcriptional factors. The RRS1 protein moves from the
cytoplasm to the plant nucleus upon physical interaction with Pop2, a bacterial type III effector
protein.
7. Recessive resistance to viruses in cereal and noncereal species: Recessively inherited
resistance is quite common for viral diseases and often confers ‘immunity’ as opposed to the
HR associated defence. Viruses are the smallest known organisms encoding only a few

12. functional proteins. To complete their life cycle they often hijack components of the host plant’s
biochemical machinery. Therefore, the recessive antiviral resistance genes are most likely to
correspond to mutations in plant host factors required at specific steps in the virus life cycle
Engineering resistance: The development of plant gene transfer systems has allowed for the
introgression of alien genes into plant genomes for novel disease control strategies, thus providing a
mechanism for broadening the genetic resources available to plant breeders. Genetic engineering offers
various options for introducing transgenic virus resistance into crop plants to provide a wide range of
resistance to viral pathogens. The first reported Agrobacterium transformed tobacco plants showed
successful tissue differentiation and functional expression of bacterial coding sequences specifying
resistance to kanamycin (KanR) and chloramphenicol (CmR).
Pathogen derived resistance (PDR): The Pathogen derived resistance approach by showing that
disease development was delayed in transgenic tobacco by expressing the coat protein (CP) gene of
Tobacco Mosaic Virus (TMV). Since then, the majority of plant virus resistance research via plant
transformation was focused on the PDR approach.
Mechanistically, PDR is divided into two categories. The 1st
category is the protein-mediated
resistance where in the expressed viralprotein itself is responsible for the observed resistance. Protein-
mediated resistance provides either broad or narrow protection. Viral coat protein (CP) is involved in
multiple aspectsofviral biology, which includes encapsulation, virus replication, cell to cell or systemic
movement, vector transmission and symptom development. Transgenic potato expressing Potato
Mosaic Virus (PMV) CP gene provides resistance to PMV and other related strains. Similarly, TMV CP
gene expressed in tobacco provides protection against TMV and TMV-related strains. However,
transgenic papaya expressing CP genes of papaya ringspot virus(PRSV) strain HAprovides protection
only against this same strain. The coat protein mediated resistance (CPMR) has been widely used and
reported in at least 35 viruses representing more than 15 different taxonomic groups including the
tobamo-, potex-, cucumo-, tobra-, carla-, poty-, luteo- and alfamo-virus groups. The CPMR mediated
resistance involves interaction between the transgenic CP and the CP of the challenging virus. To date,
severalimportant crops have been engineered using a CPMR approach (table 3).
A. Replicase and rep-protein mediated resistance: Resistance for RNA viruses utilized the
availability of viral genes that codes for RNA-dependent RNA-polymerase (RdRp). Many
researches attempted to determine the mechanism of RdRp mediated resistance with different
viruses and concluded that different mechanisms were involved for different viruses. This type of
resistance is often confused with the RNAmediated resistance.Resistance induced by replicase was
observed to be high and stable. Transgenic rice containing the RdRp of Rice yellow mottle virus
(RYMV) showed stable resistance to RYMV strains from different African locations. This
resistance,however, is very specific and limited to homologous viral strains.
B. Movement protein (MP) mediated resistance: Resistance Movement protein allows virus to
move from the site of infection to neighboring cells by modifying the gating function of the
plasmodesmata. The first MP mediated resistance was shown in tobacco plants carrying a defective
MP (dMP) which competed with the wild-type virus encoded MP for the binding sites in the
plasmodesmata. Transgenic plants showed delayed symptom expression and infection. This
strategy also manifested a broad spectrum resistance as the dMP-tobacco plants interfered with the
systemic spread of distantly related and unrelated viruses such astobra-, caulimo-, and the nepo-
viruses The 2nd
category of PDR is the RNA-mediated resistance. Here, protection is brought
about by introducing a transgenic RNAtocause degradation of transcripts or genomic RNAof plant
viruses. The puzzling observation that virus resistance of CP transgenic lines is not directly related
to the CP expression level led to the discovery of RNA-mediated resistance triggered by the
expressed CP mRNA. This phenomenon is a specific and homology dependent RNA degradation

13. mechanism that provides RNAmediated resistance to viruses.
Flow chart showing the strategies of transgenic viral resistance in plants. In RNAi mediated strategy
the dsRNA or hairpin RNA (hpRNA) is initially
generated following transcription in plant and
subsequently produces siRNA or miRNA
involving dicer protein and RISC complex. These
siRNAs or miRNAs silence sequence specific viral
RNAs and confer strong but specific resistance. In
protein product mediated strategy either the viral
genes (coat protein, replicas or movement protein)
or plant R genesare expressedand aftersubsequent
transcription and translation the protein products
govern resistance against the virus
Table 3. Transgenic strategies for virus resistance.
Mechanism/stratergies
employed Transgenic plant
Mechanism/stratergies
employed Transgenic plant
Virus
RNA interference Common bean Bean golden mosaic virus (BGMV)
Tobacco N.
Benthamiana
N. benthamiana
PMMoV
,
TSWV
, GRSV
, TCSVand WSMoV
N. benthamiana Cassava brown streak Uganda virus (CBSUV);
Cassava brown streak virus (CBSV)
Tobacco TMV
, CMV
N. benthamiana and tomato ToLCTWV
, TSWV
Coat protein meditaed
resistance
Tomato
Tobacco
N. benthamiana
TSWV
CMV
Cowpea aphid-borne mosaic virus (CABMV)
Tobacco
N. benthamiana
White clover
Chrysanthemum
CMV
, TMV
Brome mosaic virus (BMV)
Alfalfa mosaic virus (AMV)
CMV
Replicase mediated resistance Tobacco Broad spectrumressitance to Tobamoviruses
Potato PLRV
Lilium CMV
Gladiolus CMV
RNA dependent RNA
polymerase mediated resistance
Tobacco TMV
RNA satellites Pepper CMV
N. benthamiana and A.
thaliana
Bamboo mosaic virus (BaMV)
Tobacco CMV
Antisense RNAs Tobacco CMV
Ribosome-inactivating proteins
(RIP)
Tobacco
Beet
CMV
Artichoke mottled crinkle virus (AMCV)
Ribonucleases Tobacco TMV
, TEV
, CMV
, AMV
, PVY
Enhancement of HR/SAR Tobacco
Pepper
CMV
, TMV
CMV
, PMMV
Hammerhead ribozyme Tobacco CMV
///////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////

14. Question.3) Describe the mechanisms of the movement of plant values. You may explain cell to cell
and long distance movement separately. (Dr. OS. Kwon). Here the question is I am not clear movement
of plant value regarding with either “plant microbes”, “RNA movement” or “plant nutrients”. But I
explained my answer regarding with plant microbes (RNA/Virus) interactions.
Plant virus movement: Successful infection by a plant virus generally involves three basic steps:
replication, cell-to-cell movement and long distance transport through the phloem. First, the virus
(usually virions) is introduced into a wounded cell that is competent for replication. These wounds are
most commonly made by the feeding of insect vectors (e.g.,aphids, leafhoppers, thrips and whiteflies),
but are also made by other types of vectors (e.g.,fungi and mites), physical damage (e.g.,plant-toplant
contact), and pollen and seeds. Having gained access into a wounded living cell, the virus is
unencapsidated and the steps in replication proceed.
1. Cell to cell (short distance) or local movement
Cell-to-cell movement is a crucial step in plant virus infection. In many viruses, the movement function
is secured by specific virus-encoded proteins. Plant viruses have the capacity to replicate in most
individual cells, but only those that can egress from these initially infected cells and move to establish
a productive infection. Cell-to-cell movement of plant viruses occurs slowly through plasmodesmata
(PD),organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP)
modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several
distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and
provide a conduit for virion translocation. It is generally accepted that plant viruses exploit
plasmodesmata to move from initially infected cells, which are usually negligible in amount, into
neighboring healthy cells. Without cell-to-cell movement productive virus infection is not established.
Also it is known that specific movement proteins encoded by virus genomes are essential for cell-to-
cell spread. Frequently, a plant virus protein is referred to as movement protein if
i) It is not a capsid protein and
ii) Disruption of the coding sequence of this protein abolishes infection in whole plants but
has no effect on virus replication in protoplasts.
 Virus produce special protein called movement protein(MP) which help in movement by
overcome plants control of plasmodesmata
 MP are no structural protein which enables virion movement from infected cell to neighboring
cells by modifying plasmodesmata, thereby allowing local and systemic spread of viruses in
plants.
 Most plant viruses move cell to cell as complex of non-structural protein (MP) and genomic
RNA(Ribonucleoprotein-RNP)
 Coat protein is generally not need for cell to cell movement
 To move from one cell to the next, viruses exploit the channels that plant cells use to
communicate with each other.These channels are called plasmodesmata. They are linked with
proteins and can be tightly controlled by the plant.
 Relative to the diameter of plasmodesmata, virus particles are huge. Imagine it’s like trying to
pull a cat though a keyhole! Eg. CPMV,PVX,TMV
A critical step in this process is the capacity of the virus to encode one or more movement proteins
(MPs) that interact with PD, increase the PD size exclusion limit (SEL) and mediate the cell-to-cell
movement of viral nucleic acids (DNAor RNA) or virions. Numerous distinct cell-to-cell movement
mechanisms are known for plant viruses. Moreover, the factthat different types of viruses (e.g.,families
or genera) share similar cell-to-cell movement mechanisms suggests convergent evolution.
– There is emerging evidence that viral replication complex (VRCs) are closely linked to the cell-to-
cell movement complex. This can occur through the accumulation of components from the VRC to
assemble the cell-to-cell movement complex or the actualtransport of the entire VRC across PD.
– Phloem-associated and -limited viruses have effectively evolved to infect plants via multiple
mechanisms. These viruses are introduced into SEs and move in a source to sink direction to access

15. nucleate cells for replication and cell-to-cell movement. Access to nucleate cells in the phloem can
occur via cell-to-cell movement from interconnect sieve elements to companion cells, and at the shoot
and root apices, e.g., geminiviruses. Some geminiviruses can move cell-to-cell in a non-virion form;
however,for bona-fide phloem-limited geminiviruses, closteroviruses and luteoviruses this may require
virions. This area needs new research emphasis and experimental approaches.
Types and properties ofmajor cell-to-cell movement mechanisms of plant viruses
The firstinclude a large number of positive-sense single-stranded (ss)RNAviruses that are not phloem-
limited and subtly modify PD. These move cell-to-cell either as a vRNA-MP complex, not requiring
the capsid protein (CP),and are exemplified as Tobacco mosaic virus (TMV); or as a vRNA-MP-CP
complex, most likely a non-virion form, and are exemplified as Potato Virus X (PVX).
The second type is a diverse group of non-phloem-limited viruses, which include double-stranded (ds)
DNAand positive sense ssRNAviruses, and have evolved a mechanism to drastically modify PD into
MP-lined tubules through which intact virions move cell to cell.
The third type includes phloem-associated and limited viruses that include the positive sense ssRNA
closteroviruses and luteoviruses and the circular ssDNA geminiviruses. These viruses utilize the
specialized PD that interconnect sieve elements (SEs) and companion cells (CCs) for cell-to-cell
movement. Geminiviruses can move cell-to-cell via PD or cell division, induced by viral infection or
associated with normal plant development. These phloem-limited viruses may or may not require
virions for cell-to-cell movement. Although the precise mechanism(s) by which any virus moves, cell
to cell, is not known, considerable progress has been made in understanding the cell biology of the
process. The endoplasmic reticulum (ER) plays a critical role in cell-to-cell movement, especially for
viruses that do not use tubules; the cytoskeleton and myosins mediate trafficking of the movement
competent form of the virus to the plasma membrane (PM) and PD; and considerably more is known
of the host factors involved. As new and powerful tools for cell biology, microscopy and
genomics/proteomics continue to be developed, integrated studies should allow for a comprehensive
picture of the viral and host factors involved in cell-to-cell movement.
In fact, short-distance movement can best be thought of as a process of genome movement, because
many (but not all) viruses do not require virion assembly or a capsid protein to move to adjacent cells.
The cell-to-cell movement pathway as a process divided into three major steps:
(1) transfer of newly synthesized genomes from sites of replication to intracellular transport systems;
(2) directed, facilitated transport of genomes to plasmodesmata; and
(3) transit through plasmodesmata.
Basic straight ofvirus movement
I. Non tubule guided movement:
Plasmodesmata are gated open at the infection front and close afte. Thus, maintaining the integrity of
normal intracellular communication. The mechanism by which movement proteins work is by
associating with and coating the genome of the virus, causing the ribonucleoprotein complexes (RNP)
to be transported through plasmodesmata into neighboring cells. TMV forms a complex between its
MPs and the viral RNA to move between cells. (TMV)MP in targeting plasmodesmata SEL, binding
single stranded nucleic acids, interacting with the cytoskeleton, and trafficking nucleic acid between
cells.
II. Tubule guided movement:
plasmodesmata used for viral transport are permanently modified by MPs forming a tubule through
which virion moves. The movement proteins of many plant viruses from a transport tubule within the
pore of plasmodesmata that allow the transport of mature virus particles. Examples of viruses that use
this mechanism are CPMV and TSWV.
– With the emergence of new methods in cell biology, spectroscopy, protein-protein interaction,
genomics and proteomics more progress in the identification of the host factors involved in virus cell-

16. to-cell movement will be made. Hopefully, this will allow for the identification of the all of the steps
involved in viral cell-to-cell movement and additional details of the interaction of viruses and PD. For
example, the application of quantum dots, a more robust and sensitive fluorescent dye, could allow for
visualization of virus infection in vivo.
2. Systemic (long distance) movement
Plants viability requires a close and balanced co-ordination between environmental conditions, growth
and development, and pathogen defence. Information exchange between cells is an important
prerequisite for such co-ordination, and all multicellular organisms have evolved different strategies to
allow anefficient intercellular exchange of information molecules. In plants, plasmodesmata (PD)build
a cytoplasmic continuum throughout the plant body. PD allow a selective and directed transport of
molecules over short distances. In addition, higher plants contain a specialized tube system for long-
distance transport of nutrients, water,and signaling molecules, consisting of xylem and phloem. While
the xylem tubes are attributed to the apoplast, the phloem sieve elements (SEs) are interconnected with
the cytoplasmic continuum by specialized secondary PD, the pore plasmodesmal units (PPUs). PPUs
are structurally different from other PD in that they consist of multiple channels on the companion cell
(CC) side that fuse to a single pore on the SE side. Due to their specialized structure, their size
exclusion limit (SEL) substantially exceeds the SEL of PD between epidermal or mesophyll cells that
lie in the range of 1 kDa. PPUs have been shown to allow the passage of molecules larger than 60 kDa
and therefore should also allow the passage of macromolecules like proteins or smaller RNAs. The
phloem interconnects even the most distant parts of a plant and thus provides an ideal route not only for
the allocation of photo assimilates from source to sink organs, but it also enables a fast and directed
systemic information transfer. To co-ordinate development, defence, and nutrient allocation, plants
have evolved a complex battery of signaling molecules that can be translocated via the phloem. For
example, the elusive ‘florigen’ has long been known as a phloem-mobile, essential trigger for flower
induction of unknown chemical. In addition, a variety of phloem-mobile molecules are potentially
involved in defence-related signaling leading to responses like systemic-acquired resistance. Finally,
gene regulation by RNAsilencing can spreadsystemically whatis most likely mediated via the phloem.
 Viruses have evolved a diversity of mechanisms for cell-to-cell movement through PD, and
this includes viruses that can egress the phloem and those that are phloem-limited. Regardless
of their phloem-tropism all viruses encode one or more MP,and these can subtly or drastically
modify PD structure and function to mediate the cell-to-cell movement of viral nucleic acids
or virions. The diversity of MPs and cell-to-cell movement mechanisms indicates multiple
evolutionary events, including convergent evolution of the same mechanism by different
viruses and hijacking and modifying host factors involved in macromolecular trafficking.
 Phloem (fast)
 Move as particles or as protein/ nucleic acid complex (coat protein required)
 Viruses move from cell to cell through a leaf until they find a vein. Veins are used by the plant
to supply growing tissues. Food flows through the vein in the phloem.
 The flow of food through these pipe-like cells is always from mature leaves to younger leaves.
Viruses enter veins and move with the flow of food to healthy new leaf. This is much faster
than cell to cell movement.
 Companion cells provide the gateway for entry of materials into the phloem translocation
system. This is done via the plasmodesmata opening that connect the companion cell with the
sieve tube element. Companion cells supply the energyfor loading of nutrients into the phloem
and are the driving force for long distance transport between organs.
In the last decade it has been established that not only small molecules like phytohormones or
metabolites, but also macromolecules like proteins and RNAs, can be considered as potential long-
distance signaling substances. The apparent non-cell autonomous nature of some RNAs is receiving
particular attention at present, since RNA molecules were traditionally believed to act in the same cell
in which they are synthesized. However,accumulating evidence indicates that RNA can move locally
between cells, potentially as a regulator of gene expression. The homeodomain protein KNOTTED1,
for example, has been shown to facilitate the transport of its own messenger RNA (mRNA) from cell-
to-cell in the meristem in a locally restricted manner. A more recent study demonstrated that the KNOX

17. homeodomain of KNOTTED1 is necessary and sufficient to confer trafficking ability to the cell-
autonomous protein GLABROUS1 and that KNOTTED1sense RNAis translated afterits translocation.
In addition, it is now known that specific RNAs can not only move locally, but even enter the long-
distance transport route to spread systemically between different organs. Recent studies suggest that
RNA transport through the phloem is no rare phenomenon, but that it is a rather common process in
higher plants.
Types and functions ofphloem RNAs
Although RNAhad beenfound in phloem exudates of pumpkin (Cucurbita maxima) more than 30 years
ago, it was long regarded to be contamination from the sample collection procedure rather than a true
component of the transport stream. Since then it has become accepted that RNAs in the phloem are no
artefact but are an authentic constituent of the transport stream. It is known that three major types of
RNAs can be systemically transported: RNA genomesofviruses,endogenous cellular mRNAs,and
small noncoding RNAs. The phloem tubes seem to be an ideal transport route for RNA given that,
unlike other tissues, phloem sap contains no detectable RNase activity.
Viral RNAs
Long-distance movement of RNA was first observed during the spread of viral infections. The majority
of plant viruses are single-stranded RNA viruses that are replicated via double-stranded RNA
intermediates produced by an RNA-dependentRNApolymerase. These unusual double stranded RNAs
allow plants to recognize viral RNA as being foreign. The movement of viruses is thought to occur in
two phases: (1) local cell-to-cell and (2) systemic movement through the phloem. After infection,
viruses normally spread locally from cell to cell through plasmodesmata (PD) until they reach the
vascular tissue. Viruses contain special movement proteins (MPs) that have the ability to bind and
unfold single-stranded RNAs and facilitate the intercellular translocation of viral nucleic acids by
building protein–RNA transport complexes. Additionally, MPs have the ability to increase the SEL
of PD up to 10-fold in mature leaves allowing the movement of MP–RNA complexes between host
cells. Another important factor for virus movement is the coat protein (CP) and viruses can be
categorized into three groups depending on whether the CP is not required (type I) or required (type II)
for movement, or virus particles are translocated from cell to cell (type III).
Some viral mutants exist that are inhibited in systemic but not in local spread, indicating that the
mechanism of phloem import is different from that of cell-to-cell movement. Whether MPs are also
involved in the phloem-dependent movement is currently unknown. The MP of tobacco mosaic virus,
for example, is essential for cell to- cell movement but not for vascular transport of the virus, while
replicas, functional CP, and the origin of- assembly are critical for phloem long-distance transport of
tobacco mosaic virus in a host-dependent manner. While CPs are dispensable for cell-to-cell trafficking
in some virus types, CPsseemtoconstitute a factorwidely required for virus long-distance translocation.
However, virus RNAs from specific virus families also seem to be able to traffic from cell to cell and
even over long distances without the presence of MPs or CPs. Also endogenous phloem proteins,
namely different phloem lectins (CmPP2,CsPP2,CmmLec17) and the phloem protein CmPP16,have
been found to interact with viral RNAs as well as endogenous mRNAs and could thus be involved in
virus import or translocation within the phloem stream. Interestingly, cadmium at non-toxic
concentrations specifically blocks systemic but not local movement of to bamoviruses, suggesting that
plant factors,impaired by cadmium, are involved in the control of systemic virus movement.
Small, non-coding RNAs
Small non-coding RNAs have recently emerged as important transcriptional and post-transcriptional
regulators of gene expression. Interestingly, plants seem to have developed different, partially
overlapping small RNAbiosynthesis pathwaysresulting in two major classesof small regulatory RNAs,
short interfering and micro RNAs (siRNAs and miRNAs, respectively), that can be distinguished by

18. their way of biogenesis and their mode of function. siRNAs mediate a process called post-
transcriptional gene silencing (PTGS), an innate plant defence mechanism that is a widespread defence
mechanism against the activity of transposable elements and viruses. Early work using grafting
experiments on transgenic plants showed that a signal from silenced rootstocks is transmitted to non-
silenced scions expressing the respective transgene, leading to PTGS in scions. Recently, siRNA
originating from a transgene or a virus infection could be detected in the phloem of silenced but not in
unsilenced plants, suggesting that siRNAs could be the signaling molecules themselves. Meanwhile, it
is known that siRNAs can spread from their site of production, probably in the form of 21 nt RNAs.
Larger siRNAs (24–26 nt), on the other hand, are supposed to be involved in propagating the systemic
signal. In accordance with virus transport, non-toxic levels of cadmium also lead to an uncoupling of
local and systemic spread of PTGS. This indicates that long-distance transport of PTGS signals and
viruses relies on common mechanisms.
Import of phloem sap RNAs into SEs
The pore plasmodesmal units (PPUs) connecting companion cells (CCs) with sieve elements have an
unusually large size exclusion limit (SEL), opening the question whether macromolecular transport is
really specific or only restricted by the size of the molecules. Non-plant proteins like GFP (green
fluorescent protein) can move between CCs and SEs through PPUs, indicating a non-selective
trafficking of macromolecules between these two cell types. Recent study reports that GFP fusion
proteins as large as 67 kDa can move between CCs and SEs and are only retained in CCs when targeted
to membranes or the endoplasmic reticulum. Indeed, it seems as if all cytosolic proteins can enter SEs
when coupled to GFP. This suggests that proteins (and probably also nucleic acids) reach the SEs by
unspecific loss from CCs rather than by specific import mechanisms. Because of their small size, it has
been assumed that especially small RNAs are able to move between cells and reach SEs through PD by
simple diffusion. Microinjection experiments in pumpkin have demonstrated, however, that specific
miRNAs cannot move betweencells by diffusion alone but need the presence of a specific 27 kDa small
RNA-binding protein that facilitates the passage betweenCells perhaps by acting asan RNAchaperone.
Also other studies have identified severalphloem sapproteins in different speciesthat have the capacity
to bind and translocate RNA. These proteins are thought to be part of a specific phloem import and
transport machinery.
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