Plant Physiology Preview. Published on August 10, 2012, as ... · 8/10/2012 · Rothamsted Research, UK on 22-24 June 2011) advances and developments. ADVANCES IN BSMV-INDUCED PLANT

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Running head: UPDATE ON BSMV-MEDIATED FUNCTIONAL GENOMIC TOOLS

Corresponding author:

Kostya Kanyuka Wheat Pathogenomics Team Plant Biology and Crop Science Department Rothamsted Research Harpenden, AL5 2JQ United Kingdom tel: +44 (0)1582 763133 e-mail: [email protected] Article category: UPDATE

Plant Physiology Preview. Published on August 10, 2012, as DOI:10.1104/pp.112.203489

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Barley stripe mosaic virus-mediated tools for investigating gene function in cereal plants and

their pathogens: VIGS, HIGS and VOX

Wing-Sham Lee, Kim E. Hammond-Kosack, and Kostya Kanyuka*

Wheat Pathogenomics Team, Plant Biology and Crop Science Department, Rothamsted Research,

Harpenden, AL5 2JQ, UK



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Footnotes

1 W-S.L., K.K. and K.H.K. are supported by the Biotechnology and Biological Sciences Research

Council of the UK (BBSRC) through the Institute Strategic Programme 20:20 Wheat®.

* Corresponding author; e-mail [email protected].



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ABSTRACT

Food security is now considered to be a worldwide global priority. It necessitates research aimed to

increase productivity of our major monocot crops including wheat, maize and barley. This aim can

only be achieved through better understanding of crop plant biology, physiology and gene function.

DNA and RNA sequencing is undergoing a revolution, rapidly providing vast amount of novel data for

downstream functional genomics analyses. Here we review the most recent significant and exciting

advances in the development and deployment of Barley stripe mosaic virus vector derived tools,

namely Virus-induced gene silencing (VIGS), Host-mediated gene silencing (HIGS), and Virus-

mediated overexpression of heterologous protein (VOX). These tools have high potential to facilitate

identification and functional characterisation of genes in cereal crops that play key roles in various

sustainability traits, as well as genes in crop-associated organisms, such as plant pathogenic fungi,

that play key roles during plant colonisation.



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INTRODUCTION

Barley stripe mosaic virus (BSMV), the Hordeivirus type member, is the most intensely studied virus

that naturally infects two major monocot crops, namely wheat (Triticum aestivum) and barley

(Hordeum vulgare). The tripartite genome of this virus, comprising of RNAα, RNAβ and RNA�, was

sequenced and cloned in the late 1980’s. Since then there have been significant advances in the

understanding of the molecular biology of BSMV and the function of the seven main proteins encoded

by its genome (Jackson et al., 2009) (Figure 1A).

In a field situation, BSMV is easily transmitted from infected to healthy crop through plant-to-plant

contact and on most cultivars it causes mild to moderate mosaic symptoms. The virus is also known

to be efficiently transmitted via the seed (Jackson et al., 2009). In the laboratory, BSMV can be

mechanically transmitted by rub inoculation to maize, oat, Brachypodium and over 250 other species

mostly in the Poaceae (Jackson and Lane, 1981). However, amongst these are several dicot species

including Nicotiana benthamiana, in which BSMV causes mild systemic mosaic, and Chenopodium

spp., a local lesion host.

In recent years, BSMV has become a popular vector for Virus-Induced Gene Silencing (VIGS) in

barley and wheat (Scofield and Nelson, 2009; Cakir et al., 2010). This arose primarily because of the

availability of full-length infectious BSMV clones and an increasingly detailed knowledge of molecular

and biological function of its various genome components (Figure 1A). VIGS is a powerful functional

genomics tool for the rapid targeted down-regulation of host plant genes (Becker and Lange, 2010;

Senthil-Kumar and Mysore, 2011). It exploits the fact that infection of plants by viruses activates Post-

Transcriptional Gene Silencing (PTGS) defence response (Waterhouse et al., 2001). In VIGS, a short

fragment of a transcribed sequence of a plant gene is inserted into a cloned virus genome and the

recombinant virus is then inoculated onto test plants (Figure 1B). The introduced virus multiplies and

spreads from the site of infection into newly developing regions of the plant and triggers PTGS. The

inserted plant gene sequence also becomes the target for silencing, and so does the corresponding

endogenous gene. This leads to a reduction or in some cases the complete abolition of target plant

gene function, which in turn results in phenotype changes.

Bread wheat is an allohexaploid, containing three copies of each gene representing ancestrally

distinct homoeologous genomes A, B and D. The BSMV-based VIGS system (hereafter referred to as

BSMV-VIGS) is effective in simultaneous silencing of all functionally active homoeologous gene

copies in wheat. It can also target simultaneously at least two unrelated genes (Cakir and Scofield,

2008; Campbell and Huang, 2010). This increases efficiency and is highly cost effective. In these

aspects BSMV-VIGS is similar to the stable transformation-based RNA interference (RNAi), a more

commonly used technique for obtaining information about the function of individual genes in cereals

(Travella et al., 2006; McGinnis, 2010). However, the major advantage of BSMV-VIGS is that it is very

rapid. In addition, it has a moderately high throughput compared to RNAi that requires genetic

transformation. An appropriately replicated single BSMV-VIGS experiment is usually completed in 5-6



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weeks and many different plant genotypes can be tested simultaneously. By comparison, stable

cereal transformation requires a dedicated facility, well trained staff and is also relatively slow. At least

8 months are required to produce the primary transgenic barley or wheat lines (T0 generation), usually

in just a single genetic background. Stable transformation is only low throughput and true breeding

RNAi lines are not available until at least the T3 generation.

Due to its speed and moderately high throughput, BSMV-VIGS allows rapid pre-screening of

candidate genes prior to the use of other, more time consuming techniques to assess gene function

such as the stable RNAi transformation or TILLING (Targeting Induced Local Lesions IN Genomes)

(Comai and Henikoff, 2006).

With food security becoming one of the key strategic research priorities globally, there has been a

recent deluge of available sequence information for the transcriptomes and genomes of major cereal

crop species including rice, wheat, barley and maize, and a model grass species Brachypodium

distachyon (Berkman et al., 2012). The importance of BSMV-VIGS as a research tool for all aspects

of cereal plant research in conjunction with comparative genomics, computational analyses

(bioinformatics) and other currently available reverse genetic tools cannot be underestimated.

BSMV-VIGS already has an excellent track record for unravelling the function of leaf-expressed

genes in barley and wheat at the vegetative (seedling) stage of development. This research tool has

been used almost exclusively by plant pathologists, for determining the function of plant defence

signalling genes in multiple host-pathogen interactions. Since the two most recent comprehensive

reviews were written (Scofield and Nelson, 2009; Cakir et al., 2010), there have been significant

advances in the development and deployment of BSMV-VIGS in several additional monocot species.

Furthermore, two new applications of the BSMV vector have emerged. The first, named Host-Induced

Gene Silencing (HIGS) (Nowara et al., 2010), permits trans-species silencing and has been

successfully used for analysing the function of genes in plant pathogenic fungi that are expressed

during plant infection. The second enables heterologous protein overexpression in planta and has

been coined ‘VOX’ (Virus-mediated Overexpression) (Roger Wise, personal communication).

Moreover, there has recently been a substantial interest in adoption of BSMV-derived tools by

scientists in other plant disciplines. Here we review all these exciting, very recent published and

unpublished (presented and discussed at the International Cereals VIGS Workshop (ICVW),

Rothamsted Research, UK on 22-24 June 2011) advances and developments.

ADVANCES IN BSMV-INDUCED PLANT GENE SILENCING

Expansion of the host range for BSMV-VIGS

The success of BSMV-VIGS as a tool for functional genomic studies in barley and bread wheat has

led to its adoption and deployment in a number of other monocots. Silencing of phytoene desaturase

(PDS), a gene commonly used as a visual marker in VIGS experiments due to the resulting



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characteristic photobleached phenotype (Figure 2A), has been demonstrated in B. distachyon

(Demircan and Akkaya, 2010; Pacak et al., 2010; Yuan et al., 2011). In Haynaldia villosa, a wild

grass, BSMV-VIGS has already been used for the functional analysis of powdery mildew resistance-

related genes (Wang et al., 2010). Other desirable traits of interest in these species including multiple

disease resistance, strong tillering ability, and high protein seed content can be tested and then

exploited by introgression breeding in commercial cereal crops.

BSMV-VIGS is currently being optimised for other valuable cereals such as millet (Setaria italic),

maize (Zea mays), and several members of the genus Triticum (D. Li, personal communication),

although similar experiments with oats have so far met with only limited success (Pacak et al., 2010).

There has been little work as yet with non-cereal monocots, however a study in culinary ginger

(Zingiber officinale) has successfully targeted PDS for silencing thereby demonstrating the potential

for the extension of BSMV-VIGS to a wider range of monocot hosts (Renner et al., 2009).

Application of BSMV-VIGS to disciplines beyond plant pathology

The BSMV-VIGS tool has been used to investigate the involvement of the WRKY53 transcription

factor and the phenylpropanoid pathway in resistance to aphids in wheat (van Eck et al., 2010). This

was a natural first step because plant virologists frequently collaborate with entomologists to study

insect virus vectors. A number of recent studies have used BSMV-VIGS to explore gene function in

other areas of plant science. In one study, silencing of the monocot-specific P23k gene in barley

resulted in morphological abnormalities such as strong asymmetry and cracks along the leaf margins,

confirming involvement of this gene in secondary cell wall biosynthesis (Oikawa et al., 2007). BSMV-

VIGS was also utilised to investigate the effect of silencing the barley cellulose synthase CesA (Held

et al., 2008), another gene predicted to be involved in cell wall biosynthesis. Interestingly, whilst the

gene silencing construct was designed to specifically target the cellulose synthase gene family, it also

triggered down-regulation of several homologous cellulose synthase-like genes, possibly due to

transitive silencing induced by secondary small interfering RNAs (siRNAs). A third study (Pacak et al.,

2010) demonstrated BSMV-induced silencing of three barley homologs of the Arabidopsis genes

involved in regulation of inorganic phosphate (Pi) uptake and translocation. Silencing of one of these

genes, under conditions of abundantly supplied Pi resulted in excessive Pi accumulation in the leaf

tissues compared to that in control barley plants. This silencing phenotype is reminiscent of the

Arabidopsis pho2 phosphate-accumulator mutant (Delhaize and Randall, 1995), indicating that PHO2

is functionally conserved in barley and Arabidopsis.

Silencing beyond the leaf

BSMV is readily transmitted through seed (Jackson et al., 2009) and also invades the root system (Lin

and Langenberg, 1984). Therefore, due to the biology of this virus, VIGS in flowering tissues, seeds



8

and roots should be possible. The first report on successful deployment of BSMV-VIGS in wheat roots

has just been published (Bennypaul et al., 2011). In this work the coronatine insensitive1 (COI1) gene

was targeted, and the silencing (85% decrease in transcript level) resulted in a 26% reduction in root

length.

At the ICVW several teams, including our own, already employing BSMV-VIGS in their research

convincingly reported the successful silencing of target genes in floral tissue in a range of different

wheat cultivars following inoculation of upper leaves at pre-heading (Figure 2B-D). A number of these

teams are interested in identifying host genes involved in susceptibility to Fusarium species that

cause Fusarium ear blight (also known as Fusarium head blight or scab), the globally important

disease of wheat and barley, which leads to deoxynivalenol (DON) mycotoxin contaminated grain at

harvest (Dean et al., 2012). In addition, Kulvinder Gill reported successful deployment of BSMV-VIGS

for analysing function of genes expressed in developing wheat grain and meiotic tissues. Silencing of

the granule-bound starch synthase (GBSS or waxy) gene resulted in substantial reductions in the

amylose content in seeds, whereas silencing of DMC1 (disrupted meiosis cDNA1), a gene involved in

early meiosis in pollen mother cells, led to the disruption of chromosome pairing at metaphase I.

Results of this pioneering study have since been published (Bennypaul et al., 2011). Gene silencing

in floral and reproductive tissues and the grain of wheat has been achieved by applying the

corresponding BSMV-VIGS constructs to older plants with the fully expanded flag leaf. We and others

(Patrick Schweizer, personal communication) have recently discovered that same can also be

achieved in rapid cycling wheat genotypes such as USU-Apogee (Bugbee, 1999) by BSMV vector

delivery to young plants (3-5 leaf stage) (Figure 2E-F).

Silencing in the next plant generations

BSMV is able to invade the reproductive tissues prior to fertilisation and the spread and survival of

this virus in nature is due to its ability to be transmitted through seed in barley and wheat (Jackson et

al., 2009). The inheritance of BSMV-induced gene silencing in the progeny of infected plants was

initially demonstrated for barley (Bruun-Rasmussen et al., 2007). This same phenomenon was

reported for wheat at the ICVW by Kulvinder Gill. This opens up the possibility of using VIGS to target

genes that are expressed during seed dormancy, germination, and early seedling development. The

presence of BSMV appears not to impair seed development and / or appearance (Figure 2G-H).

Recent work from both the Kulvinder Gill laboratory (Bennypaul et al., 2011) and our laboratory on

silencing of PDS indicate that ~ 10-15% of progeny from the inoculated wheat plants display

photobleaching and therefore retain silencing (Figure 2I-J). The efficiency of silencing in next

generations is highly influenced by the genotype (Bennypaul et al., 2011; Lee et al., unpublished). All

the next generation plants showing photobleaching contain BSMV with either the original or shortened

PDS-specific insert (Bruun-Rasmussen et al., 2007; Lee et al., unpublished). The silencing phenotype

in the next generation plants is therefore likely to be initiated by the recombinant BSMV being carried

within the seed rather than involving an epigenetic-based mechanism. Interestingly, the proportion of



9

plants showing PDS gene silencing increases with each subsequent generation of self-pollinated

plants and up to 90-100% of the third generation plants display photobleaching (Bennypaul et al.,

2011). Again, striking differences between wheat genotypes to exhibit photobleaching in later

generations were noted.

Vector improvements

Several variations have been made to the original BSMV-VIGS system. Each variation has important

implications for the throughput and ease of use of this research tool. The originally described system

used BSMV cDNA clones under the control of the strong bacteriophage T7 promoter. Capped

transcripts for the three BSMV genomes, RNAα, RNAβ and RNAγ, are produced in vitro,

subsequently mixed together and then rub-inoculated onto individual host plants. Fragments of target

plant genes are introduced into the BSMV RNAγ genome vector via restriction digestion with multiple

restriction enzymes and ligation-based cloning (Holzberg et al., 2002).

In vitro transcription is costly and prompted innovation. One variant system from the Roger Wise

laboratory avoids this expense by replacing the T7 promoter with the 35S promoter from Cauliflower

mosaic virus (CaMV) and introducing a ribozyme sequence downstream of each viral cDNA to

generate the correct 3’-end after transcription. These modifications allow in planta transcription of the

DNA plasmids following their delivery into barley leaf tissues using microprojectile particle

bombardment (Meng et al., 2009). Sap extracted from the transfected barley leaves containing

reconstituted virus can then be used to infect more plants for VIGS studies.

Another variant of the BSMV-VIGS vector retains the T7 promoter but replaces the original cloning

site immediately 3’ to the coding region in the BSMV RNAγ genome with a Ligation-Independent

Cloning (LIC) site in order to facilitate efficient insertion of target gene fragments (Pacak et al., 2010).

The latest report unites the advantages of both systems by coupling a LIC strategy for cloning target

gene fragment inserts with an Agrobacterium-mediated delivery system (Yuan et al., 2011). Here,

BSMV genomes cloned into a binary Ti vector under control of CaMV 35S promoter are first delivered

via agroinfiltration into the leaves of N. benthamiana, an intermediate host susceptible to both A.

tumefaciens and BSMV. Sap is then extracted from the infiltrated leaves and used to inoculate a large

number of monocotyledonous plants. Since this system was first described at the ICVW, there has

been significant interest in its uptake by members of the cereal VIGS community due to its relative

simplicity and low set-up costs.

BSMV FOR GENE SILENCING IN PLANT-ASSOCIATED ORGANISMS

An emerging and exciting new technical advancement is Host-Induced Gene Silencing (HIGS). In

HIGS, silencing that is triggered directly within the plant targets genes in plant pathogens or pests



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(Figure 1Biii). The triggers, as in VIGS, are short double-stranded RNAs (dsRNAs). However, in the

case of HIGS, the dsRNAs target pathogen or pest transcripts rather than plant transcripts. Silencing-

inducing dsRNAs are produced in the host plant cells following one of the three delivery methods: (i)

microprojectile particle bombardment of RNAi constructs into plant leaves, (ii) stable plant

transformation with RNAi constructs, or (iii) using a virus vector such as BSMV (Nunes and Dean,

2011). Plants either transiently or stably expressing the dsRNA triggers are then inoculated with the

relevant organism, and the role of a target pathogen or pest gene during the infection can be studied.

Very recently, HIGS has been successfully applied to the investigation of molecular interactions

between obligate biotrophic fungi and cereal crops. In one study (Nowara et al., 2010) both stable and

transient methods of delivery of silencing constructs have been explored. First, seventy-six RNAi

constructs, each targeting one of the genes of the barley powdery mildew fungus Blumeria graminis f.

sp. hordei known to be expressed in planta during infection, were bombarded into the individual

epidermal cells of barley leaves. The ability of the fungus to form a haustorium, a specialised post

infection feeding structure required for absorbing nutrients from the plant, within these epidermal host

cells was then analysed. Remarkably, nearly a quarter of the tested RNAi constructs induced

significant reduction of haustorium formation. The function of two fungal genes, GTF1 and GTF2,

targeted by these constructs was further investigated in transgenic RNAi barley lines (GTF1 only) and

also using BSMV-HIGS. Both approaches proved to be successful, and revealed different roles for

these two genes in fungal development, with GTF1 likely to be involved in initial haustorium formation

and GTF2 in elongation of secondary hyphae, necessary for colony formation. In the second study

(Yin et al., 2011) BSMV-HIGS was used to silence twelve genes of another obligate biotroph, the

stripe rust fungus Puccinia striiformis f. sp. tritici. The genes selected were known to be expressed

during infection of wheat leaves. In these experiments silencing was only observed for genes that

were preferentially highly expressed in haustoria and not in the bulk of the fungal mycelial colony. For

both species, fungal haustoria penetrate mesophyll cells in host leaf tissue and represent the interface

for signal exchange between the fungus and the invaded plant. Presumably RNA silencing was most

efficient in fungal cells in very close proximity to plant cellular cytoplasm and less so in fungal hyphae

which have an extracellular localisation.

The mechanism(s) by which RNA silencing signals are delivered from the plant cell into fungal cells is

currently a mystery. For obligate biotrophic fungal pathogens like barley powdery mildew it has been

hypothesised that these signals are transported from the plant cell into the haustorial cells (Nowara et

al., 2010). However, these fungal cells are separated from the host cells by the fungal cell wall and

plasma membrane as well as by the extrahaustorial matrix and extrahaustorial membrane derived

from the plant plasma membrane (Panstruga and Dodds, 2009) (Figure 1Biii). Therefore, the silencing

signal (which most probably is a short dsRNA or single-stranded RNA) needs to cross multiple

physical barriers in order to initiate HIGS. One of the suggested transport routes is the exosomal

secretory pathway (Nowara et al., 2010). But clearly further research is required to answer this

intriguing question.



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HIGS of fungal genes in stably transformed plant RNAi lines in the arbuscular mycorrhizal fungus

Glomus intraradices - Medicago truncatula interaction, where the fungal and host cellular membranes

are again in close proximity, has also been demonstrated successfully (Helber et al., 2011). Recently,

another study demonstrated that expression of a β-glucuronidase (gus)-derived dsRNA hairpin in

tobacco (Nicotiana tabacum) could trigger specific silencing of this gene in the mycelia of a transgenic

GUS-expressing strain of Fusarium verticillioides during infection (Tinoco et al., 2010). This result is

particularly important because F. verticillioides growth is exclusively extracellular, and this fungus is

not known to produce an intracellular feeding structure.

Within the past two years, a BSMV-based HIGS system has emerged as a viable alternative, or at

least a complementary approach to that of generating numerous fungal gene knock-outs to

investigate the role of fungal genes in plant-fungus interactions (Nunes and Dean, 2011). This method

is particularly useful for studies of important plant pathogens and symbionts for which stable

transformation methods are not yet available, or for obligate plant-associated organisms that cannot

be cultured in vitro.

BSMV FOR OVEREXPRESSION OF HETEROLOGOUS PROTEINS (BSMV-VOX)

Virus-mediated overexpression of heterologous proteins (VOX) using the BSMV vector was first

demonstrated in studies involving the Green Fluorescent Protein (GFP) reporter and barley (Haupt et

al., 2001; Lawrence and Jackson, 2001). Although the overall levels of GFP expression from BSMV

was shown to be strong, expression was often reported to be patchy in the systemically-infected

vegetative tissue of monocotyledonous plant hosts. Whilst Lawrence and Jackson (2001) initially

suggested that this may be due to BSMV exiting the vasculature at more than one location in

systemically-infected leaves, forming multiple infection loci, it is probably also due in part to the

relatively large size of GFP (720-nt), and subsequently, the instability of the GFP coding sequence in

the viral genome.

The community consensus is that larger fragments appear to be lost from the BSMV vector more

frequently than smaller fragments (Bruun-Rasmussen et al., 2007). Mutants with a reduced insert size

stand higher chances of establishing systemic infection because they are generally more fit (i.e. they

replicate faster and / or move within the infected plant faster) than their originally sized recombinant

counterparts. As reported by different authors, inserts within the range of 140-bp to 500-bp when

integrated into the RNAγ genome of BSMV are relatively more stable than larger inserts (Holzberg et

al., 2002; Scofield and Nelson, 2009). Indeed, a flavin-based fluorescent reporter protein iLOV that is

approximately half the size of GFP (Chapman et al., 2008), appears to be more stably and more

uniformly expressed compared to GFP when delivered to wheat and barley leaves using BSMV vector

(Figure 3A and Kanyuka et al., unpublished).



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Despite this significant size constraint on BSMV-VOX, it is already a viable strategy for investigating

the function of small proteins, such as those predicted to be fungal effectors. The majority of known

fungal effectors are small (i.e. comparable in size with iLOV), usually cysteine-rich secreted proteins

containing no conserved functional domains (Stergiopoulos and de Wit, 2009). Confirmed fungal

effector proteins are of particular interest, because for some their production in planta is known to be

essential for successful pathogen invasion and colonisation, whilst others are the cues detected by

plants that lead to the triggering of resistance (R) gene mediated defence (Jones and Dangl, 2006).

BSMV-VOX has already been used to express the ToxA effector protein from Pyrenophora tritici-

repentis, the causal agent of tan spot disease in wheat (Tai et al., 2007; Manning et al., 2010).

Previously, it was known that ToxA produced in vitro and purified from the culture filtrate induces

necrosis on wheat cultivars susceptible to the disease when infiltrated into the extracellular spaces of

the leaf (Ballance et al., 1989).

Some fungal effectors may be expected to either suppress plant innate immunity during early

invasion, or stimulate cell death or induction of necrosis at later stages of disease development.

BSMV-VOX has a major advantage over microprojectile particle bombardment, another methodology

for delivery of constructs for transient protein expression in plant leaf cells, in that it avoids tissue

damage caused by bombardment that could be confused with effector-induced cell death. The ease

of inserting protein coding sequences into the BSMV vector also means that this approach could

potentially be used in a forward genetic screen to characterise the large numbers of putative effectors

now predicted from interrogation of the recently sequenced fungal genomes, for example Blumeria

graminis (Godfrey et al., 2010) and Fusarium graminearum (Brown et al., 2012).

In all BSMV-VOX studies to date involving cereal infecting fungal species, heterologous proteins were

expressed as direct C-terminal fusions to the viral γb protein. This approach, at least in some cases,

may be expected to compromise functionality or to affect the cellular localisation pattern of the

expressed protein and / or the viral γb protein. An alternative and more preferable approach involves

the use of self-processing peptide bridges, such as the 18-amino-acid-long catalytic autoproteolytic

2A peptide sequence of picornaviruses (El Amrani et al., 2004), between the fused proteins. This

approach has been successfully used in the past for the expression of GFP as an N-terminal fusion to

the BSMV γb protein (Torrance et al., 2006). We modified the BSMV RNAγ vector by inserting a

synthetic 2A gene at the 3’-terminus of the viral γb for production of C-terminal protein fusions (Figure

1Bii). This, in theory, should allow co-translational self-processing of the fusion protein resulting in

some free heterologous protein, and also permit expression of heterologous proteins with their native

N-termini e.g. proteins such as fungal effectors containing N-terminal signal peptides for secretion into

the plant apoplast. Our preliminary experiments on BSMV-VOX of NIP1, the necrosis-inducing small

secreted effector protein from Rhynchosporium commune (previously known as R. secalis) that

operates in the plant apoplast (Wevelsiep et al., 1991), confirm the above theory, albeit indirectly. R.

commune NIP1 is known to elicit defence reactions specifically in barley genotypes carrying the

cognate Rrs1 resistance gene (Rohe et al., 1995). Indeed, expression of the full-length NIP1 using a

modified BSMV as explained above resulted in genotype-specific Rrs1-dependent cell death. By



13

contrast, a truncated version of NIP1 (with the signal peptide removed) incapable of being secreted

failed to induce cell death on barley genotypes with or without Rrs1 (Figure 3B-C and Kanyuka et al.,

unpublished).

Recently, there has been a preliminary (conference) report of a new four-component BSMV vector

system that potentially enables much larger proteins to be stably expressed in planta (Robert

Brueggeman, personal communication). This system is outlined in Figure 4. If this system is indeed

proven to work, it has major implications for the use of BSMV-VOX in gene function studies. For

example, R gene candidates identified in mapping studies could be expressed directly in susceptible

genotypes, and genes from major resistance QTLs screened and identified. This type of vector would

also be highly applicable to gene function investigations through overexpression for a wide range of

predicted cereal proteins.

FINAL REMARKS AND NEW OPPORTUNITIES

The advent of the VIGS technology has revolutionised gene function discovery in many dicots and

has now become a powerful tool for plant science research. For monocots, VIGS is not yet centre

stage. However, recent significant improvements and developments in BSMV-VIGS, which include (i)

marked reduction in cost, (ii) expanded host range, (iii) ability to silence not only leaf genes but also

those expressed in root, floral tissues and the grain, and (iv) maintenance of silencing in the next

generation of plants, will certainly make this research tool more attractive to plant scientists and

present an exciting opportunity for new researchers entering the field. For example, in the near future

we expect to see BSMV-VIGS commonly used in plant research for rapid pre-screening / selecting

candidate genes from various gene cloning projects. BSMV-VIGS will also be very useful in a rapidly

growing number of genomics projects aiming to translate knowledge of gene function from model

plants to crops, such as wheat and barley. In coming years we also foresee expansion of usage of

BSMV as a vector for host-induced silencing of genes in various cereal-infecting plant pathogenic

species, especially for obligate biotrophs that cannot be grown in culture and other species that are

not yet amenable to stable transformation. Effector biology of plant-associated organisms is a

dynamic, new and exciting research area. No doubt BSMV-VOX will play an important role in

identification and functional characterisation of small effector proteins from plant-associated

organisms. Next generation derivatives of BSMV vectors allowing stable expression of larger proteins

are rumoured to become available in near future. When this happens, it will open enormous number

of new possibilities for functional protein analyses through overexpression in planta.

ACKNOWLEDGEMENTS

We thank the Biotechnology and Biological Sciences Research Council of the UK (BBSRC) for

sponsoring the ICVW (International Partnering Award, ref. BB/I025077/1), and the ICVW co-organiser



14

Steve Scofield (USDA-ARS & Purdue University, USA) and other participants for freely sharing their

ideas and unpublished information used in this manuscript. All experiments involving recombinant

BSMV at Rothamsted Research were conducted in biological containment facilities under Defra Fera-

PHSI licence number PHSI 181/6786. We thank Julian Franklin (Rothamsted Biosecurity Officer) as

well as our colleagues Martin Urban, Juliet Motteram, Jason Rudd and Hai-Chun Jing for their help

with experimental and desk work required for obtaining above mentioned plant health licence. We

also thank John Lucas, Alexandre Amaral and Martin Urban for critical reading of this manuscript.

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FIGURE LEGENDS

Figure 1. The Barley stripe mosaic virus (BSMV) genome and mechanistic models for BSMV-

VIGS, BSMV-VOX and BSMV-HIGS.

The BSMV genome is comprised of three RNAs that are capped at the 5’-end and form a tRNA-like

hairpin secondary structure at the 3’-terminus. RNAα encodes the αa replicase protein containing

methyl transferase (MT) and helicase (HEL) domains. RNAβ encodes βa (coat protein), and the βb,

βc and βd movement proteins (also known as TGB1, TGB3 and TGB2, respectively). RNAβ also

encodes a minor protein βd’ (alias TGB2’), which is expressed by translational read-through of the βd

ORF. RNA� encodes �a, the polymerase (POL) component of replicase, and the cysteine-rich �b

protein involved in viral pathogenicity. B) For BSMV-VIGS, BSMV-VOX and BSMV-HIGS,

heterologous sequences of interest are typically inserted directly downstream of the stop codon of the

�b ORF. For each experimental scenario, the modified RNA� is mixed with RNAα and RNAβ (not

shown) and inoculated onto host plants. Bi) In BSMV-VIGS, a fragment from the plant gene of interest

is cloned into BSMV RNA� usually in the antisense orientation, downstream of the stop codon of the

�b ORF. After the virus enters a host plant cell, long double-stranded RNA (dsRNA) formed during

viral replication are recognised by host Dicer-like enzymes (DCLs) that cleave it into 21-22-nt small

interfering RNAs (siRNAs). One strand of each siRNA is then incorporated into host RNA-induced

silencing complexes (RISC). RISC mediates endonucleolytic cleavage of single stranded RNAs with

sequence complementary to the incorporated siRNA strand. As some of these siRNAs will have been

generated from the plant gene fragment inserted in the BSMV genome, they will also guide RISC to

plant mRNAs with sequence complementarity, resulting in silencing of the target endogenous gene(s).

Bii) For BSMV-VOX, the coding sequence for the protein of interest is inserted immediately upstream

the in-frame stop codon of the �b ORF. In a new system, a small synthetic 2A gene encoding an

autoproteolytic peptide has been inserted between the 3’-terminus of the �b ORF and the gene

sequence coding for the heterologous protein. This enables self-processing (not shown) of the

ensuing �b fusion protein during translation of the virally-encoded proteins, which occurs very soon

after the entry of BSMV to the plant cell, thus releasing the free heterologous protein. Biii) In BSMV-

HIGS, a fragment of a fungal gene of interest is inserted in antisense orientation into RNA�

downstream of the stop codon of the �b ORF. RNA silencing signals generated within the plant cell

can then trigger gene silencing in fungal cells in intimate contact to these modified host cells, such as

fungal haustorial cells separated from plant cell membranes by only the extrahaustorial matrix (EHM).

The mechanism(s) by which HIGS occurs are not yet known, but fungal-specific siRNAs generated by

plant DCLs activity are likely to be involved.

Figure 2. Silencing of phytoene desaturase (PDS) in wheat leaves, ears and in the second plant

generation.



19

A) Upper uninoculated leaves taken from plants infected with either BSMV:asGFP control (top) or the

BSMV:asPDS showing the resulting photobleached phenotype (bottom) at 28 days post inoculation

(dpi). B-D) Inoculating the flag and / or the penultimate leaf of pre-heading wheat plants can induce

silencing in the ears. The control BSMV:asGFP infected ears appear to develop normally (panel B).

Silencing of PDS in the ears (panels C and D) results in visible photobleaching of the glumes, awns

and rachis. Photographs were taken at 28 dpi. E-F) Inoculation of lower leaves of younger wheat cv.

Apogee plants at the 3-5 leaf stage often induces silencing in the flag leaf and ear. The control

BSMV:asGFP infected plants appear to develop normally (panel E), whereas the BSMV:asPDS

inoculations (panel F), induces almost complete photobleaching in the flag leaf, and partial

photobleaching in the ear. The plants were photographed at 28 dpi. G-H) Grain from uninfected plants

(panel G) and the BSMV:asPDS inoculated plants (panel H) are comparable in size and appearance.

I-J) A proportion (10-15 %) of the progeny from BSMV:asPDS infected plants also display partial

photobleaching of the leaves within 12 days of seed germination.

Figure 3. Barley stripe mosaic virus mediated over-expression of small heterologous proteins.

A) A fragment of wheat leaf infected with recombinant BSMV expressing a fluorescent reporter

protein iLOV. Image was obtained using confocal laser microscopy. Plant cell walls are coloured

purple and chloroplasts are in blue, whereas cells containing iLOV fluoresce green. B) Recombinant

BSMV expressing full-length Nip1 protein of Rhynchosporium commune induces cell death on leaves

of barley cv. Atlas 46 expressing the cognate resistance gene Rrs1. C) Recombinant BSMV

expressing full-length Nip1 protein of R. commune induces cell death on leaves of cv. Atlas 46

containing the cognate resistance gene Rrs1, but not on a near isogenic line Atlas devoid of Rrs1

(top). Truncated Nip1, with the N-terminal signal peptide removed (no SP), is unable to induce cell

death on barley. All photographs were taken at 7 days post inoculation.

Figure 4. A new, four component BSMV vector system for expression of larger sized

heterologous proteins.

The system comprises the wild-type BSMV RNAα and RNAβ, and two differently modified RNA�

(RNA�1 and RNA�2). RNA�1 has retained the functional �a replicase gene but a large deletion in

the �b coding region renders this gene non-functional. The almost complete deletion of the �b gene

is required to ensure the retention of, and heterologous protein expression from, the recombinant

RNA�2. The latter has large deletion in the �a coding region, which allows sequences of up to 2-kb

to be inserted into this region, whilst sequences downstream of �a including the �b gene have been

left intact. During plant infection it is anticipated that the intact ya coding region and yb coding region

expressed from the two differently modified RNA� will be able to function in this ‘trans’ configuration

and thereby generate infectious BSMV expressing larger sized, potentially up to approximately 65

kDa, heterologous proteins of interest.



Figure 1



Figure 1. The Barley stripe mosaic virus (BSMV) genome and mechanistic models for BSMV-VIGS, BSMV-VOX and BSMV-HIGS.

A) The BSMV genome is comprised of three RNAs that are capped at the 5’-end and form a tRNA-like hairpin secondary structure at the 3’-terminus. RNAα encodes the αa replicase protein containing methyl transferase (MT) and helicase (HEL) domains. RNAβ encodes βa (coat protein), and the βb, βc and βd movement proteins (also known as TGB1, TGB3 and TGB2, respectively). RNAβ also encodes a minor protein βd’ (alias TGB2’), which is expressed by translational read-through of the βd ORF. RNAɣ encodes ɣa, the polymerase (POL) component of replicase, and the cysteine-rich ɣbprotein involved in viral pathogenicity. B) For BSMV-VIGS, BSMV-VOX and BSMV-HIGS, heterologous sequences of interest are typically inserted directly downstream of the stop codon of the ɣb ORF. For each experimental scenario, the modified RNAɣ is mixed with RNAα and RNAβ (not shown) and inoculated onto host plants. Bi) In BSMV-VIGS, a fragment from the plant gene of interest is cloned into BSMV RNAɣ usually in the antisense orientation, downstream of the stop codon of the ɣb ORF. After the virus enters a host plant cell, long double-stranded RNA (dsRNA) formed during viral replication are recognised by host Dicer-like enzymes (DCLs) that cleave it into 21-22-nt small interfering RNAs (siRNAs). One strand of each siRNA is then incorporated into host RNA-induced silencing complexes (RISC). RISC mediates endonucleolytic cleavage of single stranded RNAs with sequence complementary to the incorporated siRNA strand. As some of these siRNAs will have been generated from the plant gene fragment inserted in the BSMV genome, they will also guide RISC to plant mRNAs with sequence complementarity, resulting in silencing of the target endogenous gene(s). Bii) For BSMV-VOX, the coding sequence for the protein of interest is inserted immediately upstream the in-frame stop codon of the ɣb ORF. In a new system, a small synthetic 2A gene encoding an autoproteolytic peptide has been inserted between the 3’-terminus of the ɣb ORF and the gene sequence coding for the heterologous protein. This enables self-processing (not shown) of the ensuing ɣb fusion protein during translation of the virally-encoded proteins, which occurs very soon after the entry of BSMV to the plant cell, thus releasing the free heterologous protein. Biii) In BSMV-HIGS, a fragment of a fungal gene of interest is inserted in antisense orientation into RNAɣ downstream of the stop codon of the ɣb ORF. RNA silencing signals generated within the plant cell can then trigger gene silencing in fungal cells in intimate contact to these modified host cells, such as fungal haustorial cells separated from plant cell membranes by only the extrahaustorial matrix (EHM). The mechanism(s) by which HIGS occurs are not yet known, but fungal-specific siRNAs generated by plant DCLs activity are likely to be involved.



Figure 2



Figure 2. Silencing of phytoene desaturase (PDS) in wheat leaves, ears and in the second plant generation.

A) Upper uninoculated leaves taken from plants infected with either BSMV:asGFP control (top) or the BSMV:asPDS showing the resulting photobleached phenotype (bottom) at 28 days post inoculation (dpi). B-D)Inoculating the flag and / or the penultimate leaf of pre-heading wheat plants can induce silencing in the ears. The control BSMV:asGFP infected ears appear to develop normally (panel B). Silencing of PDS in the ears (panels C and D) results in visible photobleaching of the glumes, awns and rachis. Photographs were taken at 28 dpi. E-F) Inoculation of lower leaves of younger wheat cv. Apogee plants at the 3-5 leaf stage often induces silencing in the flag leaf and ear. The control BSMV:asGFP infected plants appear to develop normally (panel E), whereas the BSMV:asPDS inoculations (panel F), induces almost complete photobleaching in the flag leaf, and partial photobleaching in the ear. The plants were photographed at 28 dpi. G-H)Grain from uninfected plants (panel G) and the BSMV:asPDS inoculated plants (panel H) are comparable in size and appearance. I-J) A proportion (10-15 %) of the progeny from BSMV:asPDS infected plants also display partial photobleaching of the leaves within 12 days of seed germination.



Figure 3

Figure 3. Barley stripe mosaic virus mediated over-expression of small heterologous proteins.

A) A fragment of wheat leaf infected with recombinant BSMV expressing a fluorescent reporter protein iLOV. Image was obtained using confocal laser microscopy. Plant cell walls are coloured purple and chloroplasts are in blue, whereas cells containing iLOV fluoresce green. B) Recombinant BSMV expressing full-length Nip1 protein of Rhynchosporium commune induces cell death on leaves of barley cv. Atlas 46 expressing the cognate resistance gene Rrs1. C) Recombinant BSMV expressing full-length Nip1 protein of R.commune induces cell death on leaves of cv. Atlas 46 containing the cognate resistance gene Rrs1, but not on a near isogenic line Atlas devoid of Rrs1(top). Truncated Nip1, with the N-terminal signal peptide removed (no SP), is unable to induce cell death on barley. All photographs were taken at 7 days post inoculation.



Figure 4

Figure 4. A new, four component BSMV vector system for expression of larger sized heterologous proteins.

The system comprises the wild-type BSMV RNAα and RNAβ, and two differently modified RNAɣ (RNAɣ1 and RNAɣ2). RNAɣ1 has retained the functional ɣa replicase gene but a large deletion in the ɣb coding region renders this gene non-functional. The almost complete deletion of the ɣbgene is required to ensure the retention of, and heterologous protein expression from, the recombinant RNAɣ2. The latter has large deletion in the ɣa coding region, which allows sequences of up to 2-kb to be inserted into this region, whilst sequences downstream of ɣa including the ɣb gene have been left intact. During plant infection it is anticipated that the intact ya coding region and yb coding region expressed from the two differently modified RNAɣ will be able to function in this ‘trans’ configuration and thereby generate infectious BSMV expressing larger sized, potentially up to approximately 65 kDa, heterologous proteins of interest.



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Plant Physiology Preview. Published on August 10, 2012, as ... · 8/10/2012 · Rothamsted Research, UK on 22-24 June 2011) advances and developments. ADVANCES IN BSMV-INDUCED PLANT