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MPMI Vol. 21, No. 8, 2008, pp. 1015–1026. doi:10.1094/MPMI-21-8-1015. © 2008 The American Phytopathological Society
Nicotiana benthamiana: Its History and Future
as a Model for Plant–Pathogen Interactions
Michael M. Goodin,1 David Zaitlin,2 Rayapati A. Naidu,3 and Steven A. Lommel4
1Department of Plant Pathology and 2Kentucky Tobacco Research and Development Center (KTRDC), University
of Kentucky, Lexington 40546, U.S.A.; 3Department of Plant Pathology, Irrigated Agriculture Research & Extension Center,
Washington State University, Prosser 99350, U.S.A.; 4Department of Plant Pathology, North Carolina State University,
Raleigh 27695, U.S.A.
Submitted 27 November 2007. Accepted 11 April 2008.
Nicotiana benthamiana is the most widely used experimen-
tal host in plant virology, due mainly to the large number
of diverse plant viruses that can successfully infect it. Addi-
tionally, N. benthamiana is susceptible to a wide variety of
other plant-pathogenic agents (such as bacteria, oomycetes,
fungi, and so on), making this species a cornerstone of
host–pathogen research, particularly in the context of
innate immunity and defense signaling. Moreover, because
it can be genetically transformed and regenerated with
good efficiency and is amenable to facile methods for virus-
induced gene silencing or transient protein expression, N.
benthamiana is rapidly gaining popularity in plant biology,
particularly in studies requiring protein localization, inter-
action, or plant-based systems for protein expression and
purification. Paradoxically, despite being an indispensable
research model, little is known about the origins, genetic
variation, or ecology of the N. benthamiana accessions cur-
rently used by the research community. In addition to ad-
dressing these latter topics, the purpose of this review is to
provide information regarding sources for tools and reagents
that can be used to support research in N. benthamiana.
Finally, we propose that N. benthamiana is well situated to
become a premier plant cell biology model, particularly for
the virology community, who as a group were the first to
recognize the potential of this unique Australian native.
Additional keywords: AFLP, agroinfiltration, Arabidopsis, VIGS.
Nicotiana benthamiana: history, taxonomy,
and basic biology.
As currently circumscribed, the genus Nicotiana (Solanaceae:
Linnaeus 1753) includes 76 species of mainly tropical and
subtropical distribution from four continents, with the majority
occurring in South America and Australia (Knapp et al. 2004).
Although the base haploid chromosome number is considered
to be 12, amphiploidy is common in the genus, with many ex-
tant species (or their recent successors) featuring in the reticu-
late evolution of the allopolyploids. Haploid chromosome num-
bers range from 9 and 10 (species in section Alatae) to 24
(Nicotiana tabacum and N. rustica) and even 32 in some col-
lections of N. suaveolens, and there is a large variation in both
chromosome size (Goodspeed 1954) and genome size (Angio-
sperm DNA C-values database). In his seminal monograph on
the genus, Thomas Harper Goodspeed (1954) recognized 13
infrageneric sections, grouping the 61 then-recognized species
by common morphological and cytogenetic characters. Mod-
ern molecular phylogenetic analyses have largely supported
the Goodspeed view of Nicotiana, although the positions of
several species have been moved to more correctly reflect their
true affinities (Chase et al. 2003; Knapp et al. 2004). These latter
authors now recognize 13 sections, with many of the original
Goodspeed names preserved.
The subject of this review is N. benthamiana Domin, a
unique species endemic to Australia. N. benthamiana was first
discovered and collected on the “N.W. Coast” of New Holland
(Australia) by ship’s surgeon Benjamin Bynoe on the third
voyage of the 10-gun brig HMS Beagle, of Charles Darwin
fame. For its final voyage of exploration, the Beagle was com-
manded by J. C. Wickham (1837 to 1841) and J. Lort Stokes
(1841 to 1843), both of whom had sailed with Darwin under
Captain Robert FitzRoy. Mr. Bynoe was a highly experienced
and valued medical officer and sailor, and he is mentioned
many times in Stokes’ lengthy two-volume account of this
voyage (Stokes 1846). We know that he was also an avid natu-
ralist, having collected with Darwin in the Galapagos and else-
where on the second Beagle voyage from 1831 to 1836. Bynoe
discovered many species new to science, most of which he col-
lected with a rifle (and some of which then were eaten). In be-
tween Stokes’ excruciatingly detailed accounts of weather,
ocean depth, and tides (6 years of observations) are discus-
sions of the native peoples and the flora and fauna encountered
on land excursions along the coast and nearby islands. The sin-
gle reference made to collecting plants came from 7 November
1839, shortly after the discovery of the Victoria River: “Mr.
Bynoe, as he had done yesterday, added to his valuable collec-
tion a few rare birds, and strange plants;…” (Stokes 1846, vol-
ume 2). However, it is not known whether N. benthamiana was
discovered on that occasion. In a letter to Sir William Hooker,
dated 9 October 1843, Bynoe wrote, “I have taken advantage
of every opportunity that offered on the Coast of New Holland
and indeed the whole collection in Plants, Birds, Shells, Spirit
preparations &c [etc.] were obtained for Sir. Wm. Burnett,
however, as there are duplicates of most of them, no doubt I
shall be able to contribute towards your Herbarium.” (Keevil
Corresponding author: M. Goodin; Telephone: +1.859.257.7445 ext. 80725;
Fax: +1.859.323.1961; E-mail: firstname.lastname@example.org
*The e-Xtra logo stands for “electronic extra” and indicates that supple-
mental materials documenting detection of TSWV by ELISA and the
method used for AFLP analysis are published online.
1016 / Molecular Plant-Microbe Interactions
1949). Among the collections donated to the Herbarium Hook-
erianum are samples of what is now known to science as N.
benthamiana. Bynoe’s original specimen, the holotype, was
transferred to the Royal Botanic Gardens, Kew sometime after
the death of Sir William Hooker in 1865, and is reproduced as
Plate 1 in Burbidge (1960) and here as Figure 1. Written on the
original herbarium sheet in an unknown hand is the first classi-
fication (the basionym), N. suaveolens var. cordifolia, and the
reference to its original publication in Volume IV of the seven-
volume series Flora Australiensis (Bentham 1869). The sheet
also bears the mark of Czech botanist and politician Karel
Domin, who honored George Bentham with the publication of
N. benthamiana as a new taxon in 1929 (Goodspeed 1954).
The 20 Australasian species of Nicotiana all belong to N.
sect. Suaveolentes and all are of allopolyploid origin. Haploid
chromosome numbers within this section can be 16 (32), 18,
19, 20, 21, 22, 23, and 24, depending on the species. N. ben-
thamiana is one of two species with 19 pairs of chromosomes,
the other being N. excelsior from central Australia (Burbidge
1960), to which it is closely related (Chase et al. 2003). The
size of the haploid genome (1 C-value) of N. benthamiana is
estimated to be 3.2 pg (= 3,136 Mbp) (Bennett and Leitch
1995; Narayan 1987). In contrast, the Arabidopsis thaliana
genome is 157 Mbp, nearly 20-fold smaller than that of N.
benthamiana (Bennett and Leitch 2005; Bennett et al. 2003).
N. benthamiana has a broad, somewhat discontinuous distri-
bution from extreme Western Australia east to western Queens-
land, with a single documented collection from northern South
Australia (for a map see Australia’s Virtual Herbarium
website). In her examination of both living and preserved speci-
mens, Burbidge (1960) observed that, “A considerable range of
variation is at present accepted under this name [although the]
differences between specimens are in part due to whether the
plants were collected in a juvenile, mature or secondary
regrowth state as well as to differences between populations”
(page 351). Like the desert tobacco (N. obtusifolia) of North
America, N. benthamiana is distributed widely and occupies
seasonally arid habitats that are unfavorable to other native
species of Nicotiana. Because of this, and because N. bentha-
miana is considered to have evolved in isolation from the other
Australian species in the genus (Goodspeed 1954), we would
expect to find considerable morphological, physiological, ge-
netic, and genomic diversity present in natural populations of
this species. Partial support for this hypothesis comes from
field notes accompanying N. benthamiana herbarium specimens
collected from 1994 to 2004 (Nicholas S. Lander, Western
Australia Herbarium, personal communication). Collections of
the species made in Western Australia from 15°16′ to 25°30′ S
and 115°29′ to 128°41′ E, a land area of approximately
300,000 square miles (approximately 777,000 km2), are re-
corded to vary in plant size, plant form, flower color, flower
scent, habitat preference, soil type, and floristic association.
N. benthamiana has become an extremely important subject
for the study of host–pathogen interactions, particularly those
Fig. 1. A, Color image of the original herbarium sheet bearing the type specimen of Nicotiana benthamiana, collected by Benjamin Bynoe and housed at the
Royal Botanic Gardens, Kew. Note the flowers, which average approximately 5 cm in length. B, Detail from A showing an attached handwritten note by
Karel Domin designating these plants as the type specimen. C, Photograph of Karel Domin.
Vol. 21, No. 8, 2008 / 1017
involving plant viruses. A search of the PubMed database
(through November 2006) finds 1,743 citations containing the
term “nicotiana benthamiana” since 1995, with 38% (667)
having accumulated in 2006 alone (Fig. 2). Examination of the
titles reveals the fact that most of these articles are concerned
with studies of viral or bacterial phytopathology or gene silenc-
ing. Given this statistic, it is quite surprising that so little is
known about the basic genetics or genomics and phylogeogra-
phy of this important plant. As stated earlier, N. benthamiana
was first collected >160 years ago, but there are scant records
of its repeated collection from the wild (three are mentioned
by Goodspeed) (1954, page 487). A survey of accessible
online seed and gene banks reveals the paucity of publicly
available genetic resources for this species: the United States
Department of Agriculture–Agricultural Research Service
(USDA-ARS) National Plant Germplasm System has two ac-
cessions (plant introduction [PI] numbers 555478 and 555684),
the Botanical Garden of Nijmegen (Netherlands) lists two ac-
cessions, the Institut fur Pflanzengenetik und Kulturpflanzen-
forschung Gatersleben (IPK, Germany) has one, and the Aus-
tralian Plant Genetic Resource Information Service (AusPGRIS)
lists five accessions (all donated by D. D. Wark) but only two
are presently available for distribution. With the exception of
the three unavailable Australian accessions, none of these
come with any documentation or provenance. The Kentucky
Tobacco Research and Development Center (KTRDC) at the
University of Kentucky presently maintains six accessions of
N. benthamiana, of which two are the USDA-ARS accessions
and another two are almost certainly redundant. N. Burbidge
obtained seed from the wild and grew several of these collec-
tions in order to compare them with the type (Bynoe) speci-
men (Burbidge 1960). Two of these (T.S. 139 and T.S. 318) are
listed as unavailable by AusPGRIS, and the disposition of the
other Burbidge material is unknown. Based on plant morphol-
ogy and the pattern of reticulation of the seed testa, she con-
cluded that accession T.S. 299, from near the Ord River (Kim-
berley region, Western Australia), was “nearest to the type”
(Burbidge 1960). This is highly unlikely, however; an account
of the exploration of Western Australia (Battye 1924) reveals
that the Ord River was not discovered until 1879, more than 30
years after the Beagle returned to Portsmouth on 30 September
1843 and almost 15 years after Bynoe’s death in 1865 at the
age of 61 (Keevil 1949).
As with accounts regarding its collection, records that could
shed light on how accessions of N. benthamiana made their
way into the research community are equally scant. It is most
likely that N. benthamiana was adopted as a model plant pri-
marily due to its unparalleled susceptibility to viruses, many of
which do not infect A. thaliana (Christie and Crawford 1978;
Coutts and Buck 1985; Quacquarelli 1975). This susceptibility
is often considered to be a general characteristic of N. bentha-
miana as a species. However, as we demonstrate in a later sec-
tion, this view is likely to be naive because there is quite possi-
bly only a single accession of N. benthamiana, or a collection
of closely related accessions, being used by the plant research
community today. From preliminary experiments with a previ-
ously uncharacterized accession from the USDA Tobacco Col-
lection, it appears that there could be significant variation in
susceptibility to virus infection within this species. Thus, there
is a compelling need for collection and characterization of new
accessions of N. benthamiana from its natural range in north-
western Australia in order to expand the genetic diversity
available to researchers worldwide. That said, despite wide-
spread ignorance of the provenance of the accessions currently
in use, the genetic uniformity of these plants has potentially
contributed positively to the rapid increase in research con-
ducted with this species. Tools and resources to support pro-
jects with N. benthamiana can be readily exchanged and used
across laboratories, in many cases without the need to consider
plant effects other than those influenced by environmental and
growth conditions (Fig. 2).
Distance-based phenetic analysis of N. benthamiana.
In addition to documenting the history of N. benthamiana,
we are also concerned with establishing standards for defining
accessions of this species. To this end, all of our transgenic
lines (Chakrabarty et al. 2007; Goodin et al. 2007a) have been
produced with the same accession used to develop the widely
utilized ‘16c’ line, which expresses GFP targeted , and which
has been instrumental in the identification and characterization
of suppressors of RNA silencing and related research (Brigneti
et al. 1998; Cao et al. 2005; Roth et al. 2004; Ruiz et al. 1998;
Segers et al. 2006). Our decision to use the 16c parental line
was made primarily to avoid the possibility of producing trans-
genic plants with a significantly to the endoplasmic reticulum
(ER) different genetic background. However, this begged the
question of how much genetic variation actually exists among
the N. benthamiana accessions being used by the research
community. An informal e-mail survey, conducted by us in
2005, canvassed 236 plant virologists to obtain information
about the research use of this species. This survey failed to
identify the number of accessions in use or how N. bentha-
miana came to be adopted by the research community. Al-
though some virologists were familiar with early papers deal-
ing with N. benthamiana as a research model, it could not be
established where the actual accessions now in use were col-
lected, in whose laboratory they were first used, or the order of
distribution of this species among researchers. Therefore, we
conducted an amplified fragment length polymorphism
(AFLP)-based cluster analysis in an attempt to determine
whether any genetic variation existed among research acces-
sions. We used DNA obtained from plants grown from seed
submitted by researchers in five countries (Supplementary
Data 1). When grown to maturity, no obvious differences in
growth habit, foliage, or flowers were noted. However, a novel
accession of N. benthamiana in the KTRDC collection was a
much larger, coarser plant with larger flowers than the research
accessions. This same accession (USDA PI no. 555684), here
Fig. 2. Rapid increase in the number of research publications using Nico-
tiana benthamiana. Data were obtained from the PUBMED database on
the search term “nicotiana benthamiana”.
1018 / Molecular Plant-Microbe Interactions
referred to as ‘KTRDC-4’, stood alone in the AFLP cluster
analysis (Fig. 3). Pairwise comparisons between it and 10
other accessions (5 from KTRDC and 5 survey submissions)
gave similarity coefficients (Jaccard 1908; Lara-Cabrera and
Spooner 2004) that averaged 0.404. Comparisons among the
other 10 accessions averaged 0.924, indicating that they are
very closely related and could possibly be derived from one
source. To put this into perspective, similarity coefficients cal-
culated between the 11 accessions of N. benthamiana and an
accession of N. excelsior, the only other n = 19 species from
Australia, averaged 0.359 (Fig 3A). Interestingly, all accessions
of N. benthamiana submitted by researchers had the small-
flower phenotype, whereas KTRDC-4 had large flowers, a
characteristic it shares with the type specimen (Fig. 1A and B).
Variation in susceptibility to virus infection
in N. benthamiana ecotypes.
Given the gross morphological variation between the large-
and small-flowered plants, we hypothesized that they may also
differ in their susceptibility to pathogens. Our initial experi-
ments suggest that KTRDC-4 was significantly more resistant
to infection by Tomato spotted wilt virus (Fig. 4; Supplemen-
tary Materials 1) and other viruses (data not shown), than was
the ‘standard’ accession of N. benthamiana. These data are
particularly intriguing because N. benthamiana is generally
considered to be almost universally susceptible to plant viruses.
This opinion is clearly biased given the genetic uniformity
among accessions used for research and the absence of any
data in which variation in resistance among accessions has
been addressed, as has been done for A. thaliana (Callaway et
al. 1996; Martin et al. 1999).
The above comparison raises a question that has long been
of interest to plant virologists; namely, why is N. benthamiana
seemingly susceptible to the majority of plant viruses? To ad-
dress this issue, Yang and associates (2004) determined that
the near universal virus susceptibility is, at least in part, linked
to a naturally occurring mutation in an RNA-dependent RNA
polymerase gene (NbRdRP1m) present in the N. benthamiana
genome. Enhanced virus resistance is conferred to plants ex-
pressing the corresponding gene from Medicago truncatula.
However, in the absence of a diverse representative collection
of N. benthamiana accessions, it cannot be ruled out that this
species is universally susceptible to viruses given that
Fig. 3. A, Phenetic relationships among 11 accessions of Nicotiana ben-
thamiana based upon amplified fragment length polymorphism cluster
analyses. Five research accessions (RAs) were submitted by laboratories in
Spain (RA-2), the United Kingdom (RA-4), and the United States (RA-1,
RA-3, and RA-5). Six accessions were from the collection of the Kentucky
Tobacco Research and Development Center (KTRDC). N. excelsior was
included in these analyses because it is the only other n = 19 Nicotiana sp.
from Australia. B, Examples of floral phenotypes from plants included
in this study. All RAs had a small flower phenotype (top). KTRDC-4 had
large flowers reminiscent of the type specimen (middle). The limb on
some flowers of N. excelsior (bottom) never fully expand, unlike those of
Fig. 4. A through C, Mature plants of the research accession(RA-4) and
Kentucky Tobacco Research and Development Center (KTRDC-4) acces-
sions show differential susceptibility to infection by Tomato spotted wilt
virus (TSWV)-T. A, Visible symptoms of leaf chlorosis are observed only
on RA-4 2 weeks post inoculation (wpi). B, Symptoms on plants at 3 wpi.
Symptoms are more severe on RA-4 when compared with those produced
in KTRDC-4. Both types of plants show mortality by 6 and 10 wpi, re-
spectively (data not shown). C, Enzyme-linked immunosorbent assay data
show that virus was detected in symptomatic leaves of RA-4 but not in
KTRDC-4 at 2 wpi (shown in A), whereas virus was detected in sympto-
matic leaves of both RA-4 and KTRDC-4 at 3 wpi (shown in B). The re-
sults indicate low levels of replication or delayed systemic spread of virus
in KTRDC-4. 1 = Mock inoculated RA-4, 2 = TSWV-inoculated RA-4, 3 =
TSWV-inoculated KTRDC-4, and O.D. = optical density at 405 nm. Tissue
samples were pooled from six inoculated plants at each time point. The
experiments were repeated three times.
Vol. 21, No. 8, 2008 / 1019
KTRDC-4 appears to have enhanced resistance compared with
the widely used research accession, which differs so much
from the type (Bynoe) specimen. Therefore, it is clear that a
significant effort must be made to collect and characterize
novel accessions of N. benthamiana in order to resolve issues
about its susceptibility to viruses and, more importantly, to
provide a collection of genetically diverse ecotypes, which is
essential for any legitimate model system.
The rise of N. benthamiana.
Despite its early adoption by the plant virology community,
N. benthamiana did not enter the wider arena of plant biology
until the advent of three major technical advances. First was
the ability to express foreign genes from a plant virus vector.
This technology not only provided a means to track viral move-
ment in living cells but also revealed new insights into
fundamental aspects of plant biology such as plasmodesmatal
gating and macromolecular movement between cells, and also
allowed definition of the proteins targeted to them (Chapman
et al. 1992; Cruz et al. 1996; Escobar et al. 2003; Lucas 2006).
Second, the development of plant virus-based vectors
quickly led to the invention of a technology now known as
virus-induced gene silencing (VIGS) (Kumagai et al. 1995;
Thomas et al. 2001). VIGS enabled directed, systemic down-
regulation of virtually any gene-of-interest in plants, thereby
transforming N. benthamiana into a powerful reverse-genetics
system (Dong et al. 2007; Fu et al. 2005; Liu et al. 2002;
Ratcliff et al. 2001). One of the great advantages of VIGS is its
ability to reduce the effects of genetic redundancy if the cDNA
used for silencing is homologous to more than a single mem-
ber of a multiple gene family. Alternatively, by prudent selec-
tion of cDNAs to be used for VIGS, either individual or multi-
ple members of a gene family can be targeted (Burch-Smith et
al. 2004). Interestingly, for many years the viral vectors used
for VIGS were particularly well suited for use in N. bentha-
miana and had little utility in A. thaliana, the flagship of plant
molecular genetics although, more recently, this limitation has
eased (Burch-Smith et al. 2006; Deleris et al. 2006). Plant viral
vectors, RNA silencing, and VIGS have all been the subjects of
excellent reviews published recently; therefore, we direct
readers to these articles for elaboration on these topics (Burch-
Smith et al. 2004; Ding and Voinnet 2007; Earley et al. 2006;
The third technology that served to popularize N. bentha-
miana as a research model was agroinfiltration, which permits
proteins of interest, often expressed as fusions to autofluores-
cent proteins, to be produced transiently in plant cells in a
straightforward manner that is particularly well suited to high-
throughput studies (Fig. 5) (Goodin et al. 2002; Schob et al.
1997; Voinnet et al. 2003). Methodologically, agroinfiltration
may at first glance seem too simple to be useful, but it is pres-
ently the most facile means to transiently express proteins in
plant cells. Briefly, a suspension of Agrobacterium tumefa-
ciens cells carrying binary plasmid vectors designed for pro-
tein expression is infiltrated into the intercellular space within
a leaf using nothing more sophisticated than a 1-ml disposable
syringe (sans needle). After approximately 24 to 48 h of incu-
bation, sections of infiltrated leaves can be sampled for micros-
copy or biochemical analyses. Interestingly, as for VIGS, agro-
infiltration works exceptionally well in N. benthamiana but
poorly in other plants, including Arabidopsis thaliana. Thus,
the three major advances for manipulating protein and gene
expression in plant cells are best suited to N. benthamiana.
Taken together, it is not surprising that, in addition to the
increase in research publications in which N. benthamiana was
the sole model plant used, there has been a parallel increase in
the use of this species to compliment studies initiated in A.
thaliana (Fig. 2, inset) (Heese et al. 2007; Ohad et al. 2007;
Strasser et al. 2007; Thomas et al. 2008), wheat (Tardif et al.
2007), and other plants (Gabriëls et al. 2007; Messinese et al.
2007; Xiao et al. 2007).
The further utility of N. benthamiana is underscored by
studies that combine the methods described above. For exam-
ple, agroinfiltration and VIGS have been used simultaneously
to investigate signal transduction (Gabriëls et al. 2006) and
protein trafficking (Kanneganti et al. 2007).
By taking advantage of fact that elevated temperatures
(33°C) prevent the hypersensitive response (HR) in transgenic
tomato plants co-expressing the Cf-4 resistance gene and the
cognate HR-inducing gene Avr4, Gabriëls and associates
(2006) were able to synchronize the appearance of HR in
plants when the temperature was reduced to 25°C. They then
used AFLP analyses to identify 442 mRNA transcripts that
were differentially expressed in the early stages of the HR.
VIGS was then used to silence some of these transcripts in N.
benthamiana. This is a legitimate approach given the extensive
homology between cDNAs of Solanaceous species (Liu et al.
2002; Rensink et al. 2005). Following the onset of VIGS, agro-
infiltation was used to co-express avirulence proteins in Cf-4
tranasgenic N. benthamiana leaves. Using this combined ap-
proach, a heat-shock protein, a nuclear GTPase, an L19 ribo-
somal protein, and, most interestingly, a nucleotide-binding
leucine-rich repeat (NB-LRR)-type protein were implicated in
In a related approach, Kanneganti and associates (2007)
used VIGS to silence importin-α homologues in N. bentha-
miana. This resulted in plants that could be used to character-
ize the nuclear localization of yellow fluorescent protein fusions
that were expressed in leaves by agroinfiltration (Kanneganti
et al. 2007). Taken together, these and similar studies exem-
plify the rapidity with which N. benthamiana can be manipu-
lated in a manner that is, by and large, without equal in plant
biology. It is beyond the scope of this article to describe the
Fig. 5. Agroinfiltration can be used for a variety of purposes. Suspensions
of Agrobacterium tumefaciens cells transformed with a binary vector for
expression of genes of interest are infiltrated into Nicotiana benthamiana
leaves (Note; only the portion between the left [LB] and right [RB] bor-
ders of a generic binary vector is shown). Depending on the particular
study, these plants may be either wild-type or expressing a transgene; for
example, fluorescent markers for the endoplasmic reticulum, nucleolus
(Fib1), or chromatin (H2B). For microscopy and biochemical studies, leaf
tissues are typically sampled within 24 to 94 h post infiltration. For virus-
induced gene silencing (VIGS), plants may be incubated for several weeks
until the gene of interest is silenced, such as the knockdown of the phy-
toene desaturase gene resulting in the bleached leaf phenotype shown here.
Micrographs are reprinted from Goodin and associates (2007a,b). The pro-
tein arrays are reprinted from Burch-Smith and associates (2007).
1020 / Molecular Plant-Microbe Interactions
many variants of VIGS vectors, agroinfiltration methods, or
the combinations in which these tools have been applied.
However, it suffices to say that advances continue to be made
both with respect to novel vector systems as well as their use
alone or in combination with other techniques (Chakrabarty et
al. 2007; Dong et al. 2007; Kamoun et al. 2003; Lindbo 2007;
Liu and Page 2008; Robertson 2004; Ryu et al. 2004).
N. benthamiana: second fiddle or virtuoso?
The importance of N. benthamiana as an indispensable re-
search model in plant virology is underscored by its role in
seminal findings that have been the subject of several recent
reviews (Bisaro 2006; Briddon and Stanley 2006; Dreher and
Miller 2006; Lucas 2006; Nagy and Pogany 2006). Recently,
N. benthamiana proved instrumental in demonstrating that
host factors required for replication of Tombusvirus spp., iden-
tified in a yeast model are also required by these viruses in
plants (Wang and Nagy 2008). Briefly, these authors demon-
strated that, in yeast cells, Tomato bushy stunt virus (TBSV), a
plus-stranded RNA virus, incorporates the host metabolic en-
zyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
into the viral replicase complex. Using Tobacco rattle virus
(TRV)-mediated VIGS, the authors further demonstrated that
downregulation of GAPDH in N. benthamiana decreased rep-
lication of TBSV but not the distantly related Tobacco mosaic
virus (Wang and Nagy 2008). Interestingly, A. thaliana is a
nonhost for TBSV, making the development of N. benthamiana
as a research model essential for advancing the groundbreaking
research established in yeast (Panavas et al. 2008).
But how does N. benthamiana feature into the larger context
of plant biology? With the rapid increase in the number of se-
quenced plant genomes, we posit that a common plant model
for advancing the derivative functional and systems biology
analyses is essential. In Table 1, and in brief descriptions be-
low, we identify some of the necessary resources available to
support research with N. benthamiana, which we fully expect
to play a major role in the post-genomics era.
Vectors for expression of autofluorescent protein fusions. At
present, N. benthamiana is the most tractable plant system for
conducting high-throughput protein localization studies, given
the ease of agroinfiltration and the ability to counter-stain tis-
sues with dyes for labeling nuclei, endomembranes, and other
relevant structures within cells (Goodin et al. 2005, 2007a). In
addition to facilitating acquisition of higher quality micro-
graphs, agroinfiltration permits large numbers (hundreds to
thousands) of cells to be transfected simultaneously. This not
only increases confidence that micrographs are correctly repre-
sentative of the true localization patterns but also that the data
can be readily quantified. Additionally, sufficient tissue can be
infiltrated to permit small-scale protein purification or other
analyses on the same tissue used for microscopy. Although any
appropriately constructed binary vector can be used for agroin-
filtration, the most recently described improved systems rely
upon Gateway or other ligation-independent cloning technolo-
Table 1. Resources for research conducted in Nicotiana benthamianaa
URL or e-mail
Kim Lab email@example.com
Kentucky Tobacco Research &
Development Center (KTRDC)
Boyce Thompson Institute
The Institute for Genomic Research
The Tobacco Genome Initiative
a VIGS = virus-induced gene silencing, EST = expressed sequence tag sequence, PVX = Potato virus X, TRV = Tobacco rattle virus, BiFC = bimolecular
fluorescence complementation, Nb = N. benthamiana, Sl = Solanum lycopersicum (tomato), TRV der. = TRV derivatives.
Vol. 21, No. 8, 2008 / 1021
gies (Chakrabarty et al. 2007; Dong et al. 2007; Earley et al.
2006; Karimi et al. 2007). Continued improvements in expres-
sion vectors, autofluorescent proteins, and imaging technolo-
gies (Goodin et al. 2007b) suggest that N. benthamiana will play
a significant role in the task of localizing plant proteomes, as
has been done for other essential research models (Laketa et al.
2007; Pepperkok and Ellenberg 2006; Simpson and Pepperkok
2006). Importantly, it has been our experience that protein lo-
calization via agroinfiltration generally parallels that observed
in the context of stable transformation (Goodin et al. 2007a).
Therefore, this favors transient expression for large-scale lo-
calization projects, which can then be followed by smaller,
more directed studies using transgenic plants if necessary.
Construction of protein interaction maps. In addition to be-
ing an exceptional system for protein localization in plant
cells, N. benthamiana has been instrumental as a platform for
protein–protein interaction studies, particularly with respect to
characterization of the proteome of Arabidopsis and other spe-
cies (Citovsky et al. 2006; Ohad et al. 2007; Popescu et al.
2007; Tardif et al. 2007). In a recent review of the method,
Ohad and associates (2007) provided a table describing exam-
ples where bimolecular fluorescence complementation (BiFC)
was used to study protein interactions and localization in plant
cells. Of the 40 examples given, N. benthamiana was, by far,
the most frequently employed of the six different plant species
used for conducting BiFC experiments. Interestingly, of the 25
examples where Arabidopsis proteins were characterized, het-
erologous expression systems were used in all cases except
five, again with N. benthamiana serving as the predominant
plant for BiFC analyses.
In addition to BiFC and other protein localization studies,
the large leaves of N. benthamiana make it convenient for use
in situations where proteins of interest need to be purified in
yields sufficient to support proteomics and biochemical studies
(Popescu et al. 2007) (Fig. 5). Most recently, in order to iden-
tify targets on an A. thaliana protein array containing 1,133
proteins, calmodulin-related proteins used to probe the array
were first purified from N. benthamiana following agroinfiltra-
tion. The proteins expressed in N. benthamiana proved to be
superior to probes purified from yeast cells, particularly with
respect to the autophosphorylation activities of 96 protein
kinases (Fig. 5). These and similar plant proteomics studies
should greatly benefit from the recent development of “launch
vectors” that express replicons derived from Tobacco mosaic
virus, into which transgenes of interest are cloned that, in turn,
are introduced into plant cells via agroinfiltation (Lindbo
2007; Musiychuk et al. 2007).
Microarrays. Currently, at least four array platforms suitable
for RNA profiling in N. benthamiana are available. These in-
clude the Tom1 (cDNA) and Tom2 (oligonucleotide) arrays
that are derived from tomato expressed sequence tag (EST)
data and which have been used for profiling RNA from a num-
ber of different Solanaceous species (Moore et al. 2005). Addi-
tionally, cDNA arrays derived from potato EST have been used
for profiling N. benthamiana RNA to determine changes in
gene expression in response to virus infection (Dardick 2007;
Senthil et al. 2005). Most recently, in collaboration with
NimbleGen (Madison, WI, U.S.A.), S. Lommel and colleagues
have developed and validated a microarray derived entirely
from N. benthamiana EST sequences (the Nb-array). The cur-
rent array is composed of oligonucleotide probes (60-mers)
corresponding to 13,014 unique N. benthamiana EST. In order
to provide a more stringent test of the hypothesis that RNA vi-
ruses, regardless of the host species (plant or animal), regulate
a common “core” suite of genes, an additional 401 probes cor-
responding to plant homologues of genes shown to be differen-
tially expressed in response to animal viruses were included on
the array. In order to identify and design probes for these
genes, a comprehensive list of host genes that are differentially
expressed in response to virus infection was developed from
the literature. After filtering the data set to remove genes ab-
sent in plant genomes (e.g., immune system-type genes), the
list was used to search plant EST databases via BLAST align-
ments. Selection of sequences to be included on the array was
prioritized such that i) they matched N. benthamiana sequence,
ii) they matched an EST from a dicot species, and iii) they
matched an EST from a monocot species. Control experiments
demonstrated that there were N. benthamiana transcripts that
hybridized to the 401 selected probes. Taken together, the Nb-
array contains probes for 13,415 transcripts, which corresponds
to an estimated coverage of approximately 38% of the N. ben-
thamiana transcriptome. Enquiries regarding these microarrays
should be directed to S. Lommel (Table 1).
Transgenic marker lines. The most frequently used trans-
genic N. benthamiana line is 16c that is homozygous for
mGFP5-ER (Siemering et al. 1996), expressing green fluores-
cent protein (GFP) targeted to the ER (Brigneti et al. 1998;
Ruiz et al. 1998). This line has been instrumental in elucidat-
ing the mechanism of RNA silencing and for the identification
of virus-encoded suppressors of silencing. Seed for the 16c
line are available from the Baulcombe lab.
Primarily to support the study of plant–virus interactions,
M. Goodin and colleagues have recently generated several ad-
ditional lines that express GFP or red fluorescent protein
(RFP) fusions targeted to a variety of cellular loci, including
the ER, nucleus, nucleolus, actin, and Golgi (Chakrabarty et
al. 2007; Goodin et al. 2007b). Seed for these plants can be ob-
tained from the lead author upon written request.
Suspension cell lines. The availability of suspension cell
lines, such as the BY-2 line derived from N. tabacum cv.
Bright Yellow, effectively are the plant equivalent of the hu-
man-derived He-La cell line, which facilitates experiments that
require synchronous transfection or transformation of cells in
culture (Brandizzi et al. 2003; Nagata et al. 1992). Although
the utility of the BY-2 cell line is well established, its short-
comings are revealed in cases where certain viruses do not rep-
licate well in tobacco cells or when there is an essential require-
ment to generate protoplasts from a particular experimental
host. For these reasons, Sunter and Bisaro (2003) developed an
analogous N. benthamiana cell line. The N. benthamiana sus-
pension cell line can be obtained from the laboratory of G.
Sunter (Table 1).
EST databases. As of October 2007, there was sequence
information for 42,566 N. benthamiana EST in GenBank. Of
these, approximately 18,000 are unique. Three major contribu-
tors are responsible for generating this sequence information:
i) The Institute for Genomic Research (TIGR), which provides
this information through the Solanaceae Genomics Resource
as part of the National Science Foundation-funded Potato
Functional Genomics Project; ii) the John Innes Center, which
has contributed >8,000 EST; and iii) The Tobacco Genome Ini-
tiative (Table 1), which has contributed the largest number of
N. benthamiana EST (>25,000) to date and provides access to
both N. benthamiana and N. tabacum sequence data via a web-
based portal that requires an academic transfer agreement for
Although the number of N. benthamiana EST may be rela-
tively low, the effective number of EST can be greatly expanded
due to the high level of sequence homology present among
coding sequences of species in the Solanaceae family (Rensink
et al. 2005). As of October 2007, there are nearly 700,000 EST
sequences of solanaceous species available in public databases.
GenBank records include 258,448 EST for Solanum lycopersi-
cum (tomato), 227,375 for S. tuberosum (potato), 158,008 for
1022 / Molecular Plant-Microbe Interactions
N. tabacum (tobacco), 42,566 for N. benthamiana, and 14,017
for Petunia hybrida. In addition, there are ongoing genomics
projects for at least five other species in the Solanaceae family.
Importantly, these EST can be employed across species as
demonstrated recently by Dong and associates (2007), who
used tomato EST to effectively silence N. benthamiana genes
via high-throughput VIGS.
VIGS libraries. Advances are being made in the construction
and optimization of libraries to be used as sources of ‘VIGS-
ready’ constructs. Recently, Liu and Page (2008) have described
methods for constructing optimal VIGS libraries based on the
TRV vector (Liu et al. 2002; Ratcliff et al. 2001). Library con-
struction utilized RNA isolated from roots of N. benthamiana
plants treated with methyl jasmonate. Synthesis of cDNA was
conducted on a solid phase support and then digested with
RsaI to yield short cDNA fragments lacking poly(A) tails. The
resulting libraries contained 2,948 EST; 30% of the cDNA
inserts were 401 to 500 bp in length and 99.5% lacked poly(A)
tails. The efficiency of constructs derived from the VIGS-
cDNA libraries was tested, in the context of defining plant sec-
ondary metabolite biosynthesis, by silencing a nicotine biosyn-
thetic enzyme, putrescine N-methyltransferase (NbPMT), with
10 different VIGS-NbPMT constructs ranging in length from
122 to 517 bp. Leaf nicotine levels were found to be reduced
by more than 90% when NbPMT was silenced with the various
constructs. Based upon these and additional experiments, the
authors suggested the following design guidelines for con-
structs in TRV vectors: i) insert lengths should be in the range
of approximately 200 to 1,300 bp, ii) they should be positioned
in the middle of the cDNA, and iii) homopolymeric regions
(i.e., poly(A/T) tails) should not be included.
As an alternative to homologous sequences for VIGS, it may
often be more convenient to use heterologous gene sequences.
Senthil-Kumar and associates (2007) have systematically ex-
amined this issue and found that heterologous gene sequences
from distantly related plant species can be used to silence their
respective orthologs in the VIGS-efficient plant N. benthamiana.
Interestingly, a correlation was not always found between gene
silencing efficiency and percent homology between the het-
erologous and endogenous gene sequences. The authors con-
cluded that a 21-nucleotide stretch of 100% identity between
the heterologous and endogenous gene sequences is not abso-
lutely required for gene silencing (Senthil-Kumar et al. 2007).
Technologies such as EST mining, RNA profiling, and VIGS
have enabled many novel findings but they cannot readily
reveal such subtle changes as the relocalization of host pro-
teins that takes place in virus-infected cells (Burch-Smith et al.
2007; Chakrabarty et al. 2007; Goodin et al. 2007a and b), or
the recruitment of host factors to sites of virus replication
(Serva and Nagy 2006). For this aim, high-throughput local-
ization of the N. benthamiana proteome in both the presence
and absence of pathogens is required. However, there is pres-
ently an under-representation of cDNA libraries for N. bentha-
miana and, particularly, validated full-length cDNA clones for
genes from any species in the Solanaceae family to facilitate
such studies. For proteins that are highly conserved, it is possi-
ble to utilize functionally equivalent Arabidopsis proteins to
probe plant–virus interactions (Chakrabarty et al. 2007). Such
ORFeome-scale localization is required if the systems biology
of plant–pathogen interactions is ever to be fully realized
(Meganson and Fraser 2007).
N. benthamiana is the most widely used experimental host
in plant virology. In addition to viruses, this species has been
used in the study of a wide variety of plant pathogens (Fig. 6).
Importantly, N. benthamiana EST are generally highly ho-
mologous to those of agriculturally relevant Solanaceous
crops, such as tomato, potato, pepper, and petunia, which col-
lectively had a 2006 farm-gate value of US$5.4 billion in the
United States alone (National Agricultural Statistics Service).
Thus, functional genomics projects concerned with host–patho-
gen interactions conducted in N. benthamiana will most likely
reveal a cadre of genes that play similar roles in agronomically
important crops. This point is well supported by the recent
work of Dong and associates (2007), who showed that tomato
EST, using a high-throughput VIGS approach, can effectively
knock down gene expression in N. benthamiana. Moreover,
the ease of transformation, transient protein expression, and
gene-silencing systems for N. benthamiana makes this species
an extremely attractive model for plant cell biology in general.
This is helping to advance many research interests that were
initiated in Arabidopsis but which ultimately required support
from model systems better suited to facile protein expression
methods and microscopy techniques (Bernal et al. 2007;
Bracha-Drori et al. 2004; Zhao et al. 2006).
Clearly, N. benthamiana’s large haploid genome, like that of
its more popular relatives, may restrict its use as a genetics
system (Fig. 6) (Bennett and Leitch 2005; Bennett et al. 2003).
However, given its many other positive attributes, this limita-
tion should in no way be used as a rationale to restrict the de-
velopment of genomic resources for N. benthamiana. One area
in need of immediate improvement relates to the transformation
and regeneration efficiency of this plant, which is low relative
to systems such as Arabidopsis. Currently, there is no compar-
able floral-dip transformation system for N. benthamiana, a
technique that has greatly reduced the cost and tedium associ-
ated with making transgenic Arabidopsis plants (Clough and
Bent 1998). However, as for other plant systems, the efficiency
of generating transgenic N. benthamiana plants using traditional
tissue culture methods will likely be increased as it gains in
popularity as a research model.
Another area of concern when using N. benthamiana may
be the ‘overexpression artifacts’ that could potentially arise
from the use of agroinfiltration. A frequently raised objection
is that so-called pathogen-associated molecular patterns
(PAMPs) (Ma and Berkowitz 2007) or microbe-associated mo-
lecular patterns (MAMPs) (deWit 2007) in Agrobacterium
proteins may result in mislocalization of proteins expressed by
agroinfiltration. Indeed, PAMPS appear to be critical for T-
DNA transfer and Agrobacterium-mediated transformation per
se and do, indeed, result in relocalization of host proteins, such
as VIP1, upon infection (Dafny-Yelin et al. 2008; Zipfel 2008;
Zipfel and Felix 2005). However, it stands to reason that the
vast majority of proteins expressed by agroinfiltration will not
play roles in any such processes. In fact, Agrobacterium-medi-
ated localization of Arabidopsis proteins has proven to be
hugely successful (Koroleva et al. 2005; Ohad et al. 2007;
Pendle et al. 2005). Additionally, in our experiences with tar-
geting a variety of proteins to subcellular locales including the
nucleus, nuclear envelope, nucleolus, ER, chloroplasts, and
mitochondria, AFP fusions were correctly targeted in all cases
(Goodin et al. 2007b) (unpublished data). Although PAMPs
and MAMPs may, in rare cases, interfere with protein localiza-
tion, there is little indication that this is a general or wide-
spread problem, as indicated by the large body of literature in
which the genus Agrobacterium was used to study protein lo-
calization in plant cells.
On the other hand, users of agroinfiltration must be aware of
the potential for localization artifacts due to overexpression of
proteins. Common artifacts include protein aggregation or
membrane proliferation, which have been observed occasion-
Vol. 21, No. 8, 2008 / 1023
ally with integral membrane proteins (M. M. Goodin, unpub-
lished). These and other technical factors that need to be con-
sidered prior to embarking on protein localization studies have
been addressed by Goodin and associates (2007b).
However, when proteome-scale localization is required
(Laketa et al. 2007; Pepperkok and Ellenberg 2006; Simpson
and Pepperkok 2006), overexpression vectors are essential for
“first pass” classification of proteins with respect to subcellu-
lar localization. Proteins of interest to particular laboratories
can then be studied with alternate methods of analyses which,
in the case of plants, may require the generation of a series of
transgenic lines expressing autofluorescent protein fusions un-
der the control of native promoters as a means to most accu-
rately define localization and tissue specificity (Tian et al.
2004). Additionally, expression vectors that utilize promoters
other than the Cauliflower mosaic virus 35S are available
(Chung et al. 2005). A systematic comparison of protein ex-
pression via transient or transgenic means using a variety of
binary vector derivatives that use promoters other than the
ubiquitous 35S would be of tremendous value to the field.
However, the current approach is for high-throughput localiza-
tions of many proteins at a minimal cost per localized protein.
This ensures reliance on the 35S promoter, which is by far the
most convenient to use.
In conclusion, there is a rapidly increasing body of literature
that supports the need to develop additional genomics tools for
N. benthamiana in order to further advance, primarily in plant
virology, innate immunity and host–pathogen interactions.
However, it is abundantly clear that N. benthamiana is poised
to provide essential support in plant systems biology in the
post-genomics era (Caplan et al. 2008; Chisholm et al. 2006;
Mestre and Baulcombe 2006; Takahashi et al. 2007; Tameling
and Baulcombe 2007).
The authors wish to express their sincere gratitude to all who have con-
tributed to this work by responding to our survey or providing N. bentha-
miana seed. We are particularly indebted to D. Baulcombe, S. Dinesh-
Kumar, F. Garcia-Arenal, J. Moyer, R. Nelson, K. Perry, and N. Robertson
for contributing resources and data relevant to this article. We apologize to
all colleagues whose research is not mentioned here due to space limita-
tions. Special thanks goes to M. Chase, Royal Botanic Gardens, Kew, for
his assistance in obtaining the digital image of the type specimen of N.
benthamiana. We thank R. Dietzgen for reviewing the manuscript prior to
submission. The article is approved as PPNS no. 0475, Department of
Plant Pathology, College of Agricultural, Human, and Natural Resource
Sciences Agricultural Research Center Project No. WNPO 0616, Washing-
ton State University, Pullman 99164-6240, U.S.A. Research on TSWV
was funded by the Integrated Pest Management Collaborative Research
Fig. 6. Nicotiana benthamiana is the most versatile experimental host for plant viruses and a diverse range of other plant pathogens. Given the high degree of con-
servation of coding sequences within the family Solanaceae, N. benthamiana can support functional genomics projects related to many agriculturally important
crops. Finally, N. benthamiana is playing a major role in advancing cell biology and biochemical research extending from studies initiated in Arabidopsis
thaliana, shown here infected with Impatiens necrotic spot virus (left) or mock inoculated (right). The table compares the characteristics of the genomes for a
number of Solanaceous species and Arabidopsis (data reproduced here from the SOL Genomics Network and the Angiosperm DNA C-values database).
1024 / Molecular Plant-Microbe Interactions
Support Program (award no. EPP-A-00-04-00016-00 to R. A. Naidu). Ad-
ditional sources of funding include NIH, USDA, and KTRDC grants to M.
Goodin and continued KTRDC research funding to D. Zaitlin.
Battye, J. S. 1924. Western Australia: A History from its Discovery to the
Inauguration of the Commonwealth. Clarendon Press, Oxford.
Bennett, M. D., and Leitch, I. J. 1995. Nuclear DNA amounts in angio-
sperms. Ann. Bot. 76:113-176.
Bennett, M. D., and Leitch, I. J. 2005. Nuclear DNA amounts in angio-
sperms—progress, problems and prospects. Ann. Bot. 95:45-90.
Bennett, M. D., Leitch, I. J., Price, H. J., and Johnston, J. S. 2003. Com-
parisons with Caenorhabditis (~100 Mb) and Drosophila (~175 Mb)
using flow cytometry show genome size in Arabidopsis to be ~157 Mb
and thus ~25% larger than the Arabidopsis Genome Initiative estimate
of ~125 Mb. Ann. Bot. 91:547-557.
Bentham, G. 1869. Pages 469-470 in: Flora Australiensis: A Description of
the Plants of the Australian Territory, vol. IV, Styidieae to Pedalineae.
L. Reeve & Co., London,
Bernal, A. J., Jensen, J. K., Harholt, J., Sorensen, S., Moller, I., Blaukopf,
C., Johansen, B., de Lotto, R., Pauly, M., Scheller, H. V., and Willats,
W. G. 2007. Disruption of ATCSLD5 results in reduced growth, re-
duced xylan and homogalacturonan synthase activity and altered xylan
occurrence in Arabidopsis. Plant J. 52:791-802.
Bisaro, D. M. 2006. Silencing suppression by geminivirus proteins. Virol-
Bracha-Drori, K., Shichrur, K., Katz, A., Oliva, M., Angelovici, R.,
Yalovsky, S., and Ohad, N. 2004. Detection of protein–protein interac-
tions in plants using bimolecular fluorescence complementation. Plant
Brandizzi, F., Irons, S., Kearns, A., and Hawes, C. 2003. BY-2 cells:
Culture and transformation for live cell imaging. Chapter 1: Unit 1.7 in:
Current Protocols in Cell Biology. Wiley Interscience, New York.
Briddon, R. W., and Stanley, J. 2006. Subviral agents associated with plant
single-stranded DNA viruses. Virology 344:198-210.
Brigneti, G., Voinnet, O., Li, W. X., Ji, L. H., Ding, S.-W., and Baulcombe,
D. C. 1998. Viral pathogenicity determinants are suppressors of transgene
silencing in N. benthamiana. EMBO (Eur. Mol. Biol. Organ.) J. 17:6739-
Brown J. W., Shaw P. J., Shaw P., and Marshall D. F. 2005. Arabidopsis
nucleolar protein database (AtNoPDB). Nucleic Acids Res. 33 (Suppl.
Burbidge, N. T. 1960. The Australian species of Nicotiana L.
(Solanaceae). Aust. J. Bot. 8:342-380.
Burch-Smith, T. M., Anderson, J. C., Martin, G. B., and Dinesh-Kumar, S.
P. 2004. Applications and advantages of virus-induced gene silencing
for gene function studies in plants. Plant J. 39:734-746.
Burch-Smith, T. M., Schiff, M., Liu, Y., and Dinesh-Kumar, S. P. 2006.
Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol.
Burch-Smith, T. M., Schiff, M., Caplan, J. L., Tsao, J., Czymmek, K., and
Dinesh-Kumar, S.P. 2007. A novel role for the TIR domain in
association with pathogen-derived elicitors. PLoS Biol. 5(3):e68.
Callaway, A., Liu, W., Andrianov, V., Stenzler, L., Zhao, J., Wettlaufer, S.,
Jayakumar, P., and Howell, S. H. 1996. Characterization of cauliflower
mosaic virus (CaMV) resistance in virus-resistant ecotypes of
Arabidopsis. Mol. Plant-Microbe Interact. 9:810-818.
Cao, X., Zhou, P., Zhang, X., Zhu, S., Zhong, X., Xiao, Q., Ding, B., and
Li, Y. 2005. Identification of an RNA silencing suppressor from a plant
double-stranded RNA virus. J. Virol. 79:13018-13027.
Caplan, J. L., Mamillapalli, P., Burch-Smith, T. M., Czymmek, K., and
Dinesh-Kumar, S. P. 2008. Chloroplastic protein NRIP1 mediates innate
immune receptor recognition of a viral effector. Cell 132:449-462.
Chakrabarty, C., Banerjee, R., Chung, S. M., Farman, M., Citovsky, V.,
Hogenhout, S. A., Tzfira, T., and Goodin, M. M. 2007. pSITE vectors
for stable integration or transient expression of autofluorescent protein
fusions in plants: Probing Nicotiana benthamiana–virus interactions.
Mol. Plant-Microbe Interact. 20:740-750.
Chapman, S., Kavanagh, T., and Baulcombe, D. 1992. Potato virus X as a
vector for gene expression in plants. Plant J. 2:549-557.
Chase, M. W., Knapp, S., Cox, A. V., Clarkson, J. J., Butsko, Y., Joseph, J.,
Savolainen, V., and Parokonny, A. S. 2003. Molecular systematics,
GISH and the origin of hybrid taxa in Nicotiana (Solanaceae). Ann.
Chisholm, S. T., Coaker, G., Day, B., and Staskawicz, B. J. 2006. Host–
microbe interactions: Shaping the evolution of the plant immune
response. Cell 124:803-814.
Christie, S. R., and Crawford, W. E. 1978. Plant virus range of Nicotiana
benthamiana. Plant Dis. Rep. 62:20-22.
Chung, S. M., Frankman, E. L., and Tzfira, T. 2005. A versatile vector sys-
tem for multiple gene expression in plants. Trends Plant Sci. 10:357-361.
Citovsky, V., Lee, L. Y., Vyas, S., Glick, E., Chen, M. H., Vainstein, A.,
Gafni, Y., Gelvin, S. B., and Tzfira, T. 2006. Subcellular localization of
interacting proteins by bimolecular fluorescence complementation in
planta. J. Mol. Biol. 362:1120-1131.
Clough S. J., and Bent A. F. 1998. Floral dip: A simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant
Coutts, R. H., and Buck, K. W. 1985. DNA and RNA polymerase activities
of nuclei and hypotonic extracts of nuclei isolated from tomato golden
mosaic virus infected tobacco leaves. Nucleic Acids Res. 13:7881-7897.
Cruz, S. S., Chapman, S., Roberts, A. G., Roberts, I. M, Prior, D. A., and
Oparka, K. J. 1996. Assembly and movement of a plant virus carrying a
green fluorescent protein overcoat. Proc. Natl. Acad. Sci. U.S.A.
Dardick, C. D. 2007. Comparative expression profiling of N. benthamiana
leaves systemically infected with three fruit tree viruses. Mol. Plant-
Microbe Interact. 20:1004-1017.
Deleris, A., Gallego-Bartolome, J., Bao, J., Kasschau, K. D., Carrington, J.
C., and Voinnet, O. 2006. Hierarchical action and inhibition of plant
Dicer-like proteins in antiviral defense. Science 313:68-71.
De Wit, P. J. 2007. How plants recognize pathogens and defend
themselves. Cell Mol. Life Sci. 64:2726-2732.
Ding, S. W., and Voinnet, O. 2007. Antiviral immunity directed by small
RNAs. Cell 130:413-426.
Dong, Y., Burch-Smith, T. M., Liu, Y., Mamillapalli, P., and Dinesh-
Kumar, S. P. 2007. A ligation-independent cloning TRV vector for high-
throughput virus induced gene silencing identifies roles for NbMADS4-
1 and -2 in floral development. Plant Physiol. 145:1161-1170.
Dreher T. W., and Miller W. A. 2006. Translational control in positive
strand RNA plant viruses. Virology 344:185-197.
Earley, K. W., Haag, J. R., Pontes, O., Opper, K., Juehne, T., Song, K., and
Pikaard, C. S. 2006. Gateway-compatible vectors for plant functional
genomics and proteomics. Plant J. 45:616-629.
Escobar, N. M., Haupt, S., Thow, G., Boevink, P., Chapman, S., and
Oparka, K. 2003. High-throughput viral expression of cDNA-green
fluorescent protein fusions reveals novel subcellular addresses and
identifies unique proteins that interact with plasmodesmata. Plant Cell
Fu, D. Q., Zhu, B. Z., Zhu, H. L., Jiang, W. B., and Luo, Y. B. 2005. Virus-
induced gene silencing in tomato fruit. Plant J. 43:299-308.
Gabriëls, S. H., Vossen, J. H., Ekengren, S. K., van Ooijen, G., Abd-El-
Haliem, A. M., van den Berg, G. C., Rainey, D. Y., Martin, G. B.,
Takken, F. L., de Wit, P. J., and Joosten, M. H. 2007. An NB-LRR
protein required for HR signaling mediated by both extra- and
intracellular resistance proteins. Plant J. 50:14-28.
Gabriëls, S. H. E. J., Takken, F. L. W., Vossen, J. H. de Jong, C. F., Liu, Q.,
Turk, S. C., Wachowski, L. K., Peters, J., Witsenboer, H. M., de Wit, P.
J., and Joosten, M. H. 2006. cDNA-AFLP, combined with functional
analysis reveals novel genes involved in the hypersensitive response.
Mol. Plant-Microbe Interact. 19:567-576.
Goodin, M., Yelton, S., Ghosh, D., Mathews, S., and Lesnaw, J. 2005.
Live-cell imaging of rhabdovirus-induced morphological changes in
plant nuclear membranes. Mol. Plant-Microbe Interact. 18:703-709.
Goodin, M. M., Dietzgen, R. G., Schichnes, D., Ruzin, S., and Jackson, A.
O. 2002. pGD vectors: Versatile tools for the expression of green and
red fluorescent protein fusions in agroinfiltrated plant leaves. Plant J.
Goodin, M. M., Chakrabarty, R., Yelton, S., Martin, K., Clark, A., and
Brooks, R. 2007a. Membrane and protein dynamics in live plant nuclei
infected with Sonchus yellow net virus, a plant-adapted rhabdovirus. J.
Gen. Virol. 88:1810-1820.
Goodin, M. M., Chakrabarty, R., Banerjee, R., Yelton, S., and DeBolt, S.
2007b. New Gateways to discovery. Plant Physiol. 145:1100-1109.
Goodspeed, T. H. 1954. Pages 485-487 in: The Genus Nicotiana: Origins,
Relationships and Evolution of its Species in the Light of Their
Distribution, Morphology and Cytogenetics. Chronica Botanica,
Waltham, MA, U.S.A.
Heese, A., Hann, D. R., Gimenez-Ibanez, S., Jones, A. M., He, K., Li, J.,
Schroeder, J. I., Peck, S. C., and Rathjen, J. P. 2007. The receptor-like
kinase SERK3/BAK1 is a central regulator of innate immunity in
plants. Proc. Natl. Acad. Sci. U.S.A. 104:12217-12222.
Jaccard, P. 1908. Nouvelles recherches sur la distribution floral. Bull. Soc.
Vaudoise Sci. Nat. 44:223-270.
Kamoun, S., Hamada, W., and Huitema, E. 2003. Agrosuppression: A
bioassay for the hypersensitive response suited to high-throughput
screening. Mol. Plant-Microbe Interact. 16:7-13.
Vol. 21, No. 8, 2008 / 1025
Kanneganti, T. D., Bai, X., Tsai, C. W., Win, J., Meulia, T., Goodin, M.,
Kamoun, S., and Hogenhout, S. A. 2007. A functional genetic assay for
nuclear trafficking in plants. Plant J. 50:149-158.
Karimi, M., Bleys, A., Vanderhaeghen, R., and Hilson, P. 2007. Building
blocks for plant gene assembly. Plant Physiol. 145:1183-1191.
Keevil, J. J. 1949. Benjamin Bynoe, Surgeon of H.M.S. Beagle. J. Hist.
Knapp, S., Chase, M. W., and Clarkson, J. J. 2004. Nomenclatural changes
and a new sectional classification in Nicotiana (Solanaceae). Taxon
Koroleva, O. A., Tomlinson, M. L., Leader, D., Shaw, P., and Doonan, J.
H. 2005. High-throughput protein localization in Arabidopsis using
Agrobacterium-mediated transient expression of GFP-ORF fusions.
Plant J. 41:162-174.
Kumagai, M. H., Donson, J., della-Cioppa, G., Harvey, D., Hanley, K., and
Grill, L. K. 1995. Cytoplasmic inhibition of carotenoid biosynthesis
with virus-derived RNA. Proc. Natl. Acad. Sci. U.S.A. 92:1679-1683.
Laketa, V., Simpson, J. C., Bechtel, S., Wiemann, S., and Pepperkok, R.
2007. High-content microscopy identifies new neurite outgrowth
regulators. Mol. Biol. Cell 18:242-252.
Lara-Cabrera, S. I., and Spooner, D. M. 2004. Taxonomy of North and
Central American diploid wild potato (Solanum sect. Petota) species:
AFLP data. Plant Syst. Evol. 248:29-142.
Lindbo, J. A. 2007. TRBO: A high-efficiency Tobacco mosaic virus RNA-
based overexpression vector. Plant Physiol. 145:1232-1240.
Liu, E., and Page, J. E. 2008. Optimized cDNA libraries for virus-induced
gene silencing (VIGS) using tobacco rattle virus. Plant Methods 4:5.
Liu, Y., Schiff, M., and Dinesh-Kumar, S. P. 2002. Virus-induced gene
silencing in tomato. Plant J. 31:777-786.
Lucas, W. J. 2006. Plant viral movement proteins: Agents for cell-to-cell
trafficking of viral genomes. Virology 344:169-184.
Ma, W., and Berkowitz, G. A. 2007. The grateful dead: Calcium and cell
death in plant innate immunity. Cell Microbiol. 9:2571-2585.
Martin, A., Cabrera y Poch, H. L., Martinez Herrera, D., and Ponz, F.
1999. Resistances to turnip mosaic potyvirus in Arabidopsis thaliana.
Mol. Plant-Microbe Interact. 12:1016-1021.
Megason, S. G., and Fraser, S. E. 2007. Imaging in systems biology. Cell
Messinese, E., Mun, J. H., Yeun, L. H., Jayaraman, D., Rouge, P., Barre,
A., Lougnon, G., Schornack, S., Bono, J. J., Cook, D. R., and Ane, J.
M. 2007. A novel nuclear protein interacts with the symbiotic DMI3
calcium- and calmodulin-dependent protein kinase of Medicago
truncatula. Mol. Plant-Microbe Interact. 20:912-921.
Mestre, P., and Baulcombe, D. C. 2006. Elicitor-mediated oligomerization
of the tobacco N disease resistance protein. Plant Cell 18:491-501.
Moore, S., Payton, P., Wright, M., Tanksley, S., and Giovannoni, J. 2005.
Utilization of tomato microarrays for comparative gene expression
analysis in the Solanaceae. J. Exp. Bot. 56:2885-2895.
Musiychuk, K., Stephenson, N., Bi, H., Farrance, C. E., Orozovic, G.,
Brodelius, M., Brodelius, P., Horsey, A., Ugulava, N., Shamloul, A.-M.,
Mett, V., Rabindran, S., Streatfield, S. J., and Yusibov, V. 2007. A launch
vector for the production of vaccine antigens in plants. Influenza 1:19-
Nagata, T., Nemoto, Y., and Hasezawa, S. 1992. Tobacco BY-2 cell line as
the “HeLa” cell in the cell biology of higher plants. Int. Rev. Cytol.
Nagy, P. D., and Pogany, J. 2006. Yeast as a model host to dissect
functions of viral and host factors in Tombusvirus replication. Virology
Narayan, R. K. J. 1987. Nuclear DNA changes, genome differentiation and
evolution in Nicotiana (Solanaceae). Plant Syst. Evol. 157:161-180.
Ohad, N., Shichrur, K., and Yalovsky, S. 2007. The analysis of protein-
protein interactions in plants
complementation. Plant Physiol. 145:1090-1099.
Panavas, T., Serviene, E., Pogany, J., and Nagy, P. D. 2008. Genome-wide
screens for identification of host factors in viral replication. Methods
Mol. Biol. 451:615-624.
Pendle, A. F., Clark, G. P., Boon, R., Lewandowska, D., Lam, Y. W.,
Andersen, J., Mann, M., Lamond, A. I., Brown, J. W., and Shaw, P. J.
2005. Proteomic analysis of the Arabidopsis nucleolus suggests novel
nucleolar functions. Mol. Biol. Cell 16:260-269.
Pepperkok, R., and Ellenberg, J. 2006. High-throughput fluorescence
microscopy for systems biology. Nat. Rev. Mol. Cell Biol. 7:690-696.
Popescu, S. C., Popescu, G. V., Bachan, S., Zhang, Z., Seay, M., Gerstein,
M., Snyder, M., and Dinesh-Kumar, S. P. 2007. Differential binding of
calmodulin-related proteins to their targets revealed through high-
density Arabidopsis protein microarrays. Proc. Natl. Acad. Sci. U.S.A.
Quacquarelli, A. 1975. N. benthamiana Domin as a host for plant viruses.
Phytopathol. Mediterr. 14:36-39.
by bimolecular fluorescence
Ratcliff, F., Martin-Hernandez, A. M., and Baulcombe, D. C. 2001.
Technical advance. Tobacco rattle virus as a vector for analysis of gene
function by silencing. Plant J. 25:237-245.
Rensink, W. A., Lee, Y., Liu, J., Iobst, S., Ouyang, S., and Buell, C. R.
2005. Comparative analyses of six solanaceous transcriptomes reveal a
high degree of sequence conservation and species-specific transcripts.
BMC Genomics 6:124.
Robertson, D. 2004. VIGS vectors for gene silencing: Many targets, many
tools. Annu. Rev. Plant Biol. 55:495-519.
Roth, B. M., Pruss, G. J., and Vance, V. B. 2004. Plant viral suppressors of
RNA silencing. Virus Res. 102:97-108.
Ruiz, M. T., Voinnet, O., and Baulcombe, D. C. 1998. Initiation and
maintenance of virus-induced gene silencing. Plant Cell 10:937-946.
Ryu, C. M., Anand, A., Kang, L., and Mysore, K. S. 2004. Agrodrench: A
novel and effective agroinoculation method for virus-induced gene si-
lencing in roots and diverse Solanaceous species. Plant J. 40:322-331.
Corregendum in 2006. Plant J. 45:869.
Schob, H., Kunz, C., and Meins, F., Jr. 1997. Silencing of transgenes intro-
duced into leaves by agroinfiltration: a simple, rapid method for investi-
gating sequence requirements for gene silencing. Mol. Gen. Genet.
Segers, G. C., van Wezel, R., Zhang, X., Hong, Y., and Nuss, D. L. 2006.
Hypovirus papain-like protease p29 suppresses RNA silencing in the
natural fungal host and in a heterologous plant system. Eukaryot. Cell
Senthil, G., Liu, H., Puram, V. G., Clark, A., Stromberg, A., and Goodin,
M. M. 2005. Specific and common changes in Nicotiana benthamiana
gene expression in response to infection by enveloped viruses. J. Gen.
Senthil-Kumar, M., Hema, R., Anand, A., Kang, L., Udayakumar, M., and
Mysore, K. S. 2007. A systematic study to determine the extent of gene
silencing in Nicotiana benthamiana and other Solanaceae species when
heterologous gene sequences are used for virus-induced gene silencing.
New Phytol. 176:782-791.
Serva, S., and Nagy, P. D. 2006. Proteomics analysis of the Tombusvirus
replicase: Hsp70 molecular chaperone is associated with the replicase
and enhances viral RNA replication. J. Virol. 80:2162-2169.
Siemering, K. R., Golbik, R., Sever, R., and Haseloff, J. 1996. Mutations
that suppress the thermosensitivity of green fluorescent protein. Curr.
Simpson, J. C., and Pepperkok, R. 2006. The subcellular localization of
the mammalian proteome comes a fraction closer. Genome Biol. 7:222.
Stokes, J. L. 1846. Discoveries in Australia; with an Account of the Coasts
and Rivers Explored and Surveyed During the Voyage of H. M. S. Bea-
gle, in the Years 1837-38-39-40-41-42-43, Volumes I & II. T. and W.
Strasser, R., Bondili, J. S., Schoberer, J., Svoboda, B., Liebminger, E.,
Glossl, J., Altmann, F., Steinkellner, H., and Mach, L. 2007. Enzymatic
properties and subcellular localization of Arabidopsis beta-N-acetyl-
hexosaminidases. Plant Physiol. 145:5-16.
Sunter, G., and Bisaro, D. M. 2003. Identification of a minimal sequence
required for activation of the Tomato golden mosaic virus coat protein
promoter in protoplasts. Virology 305:452-462.
Takahashi, Y., Nasir, K. H., Ito, A., Kanzaki, H., Matsumura, H., Saitoh,
H., Fujisawa, S., Kamoun, S., and Terauchi, R. 2007. A high-throughput
screen of cell-death-inducing factors in Nicotiana benthamianaidenti-
fies a novel MAPKK that mediates INF1-induced cell death signaling
and non-host resistance to Pseudomonas cichorii. Plant J. 49:1030-
Tameling, W. I., and Baulcombe, D. C. 2007. Physical association of the
NB-LRR resistance protein Rx with a Ran GTPase-activating protein is
required for extreme resistance to Potato virus X. Plant Cell 19:1682-
Tardif, G., Kane, N. A., Adam, H., Labrie, L., Major, G., Gulick, P.,
Sarhan, F., and Laliberte, J. F. 2007. Interaction network of proteins
associated with abiotic stress response and development in wheat. Plant
Mol. Biol. 63:703-718.
Thomas, C. L, Jones, L., Baulcombe, D. C., and Maule, A. J. 2001. Size
constraints for targeting post-transcriptional gene silencing and for
RNA-directed methylation in N. benthamiana using a Potato virus X
vector. Plant J. 25:417-425.
Thomas, C. L., Bayer, E. M., Ritzenthaler, C., Fernandez-Calvino, L., and
Maule, A. J. 2008. Specific targeting of a plasmodesmal protein affect-
ing cell-to-cell communication. PLoS Biol. 6:e7.
Tian, G. W., Mohanty, A., Chary, S. N., Li, S., Paap, B., Drakakaki, G.,
Kopec, C. D., Li, J., Ehrhardt, D., Jackson, D., Rhee, S. Y, Raikhel, N.
V., and Citovsky, V. 2004. High-throughput fluorescent tagging of full-
length Arabidopsis gene products in planta. Plant Physiol. 135:25-38.
Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. 2003. An enhanced
transient expression system in plants based on suppression of gene si-
1026 / Molecular Plant-Microbe Interactions Download full-text
lencing by the p19 protein of Tomato bushy stunt virus. Plant J. 33:949-
Wang, R. Y., and Nagy, P. D. 2008. Tomato bushy stunt virus co-opts the
RNA-binding function of a host metabolic enzyme for viral genomic
RNA synthesis. Cell Host Microbe. 3:178-187.
Xiao, F., Giavalisco, P., and Martin, G. B. 2007. Pseudomonas syringae
type III effector AvrPtoB is phosphorylated in plant cells on serine 258,
promoting its virulence activity. J. Biol. Chem. 282:30737-30744.
Yang, S., Carter, S. A., Cole, A. B., Cheng, N., and Nelson, R. 2004. A
natural variant of a host RNA-dependent RNA polymerase is associated
with increased susceptibility to viruses by Nicotiana benthamiana.
Proc. Natl. Acad. Sci. U.S.A. 101:6297-6302.
Zhao, Q., Leung, S., Corbett, A. H., and Meier, I. 2006. Identification and
characterization of the Arabidopsis orthologs of nuclear transport factor
2, the nuclear import factor of ran. Plant Physiol. 140:869-878.
Zipfel, C. 2008 Pattern-recognition receptors in plant innate immunity.
Curr. Opin. Immunol. 20:10-16.
Zipfel, C., and Felix, G. 2005. Plants and animals: A different taste for mi-
crobes? Curr. Opin. Plant Biol. 8:353-360.
AUTHOR-RECOMMENDED INTERNET RESOURCES
Australia’s Virtual Herbarium website: www.rbg.vic.gov.au/avh
AusPGRIS database: www2.dpi.qld.gov.au/extra/asp/auspgris
National Agricultural Statistics Service website: www.nass.usda.gov
Royal Botanic Gardens, Kew Angiosperm DNA C-values database:
Sol genomics network: www.sgn.cornell.edu
TIGR Solanaceae Genomics Resource website: www.tigr.org/tdb/sol