Nt-RhoGDI2 regulates Rac/Rop signaling and polar cell growth in tobacco pollen tubes

Nt-RhoGDI2 regulates Rac/Rop signaling and polar cellgrowth in tobacco pollen tubes

Ulrich Klahre, Claude Becker, Arno C. Schmitt and Benedikt Kost*

Heidelberg Institute of Plant Sciences (HIP), University of Heidelberg, Im Neuenheimer Feld 360, 69120 Heidelberg, Germany

Received 7 February 2006; revised 28 February 2006; accepted 9 March 2006.*For correspondence (fax þ49 6221 54 5859; e-mail [email protected]).

Summary

Rac/Rop-type Rho-family small GTPases accumulate at the plasma membrane in the tip of pollen tubes and

control the polar growth of these cells. Nt-RhoGDI2, a homolog of guanine nucleotide dissociation inhibitors

(GDIs) regulating Rho signaling in animals and yeast, is co-expressed with the Rac/Rop GTPase Nt-Rac5

specifically in tobacco (Nicotiana tabacum) pollen tubes. The two proteins interact with each other in yeast

two-hybrid assays, preferentially when Nt-Rac5 is prenylated. Transient over-expression of Nt-Rac5 and

Nt-RhoGDI2 depolarized or inhibited tobacco pollen tube growth, respectively. Interestingly, pollen tubes

over-expressing both proteins grew normally, demonstrating that the two proteins functionally interact in

vivo. Nt-RhoGDI2 was localized to the pollen tube cytoplasm and effectively transferred co-over-expressed

YFP–Nt-Rac5 fusion proteins from the plasma membrane to this compartment. A single amino acid exchange

(R69A), which abolished binding to Nt-RhoGDI2, caused Nt-Rac5 to be mis-localized to the flanks of pollen

tubes and strongly compromised its ability to depolarize pollen tube growth upon over-expression. Based on

these observations, we propose that Nt-RhoGDI2-mediated recycling of Nt-Rac5 from the flanks of the tip to

the apex has an essential function in the maintenance of polarized Rac/Rop signaling and cell expansion in

pollen tubes. Similar mechanisms may generally play a role in the polarized accumulation of Rho GTPases in

specific membrane domains, an important process whose regulation has not been well characterized in any

cell type to date.

Keywords: polar cell growth, pollen tube, Rac/Rop GTPase, RhoGDI, signal transduction, tobacco.

Introduction

Pollen tubes grow rapidly through flower tissues and

mediate fertilization by transporting sperm cells to ovules

(Bedinger et al., 1994). Vegetative pollen tube cells elongate

by tip growth, during which polarized apical secretion re-

sults in unidirectional cell expansion (Hepler et al., 2001).

Because pollen tubes grow well in culture and are amenable

to experimental manipulation, they are widely employed as

a model system to investigate polar cell expansion in plants.

Evidence accumulated during the past decade demonstrates

that Rac/Rop GTPases, plant homologs of the Rho family of

small GTPases, associate with the plasma membrane spe-

cifically in the tip of pollen tubes and are key regulators of

the polar growth of these cells (Kost et al., 1999; Li et al.,

1999; Zheng and Yang, 2000). However, the molecular

mechanisms that control the localization and activity of

pollen tube Rac/Rop are not well understood.

Rho-family GTPases constitute a group of highly con-

served signaling proteins that have key functions in the

regulation of cellular polarization and polar cell growth in

yeast and animals (Etienne-Manneville and Hall, 2002). Rho

GTPases act as molecular switches that transduce signals in

the GTP-bound conformation by interacting with a variety of

effectors, and are inactive after GTP hydrolysis. Inter-

conversion between active GTP-bound and inactive

GDP-bound forms of Rho GTPases is regulated by GTPase-

activating proteins (RhoGAPs), which stimulate GTP hydro-

lysis, and guanine nucleotide exchange factors (RhoGEFs),

which catalyze GDP release to promote GTP binding. Point

mutations can be introduced into the particularly highly

conserved nucleotide binding domain of Rho GTPases to

generate constitutively active or dominant negative mutant

versions of these proteins. Constitutively activeRhoGTPases

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The Plant Journal (2006) 46, 1018–1031 doi: 10.1111/j.1365-313X.2006.02757.x

display little GTPase activity even in the presence of

RhoGAPs, and show a significantly increased association

with GTP in vivo and in vitro (Read et al., 2000; Trahey and

McCormick, 1987). In contrast, dominant negative forms of

these proteins do not bind nucleotides well and preferen-

tially assume a conformation that mimics the nucleotide-

free transition state in which Rho GTPases interact most

strongly with RhoGEFs. Over-expressed dominant negative

mutant Rho GTPases are thought to inhibit endogenous Rho

signaling by forming inactive complexes with available

RhoGEFs (Feig, 1999).

Most Rho GTPases are post-translationally modified by

the attachment of an isoprenoid lipid to the cysteine residue

within a C-terminal CAAX box. Prenylation is responsible

for the association of Rho GTPases with membranes, which

is thought to be essential for the activity of these proteins

(Wennerberg and Der, 2004). Membrane-bound Rho GTP-

ases are typically in equilibrium with a cytoplasmic pool

that is maintained by another group of regulatory proteins,

the guanine nucleotide dissociation inhibitors (RhoGDIs;

Sasaki and Takai, 1998). A key biochemical activity of these

proteins is their ability to remove prenylated Rho GTPases

from membranes and to form cytoplasmic heterodimers

with them (Michaelson et al., 2001; Read et al., 2000).

Consistent with these activities, RhoGDIs are most com-

monly thought to act as negative regulators of Rho

signaling in vivo (Etienne-Manneville and Hall, 2002; Olo-

fsson, 1999; Sasaki and Takai, 1998), although recent

studies have indicated that the translocation of activated

Rho GTPases to specific membrane domains (Del Pozo

et al., 2002) and the ability of these proteins to control

certain cellular processes may depend on RhoGDI interac-

tions (Lin et al., 2003). Membrane-associated RhoGDI dis-

sociation factors (RhoGDFs), which promote membrane

translocation of Rho GTPases by destabilizing the interac-

tion of these proteins with RhoGDIs (DerMardirossian and

Bokoch, 2005), may be involved in the RhoGDI-mediated

positive control of Rho signaling.

Plants contain large gene families encoding Rac/Rop

GTPases (e.g. 11 members in Arabidopsis). Some of these

proteins are specifically expressed in pollen tubes and

control the growth of these cells, whereas others regulate

various aspects of cellular behavior in different cell types

(Gu et al., 2004). Plant Rac/Rop GTPases seem to function in

a very similar manner to their yeast and animal homologs,

but form a distinct subgroup of the Rho family of small

GTPases based on amino acid sequence comparison

(Valster et al., 2000), and are controlled in part by plant-

specific regulatory mechanisms. Sequences showing

homology to genes encoding Dbl-family RhoGEFs, key

regulators of Rho GTPases in other organisms, are absent

from the Arabidopsis and rice genomes (Valster et al.,

2000). A novel plant-specific family of proteins unrelated

to previously characterized RhoGEFs has recently been

demonstrated to act as GEFs of Rac/Rop GTPases (Berken

et al., 2005; Gu et al., 2006). Plant homologs of animal

and yeast RhoGAPs or RhoGDIs have been identified, but

display unique structural features (e.g. CRIB domains in

plant RhoGAPs, Wu et al., 2000; N-terminal extension of

plant RhoGDIs, Bischoff et al., 2000; see below).

Although the molecular and cellular functions of plant

RhoGAPs (Wu et al., 2000), RhoGEFs (Gu et al., 2006; Kaoth-

ien et al., 2005) and RhoGDIs (Carol et al., 2005) have begun

to be investigated, they are not well understood to date.

Arabidopsismutants disrupted in a gene encoding a RhoGDI

homolog (At-RhoGDI1/SCN1, At3g07880) display defects in

restricting sites of Rac/Rop accumulation and cell expansion

in root epidermal cells that normally form single root hairs

(Carol et al., 2005). An N-terminally truncated version of this

Arabidopsis RhoGDI homolog (missing the first 43 amino

acids) was found to interact in yeast two-hybrid assays with

wild-type and constitutively active, but not with dominant

negative, forms of the Arabidopsis Rac/RopGTPases AtRop4

and AtRop6 (Bischoff et al., 2000). Expression of the same

Arabidopsis RhoGDI isoform in tobacco (Nicotiana tabacum)

pollen tubes prevented the formation of transversal actin

bands induced by co-expression of the Arabidopsis Rac/Rop

GTPase At-Rop1 (Fu et al., 2001), and reduced ballooning at

the tip caused by over-expression of the tobacco Rac/Rop

GTPase Nt-Rac1 (Chen et al., 2003). When expressed in

tobacco protoplasts, this Arabidopsis RhoGDI isoform inter-

fered with the activation of an auxin-responsive promoter by

over-expressed Nt-Rac1, and with the accumulation of Nt-

Rac1 in the cell cortex (Tao et al., 2002).

AmoredetailedfunctionalcharacterizationofplantRhoGDIs

and other regulators of Rac/Rop GTPases is clearly essential

to understand how Rac/Rop signaling controls cell behavior.

Because different isoforms of Rac/Rop GTPases and their

regulators are likely tohavedistinct features, it is important to

investigate functional interactions between isoforms that

display overlapping expression patterns, and to perform

in vivo experiments using cell types in which these isoforms

are co-expressed during normal development. Pollen tubes,

in which activated Rac/Rop GTPases specifically accumulate

in a restricted apical domainof theplasmamembrane (Zheng

andYang, 2000), represent an excellent system to investigate

regulatory mechanisms controlling the activity of these

proteins, potentially in a spatially resolvedmanner. Polarized

activation of Rho family GTPases does not only occur in

pollen tubes, but is also essential for fibroblastmigration and

yeast cell expansion, for example (Etienne-Manneville and

Hall, 2002). Investigation of the control of Rac/Rop signaling

in pollen tubes can potentially provide significant insights

into the molecular mechanisms underlying this process,

which are currently not well understood in any cell type

(Etienne-Manneville, 2004).

Here, we present a functional characterization of Nt-

RhoGDI2, which was identified in a yeast two-hybrid screen

Nt-RhoGDI2 regulates pollen tube Rac/Rop 1019

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for proteins interacting with the pollen tube Rac/Rop GTPase

Nt-Rac5. Nt-RhoGDI2 and Nt-Rac5 are both highly and

specifically expressed in elongating tobacco pollen tubes.

Analysis of interactions of Nt-RhoGDI2 with wild-type or

mutant forms of Nt-Rac5 in two-hybrid assays and in co-

over-expression experiments has demonstrated that Nt-

RhoGDI2 is a key regulator of Nt-Rac5 signaling and polar

growth in pollen tubes. Our data strongly suggest that Nt-

RhoGDI2-mediated recycling of Nt-Rac5 from the flanks to

the apex of the pollen tube tip controls the intracellular

distribution and activity of this GTPase.

Results

Identification of Nt-RhoGDI2 as an interaction partner of

Nt-Rac5

The tobacco Rac/Rop GTPase Nt-Rac5 (AJ250174) has been

reported to be specifically expressed at high levels in pollen

(Kieffer et al., 2000), and is very closely related (93.9–95.4%

identical amino acids) to Rac/Rop GTPases known to control

pollen tube growth in other plant species (At-Rop1, At-Rop5/

Rac2; Ps-Rop1; Gu et al., 2004). We have cloned the coding

sequence of Nt-Rac5 by RT-RCR using RNA from tobacco

pollen tubes grown in vitro as a template. Transcripts with

the expected length of 1.4 kb hybridizing under stringent

conditions to the Nt-Rac5 coding sequence were found to

accumulate to high levels specifically in tobacco flowers,

pollen grains and pollen tubes (Figure 1a). The effects of

over-expressing wild-type or mutant Nt-Rac5 on tobacco

pollen tube growth, as well as the intracellular localization of

these different forms of Nt-Rac5 fused to YFP, were essen-

tially the same as those observed with other pollen tube Rac/

Rop GTPases (see Figures 5a and 7) (Chen et al., 2003; Kost

et al., 1999; Li et al., 1999). Together, these data establish

that Nt-Rac5 regulates polar pollen tube growth in a similar

manner to its closest homologs.

To identify proteins involved inRac/Ropsignaling inpollen

tubes, a cDNA library representing genes expressed in

tobacco pollen tubes 3 h after germination was screened

for sequences encoding polypeptides that show yeast two-

hybrid interaction with constitutively active Nt-Rac5 (Nt-

Rac5G15V) carrying an additional point mutation (C194S) that

disrupts the C-terminal CAAX domain and prevents prenyla-

tion. A screen of approximately 3 · 105 yeast transformants

resulted in the selection of six cDNA clones encoding pollen

tube polypeptides that showed specific interactions with Nt-

Rac5 (unpublished data). These polypeptides were similar to

hypothetical proteins (n ¼ 3), protein kinases (n ¼ 2) or

RhoGDIs (n ¼ 1). The RhoGDI cDNA clone coded for an

N-terminally truncated polypeptide of 223 amino acidswith a

lysine residue at position one. Colony hybridization screen-

ingof the tobaccopollen tube library describedaboveusing a

probe derived from the partial RhoGDI cDNA resulted in the

identification of a corresponding full-length cDNA clone

(DQ416769). This cDNA clone contained stop codons in all

reading frames upstream of an open reading frame coding

for a 235 amino acid protein, called Nt-RhoGDI2 hereafter.

Nt-RhoGDI2 interacts specifically with wild-type and

constitutively active Nt-Rac5 in two-hybrid assays

In yeast two-hybrid assays, full length Nt-RhoGDI2 fused to

the activation domain of the GAL4 transcription factor

(GAL4-AD) interacted specifically with Nt-Rac5 and consti-

tutively active Nt-Rac5G15V fused to the GAL4 DNA binding

domain (GAL4-DB). In contrast, no two-hybrid interaction

was detected between Nt-RhoGDI2 and dominant negative

Nt-Rac5 (Nt-Rac5T20N, Figure 2 and Figure S1). In these as-

says, growth of yeast co-transformants on medium without

histidine was analyzed, which indicates reporter gene

expression resulting from two-hybrid interactions. Co-

transformants expressing the GAL4-AD:Nt-RhoGDI2 fusion

together with free GAL4-DB, or free GAL4-AD together with

wild-type or mutant Nt-Rac5 fused to GAL4-DB, did not grow

on histidine-free medium.

Data presented in Figure 2 show two-hybrid interactions

between Nt-RhoGDI2 and wild-type or mutant forms of Nt-

Rac5 with an unmodified C-terminal CAAX domain, which

are presumably prenylated when expressed in yeast (Read

et al., 2000). Prenylation is expected to interfere with nuclear

targeting, which is required for the detection of two-hybrid

interactions. On the other hand, a large proportion of the

free energy of RhoGDI binding to Rho GTPases is thought to

be provided by interactions between a hydrophobic pocket

at the C-terminus of RhoGDIs and the prenyl tail attached to

Rho GTPases (Hoffman et al., 2000; Scheffzek et al., 2000).

The strength of yeast two-hybrid interactions between Nt-

RhoGDI2 and different forms of prenylated or unprenylated

(C194S) Nt-Rac5was estimated based on the growth of yeast

co-transformants on medium containing the histidine bio-

synthesis inhibitor 3-amino-1,2,4-triazole (3-AT) at different

concentrations (Aguilar et al., 1997). In these experiments,

(a) (b)

Figure 1. Analysis of gene expression patterns by Northern blotting.

Aliquots of 10 lg (a) or 5 lg (b) total RNA per lane were separated by gel

electrophoresis (lower panels), blotted and hybridized with DIG-labeled PCR

fragments corresponding to the Nt-Rac5 (a) or Nt-RhoGDI2 (b) coding

sequences (upper panels). Groups of lanes (separated by vertical lines)

shown in (a) have been rearranged to facilitate comparison with (b). R, root; S,

stem; L, leaf; Fb, flower bud; Fm, mature flower; P, mature pollen; PT, pollen

tube; *, 1.4 kb; �, 1 kb.

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ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 1018–1031

Nt-RhoGDI2 interacted most strongly with prenylated wild-

type Nt-Rac5 (Figure S1).

Nt-RhoGDI2 is closely related to well characterized RhoGDIs,

and is specifically expressed at high levels in pollen and in

pollen tubes

Nt-RhoGDI2 shows significant similarity (Figure S2) to bo-

vine Bt-RhoGDI1 (S12121, 26.7% identical amino acids) and

yeast Sc-RDI1 (BAA06499, 27.1% identical amino acids), two

RhoGDIs sharing 34.9% identical amino acids that have

been extensively characterized structurally (Hoffman et al.,

2000) and/or biochemically (Masuda et al., 1994; Sasaki

et al., 1993).

ProbingNorthernblots under stringent conditionswith the

Nt-RhoGDI2 coding region demonstrated that hybridizing

transcripts with the expected length of 1 kb were detectable

in flowers, and accumulated to high levels specifically in

pollen and in pollen tubes (Figure 1b). The data presented in

Figure 1 indicate that Nt-Rac5 and Nt-RhoGDI2 show sub-

stantial co-expression specifically in tobacco pollen and

pollen tubes. Together with the significant binding between

Nt-Rac5 and Nt-RhoGDI2 observed in two-hybrid assays

(Figure 2 and Figure S1), this indicated that the two proteins

may functionally interact during pollen tube elongation.

Transiently over-expressed Nt-RhoGDI2 inhibits tobacco

pollen tube growth and accumulates in the cytoplasm

Cultured tobacco pollen tubes transiently expressing Nt-

RhoGDI2 under the control of the Lat52 promoter (Twell

et al., 1991) following gene transfer into germinating pollen

by particle bombardment remainedmuch shorter compared

to control pollen tubes expressing the non-invasive marker

protein b-glucuronidase (GUS) under the control of the same

promoter (Figure 3). YFP co-expression, also under the

control of the Lat52 promoter, was employed to identify

transformed pollen tubes. As indicated by YFP fluorescence,

the expression level of introduced genes constantly

increased during the first 24 h after particle bombardment,

and varied considerably between individual pollen tubes.

Analysis of pollen tubes readily detectable by epi-fluores-

cence microscopy using short (<500 msec) exposure times

Figure 2. Yeast two-hybrid interactions between Nt-RhoGDI2 and Nt-Rac5.

Yeast transformants co-expressing different forms of Nt-Rac5 (G15V, consti-

tutively active; T20N, dominant negative) with an intact CAAX domain fused

to the DNA binding domain of the GAL4 transcription factor (GAL4 DB),

together with Nt-RhoGDI2 fused to the GAL4 activation domain (GAL4 AD),

plated on histidine-containing medium and on histidine-free medium.

Serving as negative controls are transformants co-expressing different forms

of GAL4 DB:Nt-Rac5 or GAL4 AD:Nt-RhoGDI2, along with free GAL4 AD or

GAL4 DB, respectively. Growth of yeast transformants on histidine-free

medium indicates reporter gene activation resulting from two-hybrid inter-

actions between Nt-RhoGDI2 and Nt-Rac5.

(a) (b)

Figure 3. Effects of transient over-expression of Nt-RhoGDI2 and YFP:Nt-RhoGDI2 on tobacco pollen tube growth.

(a) Microscopic analysis of pollen tubes co-expressing the indicated proteins 24 h after gene transfer. Upper panels: high-magnification (40· lens) epi-fluorescence

and transmitted light images (scale bar: 50 lm). Lower panels: low-magnification (5· lens) epi-fluorescence images (scale bar: 500 lm). g, pollen grain; t, pollen tube

tip.

(b) Statistical analysis of pollen tube length 9 h after gene transfer (representative example of three independent data sets). GDI2, Nt-RhoGDI2; error bars: 95% CI

(n ‡ 42).

Nt-RhoGDI2 regulates pollen tube Rac/Rop 1021

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showed that Nt-RhoGDI2-expressing pollen tubes were less

than half as long as GUS-expressing pollen tubes 9 h after

gene transfer (Figure 3b), and, in contrast to the latter, did

not continue to elongate after this time (Figure 3a).

When co-expressedwith GUS, Nt-RhoGDI2 fused to the C-

terminus (Figure 3), or the N-terminus (unpublished data), of

YFP had the same effects on pollen tube growth as co-

expression of free Nt-RhoGDI2 and YFP (Figure 3), indicating

that both YFP fusionswere functional. Neither fusion protein

affected pollen tube growth when transiently expressed at

minimal levels, at which they were barely detectable by low

magnification epi-fluorescence microscopy using short

(<500 msec) exposure times (Figure 4a). Confocal analysis

of weakly fluorescent pollen tubes expressing Nt-RhoGDI2

fused to the C-terminus (Figure 4b), or the N-terminus

(unpublished data), of YFP at minimal levels demonstrated

that both fusion proteins were diffusely distributed through-

out the pollen tube cytoplasm, similar to transiently

expressed free YFP (Figure 4b). Nt-RhoGDI2 fusion proteins

expressed at minimal or at higher levels (based on fluores-

cence intensity) were never found to accumulate at the

plasma membrane, not even in the presence of co-over-

expressed Nt-Rac5 (unpublished data). In accordance with

these observations, fractionation of tobacco pollen tube

extracts by high-speed centrifugation showed that endog-

enously expressed Nt-RhoGDI2 accumulated in membrane-

depleted cytoplasm (Figure 4c). The data shown in Figures 3

and 4 are consistent with models of RhoGDI function

presented in the literature (Etienne-Manneville and Hall,

2002), which propose that RhoGDIs are cytoplasmic proteins

that act as negative regulators of Rho signaling.

Nt-RhoGDI2 and Nt-Rac5 neutralize each other’s

over-expression effects

Transient over-expression of Nt-Rac5 depolarized pollen

tube growth and resulted in the formation of large balloons

instead of elongating tips (Figures 5a and 6). The same

effects in a more pronounced form were obtained when

Nt-Rac5G15V was expressed (Figures 5a and 6). In contrast,

Nt-Rac5T20N inhibited pollen tube growth without causing

comparable ballooning (Figures 5a and 6). These effects

were essentially the same as those observed upon over-

expression of wild-type or mutant forms of related pollen

tube Rac/Rop GTPases (Chen et al., 2003; Kost et al., 1999; Li

et al., 1999).

The pollen tubes shown in Figure 5(a) expressed wild-

type or mutant Nt-Rac5 along with YFP and GUS, all under

the control of the Lat52 promoter, following bombardment

with particles coated with equal amounts of three different

expression constructs (see Experimental procedures). To

analyze effects of Nt-RhoGDI2 co-over-expression with

different forms of Nt-Rac5, a plasmid conferring Lat52-

controlled expression of Nt-RhoGDI2 was used instead of

the GUS construct (Figure 5b). Interestingly, pollen tubes co-

over-expressing Nt-Rac5 and Nt-RhoGDI2 reached a similar

length to control pollen tubes expressing only marker

proteins 7 h after gene transfer (Figure 6). Apart from some

tip-swelling occasionally detected after prolonged culture

(24 h, unpublished data), they generally showed a normal

morphology (Figure 5b) and displayed neither the massive

ballooning nor the reduced growth that were observedwhen

Nt-Rac5 (Figure 5a) or Nt-RhoGDI2 (Figure 3), respectively,

were over-expressed individually. These results demon-

strate that Nt-RhoGDI2 and Nt-Rac5 functionally interact in

tobacco pollen tubes, and indicate, together with the two-

hybrid data shown in Figure 3 and Figure S1, that the two

(a)

(b)

(c)

Figure 4. Intracellular localization of Nt-RhoGDI2 in tobacco pollen tubes.

(a) Low-magnification (5· lens) epi-fluorescence images of pollen tubes

expressing YFP:Nt-RhoGDI2 at moderate (upper panels) or minimal (lower

panels) levels 6 h after gene transfer (scale bar: 500 lm).Moderate expression

inhibited pollen tube growth, caused image saturation after 5 sec of exposure

and was optimally imaged with an exposure time of 250 msec. Minimal

expression did not affect pollen tube elongation and was visible after 5 sec of

exposure, but barely detectable with an exposure time of 250 msec (repre-

sentative example of 15 similar data sets obtained in three independent

experiments).

(b) Single confocal optical sections through the tips of pollen tubes expres-

sing YFP:Nt-RhoGDI2 or YFP at minimal levels 6 h after gene transfer (each

representing at least 20 similar images collected in three independent

experiments; scale bar: 10 lm). Central sections through pollen tubes lying

flat on the cover-slip surface were imaged.

(c) Immunoblot showing accumulation of endogenously expressed Nt-

RhoGDI2 (detected using a polyclonal antibody prepared against this protein)

in the cytoplasmic fraction (S100k) of tobacco pollen tube extracts, which was

separated from membrane fractions (P10k, P100k) by centrifugation. S,

supernatant; P, pellet; 10k/100k, 10 000/100 000 g.

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ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 1018–1031

proteins may form an inactive complex in the pollen tube

cytoplasm.

This interpretation, which is consistent with models of

RhoGDI function proposed in the animal and yeast literature

(Etienne-Manneville and Hall, 2002), was supported by

titration experiments (Figure S3). In these experiments,

pollen tubes were bombarded with particles coated with

plasmid mixtures containing Nt-Rac5 and Nt-RhoGDI2

expression vectors at different ratios. The combined amount

of both expression vectors was kept constant. Normal pollen

tube growth was obtained at ratios of roughly 1:1. In

contrast, bombardment with an excess of Nt-Rac5 vector

(2:1, 3:1 or 5:1) or NtRhoGDI2 vector (1:2, 1:3 or 1:5) induced

ballooning or inhibited growth, respectively (Figure S3).

Nt-RhoGDI2 had no effect on the ballooning induced by

co-expressed Nt-Rac5G15V (Figures 5b and 6), conceivably

because this Nt-Rac5 mutant interacts weakly with Nt-

RhoGDI2, as indicated by two-hybrid assays (see Figure S1),

and has a higher potential to depolarize pollen tube growth

than Nt-Rac5 (Figures 5a and 6). The length of pollen tubes

expressing Nt-Rac5T20N was further reduced upon co-

expression of Nt-RhoGDI2 (Figures 5 and 6), which is

consistent with the expectation that each of the two proteins

inhibits pollen tube growth by a different mechanism when

over-expressed. Nt-Rac5T20N is likely to inhibit putative Rac/

Rop GEFs by forming inactive complexes with them (Feig,

1999), whereas Nt-RhoGDI2 appears to do the same to

endogenous Rac/Rop GTPases.

(a)

(b)

Figure 5. Morphology of tobacco pollen tubes transiently over-expressing wild-type or mutant Nt-Rac5, in the absence or presence of co-over-expressed

Nt-RhoGDI2.

Microscopic analysis of pollen tubes co-expressing 24 h after gene transfer different forms of Nt-Rac5 (a) with GUS and YFP, or (b) with Nt-RhoGDI2 and YFP. Upper

panels in (a) and (b): high-magnification (40· lens) epi-fluorescence and transmitted light images (scale bar: 50 lm). Lower panels in (a) and (b): low-magnification

(5· lens) epi-fluorescence images (scale bar: 500 lm). g, pollen grain; t, pollen tube tip.

Nt-RhoGDI2 regulates pollen tube Rac/Rop 1023

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Nt-RhoGDI2 differentially affects the intracellular

localization of wild-type and mutant Nt-Rac5

As observed on confocal optical sections through weakly

fluorescent elongating pollen tubes, Nt-Rac5 or Nt-Rac5G15V

fused to the C-terminus of YFP (YFP:Nt-Rac5/Nt-Rac5G15V)

accumulated at the plasma membrane at the tip of these

cells when transiently expressed at low levels 4–8 h after

gene transfer, before significant growth depolarization was

induced (Figure 7, left column, and Figure S4). Wild-type

and constitutively active forms of a related pollen tube Rac/

Rop GTPase (At-Rac2/Rop5) fused to YFP showed essentially

the same intracellular distribution (Kost et al., 1999).

Co-expression of Nt-RhoGDI2 prevented YFP:Nt-Rac5

from detectably accumulating at the plasma membrane

(Figure 7, right column), which confirms that Nt-RhoGDI2

functionally interacts with Nt-Rac5 in vivo and demonstrates

that it can translocate associated Rho GTPases from the

plasma membrane to the cytoplasm. This ability is charac-

teristic of all RhoGDIs, and is thought to be essential for the

regulation of Rho signaling by these proteins (Etienne-

Manneville and Hall, 2002). YFP:Nt-Rac5G15V was only par-

tially removed from the plasma membrane by co-over-

expressed Nt-RhoGDI2 (Figure 7), which is consistent with

the comparably weak interaction between Nt-RhoGDI2 and

Nt-Rac5G15V detected in two-hybrid assays (Figure S1), as

well as with the absence of an effect of Nt-RhoGDI2 on

ballooning induced by Nt-Rac5G15V in co-over-expression

experiments (Figures 5 and 6).

Particularly informative was the analysis of the intracel-

lular localization of Nt-Rac5T20N fused to the C-terminus of

YFP, both in the absence and in the presence of co-over-

expressed Nt-RhoGDI2, with which this form of Rac5 does

not detectably interact in two-hybrid assays (Figure 2 and

Figure S1). In the absence of Nt-RhoGDI2 over-expression,

YFP:Nt-Rac5T20N clearly displayed a much stronger overall

association with the plasma membrane than YFP:Nt-Rac5

and YFP:Nt-Rac5G15V (Figure 7 and Figure S4), indicating

that endogenous pollen tube RhoGDI activity partially

transferred YFP:Nt-Rac5 and YFP:Nt-Rac5G15V, but not

YFP:Nt-Rac5T20N, from the plasma membrane to the cyto-

plasm. Under these conditions, in contrast to YFP:Nt-Rac5

and YFP:Nt-Rac5G15V, YFP:Nt-Rac5T20N did not significantly

accumulate at the plasmamembrane in the pollen tube apex

(Figure 7, arrow head, and Figure S4), where endogenously

expressed pollen tube Rac/Rop GTPases are presumed to

be localized (Zheng and Yang, 2000). These observa-

tions suggest the exciting possibility that RhoGDI interac-

tion may promote tip localization of pollen tube Rac/Rop

GTPases.

Figure 7. Intracellular localization of transiently expressed YFP fused to wild-

type or mutant Nt-Rac5 in tobacco pollen tubes in the presence or absence of

co-over-expressed Nt-RhoGDI2.

Single confocal optical sections through the tips of weakly fluorescent pollen

tubes co-expressing wild-type or mutant YFP:Nt-Rac5 with GUS (left column)

or Nt-RhoGDI2 (right column) at low levels. Images were taken 4–6 h after

gene transfer, before significant growth depolarization was induced, and

represent at least 20 similar images collected in three independent experi-

ments. In all cases, central sections through pollen tube tips lying flat on the

cover-slip surface were imaged (scale bar: 10 lm). WT, Nt-Rac5; G15V, Nt-

Rac5G15V; T20N, Nt-Rac5T20N; R69A, Nt-Rac5R69A; arrow heads, weak associ-

ation of YFP:Nt-Rac5T20N and YFP:Nt-Rac5R69A with the plasma membrane at

the tip. Serial confocal sections through pollen tubes expressing YFP:Nt-Rac5

or YFP:Nt-Rac5T20N are shown in Figure S4.

Figure 6. Growth of tobacco pollen tubes transiently over-expressing wild-

type or mutant Nt-Rac5, in the absence or presence of co-over-expressed

Nt-RhoGDI2.

Statistical analysis of pollen tube length 7 h after gene transfer (representative

example of three independent data sets). GDI2, Nt-RhoGDI2; WT, Nt-Rac5;

G15V, Nt-Rac5G15V; T20N, Nt-Rac5T20N; R69A, Nt-Rac5R69A; error bars: 95% CI

(n ‡ 44).

1024 Ulrich Klahre et al.

ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 1018–1031

Co-over-expressed Nt-RhoGDI2 failed to effectively trans-

fer YFP:Nt-Rac5T20N to the cytoplasm, but detectably re-

duced the overall plasma membrane association of this

fusion protein and induced its partial redistribution to the

pollen tube tip (Figure 7). Residual low-affinity binding of Nt-

RhoGDI2 to Nt-Rac5T20N, which is beyond the detection limit

of two-hybrid assays (Figure 2 and Figure S1), may have

been responsible for these effects. However, it cannot be

excluded that the strong inhibition of pollen tube growth

resulting from Nt-RhoGDI2 co-over-expression indirectly

affected YFP:Nt-Rac5T20N localization in these experiments.

Nt-Rac5R69A is impaired in its ability to interact with Nt-

RhoGDI2, to accumulate at the pollen tube tip and to affect

pollen tube growth when over-expressed

To clarify the role of interactions with Nt-RhoGDI2 in the

control of the intracellular localization of Nt-Rac5, we have

replaced the arginine at position 69 of Nt-Rac5 by alanine

using site-directed mutagenesis. Corresponding mutations

have been shown to specifically disrupt the interaction of

animal Rho GTPases with RhoGDIs, without affecting their

binding to effectors or other regulators (Gibson and Wilson-

Delfosse, 2001; Lin et al., 2003). As demonstrated using

yeast two-hybrid assays, the R69A mutation abolished the

interaction of Nt-Rac5 with Nt-RhoGDI2, but did not signifi-

cantly affect binding of Nt-Rac5 to Nt-RhoGAP, Nt-Kinase1

or Nt-Ric1, three putative Nt-Rac5 regulators or effectors that

we are currently functionally characterizing (Figure S5). To-

gether with the observation that recombinant Nt-Rac5 and

Nt-Rac5R69A displayed identical in vitro GTPase activities

(Figure S5), this demonstrates that the R69A mutation dis-

rupts binding of Nt-Rac5 to Nt-RhoGDI2 without affecting

other key biochemical functions of this protein.

Interestingly, transiently over-expressed Nt-Rac5R69A

failed to induce the massive ballooning at the pollen tube

tip that is induced by over-expressed Nt-Rac5 (Figure 5a).

Nt-Rac5R69A over-expression had no effect on the length of

tobacco pollen tubes 7 h after gene transfer (Figure 6), and

generally did not affect pollen tube morphology (Figure 5a),

although some tip-swelling was occasionally observed after

prolonged culture (24 h, unpublished data). These data

strongly suggest that the activation of Nt-Rac5 in pollen

tubes requires interaction with Nt-RhoGDI2, and that Nt-

RhoGDI2 promotes rather than downregulates Rac/Rop

signaling in these cells. In contrast to NtRac5, Nt-Rac5R69A

did not prevent the inhibition of pollen tube growth resulting

from transiently co-over-expressed Nt-RhoGDI2 (Figures 5b

and 6), which confirmed that the R69A mutation disrupted

the functional interaction between Nt-Rac5 and Nt-RhoGDI2

in living pollen tubes.

Both in the presence and in the absence of co-over-

expressed Nt-RhoGDI2, the intracellular localization in pol-

len tubes of transiently expressed Nt-Rac5R69A fused to the

C-terminus of YFP was indistinguishable from that of YFP

fused to Nt-Rac5T20N (Figure 7), which does not detectably

interact with Nt-RhoGDI2 in yeast two-hybrid assays either

(Figure 2 and Figure S1). As shown in Figure 7 (left column),

YFP:Nt-Rac5R69A strongly associated with the plasma mem-

brane in the shank and in the flanks of the tip, but not in the

apex (arrow head), of normally growing pollen tubes that did

not co-over-express Nt-RhoGDI2. This strongly suggests

that Nt-RhoGDI2 interaction is required for the tip localiza-

tion of wild-type Nt-Rac5. Nt-Rac5R69A is not only mis-

localized but also fails to depolarize pollen tube growth

when over-expressed (see above), which indicates that Nt-

RhoGDI2-dependent transport of Nt-Rac5 to the tip may be

essential for activation of this protein.

As discussed above, the intracellular distributions of

YFP:Nt-Rac5T20N and YFP:Nt-Rac5R69A in pollen tubes co-

over-expressing Nt-RhoGDI2 are consistent with low-affinity

interactions between these fusion proteins and Nt-RhoGDI2,

which are not detectable in two-hybrid assays. However, it is

possible that these distributions were indirectly affected by

the strong inhibition of pollen tube growth resulting from

Nt-RhoGDI2 co-over-expression.

Discussion

Functional interactions between Nt-RhoGDI2 and Nt-Rac5

play an import role in the control of tobacco pollen tube

growth

Several lines of evidence demonstrate that Nt-Rac5 and Nt-

RhoGDI2 are specifically co-expressed in tobacco pollen

tubes. The Nt-Rac5 coding sequence was cloned by RT-PCR

using RNA isolated from tobacco pollen tubes as a template.

Colony hybridization screening of a tobacco pollen cDNA

library using the Nt-Rac5 coding sequence as a probe

resulted in the identification of two cDNA clones encoding

Nt-Rac5 and a novel, highly similar pollen tube Rac/Rop

GTPase, respectively (U. Klahre and B. Kost, unpublished

data). cDNAs encoding Nt-RhoGDI2 were identified in the

same pollen tube library using yeast-two hybrid and colony

hybridization screening. Transcripts hybridizing to the Nt-

Rac5 or the Nt-RhoGDI2 coding sequences were detectable

in flowers containing developing or mature pollen, and

accumulated to high levels specifically in pollen and in pol-

len tubes (Figure 1). Similar expression patterns are dis-

played by other genes with important functions during

pollen tube growth (e.g. Chen et al., 2002; Cheung et al.,

2002). Mature pollen grains are known to store large

amounts of transcripts of such genes to prepare for rapid

protein production upon germination (Becker et al., 2003;

Honys and Twell, 2003).

Nt-Rac5 and Nt-RhoGDI2 dramatically affected the growth

of tobacco pollen tubes when over-expressed individually,

in the case of Nt-Rac5 in a manner that is characteristic of

Nt-RhoGDI2 regulates pollen tube Rac/Rop 1025

ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 1018–1031

pollen tube Rac/Rop GTPases. In transient over-expression

experiments, pollen tube elongation was depolarized by Nt-

Rac5 or Nt-Rac5G15V, and strongly inhibited by Nt-Rac5T20N

or Nt-RhoGDI2, indicating that Nt-Rac5 and Nt-RhoGDI2

both have important functions in the control of this process.

Interestingly, the two proteins showed significant interac-

tionwith each other in yeast two-hybrid assays (Figure 2 and

Figure S1) and functionally interacted in vivo (Figures 5–7

and Figure S3). When Nt-RhoGDI2 was co-over-expressed

with wild-type Nt-Rac5, with which it interacts most strongly

(Figure 7 and Figure S1), under conditions that are expected

to result in similar expression levels of the two proteins, they

prevented each other from affecting pollen tube growth

(Figures 5 and 6 and Figure S3). Rac/Rop signaling appar-

ently remained unperturbed and pollen tubes were able to

elongate normally, conceivably because excess Rac/Rop and

RhoGDI proteins were sequestered into inactive cytoplasmic

heterodimers, without disturbing the critical equilibrium

between the activities of these proteins. A minor increase or

decrease of the ratio between Nt-Rac5 and Nt-RhoGDI2 over-

expression levels in titration experiments depolarized or

inhibited pollen tube growth, respectively, hinting at a rather

delicate nature of this equilibrium (Figure S3). When co-

expressed in normally elongating pollen tubes with Nt-

RhoGDI2, YFP:Nt-Rac5 did not detectably accumulate at the

plasma membrane in the apex (Figure 7), although the

presence of Rac/Rop GTPases at this location is thought to

be essential for pollen tube elongation (Zheng and Yang,

2000). Conceivably, excess YFP:Nt-Rac5 forming an inactive

complex with Nt-RhoGDI2 in the cytoplasm (see above) has

prevented the detection of low-level accumulation of this

fusion protein at the plasma membrane.

Together, these data establish that functional interactions

between Nt-RhoGDI2 and Nt-Rac5 play an important role in

the control of tobacco pollen tube growth. This conclusion is

strongly supported by the observation that the R69A muta-

tion, which disrupted the interaction of Nt-Rac5 with Nt-

RhoGDI2 in two-hybrid assays (Figure S5) and in co-over-

expression experiments (Figures 5–7), dramatically reduced

the ability of this protein to depolarize pollen tube growth

upon transient over-expression.

Nt-RhoGDI2 can translocate Nt-Rac5 from the pollen tube

plasma membrane to the cytoplasm

In accordance with the considerable sequence conservation

betweenwell-characterized animal or yeast RhoGDIs and Nt-

RhoGDI2, these proteins share a number of important fea-

tures. They interact with nucleotide-bound, but not with

nucleotide-free, Rho GTPases (Figure 2 and Figure S1)

(Gibson and Wilson-Delfosse, 2001; Michaelson et al., 2001;

Strassheim et al., 2000). Curiously, animal RhoGDIs have

been reported in some cases to bind with a strong prefer-

ence to GDP-bound Rho GTPases (Ueda et al., 1990),

whereas other studies have indicated that they interact

equally well with GDP- and GTP-bound forms of these pro-

teins (Nomanbhoy and Cerione, 1996). In vitro interaction

assays using nucleotide-loaded recombinant Nt-Rac5 would

be needed to firmly establish possible differences in the

affinities with which this protein in the GDP- or GTP-bound

conformations binds to Nt-RhoGDI2. Such assays could not

be performed, because they require significant amounts of

prenylated recombinant Nt-Rac5 (Nomanbhoy and Cerione,

1996), which we were unable to generate using various

methods that work for animal and yeast homologs (over-

expression in yeast, in vitro translation in reticulocyte ex-

tracts, prenylation using cellular extracts or purified gera-

nylgeranyl transferase). However, our data show that Nt-

RhoGDI2 interacts more strongly with Nt-Rac5 than with Nt-

Rac5G15V in two-hybrid assays, which is consistent with

preferential binding of Nt-RhoGDI2 to GDP-bound Nt-Rac5

(Figure S1).

The interaction of animal RhoGDIs (Gibson and Wilson-

Delfosse, 2001; Hori et al., 1991; Nomanbhoy and Cerione,

1996), as well as of Nt-RhoGDI2 (Figure S1), with their target

Rho GTPases is promoted by prenylation of the latter.

RhoGDIs, including Nt-RhoGDI2, are localized to the cyto-

plasm (Figure 4) (Koch et al., 1997; Ueda et al., 1990), can

remove target Rho GTPases from membranes and are able

to retain these proteins in the cytoplasm (Figure 7) (Isomura

et al., 1991; Michaelson et al., 2001; Read et al., 2000),

presumably by forming heterodimers with them. Consistent

with these activities and the notion that membrane localiza-

tion of Rho GTPases is considered essential for their activity

(Wennerberg and Der, 2004), over-expression of RhoGDIs

was generally found to inhibit cellular processes that

depend on Rho signaling (e.g. Li et al., 2003; Masuda et al.,

1994). The inhibition of pollen tube elongation by over-

expressed Nt-RhoGDI2 (Figure 3) represents a typical exam-

ple of this effect. Based on the observations summarized in

this paragraph, RhoGDIs are most commonly thought of as

negative regulators of Rho GTPases, which remove inactive

GDP-bound forms of these proteins from the plasma mem-

brane and maintain them in this state in the cytoplasm

(Etienne-Manneville and Hall, 2002; Olofsson, 1999; Sasaki

and Takai, 1998).

Nt-RhoGDI2 interaction is essential for the normal

localization and activity of Nt-Rac5

Recent observations have indicated that RhoGTPase signa-

ling in some cell types is promoted rather than downregu-

lated by RhoGDIs. RhoGDIs that appear to strongly interact

with GTP-bound RhoGTPases have been proposed to have a

function in translocating these proteins in the activated form

to specific membrane domains (Del Pozo et al., 2002). A

mammalian Rho GTPase carrying a mutation corresponding

to the R69A exchange in Nt-Rac5 was specifically disrupted

1026 Ulrich Klahre et al.

ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 1018–1031

in its ability to interact with RhoGDI and failed to induce the

same effects as its wild-type counterpart when over-ex-

pressed in cell cultures, indicating that the functions of this

GTPase depend at least in part on RhoGDI interaction (Lin

et al., 2003).

We have demonstrated that Nt-Rac5R69A does not signi-

ficantly interact with Nt-RhoGDI2 in yeast two-hybrid assays

(Figure S5a) or in co-over-expression experiments (Fig-

ures 5b, 6 and 7), but shows the same GTPase activity as

Nt-Rac5 (Figure S5b) and displays normal two-hybrid inter-

actions with three different putative Nt-Rac5 regulators or

effectors (Figure S5a). Interestingly, the R69A mutation

caused Nt-Rac5 to show enhanced overall membrane

association (Figure 7), to accumulate at the plasma mem-

brane in the shank and in the flanks of the pollen tube tip, but

not in the apex (Figure 7), and to be significantly compro-

mised in its capability to induce growth depolarization upon

over-expression (Figure 5a). These data strongly suggest

that interaction with Nt-RhoGDI2 is essential for the normal

localization and activity of Nt-Rac5 at the tip of tobacco

pollen tubes.

Consistent with the results summarized above andwith all

our other data, we propose that Nt-RhoGDI2 mediates

recycling of inactive Nt-Rac5 from the flanks of the tip to

the pollen tube apex, where this GTPase is activated

(Figure 8). Our data provide compelling evidence for a key

role of this mechanism in the maintenance of the spatially

restricted Rac/Rop signaling that controls polarized cell

expansion at the tip of tobacco pollen tubes. Although the

recently observed accumulation of Rac/Rop GEFs at the

pollen tube apex (Gu et al., 2006) strongly supports the

model shown in Figure 8, the proposed association of

RhoGDF and RhoGAP activities with the apex and the flanks

of the tip, respectively, remains to be demonstrated.

In accordance with a key function of GDI-mediated

recycling in the control of Rac/Rop localization and activity,

it has recently been shown that an Arabidopsis GDI homolog

(At-RhoGDI1/SCN1, At3g07880) is essential for the spatial

restriction of Rac/Rop-dependent root hair growth (Carol

et al., 2005), a process that is closely related to pollen tube

elongation (Hepler et al., 2001). From individual root epider-

mal cells of mutant plants disrupted in the At-RhoGDI1/scn1

gene, instead of a single highly elongated unbranched root

hair, several short root hairs emerge, which often split at the

tip to form multiple growth sites. As may be expected from

the disruption of a GDI-dependent Rac/Rop recycling mech-

anism similar to that illustrated in Figure 8, enhanced and

delocalized accumulation of a Rac/RopGTPase at the plasma

membrane is associated with the cell expansion defects in

the root epidermis of At-RhoGDI1/scn1 mutants.

RhoGDI-mediated recycling of Rho GTPases may specif-

ically be required in elongating pollen tubes and root hairs,

in which massive apical secretion is expected to cause a

constant retrograde flow of plasma membrane material

away from the tip. This assumption is consistent with the

observation that pollen tube RhoGDIs are highly conserved

among each other, but diverge more significantly from

sporophytic and non-plant isoforms (Figure S2). However,

accumulation of activated Rho GTPases in specific plasma

membrane domains plays a crucial role in polarizing not

only pollen tubes but also yeast and animal cells (Etienne-

Manneville and Hall, 2002). It is possible that Rho GTPase

recycling by RhoGDIs is also involved in the polarization of

Rho signaling in these cells, a process that is not well

understood to date (Etienne-Manneville, 2004).

Experimental procedures

Plant material

As a source of pollen, tobacco (N. tabacum cv. Petit Havana SR1)plants were grown from seeds at monthly intervals and maintainedin a greenhouse.

cDNA library construction and cloning by RT-PCR

Total RNA was isolated using repeated phenol–chloroform extrac-tion and selective lithium chloride precipitation based on a pub-lished protocol (Soni and Murray, 1994) from tobacco pollen tubesthat were grown in liquid culture medium (Read et al., 1993) for 3 h,collected by centrifugation (700 g) and washed with buffer (0.4 M

mannitol, 50 mM Tris–Cl pH 6.0). Freezing or grinding were notrequired for RNA extraction. PolyAþ RNA was prepared byoligo(dT)-cellulose (#27-5543-0227; Amersham Biosciences, Piscat-away, NJ, USA) affinity purification. cDNA generated from 5 lgpolyAþ RNA using a cDNA synthesis kit (#200400; Stratagene, LaJolla, CA, USA) was cloned into the yeast two-hybrid prey vectorpGAD-GH (BD Biosciences-Clontech, Mountain View, CA, USA)restricted with EcoRI and XhoI. The resulting primary cDNA libraryconsisted of 1.3 · 106 Escherichia coli clones, of which more than90% contained plasmids with cDNA inserts that were in roughlyone-third of the cases larger than 1 kb. The primary library wasamplified for 2 h in liquid LB medium before plasmid purificationusing a large-scale kit (#12181; Qiagen, Valencia, CA, USA).

Figure 8. Nt-RhoGDI2-mediated recycling is required for normal Nt-Rac5

localization and activity in tobacco pollen tubes.

Nt-RhoGDI2 is proposed to transfer inactive GDP-bound Nt-Rac5 from the

plasma membrane at the flanks of the pollen tube tip to the cytoplasm, and to

be essential for the transport of this GTPase back to the apex, where it is re-

inserted into the plasma membrane and turned into the GTP-bound active

form by nucleotide exchange. Asterisks indicate hypothetical activities whose

association with the pollen tube plasma membrane at the indicated locations

remains to be demonstrated.

Nt-RhoGDI2 regulates pollen tube Rac/Rop 1027

ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 1018–1031

A cDNA corresponding to the coding sequence of Nt-Rac5 wasamplified from total pollen tube RNA prepared as described aboveusing a one-step reverse transcription PCR kit (#210210; Qiagen).The nucleotide sequence of the amplification product was identicalto the Nt-Rac5 sequence reported in the literature (Kieffer et al.,2000).

cDNA isolation by colony hybridization

Colony hybridization was performed using DIG (digoxigenin)-labe-led probes as described in the DIG user manual supplied by RocheInc. (Basel, Switzerland). Approximately 100 000 colonies of thecDNA library described above were screened on large agar platesand re-screened using this procedure. Membranes were hybridizedas described below for RNA blots.

Recombinant DNA construction

Site-directed mutagenesis of the Nt-Rac5 coding sequence wasperformed using a PCR-based mutagenesis kit (#200519 quick-change; Stratagene), or regular PCR with primers carrying mis-matches. Mutagenized fragments were sub-cloned into expressionvectors, and sequenced to confirm the absence of PCR errors as wellas the presence of introduced mutations.

Standard recombinantDNAmethodology (Sambrook andRussell,2001) was employed to clone coding regions of cDNAs encodingwild-type or mutant Nt-Rac5, or Nt-RhoGDI2, into the multiplecloning sites (MCS) of various expression vectors: (i) a pUCAP-basedvector (van Engelen et al., 1995; Kost et al., 1998) containing anMCSbetweenaLat52promoter (Twellet al., 1991) andanospolyAþ signalfor transient expression in pollen tubes, (ii) the same vectorcontaining, before or after a modified MCS, a sequence encodingEYFP (BD Biosciences-Clontech) fused at the C- or the N-terminus,respectively, to a 5 · Gly–Ala linker for transient expression of YFPfusion proteins in pollen tubes, (iii) pGBK-T7 (BD Biosciences-Clontech) to express the DNA binding domain of the GAL4 transcrip-tion factor fused todifferent formsofNt-Rac5 in yeast cells as abait intwo-hybrid screens and assays, (iv) pGAD-GH (BD Biosciences-Clontech) to express the activation domain of the GAL4 transcriptionfactor fused to Nt-RhoGDI2 as prey in yeast for two-hybrid assays,and (v) pGEX-4T (Amersham Biosciences) to express GST (glutathi-one S-transferase) fusion proteins in E. coli for purification. Expres-sion constructs containing the coding sequences of EYFP, or b-glucuronidase (Kost et al., 1999), between the Lat52 promoter andthe nos polyAþ signal were also generated based on the pUCAP-derived vector described above for transient expression in pollentubes. PCR-amplified fragments introduced into expression vectors,as well as junctions between sequences linked to express transla-tional fusions, were sequenced to confirm the absence of PCR errorsand the generation of in-frame fusions, respectively.

RNA isolation and blotting

RNA employed in blotting experiments was isolated using Trizolaccording to the manufacturer’s recommendations (Invitrogen,Carlsbad, CA, USA). RNA blotting was essentially performed asdescribed by Sambrook and Russell (2001). Aliquots of 5–10 lg RNAper lane were loaded on a denaturing agarose gel (1.5% agarose,20 mM MOPS, 10 mM Na-acetate, 1 mM EDTA, pH 7), electro-phoresed in MOPS buffer and blotted onto Duralon-UV membranes(Stratagene). RNA was cross-linked to membranes using a Strata-linker (Stratagene). Probes were prepared using DIG-nucleotides

according to themanufacturer’s method (Roche Inc.) and hybridizedin 47% formamide, 540 mM NaCl, 30 mM Na-phosphate, 3 mM

EDTA, 1% SDS, 0.005% each of BSA, Ficoll and polyvinyl-pyrroli-done at 42�C overnight. Membranes were rinsed once in 2· SSC atroom temperature followed by three washes in 0.1% SDS, 0.1· SSCat 55�C. DIG-labeled probes were detected according to the manu-facturer’s recommendation using CDP-star as a substrate for AP-linked anti-DIG antibodies (Roche Inc.). Blots were exposed toHyperfilm ECL (Amersham Biosciences).

Yeast two-hybrid screen and assays

Yeast two-hybrid screening was performed using materials andprotocols provided by the ‘Matchmaker’ GAL4 system (manualPT3061-1; BD Biosciences-Clontech). Saccharomyces cerevisiaeHF7c cells containing intact bait constructs (Nt-Rac5G15V/C194S codingsequence in pGBK-T7, see above), as verified by restriction analysisand partial sequencing of plasmid isolated from these cells, weretransformed using a large-scale lithium acetate method with 200 lgplasmid purified from a tobacco pollen tube cDNA library (cDNAinserts in pGAD-GH; prey constructs, see above). Transformed cellswere plated on medium lacking histidine (#4027-012 and #4530-122;MP Biomedicals, Irvine, CA, USA) to screen for two-hybrid inter-actions. Simultaneous plating of a small aliquot of transformed cellsonmediumsupplementedwith histidine (#24842; Serva,Heidelberg,Germany) indicated the total number of co-transformants containingbait and prey constructs screened. Prey constructs were purifiedfrom yeast colonies appearing on histidine-free medium 3–14 daysafter gene transfer and amplified in E. coli. Specific interactionsbetween Nt-Rac5 and polypeptides encoded by cDNA inserts inpurified prey constructs were verified using two-hybrid assays.

To perform yeast two-hybrid assays, prey constructs (pGAD-GHwith cDNA inserts) were simultaneously co-transformed into HF7ccells with different bait constructs (pGBK-T7 containing cDNAsencoding wild-type or mutant Nt-Rac5) using a small-scale lithiumacetate method (manual PT3061-1; BD Biosciences-Clontech). Allbait and prey constructs were also co-transformed with emptypGAD-GH and pGBK-T7, respectively, to generate negative controlsamples. Equal volumes of each batch of co-transformed cells wereplated on histidine-containing medium to determine co-transfor-mation efficiencies, and on histidine-free medium supplementedwith 0, 1 or 2 mM 3-AT (3-amino-1,2,4-triacole; #A-8056, Sigma, StLouis, MO, USA) to detect two-hybrid interactions. If co-transfor-mation of all samples was successful (several hundred yeastcolonies visible on histidine-containing medium 3 days after genetransfer), specific two-hybrid interactions were demonstrated bygrowth on histidine-free medium of HF7c cells transformed withbait and prey constructs, and by the absence of growth on thismedium of HF7c cells containing only bait or prey constructs alongwith empty pGAD-GH or pGBK-T7, respectively. To obtain the datashown in Figure 2 and Figures S1 and S5, HF7c co-transformantsgrowing on histidine-containing medium (several colonies fromeach plate) were transferred to 5 ml liquid histidine-containingmedium and cultured. After 48 h, 10 ll aliquots of each culture wereplated onmedia with and without histidine, the latter supplementedwith 0, 1 or 2 mM 3-AT. Plates were incubated for 3 days beforephotographs were taken.

Transient gene expression

Expression vectors were transferred into tobacco pollen grainsgerminating on solid culture medium (Read et al., 1993) by particlebombardment using a helium-driven particle accelerator (PDS-

1028 Ulrich Klahre et al.

ª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), 46, 1018–1031

1000/He; Bio-Rad, Hercules, CA, USA) as previously described (Kostet al., 1998). When two or three plasmids were co-transformed,respectively, particles were coated with 5 lg (2.5 lg of each plas-mid) or 6 lg (2 lg of each plasmid) plasmid DNA (unless statedotherwise: see titration experiments). All expression vectors usedranged in size between 4.3 and 5.3 kb.

Microscopy and image analysis

At the indicated times after gene transfer, transiently transformedfluorescent pollen tubes were transferred as previously described(Kost et al., 1998) onto cover slips for microscopic analysis. Epi-fluorescence and transmitted light images were recorded using aninvertedmicroscope (DM IRB; Leica, Bensheim, Germany) equippedwith DIC (differential interference contrast) optics, a 100 W mercurylamp, an FITC filter block (excitation: 450–490 nm, dichroic: 510 nm,emission: 515 long pass; I3 S, Leica), 5· and 40· lenses (N PLAN 5·/0.12 andHCXPL FL L 40 ·/0.6, Leica), and a digital camera (DFC350FXR2, Leica). Unless stated otherwise, epi-fluorescence images shownand/or employed to analyze pollen tube length (using publiclyavailable image analysis software: IMAGEJ; http://rsb.info.nih.gov/ij/)were taken with exposure times shorter than 500 msec. A laserscanning microscope (#1220004 LSM510Meta; Zeiss, Jena,Germany) and a 100 ·/1,45 NA oil immersion lens (#1084514; Zeiss)were employed for confocal analysis. YFP fluorescence excited withthe 514 nm line of an argon laser was imaged through a 405/514 nmdichroic mirror and a 530–600 nm band pass emission filter. Epi-fluorescence and confocal images were contrast-enhanced byadjusting brightness and gamma settings using image-processingsoftware (PHOTOSHOP; Adobe Systems Inc., San Jose, CA, USA).

GTPase assays

Recombinant wild-type andmutant versions of Nt-Rac5 fused to theC-terminus of GST were purified from E. coli BL-21 using standardprocedures (Sambrook and Russell, 2001) and assayed for GTPaseactivity essentially as described previously (Self and Hall, 1995). Inbrief, subsequent to preloading recombinant fusion proteins with[c-32P]GTP (Hartmann Analytic, Braunschweig, Germany), radioac-tivity remaining associated with these proteins was measured afterdifferent periods of incubation in assay buffer.

Cell fractionation and immunoblotting

Preparation and fractionation of pollen tube extracts was performedaccording to the method described by Potocky et al. (2003) withminor modifications. Pollen of 50 flowers was transferred to 10 mlculture medium (Read et al., 1993). After 5 h, pollen tubes werecollected by vacuum filtration, ground in liquid nitrogen and re-suspended in 600 ll homogenization buffer (0.25 M sucrose, 3 mM

EDTA, 5 mM DTT, 70 mM Tris–Mes pH 8.0) containing proteaseinhibitors (Serva). Extracts were centrifuged sequentially at 3000 g

for 5 min, at 10 000 g for 5 min, and at 100 000 g for 60 min toseparate cytoplasmic and various membrane fractions. Proteinconcentrations were determinedwith Bradford solution (Bio-Rad) toensure equal loading. Blots were probed with an antibody gener-ated against a GST:Nt-RhoGDI2 fusion protein.

Acknowledgements

The authors would like to thank Katja Piiper for excellent technicalsupport. Funding was received from the German Research Council

(DFG; KO 2278) and the state of Baden-Wurttemberg (Fors-chungsschwerpunktprogramm).

Supplementary Material

The following supplementary material is available for this articleonline:Figure S1. Estimation of the strength of yeast two-hybrid interac-tions between Nt-RhoGDI2 and different forms of prenylated andunprenylated Nt-Rac5.Figure S2. Alignment of the amino acid sequences of Nt-RhoGDI2and related RhoGDIs.Figure S3. Effects of transient co-over-expression of Nt-Rac5 and Nt-RhoGDI2 at different ratios on tobacco pollen tube growth.Figure S4. Analysis of the intracellular localization of transientlyexpressed YFP:Nt-Rac5 and YFP-NtRac5T20N in tobacco pollen tubesby serial confocal sectioning.Figure S5. Analysis of yeast two-hybrid interaction with Nt-RhoGDI2and GTPase activity of Nt-Rac5R69A.This material is available as part of the online article from http://www.blackwell-synergy.com

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