A Bacterial Effector Acts as a Plant Transcription Factor and Induces a Cell Size Regulator

See allHide authors and affiliations

Science  26 Oct 2007:
Vol. 318, Issue 5850, pp. 648-651
DOI: 10.1126/science.1144956


Pathogenicity of many Gram-negative bacteria relies on the injection of effector proteins by type III secretion into eukaryotic cells, where they modulate host signaling pathways to the pathogen's benefit. One such effector protein injected by Xanthomonas into plants is AvrBs3, which localizes to the plant cell nucleus and causes hypertrophy of plant mesophyll cells. We show that AvrBs3 induces the expression of a master regulator of cell size, upa20, which encodes a transcription factor containing a basic helix-loop-helix domain. AvrBs3 binds to a conserved element in the upa20 promoter via its central repeat region and induces gene expression through its activation domain. Thus, AvrBs3 and likely other members of this family provoke developmental reprogramming of host cells by mimicking eukaryotic transcription factors.

Gram-negative phytopathogenic bacteria of the genus Xanthomonas cause a broad variety of diseases in crop plants (1). Pathogenicity depends on the translocation of effector proteins directly into the plant cell cytosol by a type III secretion (T3S) system (2). The AvrBs3 family is a prominent effector class in Xanthomonas spp. (3), comprising major virulence determinants (46). These effectors are characterized by a central repeat region, nuclear localization signals (NLSs), and an acidic transcriptional activation domain (AD) (3). AvrBs3 was isolated from X. campestris pv. vesicatoria (Xcv), the causal agent of bacterial spot disease on pepper and tomato (7). In susceptible host plants and other solanaceous species, AvrBs3 elicits hypertrophy (i.e., enlargement) of mesophyll cells (8) and also contributes to the dispersal of Xcv between pepper plants under field conditions (9). Cell enlargement is also found in other complex disease phenotypes [e.g., citrus canker elicited by the AvrBs3-like effector PthA from X. axonopodis pv. citri (10) and Pantoea agglomerans–induced gall formation (11)].

AvrBs3 has a central region consisting of 17.5 nearly identical 34–amino acid repeats [which determine the specificity of protein activity in plants (8, 12)], functional NLSs, and an AD in its C terminus (1315)—all of which are essential for AvrBs3 activity. Furthermore, AvrBs3 forms homodimers (16) and appears to be a transcription factor localized to the plant cell nucleus (8, 1315). Pepper upa (upregulated by AvrBs3) genes that are induced by AvrBs3 encode, amongst others, putative α-expansins and auxin-induced proteins that might be involved in the induction of hypertrophy (8). However, most upa genes are indirect targets of AvrBs3 because their induction requires de novo synthesis of plant proteins (8).

To isolate upa genes that are direct targets of AvrBs3, we infected susceptible pepper plants [cultivar Early Calwonder (ECW)] with Xcv strain 85-10 expressing avrBs3 or carrying an empty vector in the presence of cycloheximide, which blocks eukaryotic protein synthesis. cDNA fragments corresponding to AvrBs3-induced pepper genes were identified by suppression-subtractive hybridization and confirmed by reverse Northern analysis and reverse transcription polymerase chain reaction (RT-PCR). Among these cDNAs, we detected the previously identified upa10 and upa11 genes (8), validating our approach, as well as previously unrecognized AvrBs3-induced genes. One gene, designated upa20, encodes a putative transcription factor of the basic helix-loop-helix (bHLH) family and was chosen for further analysis.

We isolated the full-length cDNA of upa20 by rapid amplification of cDNA ends (RACE)–PCR and determined the corresponding genomic sequence from a pepper BAC (bacterial artificial chromosome) library (17). upa20 has a complex gene structure comprising eight exons and seven introns (Fig. 1A) and encodes a putative 340–amino acid bHLH transcription factor with a predicted molecular mass of 37.8 kD. The bHLH domain, which generally serves as DNA binding and dimerization domain (18), is located in the region from amino acids 167 to 225 (Fig. 1B). BLASTP analyses show that Upa20 is most related to an uncharacterized protein from rice (Os09g0510500; accession BAF25548.1; 48% identity and 63% similarity over 240 amino acids) and is also similar to BIGPETALp (BPEp) from Arabidopsis (accession CAK32499.1; 55% identity and 70% similarity over 134 amino acids), which is involved in the control of petal size (19).

Fig. 1.

(A) Structure of upa20 in pepper. Coding and noncoding exons are indicated as black and white rectangles, and introns are indicated as black lines between the exons. (B) Amino acid sequence (30) of the Upa20 bHLH region (amino acids 167 to 225). The amino acids that define the bHLH motif (18) are color-coded (basic region, blue; first helix, yellow; loop, pink; and second helix, green). Conserved amino acids were changed to alanine in the basic region (U20B) and in the HLH dimerization motif (U20H). (C and D) Confocal laser scanning microscopy of N. benthamiana 2 days after Agrobacterium-mediated transfer of upa20::gfp (C) or gfp (D). 4′, 6′-Diamidino-2-phenylindole (DAPI) staining indicates nuclei. Scale bars, 20 μm. (E) N. benthamiana transiently expressing gfp (1), upa20::c-myc (2), U20B::c-myc (3), and U20H::c-myc (4), 8 days post-infiltration (dpi). (F) Upa20 induces the expression of upa7, encoding a putative α-expansin. RT-PCR analysis of susceptible pepper plants 1 and 2 dpi of Agrobacterium delivering avrBs3, gfp, upa20, U20B, and U20H, respectively, is shown. EF1α was used as control for equal cDNA amounts.

To address the role of upa20 in the induction of hypertrophy, we performed virus-induced gene silencing of upa20 in Nicotiana benthamiana, which severely reduced the normally strong AvrBs3-dependent hypertrophy, the result of which was visible as pustules on the lower leaf surface (Fig. 2, A and B). Agrobacterium-mediated upa20 expression demonstrated that Upa20 alone induces hypertrophy in N. benthamiana and other solanaceous plants (Fig. 2C and fig. S1). In upa20-expressing tissue of N. benthamiana, palisade and spongy parenchyma cells were strongly enlarged as compared with those in tissue of the control (Fig. 2, D and F). The hypertrophy reaction was phenotypically similar to tissue transiently expressing avrBs3 (Fig. 2E) but was faster and stronger. Further observations revealed cell wall invaginations exclusively in upa20-expressing tissue, suggesting that cell wall synthesis is increased (Fig. 2, G and H). In addition, chloroplasts of upa20-expressing cells showed a decrease in starch content (Fig. 2, I and J) in accordance with cell enlargement being a highly energy-consuming process. Therefore, our results suggest that Upa20 is a master regulator of cell enlargement that stimulates cell growth, in contrast to BPEp from Arabidopsis, which reduces cell size (19). Consistent with its predicted function as a transcription factor, N- and C-terminal green fluorescent protein (GFP) fusions of Upa20 were exclusively localized to the nucleus, where they often concentrated in distinct foci (Fig. 1C and fig. S2). These foci are reminiscent of nuclear speckles, which may be storage and assembly areas of transcription and splicing factors, supplying them to active transcription sites (20). When the basic region (U20B) and the dimerization domain (U20H) of Upa20 were altered (Fig. 1B), both mutants failed to cause hypertrophy in N. benthamiana (Fig. 1E), although all proteins were expressed in similar amounts (fig. S3).

Fig. 2.

(A and B) Transient expression of avrBs3 elicits a hypertrophy in gfp-silenced (A), but not in upa20-silenced (B), N. benthamiana leaves, 11 dpi. (C) Agrobacterium-mediated expression of avrBs3 (1) and upa20 (2) causes hypertrophy in N. benthamiana 7 dpi relative to control [empty transferred DNA (T-DNA) (3)]. (D to F) Light microscopy of N. benthamiana leaves 4 days after Agrobacterium-mediated delivery of empty T-DNA (D), avrBs3 (E), and upa20 (F). (G) Higher magnification of a sector in (F). Arrows indicate cell wall invaginations. (H) Electron micrograph of a cell wall invagination of an N. benthamiana palisade cell expressing upa20, 4 dpi. Asterisks in (G) and (H) mark apoplastic spaces. (I and J) Chloroplasts of N. benthamiana cells 3 dpi with Agrobacterium delivering an empty T-DNA (I) or upa20 (J). Scale bars correspond to 100 μm [(D) to (F)], 30 μm(G), and 2 μm [(H) to (J)], respectively. Ch, chloroplast; CW, cell wall; V, vacuole.

Together, these results suggest that Upa20 is a bona fide bHLH transcription factor. We wondered whether any of the previously identified upa genes are target genes of Upa20. Induction of upa1 to upa9, expression of which requires de novo protein synthesis, starts 6 to 9 hours after infection of pepper plants with Xcv delivering AvrBs3 (8), whereas upa20 induction starts within 3 to 4 hours post-infection (hpi) (fig. S4). RT-PCR was performed with RNA from susceptible pepper plants transiently expressing upa20 and the upa20 bHLH mutants along with controls. Upa20 activated the expression of upa7, encoding a putative α-expansin, which was shown to be dependent on the bHLH domain of Upa20 (Fig. 1F). However, the other genes tested were not consistently induced by Upa20, suggesting that AvrBs3 activates additional genetic pathways.

Our results indicate that the visible consequence of AvrBs3 virulence activity in susceptible plants, the induction of cell hypertrophy, is caused by AvrBs3 targeting upa20, a regulator of cell enlargement. To elucidate the mechanism of upa20 induction, we tested for the requirement of the eukaryotic motifs in AvrBs3. Pepper plants infected with Xcv expressing avrBs3 and mutant derivatives clearly showed that both the NLSs and AD are required for gene activation (fig. S5). In addition, deletion of four repeats abolished upa20 induction (fig. S5), demonstrating that the central repeat region of AvrBs3 is indispensable for function. Similarly, the AvrBs3 homolog AvrBs4, which differs from AvrBs3 mainly in the repeat region (21), does not enhance upa20 expression, which is in agreement with the finding that AvrBs3 deletion derivatives and AvrBs4 do not induce a hypertrophy (8).

To identify an AvrBs3-responsive DNA element, we isolated the promoter and determined the transcription start site of upa20. Comparison of cDNA sequences revealed that, in the presence of AvrBs3, transcription of upa20 starts 37 base pairs (bp) downstream of the prevalent start site used for the basal level of upa20 transcription under non-inducing conditions (fig. S6). Upstream of both transcription start sites, a TATA box motif is located (fig. S6). These data suggest that upa20 undergoes alternative transcriptional initiation, which has been reported for a wide range of eukaryotic genes (22, 23) and is probably mediated by the action of different transcription factors.

To analyze the AvrBs3 inducibility of the upa20 promoter, we used a promoterless egfp::uidA cassette as a reporter in N. benthamiana after Agrobacterium-mediated transformation. β-Glucuronidase (GUS) activities and GFP fluorescence revealed that the upa20 promoter is AvrBs3-responsive (Fig. 3B and fig. S7). Promoter deletions from both ends limited the AvrBs3-responsive region to a 55-bp sequence (Fig. 3, A and B). We searched for sequence similarities in the promoters of other putative direct targets of AvrBs3, concentrating on upa10 (8). The upa10 promoter was isolated from pepper ECW by genome walking and revealed a motifin common with the upa20 promoter that we termed the upa box (Fig. 3C). This motif contains a TATA box followed by two stretches of cytosine residues, separated by two and three other nucleotides, respectively. We exchanged all cytosines (Cs) of the motif with guanines (Gs) (asterisks in Fig. 3C; this mutant was designated ubm1), which abolished the induction of the reporter gene by AvrBs3 without affecting the basal activity of the upa20 promoter. Notably, the upa box is also present in the promoter of the Bs3 resistance gene, whose activation by AvrBs3 leads to the induction of cell death in the resistant pepper line ECW-30R (24). In susceptible ECW plants, a 13-bp insertion between the two C stretches in the upa box also abolishes gene induction (24).

Fig. 3.

(A) Promoter fragments tested for AvrBs3 inducibility. The transcriptional start sites, in the presence of AvrBs3 (+1) and predominantly used in the absence of AvrBs3 (37 bp upstream of +1), are indicated with solid vertical lines. The transcription start of F0R2 was mapped to the attB2 site of the construct. Dashed lines and numbers above indicate positions with respect to +1. (B) GUS assays show AvrBs3 inducibility of the upa20 promoter in N. benthamiana. Promoter fragments shown in (A) and a upa box mutant derivative of F0R0, F0R0ubm1 [see (C)], were cloned in front of a egfp::uidA reporter and tested by codelivery with avrBs3 and empty T-DNA. 4-MU, 4-methylumbelliferone. Error bars indicate SD. (C) The smallest AvrBs3-responsive upa20 promoter fragment (F2R2) (top row) as compared with a corresponding region in the upa10 promoter. Numbers refer to the transcriptional start site (+1) in the presence of AvrBs3. Nucleotides that belong to the proposed upa box are indicated in italics. Asterisks mark nucleotides that were changed to G in mutant ubm1.

We performed chromatin immunoprecipitation (ChIP) with an AvrBs3-specific antibody (25) to determine whether AvrBs3 associates with the upa20 promoter in planta. Chromatin was isolated from pepper ECW infiltrated with Xcv strain 82-8 naturally expressing avrBs3 and a T3S mutant derivative (82-8ΔhrcV) that expresses avrBs3 but is not able to translocate effector proteins into the plant cell (26). AvrBs3 precipitated with the upa20 promoter containing the upa box but not with a sequence located 2 kb downstream of the upa box (Fig. 4, A and B). This demonstrates that DNA binding is specific, albeit it is not clear whether AvrBs3 binding is mediated by a plant protein.

Fig. 4.

(A) Fragments in the pepper upa20 region analyzed by ChIP. (B) ChIP analysis of pepper ECW 12 hpi with Xcv strain 82-2 (+T3SS) and the T3S system mutant 82-8ΔhrcV (–T3SS). Fragments surrounding [(1); 197 bp] and downstream of the upa box [(2); 209 bp] [see (A)] were amplified with cycle numbers as indicated. (C) EMSA with increasing molar amounts of GST::AvrBs3 incubated with 50 fmol biotin-labeled 36-bp upa20 promoter fragments containing the upa box. The control is GST. Unlabeled WT fragments and fragments with a mutated upa box (ubm1, see Fig. 3C) were used as competitor DNA. (D) EMSA of 50 fmol biotin-labeled WT promoter fragment incubated with increasing amounts of GST:: AvrBs3ΔN+C (AvrBs3 repeat region) and GST::AvrBs3Δrepeats. Unlabeled WT DNA fragments were used for competition. Arrows, bound probe; asterisks, free probe. The uppermost signals correspond to DNA in the slots. For expression of the GST fusion proteins, see fig. S9.

Although AvrBs3 does not contain a classical DNA binding domain (3), we tested whether it directly interacts with the upa20 promoter DNA in electrophoretic mobility shift assays (EMSAs) with a 36-bp biotin-labeled upa20 promoter fragment containing the upa box. Addition of GST::AvrBs3 identified a protein-DNA complex with levels that increased as increasing amounts of fusion protein were added, whereas glutathione S-transferase (GST) alone failed to bind DNA (Fig. 4C). The band shift was competed when unlabeled upa box DNA was added, whereas the ubm1 fragment (Fig. 3C) competed in binding to a much lesser extent (Fig. 4C), demonstrating that AvrBs3 is specifically binding to the upa box. In addition, EMSA with a biotin-labeled ubm1 fragment confirmed a weaker binding of AvrBs3 to the mutant as compared with that to the wild-type (WT) sequence (fig. S8A), and this binding was competed more efficiently by the unlabeled WT sequence than by the unlabeled ubm1 fragment (fig. S8B).

An AvrBs3 derivative consisting of only the repeat region bound the upa20 promoter fragment in EMSA, albeit less efficiently than the WT protein. In contrast, a protein containing the N and C termini of AvrBs3 but lacking the repeats did not bind (Fig. 4D). These data suggest that the 17.5 repeats in the central region of AvrBs3 mediate the specific interaction with DNA, which is consistent with the fact that the repeat region determines the specificity of AvrBs3 activity (8, 12), including upa20 induction (fig. S5). The AvrBs3 family member AvrXa7 shows an in vitro DNA binding activity with a preference for adenine-thymine (AT)–rich DNA (27). Our finding (that C-to-G mutations without change in AT content affected both binding of AvrBs3 in vitro and activation of the upa20 promoter in planta) demonstrates that specificity, not base composition, determines binding.

Previously, induction of host genes has been reported for the AvrBs3-like effectors PthXo1 and AvrXa27 from the rice pathogen X. oryzae pv. oryzae. Both proteins differentially induce rice genes on the basis of their promoter variations (28, 29). Sequence polymorphisms are located within 80 bp upstream of the transcription start sites (28, 29) (i.e., in a similar position relative to the upa box in AvrBs3-responsive promoters). However, the upa box is not found in the rice gene promoters, suggesting that AvrBs3-like proteins have different DNA binding specificities, which are mediated by the corresponding repeat regions. The nearly identical repeats of AvrBs3-like effectors usually differ at amino acid positions 12 and 13 (3); hence, these amino acids might be involved in the specific interaction with DNA. How the highly similar effector proteins confer different DNA binding specificities is enigmatic and awaits the structural analysis of AvrBs3 and its homologs. In light of our data, we propose that the molecular principle of AvrBs3 action may be common to other members of this large and important family of type III effectors in bacterial plant pathogens.

Supporting Online Material

Materials and Methods

Figs. S1 to S9

Table S1

References and Notes

References and Notes

View Abstract

Stay Connected to Science

Navigate This Article