How the bacterial plant pathogen Xanthomonas campestris pv

contains eukaryotic signatures such as nuclear localization signals. (NLS), and has ... of an N-terminal signal peptide (for review see Hueck, 1998). Based on an ...
Télécharger le PDF
196KB taille 2 téléchargements 154 vues
commentaire
Report
mpp010.fm Page 73 Thursday, February 10, 2000 2:43 PM

MOLECULAR PLANT PATHOLOGY (2000) 1(1), 73–76

How the bacterial plant pathogen Xanthomonas campestris pv. vesicatoria conquers the host Blackwell Science, Ltd

U L L A B O N A S , G U I D O VA N D E N A C KE R V E KE N 1 , D A N I E L A B Ü T T N E R , K A R O L I N E H A H N , ERIC MAROIS, DIRK NENNSTIEL, LAURENT NOEL, OMBELINE ROSSIER AND BORIS SZUREK Institut für Genetik, Martin-Luther-Universität, 06099 Halle, Germany

SUMMARY Xanthomonas campestris pv. vesicatoria (Xcv) is the causal agent of bacterial spot disease on pepper and tomato. Pathogenicity on susceptible plants and the induction of the hypersensitive reaction (HR) on resistant plants requires a number of genes, designated hrp, most of which are clustered in a 23-kb chromosomal region. Nine hrp genes encode components of a type III protein secretion apparatus that is conserved in Gramnegative plant and animal pathogenic bacteria. We have recently demonstrated that Xcv secretes proteins into the culture medium in a hrp-dependent manner. Substrates of the Hrp secretion machinery are pathogenicity factors and avirulence proteins, e.g. AvrBs3. The AvrBs3 protein governs recognition, i.e. HR induction, when bacteria infect pepper plants carrying the corresponding resistance gene Bs3. Intriguingly, the AvrBs3 protein contains eukaryotic signatures such as nuclear localization signals (NLS), and has been shown to act inside the plant cell. We postulate that AvrBs3 is transferred into the plant cell via the Hrp type III pathway and that recognition of AvrBs3 takes place in the plant cell nucleus.

I N T RO D U C T I O N The Gram-negative bacterium Xanthomonas campestris pv. vesicatoria (Xcv ) is the causal agent of bacterial spot of pepper and tomato. This disease occurs in many countries throughout the world and is of great economic importance in regions with a warm and humid climate (Jones et al., 1998). In natural infections, the bacteria enter the plant through stomata or wounds to reach the intercellular spaces of the tissue where they establish an intimate relationship with the plant cell. Xcv remains local*Correspondence: E-mail: [email protected] Present address: Department of Molecular Cell Biology, Utrecht University, Utrecht, the Netherlands.

1

© 2000 BLACKWELL SCIENCE LTD

ized in the intercellular spaces and does not invade the xylem. The outcome of the interaction between Xcv and the plant is determined by genes in both the pathogen and the host. In a susceptible plant, infection with a virulent strain of Xcv leads to bacterial multiplication, giving rise to small water-soaked lesions that later on become necrotic (compatible interaction). In the presence of a resistance gene (e.g. Bs3, Bs for bacterial spot resistance) in the plant and a matching avirulence gene (e.g. avrBs3) in the bacterium, a hypersensitive reaction (HR) is induced (incompatible interaction) (Minsavage et al., 1990). The HR is a rapid and highly localized cell death reaction which coincides with the arrest of bacterial growth. In an attempt to elucidate the genetic and biochemical basis of compatible and incompatible interactions, we focus on the analysis of early events: what does the bacterium need for pathogenicity, and how does the plant recognize avirulent bacteria? Two types of bacterial genes, hrp and avr, have been identified genetically. The hrp (hypersensitive reaction and pathogenicity; Bonas et al., 1991) genes are essential for any obvious interaction with the plant, i.e. for pathogenicity on susceptible plants and the induction of the HR on resistant plants. hrp mutants are not only unable to establish a ‘normal’ interaction with the plant, but they are also unable to grow in the plant tissue (see Alfano and Collmer, 1997; for review). Recognition of the pathogen by resistant host plants requires functional hrp genes and is determined by a pair of corresponding genes, one for avirulence (avr ) in the bacterium and the other for resistance (R ) in the plant. For example, Xcv expressing the avrBs3 gene specifically induces the HR in Bs3 pepper plants (Bonas et al., 1989). avrBs3 is a member of a large family of highly homologous avr/pth (pathogenicity) genes in Xanthomonas. Proteins of the AvrBs3 family display interesting features, the most striking being the presence of more than 10 nearly identical tandem repeats of 34 amino acids each in the middle of the protein. The AvrBs3 protein (122 kDa) contains 17.5 repeats which were shown to determine recognition specificity (Fig. 1; Herbers et al., 1992).

73

mpp010.fm Page 74 Thursday, February 10, 2000 2:43 PM

74

U. BONAS et al.

Fig. 1 Schematic structure of the AvrBs3 protein from Xcv. The 1164-amino acid protein consists of three main domains. The central domain contains 17.5 nearly identical 34-amino acid repeats, which determine recognition specificity by resistant plants. The C-terminal part contains two functional nuclear localization signals (NLS).

h r p G E N E S E N C O D E A T Y P E I I I P R OT E I N S E C R E T I O N S YS T E M Most hrp genes in Xcv are clustered in a 23-kb chromosomal region, and are organized in six operons, hrpA to hrpF (Fig. 2; Bonas et al., 1991). Expression of hrp genes is induced in the plant and in a particular minimal medium, XVM2 (Wengelnik et al., 1996a). So far, two regulatory genes have been characterized, hrpX and hrpG, which map outside the large hrp gene cluster. hrpG encodes an OmpR-type two-component transcription activator and, in inducing conditions, activates the expression of hrpA and of hrpX. HrpX, an AraC-type regulator, activates the other five known hrp loci, hrpB to hrpF (Wengelnik and Bonas, 1996; Wengelnik et al., 1996b). The large hrp gene cluster of Xcv contains 22 genes, nine of which encode proteins with similarity to components of type III protein secretion systems (Fig. 2). Type III secretion systems have been described for many Gram-negative pathogens of plants and animals, and allow sec-independent transport of proteins across both bacterial membranes without cleavage of an N-terminal signal peptide (for review see Hueck, 1998). Based on an overall similarity in structure, organization, and regulation the hrp gene clusters of plant pathogenic bacteria can be classified into: group I (Erwinia amylovora, Pseudomonas syringae) and group II (Ralstonia solanacearum, Xanthomonas campestris; Alfano and Collmer, 1997). The conserved hrp genes, which encode membrane proteins and a putative ATPase, have been designated hrc (hrp conserved; Bogdanove et al., 1996; Fig. 2). To study the role of the non- hrc genes in the Xcv hrp gene cluster we generated mutants carrying nonpolar mutations, mostly deletions, in individual genes (Huguet et al., 1998; unpublished data). Most mutants analysed so far are typical hrp mutants. However, some genes, designated hpa (hrp associated) are not essential for the HR induction in incompatible interactions, but play a role in pathogenicity. One example is the hpaA gene, which is located within the hrpD operon (Fig. 2) and is surrounded by true hrp genes. hpaA mutants are strongly impaired in growth and disease symptom formation on susceptible plants, but are still able to induce the HR in the resistant plant, although the reaction is partial. hpaA encodes a 30-kDa protein, which is

Fig. 2 Structural organization of the hrp gene cluster in Xcv. The 23-kb hrp region consists of six operons, hrpA-hrpF. Based on sequence analysis 9 genes are predicted to encode conserved components of a type III protein secretion system; these genes are designated hrc genes. The hpaA gene is located in the hrpD operon, downstream of hrcS.

related only to the product of the R. solanacearum hrpV gene. Interestingly, the HpaA protein contains functional NLSs which are important for pathogenicity. Taking all the data together, we believe that the HpaA protein is not a structural component of the type III machinery, but is an effector protein that is translocated into the plant cell via the Hrp pathway (Huguet et al., 1998).

H rp T Y P E I I I S E C R E T I O N I N V I T R O Under certain culture conditions, Xcv secretes proteins in an Hrp-dependent manner into the medium. To demonstrate this secretion in vitro we established special assay conditions. First, we isolated point mutations in the key regulatory gene, hrpG (e.g. an E44K exchange; designated hrpG*), which rendered hrp gene expression constitutive in normally non-inducing culture conditions (Wengelnik et al., 1999). We then ‘composed’ a secretion medium and used AvrBs3 as a ‘reporter’ protein for secretion (Rossier et al., 1999). AvrBs3 was a likely substrate of the Hrp secretion system because its function (but not expression) depends on hrp genes, and recognition of AvrBs3 had previously been shown to occur inside the plant cell (Van den Ackerveken et al., 1996). Incubation of hrpG* bacteria in minimal medium with an acidic pH triggers the Hrp-dependent secretion of a number of proteins, including AvrBs3. In a normal experiment, 10 –20% of the total AvrBs3 protein is detected in culture supernatants of the type III wild-type but not the hrcV (conserved putative inner membrane protein) mutant strain. The presence of AvrBs3 in the culture supernatants is hrp-dependent, as cytoplasmic marker proteins are not detectable in the culture supernatant (Rossier et al., 1999). In addition to AvrBs3 and AvrRxv, an avirulence protein with similarity to YopJ/YopP from Yersinia spp. (Ciesiolka et al., 1999), Xcv secretes proteins from other plant pathogenic bacteria. Examples are PopA from R. solanacearum, and AvrB from P. syringae. Even more interesting was the finding that YopE from the mammalian pathogen Y. pseudotuberculosis is secreted by Xcv in an hrp-dependent manner (Rossier et al., 1999). The occurrence of heterologous secretion suggests that the secretion signal of type III substrates from different bacterial species is conserved.

MOLECULAR PLANT PATHOLOGY (2000) 1(1), 73–76 © 2000 BLACKWELL SCIENCE LTD

mpp010.fm Page 75 Thursday, February 10, 2000 2:43 PM

Xanthomonas campestris pv. vesicatoria

75

H O W I S Av r B s 3 R E C O G N I Z E D B Y THE PLANT CELL? We previously showed by transient expression of AvrBs3 using Agrobacterium -mediated transformation that AvrBs3 recognition takes place in the plant cell, and suggested that Xcv transfers AvrBs3 into the plant cell via the Hrp type III pathway (Van den Ackerveken et al., 1996). Furthermore, AvrBs3 contains functional NLSs which are essential for its recognition by the Bs3 pepper plant (Fig. 1). Our working model for AvrBs3 therefore assumes a type III secretion (injection?) into the plant cell and transport from the plant cell cytoplasm into the nucleus. This hypothesis is supported by recent findings of Zhu et al. (1998, 1999) that the C-terminus of AvrXa10 (an AvrBs3homologue) from Xanthomonas oryzae plays a role in transcription activation. A second indication for a possible function of AvrBs3 in the plant cell nucleus is the isolation of two genes for pepper importin-α which specifically interact with AvrBs3 in the yeast two-hybrid system. Importin-α functions as an NLS receptor, which binds NLS-containing proteins in the cytoplasm. After interaction with importin- β, this complex is imported into the nucleus where cargo and importins dissociate (Mattaj and Conti, 1999).

CONCLUSION Interestingly, several avrBs3-homologues not only play a role in avirulence, i.e. recognition by resistant plants, but also in disease symptom formation. Examples include pthA, which causes hyperplasia in citrus (Duan et al., 1999), and avrBs3 (U.B. et al., unpublished results). There is accumulating evidence that many avr genes play a role in pathogenicity and that this in fact was (or is) the primary function of these genes (Bonas and Van den Ackerveken, 1999). One of the models integrating our current understanding of Hrp-type III secretion and the interaction with the plant cell is given in Fig. 3. Besides searching for the nature and targets of bacterial effector proteins, we wonder why hrp mutant bacteria do not grow in planta . Electron microscopy revealed that hrp mutants induce a local, unspecific defence reaction (e.g. callose deposition) in the plant tissue (Brown et al., 1995). As such reactions are not detectable with wild-type bacteria hrp genes could suppress unspecific defence reactions. This and all other unsolved questions provide exciting challenges for future research.

AC K N O W L E D G E M E N T S Work in our laboratory is currently funded by grants from the Deutsche Forschungsgemeinschaft (SFB363) and the EEC (BIO4-CT97-2244).

Fig. 3 Simplified model of the interaction between Gram-negative plant pathogenic bacteria and the plant cell. It is suggested that bacterial effector proteins are secreted (injected?) into the plant cytoplasm via the Hrp type III secretion system. Once in the plant cell, the effectors interfere with host metabolism for their own benefit, leading to disease. In case of specific recognition of an effector protein by the plant resistance gene (R gene) mediated surveillance system, plant defence reactions are induced resulting in resistance.

RE F E RE N C E S Alfano, J.R. and Collmer, A. (1997) The Type III (Hrc) secretion pathway of plant pathogenic bacteria: trafficing Harpins, Avr proteins, and death. J. Bacteriol. 179, 5655 – 5662. Bogdanove, A., Beer, S.V., Bonas, U., Boucher, C.A., Collmer, A., Coplin, D.L., Cornelis, G.R., Huang, H.-C., Hutcheson, S.W., Panopoulos, N.J. and Van Gijsegem, F. (1996) Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria. Mol. Microbiol. 20, 681–683. Bonas, U. and Van den Ackerveken, G. (1999) Gene-for-gene interactions: bacterial avirulence proteins specify plant disease resistance. Curr. Opin. Microbiol. 2, 94 – 98. Bonas, U., Schulte, R., Fenselau, S., Minsavage, G.V. and Staskawicz, B.J. (1991) Isolation of a gene cluster from Xanthomonas campestris pv. vesicatoria that determines pathogenicity and the hypersensitive response on pepper and tomato. Mol. Plant-Microbe Interact. 4, 81–88. Bonas, U., Stall, R.E. and Staskawicz, B. (1989) Genetic and structural characterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol. Gen. Genet. 218, 127–136. Brown, I., Mansfield, J. and Bonas, U. (1995) hrp genes in Xanthomonas

© 2000 BLACKWELL SCIENCE LTD MOLECULAR PLANT PATHOLOGY (2000) 1(1), 73–76

mpp010.fm Page 76 Thursday, February 10, 2000 2:43 PM

76

U. BONAS et al.

campestris pv. vesicatoria determine ability to suppress papillae deposition in pepper mesophyll cells. Mol. Plant-Microbe Interact. 8, 825 – 836. Ciesiolka, L.D., Hwin, T., Gearlds, J.D., Minsavage, G.V., Saenz, R., Bravo, M., Handley, V., Conover, S.M., Zhang, H., Caporgno, J., Phengrasamy, N.B., Toms, A.O., Stall, R.E. and Whalen, M.C. (1999) Regulation of expression of avirulence gene avrRxv and identification of a family of host interaction factors by sequence analysis of avrBsT. Mol. Plant-Microbe Interact. 12, 35 – 44. Duan, Y.P., Castenada, A., Zhao, G., Erdos, G. and Gabriel, D.W. (1999) Expression of a single, host specific, bacterial pathogenicity gene in plant cells elicits division, enlargement, and cell death. Mol. PlantMicrobe Interact. 12, 556 –560. Herbers, K., Conrads-Strauch, J. and Bonas, U. (1992) Race-specificity of plant resistance to bacterial spot disease determined by repetitive motifs in a bacterial avirulence protein. Nature, 356, 172–174. Hueck, C.J. (1998) Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62, 379 – 433. Huguet, E., Hahn, K., Wengelnik, K. and Bonas, U. (1998) hpaA mutants of Xanthomonas campestris pv. vesicatoria are affected in pathogenicity but retain the ability to induce host-specific hypersensitive reaction. Mol. Microbiol. 29, 1379 –1390. Jones, J.B., Stall, R.E. and Bouzar, H. (1998) Diversity among Xanthomonads pathogenic on pepper and tomato. Annu. Rev. Phytopathol. 36, 41– 58. Mattaj, I.W. and Conti, E. (1999) Snail mail to the nucleus. Nature, 399, 208 – 210. Minsavage, G.V., Dahlbeck, D., Whalen, M.C., Kearney, B., Bonas, U., Staskawicz, B.J. and Stall, R.E. (1990) Gene-for-gene relationships

specifying disease resistance in Xanthomonas campestris pv. vesicatoria– pepper interactions. Mol. Plant-Microbe Interact. 3, 41–47. Rossier, O., Wengelnik, K., Hahn, K. and Bonas, U. (1999) The Xanthomonas Hrp type III system secretes proteins from plant and mammalian bacterial pathogens. Proc. Natl Acad. Sci. USA, 96, 9368–9373. Van den Ackerveken, G., Marois, E. and Bonas, U. (1996) Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell, 87, 1307–1316. Wengelnik, K. and Bonas, U. (1996) HrpXv, an AraC-type regulator, activates expression of five out of six loci in the hrp cluster of Xanthomonas campestris pv. vesicatoria. J. Bacteriol. 178, 3462–3469. Wengelnik, K., Marie, C., Russel, M. and Bonas, U. (1996a) Expression and localization of HrpA1, a protein of Xanthomonas campestris pv. vesicatoria essential for pathogenicity and induction of the hypersensitive reaction. J. Bacteriol. 178, 1061–1069. Wengelnik, K., Van den Ackerveken, G. and Bonas, U. (1996b) HrpG, a key hrp regulatory protein of Xanthomonas campestris pv. vesicatoria is homologous to two-component response regulators. Mol. PlantMicrobe Interact. 9, 704 – 712. Wengelnik, K., Rossier, O. and Bonas, U. (1999) Mutations in the regulatory gene hrpG of Xanthomonas campestris pv. vesicatoria result in constitutive expression of all hrp genes. J. Bacteriol. 181, in press. Zhu, W., Yang, B., Chittoor, J.M., Johnson, L.B. and White, F.F. (1998) AvrXA10 contains an acidic transcriptional activation domain in the functionally conserved C terminus. Mol. Plant-Microbe Interact. 11, 824–832. Zhu, W., Yang, B., Wills, N., Johnson, L.B. and White, F.F. (1999) The C terminus of AvrXa10 can be replaced by the transcriptional activation domain of VP16 from the herpes simplex virus. Plant Cell, 11, 1665 – 1674.

MOLECULAR PLANT PATHOLOGY (2000) 1(1), 73–76 © 2000 BLACKWELL SCIENCE LTD