DE102004010023A1 - Bacterial system for protein transport into eukaryotic cells - Google Patents

Abstract

The invention is based on the problem of developing a system for targeted transport of proteins into eukaryotic cells. DOLLAR A The specific transport of proteins into eukaryotic cells is through the use of a type III secretion system using bacterial strains mutated in hpaB or homologous genes. Starting from a Gram-negative pathogenic bacterial strain, a non-pathogenic strain is generated, which is mutated in the hpaB gene or a homologous gene, which does not mediate significant translocation of bacterial effector proteins. However, overexpressed effector proteins or proteins possessing the type III secretion signal of a non-effector protein can be translocated into eukaryotic cells using the bacterial TTSS. DOLLAR A The bacterial system according to the invention is used for the transport of bacterial proteins in eukaryotic cells in order to influence or modify cellular processes such as gene expression, growth, development and defense / resistance mechanisms.

Description

The The present invention relates to the targeted transport of proteins in eukaryotic cells by the bacterial type III secretion system (TTSS, "type III secretion system ") using bacterial strains, which mutated in hpaB or homologous genes.

The TTSS is present in most Gram-negative plant and animal pathogens Bacteria for the infection and colonization of eukaryotic host organisms essential. This protein transport system spans both bacterial Membranes (inner and outer membrane) and is with an extracellular Pilus associated. The TTSS mediates both the secretion of bacterial Proteins in the extracellular Medium as well as the translocation of so-called effector proteins into the eukaryotic host cell / 1 /. In phytopathogenic bacteria is the TTSS of hrp ("hypersensitive response and pathogenicity ") genes encoded, which are usually organized as a cluster. hrp genes were for many Gram-negative plant pathogenic bacteria such as Erwinia amylovora, Ralstonia solanacearum, Pathovare of Pseudomonas syringae and species of Xanthomonas / 2 /. Based on the arrangement the genes and the hrp gene regulation one distinguishes bacteria the group 1, for example species of the genera Pseudomonas and Erwinia, and Group 2 bacteria, which include Ralstonia solanacearum and species of the genus Xanthomonas belong to / 3 /.

In pathogenic bacteria are hrp genes for bacterial pathogenicity as well for the Induction of hypersensitive response (HR) to resistant host or non-host plants essential. HR is a faster, programmed death of plant Cells and usually occurs in conjunction with plant defense reactions on. The induction of HR can be through the direct or indirect Recognition of individual bacterial effector proteins - also as Avirulence proteins (Avr proteins) called - from corresponding plant Resistance proteins triggered become / 4 /. The fact that the HR also in transient expression of individual Avr proteins in resistant plants was a first indication that Avr proteins be transported to the plant cell. Many effector proteins of plant pathogenic bacteria were initially due to their avirulence activity in plants identified with appropriate resistance proteins.

Of the in claim 1 invention was based on the problem to develop a protein transport system that supports the specific transport selected Effectors in the plant cell allows. A possibility For this purpose, the heterologous expression of hrp gene clusters in non-pathogenic Gram-negative bacteria already used for Escherichia coli and Pseudomonas fluorescens strains has been described (patent WO0002996 / US2002083489). Here was the hrp gene cluster of P. syringae pv. syringae from the cosmid pHIR11 in both bacterial strains expressed. Are by introducing a second plasmid additionally the P. syringae effector proteins expressing AvrB or AvrPto so induce the generated E. coli and P. fluorescens strains in the plant have an AvrB or AvrPto-specific HR / 5,6 /. Not possible, however, was AvrB and AvrPto in the culture medium of P. syringae, E. coli (pHIR11) or P. Fluorescens (pHIR11) to detect. In contrast, the secretion succeeded of AvrB and AvrPto in the culture medium of E. coli strains, the express the hrp gene cluster of Erwinia chrysanthemi / 7 /. in the Contrary to the P. syringae hrp gene cluster, the E. chrysanthemi hrp genes are also expressed and allowed in complex medium a cell contact independent Protein secretion.

The Expression of heterologous TTSS in non-pathogenic bacteria succeeded but so far only using the hrp gene cluster of Gram-negative Bacteria of group 1 (P. syringae and E. chrysanthemi), since only here the necessary regulatory proteins within the gene cluster are encoded. However, the TTSS of group 1 bacteria may be effector proteins from Group 2 bacteria do not translocate. So it turned out that this Effector protein AvrBs3 from X. campestris pv. Vesicatoria (group 2 bacterium) by the TTSS of P. syringae (belonging to group 1) is not translocated into plant cells. For these analyzes, avrBs3 was added the hrp gene cluster of P. syringae in P. fluorescens and the resulting bacterial strain into AvrBs3-responsive plants infiltrated.

A general disadvantage of using heterologously expressed hrp gene clusters for targeted transport of individual proteins is that non-pathogenic bacteria such. BP fluorescens and E. coli laboratory strains may develop pathogenic properties on eukaryotic organisms by endowment with a TTSS. This problem has been solved according to the invention by generating a non-pathogenic strain which has mutated in the hpaB ("hrp-associated" B) gene starting from the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. Vesicatoria hpaB mutants do not translocate significant amounts of effector proteins , but are able to mediate the translocation of proteins using their own TTSS, the secretion signal of a so-called Own non-effector protein.

The hpaB gene used by X. campestris pv. Vesicatoria (GenBank accession AF056246) is located in the hrp gene cluster downstream of hrpE1 and encodes a protein that controls TTSS-dependent protein secretion and translocation. In X. campestris pv. Vesicatoria strain 85-10, the mutation of hpaB leads to complete loss of bacterial ability to grow on susceptible pepper plants and cause disease symptoms or to induce HR on resistant pepper plants. In the X. campestris pv. Vesicatoria strain 85 *, the mutation of hpaB also leads to the loss of pathogenicity on susceptible plants, but still allows the induction of a very weak, partial HR on resistant plants (see 1 ). Strain 85 * is a derivative of strain 85-10 and contains hrpG *, which encodes a constitutively active form of the regulatory protein HrpG. HrpG is a member of the OmpR family of two-component regulators and activates the expression of the hrp genes in the plant / 8 / using the AraC-type-like transcriptional activator HrpX. The use of hrpG * mutants not only allows for hrp gene expression in vitro, but also allows the analysis of type III-dependent protein secretion in the culture medium.

The described phenotypes of hpaB mutant strains in susceptible and resistant plants are due to a greatly reduced efficiency in the transport of effector proteins. Thus, in vitro secretion analyzes showed that in the culture supernatant of hpaB mutants, the effector proteins AvrBs3, AvrBs1 and AvrBsT are only in small amounts or not detectable (see 2 ). In contrast, the secretion of HrpF and XopA is not affected by the mutation of hpaB. HrpF and XopA do not belong to the group of effector proteins, ie they are not translocated into the plant cell. Since HrpF is essential for pathogenicity and interacts with membranes, this protein is the major component of the type III translocon / 8 /. The mutation of xopA leads to a reduction of bacterial pathogenicity on susceptible plants and to the induction of reduced HR on resistant plants / 9 /. XopA is therefore involved in effector protein translocation. It has been found that a fusion protein containing the N-terminal 51 amino acids of XopA fused to AvrBs3Δ2 is secreted by strain 85 ^* but does not induce AvrBs3-specific HR. AvrBs3Δ2 is an N-terminal deletion derivative of the effector protein AvrBs3, which is no longer secreted by the type III secretion system. However, AvrBs3Δ2 still induces HR in resistant plants after transient expression in the plant or after fusion to a secretion and translocation signal and is therefore used as a reporter protein to analyze type III-dependent protein translocation / 10,11 /. The fact that the Xopa ₁ _ ₅₁ * no HR induced -AvrBs3Δ2 fusion protein by transport through the TTSS of stem 85 on AvrBs3-responsive plants shows that Xopa is not translocated from this bacterial strain. Both XopA and HrpF thus belong to the group of non-effector proteins, which refers to proteins that are secreted by TTSS, but not translocated.

In the 2 The secretory analyzes presented here show that the mutation of hpaB leads to a drastic reduction in the secretion of effector proteins, but does not affect the secretion of non-effector proteins. According to the invention, a "mutation of hpaB" characterizes all base pair exchanges and deletions in the hrp gene cluster in the region of the hpaB gene or in the region of hpaB-associated promoter elements which prevent expression of the hpaB gene or lead to the expression of a non-functional gene product According to the invention, "nonfunctional" also denotes all hpaB gene products which lead to a reduced efficiency in the in vitro secretion of effector proteins compared to the corresponding hpaB wild-type strain. For the investigation of hpaB mutants, the mutations in the bacterial genome by homologous recombination with the help of the suicide vector pOK1 / 12 / introduce.

It has thus been shown that HpaB has an essential function during the Effector protein transport through the TTSS takes over. HpaB of X. campestris pv. vesicatoria is a 162 amino acid protein with an isoelectric Point of 4.3 and with a leucine content of 13.6%. HpaB points Thus, typical features of type III secretion Schaperonen on each other Although rarely show sequence homology, but usually small, acid and Leucine-rich. Type III secretion chaperones contribute to stability and / or for the efficient secretion of one or more translocon or Effector proteins at / 13 /. HpaB as a general type III secretion schaperone allows the secretion of effector proteins, while contributing to the secretion of non-effectors not involved. The complete loss the pathogenicity hpaB mutants are due to the fact that the protein levels of effectors remaining in the absence of HpaB be translocated, for a colonization of susceptible plants and the induction of Disease symptoms are insufficient.

However, hpaB mutants are still capable of transporting significant amounts of effector proteins into the plant cell when overexpressed. Thus, X. campestris induces pv. Vesicatoria strain 85 * ΔhpaB (pDSF300), which underlies AvrBs3 Control of a strong lac promoter overexpressed in AvrBs3-responsive plants, the HR (see 4 ). According to the invention, overexpression is to be understood as meaning the gene expression of a "stronger" promoter in comparison to the wild-type promoter, wherein the "strength" of the promoter determines the transcription rate of the gene to be expressed. Promoters are DNA sequences that allow the binding of RNA polymerase and thus the mRNA synthesis. Expression vectors containing promoters may contain various combinations of transcription and translation initiation signals. According to the invention, overexpression also means the expression of effector genes from the native promoter in an independently replicating expression vector, which leads to an increased copy number of the corresponding gene compared to the wild-type strain. Examples of expression vectors containing a replication signal for E. coli and X. campestris pv. Vesicatoria and a marker gene (antibiotic resistance gene) for selection and which can be used for overexpression are pLAFR, pDSK, pUFR and pBBR derivatives. For example, the sequence to be expressed can be ligated into the vectors at appropriate restriction sites. The resulting plasmid can be introduced into E. coli by electroporation and subsequently brought into the desired X. campestris pv. Vesicatoria strains via a triparental conjugation or electroporation. E. coli and X. campestris pv. Vesicatoria strains containing the expression vector are selected on culture medium with appropriate antibiotics. The cloning of genes into expression vectors is carried out by known techniques / 14 /. The conjugation of expression plasmids in X. campestris pv. Vesicatoria is described / 15 /.

It has also been shown that hpaB mutants in addition to individual effector proteins whose overexpressing corresponding coding sequences also translocate reporter proteins, provided that they contain an N-terminal secretion signal from a non-effector protein. Thus induced strain 85 * expressing the fusion protein ΔhpaB hrpF ₁ _ ₂₀₀ -AvrBs3Δ2 which the N-terminal 200 amino acids of hrpF fused to AvrBs3Δ2 contains the AvrBs3-specific HR. A similar result was obtained for strain ΔhpaB * 85, which expresses the fusion protein Xopa ₁ _ ₅₁ -AvrBs3Δ2 containing the N-terminal 50 amino acids fused to AvrBs3Δ2 of Xopa. Both HrpF and XopA are normally non-effector proteins, ie they are secreted but not translocated in the presence of HpaB. Thus, induced stem 85 *, which hrpF ₁ _ ₂₀₀ -AvrBs3Δ2 or Xopa expressed ₁ _ ₅₁ -AvrBs3Δ2, no HR in AvrBs3-responsive plants, although both fusion proteins in the culture supernatant can be detected (see 3 and 4 ). The translocation of both proteins into hpaB mutants shows that HpaB controls type III-dependent protein translocation. Thus, HpaB inhibits the translocation of non-effectors into the host cell and simultaneously promotes the transport of effector proteins as a general type III secretion schaperone.

According to the invention is thus a bacterial strain used that has no pathogenic properties possesses more, however, is able to effector proteins after their overexpression or fusion proteins that have a type III secretion signal for this bacterial strain contained in the plant cell. The inventive method offers thus a favorable one Alternative to the use of heterologous protein transport systems, the on the expression of the hrp gene cluster of a group 1 bacterium in one non-pathogenic strain. A significant advantage of the present Invention is to provide a bacterial strain available whose TTSS do not have significant levels of endogenous effector proteins more translocated. The danger that hpaB mutant strains are pathogenic Developing properties is thus almost impossible.

The extended inventive solution also the range of bacterial transport systems and makes it possible single proteins targeted by the TTSS of group 2 bacteria into eukaryotic Infiltrate cells. This is a huge advantage, depending on protein to transport only one particular type of TTSS one ensures optimal translocation into the target cell. Thus, according to the invention for the targeted Transport of effector proteins from bacteria of the genus Xanthomonas the TTSS of a strain of the same genus are used.

The The present invention is not only applicable to the host plants The hpaB mutants used can also be used with other dicots or monocotyledonous plants or other eukaryotic target cells such as yeast cells are performed. The inventive method is used to transport bacterial proteins into eukaryotic Cells used to cellular processes to modify specifically. In general, all effector proteins find application according to the invention, the plant processes like growth, development and defense / resistance mechanisms or eukaryotic with fundamental cellular processes Host cells interfere.

In addition to bacterial effector proteins, according to the invention other proteins, if they are fused to a functional bacterial type III secretion signal, can also be transported into eukaryotic cells. The type III secretion signal is any protein or mRNA sequence that secretes a protein through it te TTSS allows. Suitable type III secretion signals are found in all TTSS-secreted proteins, typically within the N-terminal 50-100 amino acids. Thus, for example, the N-terminal 51 amino acids of XopA can be used as a type III secretion signal according to the invention for the transport of a protein without its own type III secretion signal. For expression of fusion proteins fusions between the coding sequence of the type III secretion signal and the protein to be transported z. B. produced by ligation. For fusion, the two sequences must be in the same reading frame. In addition, the ligation must be done in a manner that allows optimal and stable expression of the encoded fusion protein. There may also be an intermediate region between the type III secretion signal and the gene to be fused. The expression of the resulting fusion construct can be carried out in an expression vector under the control of an endogenous promoter of the bacterium or under the control of a constitutive or inducible promoter in the expression plasmid. The expression and secretion of the engineered fusion protein can be checked by secretion analyzes as well as by using appropriate antibodies against epitopes of the protein to be analyzed.

applications for proteins, after fusion to a type III secretion signal by the TTSS of hpaB mutants could be translocated are such proteins, in important cellular processes and intervene metabolic pathways and thereby, for example, in plants promote growth or cause resistance to pathogenic organisms or herbicides. Generally, according to the invention, the transport all those proteins of interest, the cell's own or heterologous modify expressed proteins in eukaryotic cells or the Modify DNA organization or gene expression.

A further application possibility The invention consists in the transport of proteins into the extracellular environment a host cell. It can be used for this purpose proteins that either a have their own type III secretion signal, or to one in the used Bacterial system functional type III secretion signal are fused. However, it must be secured be that the proteins to be transported, although secreted by the TTSS, but not translocated into the plant cell. A possibility for this consists in the use of hpaB / hrpF double mutants. The mutation in hrpF prevents the translocation of type III-dependent secreted proteins into the host cell, impaired whereas protein secretion by the TTSS into the extracellular medium not / 8 /. Another possibility consists in the successive shortening the type III secretion signal used, if it mediates secretion and translocation in hpaB mutants. In case of secretion and translocation signal are separated from each other locally, can by Shortenings or Deletions in the region of the translocation signal generates a signal which in hpaB mutants secretion but not translocation taught.

Next the previously mentioned applications the invention can also after in vitro cultivation of the bacteria for protein transport in the extracellular Medium can be used. Secreted proteins can be removed after separation of the bacteria be purified from the culture medium. For this purpose of application must the secretion of the protein to be transported by the TTSS guaranteed be. It can the proteins have their own type III secretion signal or to a heterologous type III secretion signal be fused, which may be signals in hpaB mutants only the Secretion through the TTSS or even mediate the translocation.

For the inventive method can all pathogenic bacteria are used as starting material, expressing an hrp gene cluster as well as hpaB or homologous genes. The sequence of hpaB from X. campestris pv. Vesicatoria is in the database available.

The Invention includes the production of hpaB gene mutants in all Gram negative Bacteria expressing hpaB or homologous genes. The invention further relates to the heterologous expression of hrp gene clusters, which are mutated in hpaB or a homologous gene, in non-pathogenic ones Bacteria. Since hpaB and homologous genes so far only in bacteria of the group 2 are found in the generation of such a heterologous Systems to note that in addition to the hrp gene cluster also the regulatory proteins required for hrp gene expression (the in Group 2 bacteria are not encoded in the hrp gene cluster) Need to become or otherwise the expression of the hrp genes must be guaranteed.

embodiments

1. Bacterial strains and plasmids

E. coli DH5α was purchased from Bethesda Research Laboratories, Bethesda, Md. The X. campestris pv. Vesicatoria strains 85-10, 85 * and 82 * are described in references / 16 / and / 17 /. X. Campestris pv. Vesicatoria strains were grown at 30 ° C in NYG medium / 18 /. The plasmid pBluescript (II) KS used for cloning was acquired by the company Stratagene, Heidelberg, Germany. The expression vectors pDSK604 and pLAFR6 are described in Escolar et al. / 19 / or in Bonas et al. / 20 / described. Plasmids were introduced into E. coli by electroporation and in X. campestris pv. Vesicatoria by conjugation / 21 /. Antibiotics were used in the following concentrations: ampicillin, 100 μg / ml; Rifampicin, 100 μg / ml; Spectinomycin, 100 μg / ml and tetracycline, 10 μg / ml.

2. Plant material and infiltrations

The paprika cultivars Early Cal Wonder (ECW), ECW-10R and ECW-30R / 22 / as described in Bonas et al. / 23 / described and infiltrated with X. campestris pv. Vesicatoria strains. Bacterial suspensions were infiltrated in peppermint leaves at concentrations of 2 x 10 ⁸ bacteria / ml (corresponding to an optical density of 0.2 at a wavelength of 600 nm) in 1 mM MgCl ₂ . The onset of disease symptoms or HR was analyzed over a period of one to three days after infiltration. For studies of bacterial growth in the plant bacteria suspensions were infiltrated in concentrations of 10 ⁴ cfu / ml in 1 mM MgCl ₂ in leaves of pepper cultivars ECW / 23 /.

3. Preparation of a hpaB mutant

to Production of a mutation in the hpaB gene became the 3.1 kb region of hrpE into the EcoRV and Xhol cleavage site of pBluescript (II) KS. Subsequently was a 420 bp big one Fragment of hpaB cut out using Csp45l and the remaining Construct religates. The deletion of the Csp45I fragment leads to Deletion of the amino acids 13 to 149 in the hpaB gene product. The resulting 2.7 kb insert was now in the BamHl / Sall interfaces of the suicide vector pOK1 / 12 / cloned and the resulting hpaB deletion construct into the X. campestris pv. vesicatoria strains 85-10, 85 * and 82 * conjugated. The replacement of the wild-type gene against the Deletion in hpaB by homologous recombination was by PCR ("polymerase chain reaction ", polymerase chain reaction) -based Analyzes were performed using primers hpaB-for (5'-CGAATTCGTCCATGTCTCACCACAGATC-3 ') and hpaB-rev (5'-CGAGCTCGGCGCGTAACCACAGATAGTT-3').

The resulting hpaB mutants were characterized by phenotypic analyzes. For this purpose, bacterial suspensions were inoculated into leaves of susceptible and resistant plants. As in 1 As shown, deletion of hpaB in strain 85-10 results in complete loss of bacterial pathogenicity, ie the ability to grow on susceptible plants and induce disease symptoms. Furthermore, strain 85-10ΔhpaB is no longer able to induce HR in resistant plants. Strain 85 * ΔhpaB is also no longer pathogenic on susceptible plants, but still induces a partial HR on resistant plants. Comparable phenotypes were observed upon inoculation of strain 82 * ΔhpaB into leaves of corresponding susceptible and resistant plants.

4. Construction of AvrBs3Δ2 Fusion Proteins

To prepare an HrpF-AvrBs3Δ2 expression construct, a 1.1 kb EcoRI fragment containing the first 387 codons of hrpF was ligated into the EcoRI site of plasmid pDSF356F / 10 /. The resulting construct pDhrpFN356 expressing hrpF ₁ _ ₃₈₇ -AvrBs3Δ2, was conjugated into the X. campestris pv. Vesicatoria strains 85 * and 85 * ΔhpaB. To prepare a second HrpF-AvrBs3Δ2 expression construct, the first 200 codons of hrpF were PCR amplified using the primers HrpF-for (5'-TACTGAATTCGCCTCTATGTCGCTC-3 ') and HrpF-200rev (5'-CTGTCGAATTCGATCTTGCCGCCGCACTTG-3') and amplified in ligated the EcoRI site of pDSF356F. The resulting construct pDS200F356F expressed hrpF ₁ _ ₂₀₀ -AvrBs3Δ2 and was conjugated also in the X. campestris pv. Vesicatoria strains 85 * and 85 * ΔhpaB.

To prepare a Xopa ₁ _ ₅₁ -AvrBs3Δ2 expression construct the first 51 codons of Xopa 680 and the region of translation initiation codon bp upstream of the Xopa by PCR and the primers were xopAfor (5'-CACCGTACCGTTGTTGTTGCGATG-3 ') and xopArev (5'-TGAAAAGATGAACTGGGTCAG -3 ') amplified. The resulting PCR product was ligated into the donor vector pENTR / D-TOPO (available from Invitrogen, Carlsbad, Calif.) Containing attL sites using a topoisomerase and then recombined into the target vector pL6GW356. pL6GW356 has an attR reading frame cassette fused to avrBs3Δ2 and was reported in Noel et al. / 11 / described. The resulting construct pL6xopA356 expressing Xopa ₁ _ ₅₁ -AvrBs3Δ2, was conjugated into the X. campestris pv. Vesicatoria strains 85 * and 85 * ΔhpaB.

5. Type III-dependent protein secretion in hpaB mutants

For the analysis of type III-dependent protein secretion in hpaB mutants, the X. campestris pv. Vesicatoria strains 82 * and 82 * ΔhpaB were incubated in secretion medium. Subsequently, the protein total extracts were separated from the culture supernatants and analyzed by SDS-polyacrylamide electrophoresis and immunoblot using antibodies against HrpF and AvrBs3 / 24,25 /. hrpF was present in comparable amounts in proteinintal extracts and culture supernatants of both strains (see 2 ). In contrast, AvrBs3 could not be detected in the culture supernatant of strain 82 * ΔhpaB, although it was detected in protein total extracts of both strains and in the culture supernatant of strain 82 *. To analyze the secretion of other effector proteins, avrBs1-c-myc and avrBsT-c-myc expression constructs / 19 / were conjugated to the X. campestris pv. Vesicatoria strains 85 * and 82 * and to the corresponding hpaB mutants. Since the proteins secreted by the TTSS under in vitro conditions can only be detected immunologically in the culture supernatant, secretory analyzes require the availability of specific antibodies to epitopes of the proteins to be analyzed. For this reason, the effector proteins AvrBs1 and AvrBsT were provided with a C-terminal c-myc epitope. Both AvrBs1-c-myc and AvrBsT-c-myc were detected in the total extracts of hpaB wild-type and hpaB mutant strains in comparable amounts from an anti-c-myc antibody (Amersham Pharmacia Biotech, Freiburg, Germany). Furthermore, both proteins were detected in the culture supernatant of the hpaB wild-type strain, but were not or only weakly detectable in the culture supernatant of hpaB mutants (see 2 ). In order to prove that the detection of proteins in the culture supernatant is not due to lysis of the bacterial cells, the immunoblots were incubated with an antibody against the intracellular protein HrcN / 26 /, which was detected only in protein total extracts.

The described experimental observations illustrate that HpaB is involved in the secretion of effector proteins, while it is believed that it does not significantly affect the export of non-effector proteins by the TTSS. Thus, in addition to HrpF, the non-effector protein XopA was detected in comparable amounts in culture supernatants of X. campestris pv. Vesicatoria strain 85 * and 85 * ΔhpaB (see 2 ). The hypothesis could also by Sekretionsanalysen of X. campestris pv. Vesicatoria strains 85 * and 85 * ΔhpaB that the fusion proteins hrpF ₁ _ ₂₀₀ -AvrBs3Δ2 and Xopa ₁ _ expressed -AvrBs3Δ2 _51, are confirmed. Thus were both hrpF ₁ _ ₂₀₀ -AvrBs3Δ2 and Xopa detected ₁ _ ₅₁ -AvrBs3Δ2 in total extracts and culture supernatants of wild-type and hpaB hpaB mutant strains (see 3 ). These results indicate that the N-terminal protein regions of HrpF and XopA contain secretion signals that mediate hpaB-independent secretion of the AvrBs3Δ2 reporter protein. Thus, the type III-dependent secretion of the reporter protein AvrBs3Δ2 per se can be independent of the presence of HpaB.

6. Analysis of protein translocation in hpaB mutants

For the analysis protein translocation in hpaB mutants were AvrBs3 and AvrBs3Δ2 fusion proteins in X. campestris pv. vesicatoria strains 85 * and 85 * ΔhpaB and the bacteria in leaves of the chili cultivar ECW-30R inoculated. ECW-30R plants included the resistance gene Bs3, which is the specific recognition of AvrBs3 mediated and subsequently induces defense reactions with a HR accompany / 22 /. Furthermore, by the resistance protein Bs3 also AvrBs3Δ2 fusion proteins if they transiently mediated, for example, via Agrobacterium-mediated Gene transfer expressed in plant cells or by X. campestris pv. vesicatoria translocated. The presence of the nuclear localization sequences in AvrBs3 or AvrBs3Δ2 fusion proteins is there for the detection by Bs3 is essential. It is assumed that AvrBs3 and AvrBs3Δ2 fusion proteins must be transported to the nucleus to the Bs3-specific HR induce.

X. campestris pv. Vesicatoria strain 85 * (pDS300F) containing the avrBs3 expression construct / 27 / induces Bs3-specific HR on ECW-30R plants. On the other hand, induce 85 * strains that hrpF ₁ _ ₃₈₇ -AvrBs3Δ2, hrpF ₁ _ ₂₀₀ -AvrBs3Δ2 or expressing Xopa ₁ _ ₅₁ -AvrBs3Δ2 disease symptoms ECW 30R plants. This shows that the N-terminal regions of HrpF and XopA have no export signal, which mediates in strain 85 * the translocation of the AvrBs3Δ2 reporter (see 4 ). If, on the other hand, HrpF ₁ _ ₃₈₇ -AvrBs3Δ2, HrpF ₁ _ ₂₀₀ -AvrBs3Δ2 or XopA ₁ _ ₅₁ -AvrBs3Δ2 are expressed in the hpaB mutant strain 85 * ΔhpaB, they induce the Bs3-specific HR after infiltration into ECW-30R plants. This demonstrates that the N-terminal protein regions of HrpF and XopA can mediate the translocation of the AvrBs3Δ2 reporter protein in the absence of HpaB. In addition to AvrBs3Δ2 fusion proteins, AvrBs3 is also translocated into the plant cell upon overexpression of Plamid pDS300F from strain 85 * ΔhpaB. Thus, strain 85 * ΔhpaB (pDS300F) induces Bs3-specific HR in ECW-30R plants (see 4 ). In summary, it has been shown that hpaB mutants are able to translocate significant amounts of an effector protein into the plant cell, provided that this effector protein is overexpressed. Significant protein levels are those that are sufficient to induce complete, non-partial HR in the corresponding resistant plant. In addition to effector proteins after overexpression, hpaB mutants can also translocate fusion proteins containing the N-terminal protein region of a non-effector protein such as HipF or XopA.

literature

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85-10

X. campestris pv. vesicatoria strain 85-10

85 *

X. campestris pv. vesicatoria strain 85 *

85-10ΔhpaB

hpaB deletion mutant of X. campestris pv. Vesicatoria strain 85-

10

85 * ΔhpaB

hpaB deletion mutant of X. campestris pv. Vesicatoria strain 85 *

85-10ΔhrpG

nonpathogenic X. campestris pv. Vesicatoria strain 85-10ΔhrpG, with

one Mutation in the regulatory gene hrpG

+

when aqueous lesions known disease symptoms

-

none visible disease symptoms

Mr

partial HR (hypersensitive reaction)

wt

hpaB wild-type strain

ΔhpaB

hpaB-mutant tribe

hrpF

Type III translocon protein, type III secreted, not translocated

in the

Wild-type strain

AvrBs3

effector Type III-dependent secreted and translocated

HrcN

intracellular protein of the TTSS, not secreted

AvrBs1-c-myc

effector with C-terminal c-myc epitope, type III dependent

secreted

and translocated

AvrBsT-c-myc

effector with C-terminal c-myc epitope, type III dependent

secreted

and translocated

Xopa

potential Type III translocon protein, type III-dependent secreted, not

translocated in the wild-type strain

Xopa ₁ _ ₅₁ -AvrBs3Δ2

fusion protein between the N-terminal 51 amino acids of XopA

and the deletion derivative AvrBs3Δ2, that in the N-terminal 152

Amino acids deleted is

HrpF ₁ _ ₃₈₇ AvrBs3Δ2

fusion protein between the N-terminal 387 amino acids of HrpF

and the deletion derivative AvrBs3Δ2

HrpF ₁ _ ₂₀₀ AvrBs3Δ2

fusion protein between the N-terminal 200 amino acids of HrpF

and the deletion derivative AvrBs3Δ2

Claims (13)

Bacterial system for protein transport into eukaryotic Cells consisting of the type III secretion system and a hpaB mutant Bacterial strain, where hpaB denotes a gene in the hrp gene cluster, or a bacterial strain that has a mutation in a hpaB homologous or functionally analogous gene. Transport system according to claim 1, characterized in that that the organism used is a Gram-negative bacterium. Transport system according to claim 1, characterized in that that the organism used is cultured in vitro. Transport system according to claim 1, characterized in that that the organism used together with eukaryotic cells is cultivated. Transport system according to claim 1, characterized in that that the organism used with plants, e.g. through infiltration, Dipping or spraying is brought into contact. Transport system according to claim 1, characterized in that that the mutation of hpaB or of a homologous gene expression of the respective gene product prevented or modified so that its functionality is reduced or lost. Transport system according to claim 1, characterized in that that the mutation of hpaB or of a homologous gene to a Reduction of effector protein secretion by the type III secretion system leads. Transport system according to claim 1, characterized in that that hpaB or a homologous gene mutates in the genome of the bacterial strain used is. Transport system according to claim 1, characterized in that that the the type III secretion system coding genes are expressed from an expression plasmid, which contains a mutation in hpaB or a homologous gene. Transport system according to at least one of claim 1 to 9, characterized in that the bacterial strain used contains a recombinant nucleic acid molecule in which an active promoter is operably linked to a nucleic acid sequence, which encodes an effector protein. Transport system according to at least one of claim 1 to 10, characterized in that the coded effector protein a type III secretion signal which is functional in the bacterial strain used contains. Transport system according to at least one of claim 1 to 9, characterized in that the bacterial strain used contains a recombinant nucleic acid molecule in which an active promoter is operably linked to a nucleic acid sequence, a fusion protein between a type III secretion signal and the protein to be transported coded. Transport system according to at least one of claim 1 to 9 and 12, characterized in that the fusion protein an amino acid sequence between the type III secretion signal and the protein to be transported may contain.