In some bacterial phytopathogens including Pectobacterium atrosepticum (P. atrosepticum), the function of type III secretion systems (T3SS) has not been clearly understood. P. atrosepticum is a bacterial pathogen which is restricted to S. tuberosum in temperate regions. It causes a blackleg in the field and a soft rot in tubers after harvesting. Extra-cellular depolymerase enzymes are the main virulence factors for pectolytic species of Pectobacteria, although there are others whose roles have not been determined yet (1). Currently, scientists (2) have been more interested in the study of T3SS and the proteins and substrates of this system because T3SS genes apart from a secretion system play a role in the induction of hypersensitivity reactions (3, 4). Harpin proteins (Hrp) have a unique ability to induce hypersensitivity reactions in the leaflet tissues of tobacco after infiltration and this is connected to the T3SS function (5). HrpN from Erwinia amylavora was the first phytopathogenic protein for which T3SS role was shown (6). It is suggested that harpins perform as helpers in the translocation of Avr-proteins but their exact role and function is still not clear (7, 8). A structural similarity has also been shown between the HrpW C-terminal protein and the pectate lyase enzyme (9). In spite of this, it has been supposed that harpins are necessary for the transportation of virulence proteins from bacteria into plant cells (10, 11). Resistance against bacterial infection in plants can be induced by a variety of elicitors (12, 13). Production of antimicrobial proteins such as pathogenesis-related (PR) proteins is a ubiquitous defense response of plants to pathogenic attack (14-16). While interacting with a resistant host plant, pathogens trigger localized hypersensitive responses which are regulated by complex mechanisms (17). Signals initiated at the site of infection lead to the induction of specific PR genes in uninfected parts of the plant by the means of a process termed ‘systemic acquired resistance’ or SAR (18). Induction of PR-proteins in some dicots tightly correlates with the onset of generally designated systemic acquired resistance (19). PR5 proteins are also called thaumatin-like proteins because of their striking sequence similarity to thaumatin, a sweet-tasting protein from Thaumatococcus daniellii (20). PR5 proteins have been characterized from a wide range of plant species in both dicotyledonous and monocotyledonous plants. HIN is as an exemplary gene for the analysis of elicitor-induced PR gene. HIN is up-regulated during the hypersensitive response, is harpin-induced and generated by an incompatible plant-pathogen interaction (21).
The objectives of the study were to investigate the effect of T3SS from P. atrosepticum on the expression of the resistance genes in S. tuberosum and thereby the influenced pathogen virulence or disease severity.
3. Materials and Methods
After the sequencing of P. atrosepticum HrpW fragment, the encoded protein was predicted and a phylogenetic tree for some phytopathogens was constructed. Also, virulence properties of P. atrosepticum strains and sensitivity of S. tuberosum varieties against the bacterium were investigated by assessing the PR-5 and HIN gene expression changes by real-time PCR. Bacterial cultures (Table 1) were stored in test-tubes containing 5 mL of 0.5% meat-peptone agar under sterile Vaseline oil at 4°C. Bacteria were grown either in Lauria and Bertani (LB, Tripton 10 g.L -1 ), Yeast extract 5 g.L -1 , NaCl 10 g.L -1 liquid, minimal medium A (22) or agar solidified media. P. atrosepticum and E. coli cells were grown at 28°C and 37°C, respectively. Bacteria in liquid cultures were grown in a shaking incubator. When required, antibiotics were added at the following concentrations: ampicillin 100 mg.mL -1 , gentamicin 10 mg.mL -1 . Preparation of plasmid DNA, restriction enzyme digestion, ligation, DNA electrophoresis and transformation, conjugation as well as western and dot blotting were carried out as previously described (23,24). pAS17 was constructed by the insertion of the 600 bps HrpW fragment from pLA16 and was digested by BglII and EcoRV and inserted into pUC18 which was restricted by SmaI and BamHI. pAS18 was constructed by the insertion of the 1000 bps HrpW fragment from pLA16; digested by SalI and BglII and inserted into pUC18 restricted by SalI and BamHI. Two subclones of pAS17 (including 600 bps HrpW fragment from P. atrosepticum) and pAS18 (including 1000 bps HrpW fragment from P. atrosepticum ) were used as plasmids carrying HrpW parts for nucleotide sequence determination by Amersham Pharmacia Biotech (UK) apparatus and Oligo-nucleotide primers and Fermentas kit (Lithuania) complex. DNA sequence analyses were performed with the PC gene software (ALFwin Software). The tree was constructed by the comparison of the HrpW sequences in various phytopathogenic bacteria using the Neighbor-joining method (25). The phylogenetic analysis was performed using the MEGA 4 software, which employed 1000 bootstrap replications and the maximum parsimony method. For measuring the degree of maceration, a S. tuberosum slice of about 1 cm thickness and 2 - 3 cm 2surface area was incubated overnight with 5 μL of bacterial cultures (OD about 2.0) at 100% and relative humidity at 28°C. Samples were studied after 24 h. The degree of tissue maceration was estimated by determining the ease at which the tissue could be pulled apart using a spatula. The macerated tissue of each slice was then weighted. Total RNA was extracted by the means of the TRIZOL method (Bio-Rad Laboratories). One gram of tissue was ground in a mortar and pestle. Tissue was put into a plastic screw-cap tube and centrifuged with 15 mL TRIzol reagent and was incubated for 5 min at room temperature or 60° C. It was washed with chloroform and finally, RNA precipitated by adding isopropanol. cDNA was synthesized using a Revert and Aid First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturer’s instructions (http://products.invitrogen.com/ivgn/product/15596026). The following RNA/primer mixture was prepared in each tube: 5 μg of total RNA, 3 μL random hexamer (50 ng.L -1 ), 1 μL of 10 mM dNTP mix, DEPC H2O to 20 μL, 4 μL of 5X RT buffer, 0.5 μL of 25 RNase inhibitor, 2 μL of RT enzyme. The tubes were incubated at 42ºC for 60 min then heated at 70ºC for 15 min and stored at -20ºC until use for real-time PCR. 0.5 μg of cDNA was added to the RT-PCR mixture containing 1X PCR buffer (Sigma), 0.2 mM dNTP, 50 nM Primers, 0.2 U Taq polymerase, 1X SYBR Green1, 2.5 mM MgCl 2 and dH 2 O up to a final volume of 30 μL. Amplification of EF-1, PR-5 and HIN genes were achieved by using the following primers: For EF-1 -5’-TTGATGCTCTTGACCAGATTAACG-3’, 5’-ACGGGCACAGTTCCAATACC-3’; for PR5 S. tuberosum (Osmotin): PR5f -5'-ATTTGAGGTCCATAACAACTGTCC-3', PR5r-5'-CAATTTAGTACGACCCCAAATACG-3' and for HIN: HIN1f -5'-GCAACTGCATTTTCCAAATCATC-3', HIN1r-5'-CACGTAGAAATTGACCTTGTTAGG-3'. Gene-specific forward and reverse primer pair concentrations were normalized and mixed (concentration of each primer in the mixture was 5 pmol.μL -1). Amplification was performed with the following protocol by programmable ABI Prism 7000 (Applied Biosystems): 1 cycle at 94°C for 5 min followed by 34 cycles at 94°C for 30 sec, 55°C for 30 sec and 72°C for 45 sec, finalized by 1 cycle at 72°C for 7 min. The specificity of the PCR products were determined by a SYBR Green1 melting curve. The real-time PCR results were analyzed by the SDS 7000 software. Experiments were in triplets and data were statistically analyzed using mean averages and their confidence intervals (P < 0.05). pLA16 and pUC18 were used as HrpW donor and vector for sub cloning, respectively. HrpW gene was divided into two parts and each part was cloned individually. Both plasmids were transformed into E. coli and the inserted fragments were sequenced. A phylogenic tree was constructed by summarizing the results and on the basis of BLAST data searches for HrpW in P. atrosepticum and some other phytopathogenic bacteria (25). The effect of T3SS wild type and mutant on S. tuberosum tuber was investigated by maceration of the infiltrated tissues in some varieties of S. tuberosum tubers including Briz, Adretta, Zhuravinka, Lasunak, and Skarb.
All strains effectively destroyed S. tuberosum tubers but the extent of tissue maceration between wild-type and mutants was significantly different. Apart from the influence of T3SS mutation on maceration amounts, comparison of maceration of tubers also revealed differences in the degree of sensitivity of various types of S. tuberosum (Table 2). Also, some other varieties were infiltrated with the normal strain (as a control) and two of the most forceful strains of T3SS mutants (Table 3). The results show that the use of T3SS mutation can be a useful marker for detecting sensitivity of different varieties of S. tuberosum . As T3SS components can elicit a hypersensitivity response, it seems that the expression of hypersensitivity genes might explain the greater amounts of tuber maceration. Therefore, the degree of PR-5 (osmotin) expression was studied in S. tuberosum tubers infiltrated by different strains of P. atrosepticum (Table 4). The greater amount of PR-5 expression was associated with mutant strains of P. atrosepticum rather than the wild type (JN42). As the HIN gene is related to hypersensitivity response and is closely related to the PR-5, its gene expression was studied (7) (Table 5).
HrpW protein characteristics have been demonstrated by previous work (26). Moreover, the complete genomic sequences of the P. atrosepticum strain SCRI1043 (Eca1043) and assessment of its similarity to other members of the Enterobacteriaceae and other phytopathogenic bacteria has been described (27); There is a higher similarity between harpins and the cloned HrpW sequence of P. atrosepticum strain (JN42) and its encoded proteins (as shown by BLAST data-base search). A phylogenetic tree was constructed by the comparison of HrpW in JN42 strain and similar proteins in other phytopathogens (25) (Figure 1). A previously constructed phylogenetic tree based on the 16S rDNA revealed varying levels of taxonomic congruence for the structure of Erwinia, Brenneria and Pectobacterium (26). Moreover, based upon the predicted HrpW sequences, a phylogenetic tree was constructed. It was concluded that the bacterial group of Pectobacteria is divided into pectolytic and nonpectolytic types. PR-5 and HIN expression amounts in S. tuberosum tubers were dependent on bacterial T3SS function. It was suggested that HrpN and DspE proteins seemed to be avirulent factors. Considering the high reliability of the present phylogenetic tree, it is possible to divide phytopathogens into three groups. The first group includes Pseudomonas syringae and Pseudomonas avellanae while the second involves Pseudomonas cichorii and Pseudomonas viridiflava but Pectobacterium falls into the third group. Studies have suggested that hypersensitivity reactions in P. atrosepticum may be related to T3SS function (3, 4, 28) as it’s been suggested that one or more proteins secreted by T3SS were responsible for this phenotypic effect (as a result of interaction between host-plant and pathogen). There was a considerable increase in maceration activity (60 - 130%) in all T3SS mutant strains (JN504, HW1, JN502, TA85, VKE) compared with the wild type strain (JN42) (Tables 2 and Table 3). This was supported by previous reports ( 27 , 29 ). It had been expected that the HrpN and HrpW mutations in P. atrosepticum would decrease bacterial virulence because they have already been shown to reduce hypersensitivity reactions (Table 3 and Table 4). To examine whether S. tuberosum varieties play role in the amount of maceration, we collected five accessible varieties (Briz, Zhuravinka, Adretta, Lasunak and Skarb) and infiltrated them with P. atrosepticum strains. The results showed that, Arkhideya and Zhuravinka varieties of S. tuberosum were more sensitive while the least vulnerable ones seemed to be Delfin and Uladar (Table 3). Over expression of PR-protein in S. tuberosum tuber tissue by P. atrosepticum pectolytic enzymes was shown previously (30). Moreover, the influence of Pectobacterium carotovorum on PR-3 regulator gene of S. tuberosum has also been reported ( 31 ). Due to the PR-5 over expression (Table 4) in tubers affected by T3SS mutant strains of bacteria compared with the wild type (JN42), it seemed that high-level of expression of PR5 in S. tuberosum tubers was related to T3SS. In spite of this, in tubers infiltrated by HrpW mutants (including HW1 and VKW strains) PR-5 expression is significantly higher than the others (Table 4). Also, HIN expression in tubers infiltrated by T3SS mutants is higher than the wild-type (Table 5). Therefore, It was suggested that high amounts of maceration for infiltrated tubers by T3SS mutants appeared to be a useful marker for the identification of resistant tuber varieties, as S. tuberosum slices were significantly less macerated by JN42 strain than T3SS mutants such as VKE and JN502. In addition it was concluded that HrpN and DspE proteins had avirulence activity (Table 1).
This study was carried out at the Department of Molecular Biology, Biology Faculty, Belarusian State University. Authors are indebted to the supervisor, Professor Anatoli Evtushenkov for providing the opportunity to work in his group. His constructive guidance and feedback have been invaluable. Also, thanks to Professor Valeri Bakaev for the scientific edition of this study and E.A. Nickolaichik, associate professor of Molecular Biology Department, for providing mutant bacterial strains.
Implication for health policy/practice/research/medical education As secretion mechanisms in P. atrosepticum is similar to secretion mechanisms in human pathogens, consequently data obtained in this research could be counted as a good background for research projects in medicine.
Please cite this paper as Aghabozorgi S. Virulence Function of Pectobacterium atrosepticum Secretion System Mutants on Evaluation of Some Solanum tuberosum Resistance Genes. Iran J Biotech. 2013: 11(2): in press. DOI: 10.5812/ijb.11211
Authors’ Contribution This work was done by contribution of Potato Research Institution in Belarus.
Financial Disclosure Research was done on the basis of biological engineering and safety in Belarus.
Funding/ Support Commission of ministries in Belarus No.512 on 10.05.2005 confirmed the financial budget for this project.