Li Chen1
, Jianke Li1,2, Msatoshi Shirota3, Kouhei Ohnishi3 1. College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi'an 710119, Shaanxi Province, China;
2. University Key Laboratory of Food Processing Byproducts for Advanced Development and High Value Utilization, Shaanxi Normal University, Xi'an 710119, Shaanxi Province, China;
3. Research Institute of Molecular Genetics, Kochi University, Nankoku, Kochi 783-8502, Japan
Received: 3 May 2017; Revised: 22 May 2017; Published online: 25 July 2017
Foundation item: Supported by the Fundamental Research Funds for the Central Universities (GK201703068) and by the University Key Laboratory of Food Processing Byproducts for Advanced Development and High Value Utilization (1111090010)
Corresponding author: Li Chen, Tel/Fax:+86-29-85310517;E-mail:chenlisp@snnu.edu.cn.
Abstract: Objective By testing the internal bacterial growth of SKWP mutants generated in Ralstonia solanacearum strain OE1-1, we evaluated the contribution of these SKWP effectors to bacterial fitness towards host plants.Methods The deletion mutant and complementation mutant of R. solanacearum OE1-1 were constructed. A competition assay in mixed infection was adopted to evaluate the contribution of the SKWP to bacterial fitness in tobacco (Nicotiana tabacum cv. Bright Yellow) and eggplant (Solanum melongena cv. Senryo-nigou).Results Eggplant was the appropriate host for competitive index (CI) assay of R. solanacearum strain OE1-1. All 6 SKWP mutants affected bacterial fitness in eggplant to some extent while SKWP4 appeared to be most important.Conclusion The SKWP effectors were important for bacterial proliferation in eggplant tissues according to the CI analysis, which paving a new way for further identification of the function on whole SKWP family.
Key words: Ralstonia solanacearum OE1-1SKWPCI assaybacterial proliferation
应用竞争力指数分析Ⅲ型效应子SKWP对青枯菌在寄主植物体内增殖能力的影响
陈立1
, 李建科1,2, 成田雅敏3, 大西浩平3 1. 陕西师范大学食品工程与营养科学学院, 陕西 西安 710119;
2. 陕西师范大学食品加工副产物深度开发与高值化利用重点实验室, 陕西 西安 710119;
3. 高知大学分子遗传研究中心, 南国市 日本 783-8502
收稿日期：2017-05-03；修改日期：2017-05-22；网络出版日期：2017-07-25
基金项目：中央高校基本科研业务费（GK201703068）；校级重点实验室创新平台（1111090010）
通讯作者：陈立, Tel/Fax:+86-29-85310517;E-mail:chenlisp@snnu.edu.cn.
摘要：目的 研究Ⅲ型效应子SKWP对青枯菌OE1-1在寄主植物体内增殖能力的影响。方法 构建青枯菌RK7197（野生型突变体，带Gm抗性）和SKWP单基因缺失突变体（带PB抗性），通过竞争力指数分析SKWP各效应子对青枯菌OE1-1在叶片组织内增殖能力的影响。结果 竞争力指数适合在寄主植物茄子上分析各效应子功能，6个SKWP效应子对OE1-1细菌增殖能力影响不同，SKWP4影响最明显。结论 竞争力指数可提供一个新视野来分析SKWP各效应子对青枯菌OE1-1在寄主茄子上增殖能力的影响。
关键词：青枯菌OE1-1SKWP竞争力指数分析细菌增殖
Ralstonia solanacearum has been established as a model bacterium for plant pathology thanks to pioneering molecular and genomic studies[1-2]. This pathogen endangers the food safety in tropical and subtropical agriculture, especially in China, Bolivia, Bangladesh and Uganda. As for many bacterial pathogens, the main virulence determinant in R. solanacearum is the type Ⅲ secretion system (T3SS) which injects a number of effector proteins into plant cell causing lethal wilting disease in hosts or a hypersensitive response (HR) in resistant plants[3-4]. It has been reported that type Ⅲ secretion effectors (T3Es) are involved in virulence development in host plants but the mechanism remains unclear.
Generally pathogenicity assays based on quantification of wilting symptoms failed to detect a significant contribution of R. solanacearum T3Es in this process, thus revealing the collective effect of T3E in pathogenesis. So far there are three methods mainly for studying the contribution of the each single T3E to bacterial fitness in planta, (ⅰ) measure the bacterial population inside a given host plant; (ⅱ) measure the growth of P. syringae heterologously expressing R. solanacearum T3E in Arabidopsis[5-8]; (ⅲ) competitive index assay (CI assay) between co-infection wild type strain and mutant strains[9]. CI was defined as the mutant/wild-type ratio within the output sample divided by the corresponding ratio in the inoculum. In such in vivo competitive assay, the wild-type and mutant strains are co-inoculated in the same plant, thus reducing plant-to-plant variation and enhancing sensitivity in the detection of differential fitness between the two strains[9]. Macho et al. has successfully evaluated 12 T3E genes of strain GMI1000 to their contributions to bacterial fitness in host plants by using CI assay[10-11]. CI assay has been proved to be a sensitive and reliable assay that allows quantifying a differential fitness of R. solanacearum strains in planta.
In this study, we evaluated the contribution of SKWP effectors of the Japanese R. solanacearum strain OE1-1 to bacterial fitness in host plant by using CI assay. Strain OE1-1 (race1, biovor3) causes lethal wilting disease on tobacco and eggplant, encodes more than 70 T3Es and shares high homologous with those effectors in model strain GMI1000. SKWP effectors (6 members) contain a novel amino acid repeat domain, designated as SKWP repeats from its conserved motif. The studies on SKWP effectors of R. solanacearum in virulence activity in host plants has been well developed while their bacterial fitness in host plant is scarce. Therefore, we provided the evidence of SKWP's contribution to bacterial fitness by using CI assay. Analysis of the contribution of SKWP effectors will prove invaluable for understanding R. solanacearum invasion strategies and seeking new ways to prevent disease.
1 Materials and Methods 1.1 Bacterial strains, plasmids and growth conditions The bacterial strains and plasmids used for this study are described in Table 1. R. solanacearum strain OE1-1 (race1, biovar3) and its derivative mutants were streak on BG medium at 28 ℃. Escherichia coli DH12S (Invitrogen Corp., Carlsbad, CA, USA) and S17-1 were grown on Luria-Bertani (LB) medium and incubate at 37 ℃ overnight. Escherichia coli DH12S was used for plasmid construction and S17-1 was used in conjugation experiments. Antibiotics were used at the following concentrations: ampicillin (Ap), 100 mg/L; kanamycin (Km), 50 μg/mL; gentamicin (Gm), 50 mg/L; polymyxin B (PB), 50 mg/L; 5-bromo-4-chloro-3-indolyl-beta-D-galactopy-ranoside (X-gal), 40 mg/L, isopropyl-beta-D-thiogalactopyranosiade (IPTG), 100 μmol/L.
Table 1. R. solanacearum stains and plasmids used in this study
| Designation | Relevant characteristics | Reference or source |
| E. coli | ||
| DH12S | araD139 Δ(ara, leu)7697 ΔlacX74 galU galK mcrA Δ(mrr-hsdRMSmcrBC) rpsL deoR ?80dlacZΔM15 nupG recA1/F9proAB1 lacIq ZΔM15 | Invitrogen |
| S17-1 | thi pro hsdR- hsdM+ recA pro hsdR RP4-2-Tc::Mu-Km::Tn7 | [24] |
| Ralstonia solanacearum | ||
| OE1-1 | wild type (race 1 biovar 3) | [25] |
| RK7197 | OE1-1derivative, Gmr | This study |
| RK7029 | OE1-1 Δrsc3401 (skwp1) | This study |
| RK7058 | OE1-1 Δrsp1374 (skwp2) | This study |
| RK7028 | OE1-1 Δrsp0930 (skwp3) | This study |
| RK7033 | OE1-1 Δrsc1839 (skwp4) | This study |
| RK7050 | OE1-1 Δrsp0296 (skwp5) | This study |
| RK7032 | OE1-1 Δrsc2130 (skwp6) | This study |
| Plasmids | ||
| pBlueScript Ⅱ ks(+) | Phagemid sequencing vector, Apr | Stratagene, La Jolla CA, U.S.A. |
| pTNS2 | T7 transposase expression vector | |
| pUC18mini-Tn7T-Gm | Gmr on mini-Tn7T; for gene insertion in Gmr bacteria | [26] |
| pK18mobsacB | Allelic exchange vector, Kmr oriT sacB | [27] |
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1.2 Construction of single deletion mutant and complementation mutant Two 500-bp fragments were PCR amplified by PrimeStar HS DNA Polymerase (TaKaRa Bio, Otsu, Japan) using two pairs of primers (Table 2): OEC(P)xxxxA51 and OEC(P)xxxxB51 primers for upstream fragment, and OEC(P)xxxxA31 and OEC(P)xxxxB31 for downstream fragment. Then the fragments were cloned on pre-digested vector pBluescriptⅡ KS(+)/EcoR V to generate pBC(P)xxxx-5 and pBC(P)xxxx-3 plasmids. The EcoR Ⅰ-BamH Ⅰ fragment of pBC(P)xxxx-5 and the BamH Ⅰ-Hind Ⅲ fragment of pBC(P)xxxx-3 were ligated to pre-digested vector pK18mobsacB/EcoR Ⅰ-Hind Ⅲ to generate pKC(P)xxxx. The sequences of the plasmids were determined with Bigdye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) with primer either M13-47 (5′-TGTAAAACG ACGGCCAGT-3′) or RV-M (5′-CAGGAAACAGCT ATGACC-3′). pKC(P)xxxx was transferred from E. coli S17-1 into R. solanacearum strain. Deletion mutant strain was generated through two consecutive homologous recombination events (Figure 1). The plasmid pUC18-mini-Tn7T-Gm was electroporated with a transposase-containing helper plasmid pTNS2 into OE1-1 derivative. The transformant was selected on BG agar medium supplemented with Gm and PB. The insertion was verified using colony PCR with primer glms and Tn7R (Figure 2). The strain of OE1-1 derivative was named as RK7197 which carried resistance to gentamicin but did not affect virulence.
Table 2. Primers used in this study
| OEC3401A51 | TCCGCTTCCGATACGGTGAGTGGTC |
| OEC3401B51 | ggatccGGTTCCTGCACGCTTTGCATTCG |
| OEC3401A31 | ggatccACGCGGCCGCGCGGCGGCAGG |
| OEC3401B31 | ATCATCCTGCGCAGCTTCCTGCGGC |
| OEP1374A51 | ACGGCGGCAAACTGCTGCGC |
| OEP1374B51 | ggatccTTAGGGTTCTGCCGATGTTTTTATG |
| OEP1374A32 | ggatccCTGCTTCAGACGATGCAG |
| OEP1374B32 | ACGGTGGCGGCATAGGTC |
| OEP0930A51 | TGATCCCGGGTGAATCCATTCGCGGAC |
| OEP0930B51 | ggatccCCCCGATCCGCTCCCCTGCGTG |
| OEP0930A31 | ggatccTCGTACTTTCCGTGCAAAATC |
| OEP0930B31 | CAGCGCATCGCCGCGCCGATG |
| OEC1839A51 | CGTARGAGGCCCGCGCCG |
| OEC1839B51 | ggatccGCGGCGAGGGAGCGACCTG |
| OEC1839A31 | ggatccCCCAGGCGCGGCAACGGGC |
| OEC1839B31 | GTCATCGAGGTCGTCGCG |
| OEP0296A51 | TTCGCCGGTGGCCGCCTT |
| OEP0296B51 | ggatccAGGTTGTATTCACAATAGTTC |
| OEP0296A31 | ggatccTGACCCGGTACGCCGGCA |
| OEP0296B31 | CACCAACTACGGCGGGCAG |
| OEC2130A51 | GCGGCGATCATTGCGTCG |
| OEC2130B51 | ggatccTTCCAAAGCGTTGCGTGAGCG |
| OEC2130A31 | ggatccGCGCGTGCGGCCCCACG |
| OEC2130B31 | GTATCGATGAGCGCGGCAAG |
| glms | GCGCTCAAGCTCAAGGAGATC |
| Tn7R | CACAGCATAACTGGACTGATTTC |
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| Figure 1 Construction of single SKWP deletion mutants. A: schematic diagram of single skwp deletion using pK18mobSacB based on homologous recombination; B: colony PCR results of single skwp deletions. M: 10 kb DNA ladder. |
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| Figure 2 Construction of RK7197. A: structure of pUC18-mini-Tn7T-Gm; B: schematic diagram of site-specific insertion into the chromosome of R. solanacearum OE1-1; C: Colony PCR of RK7197. M: 1 kb DNA Ladder; lane 1-5: RK7197. |
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1.3 Plant tests R. solanacearum cells were incubated overnight in B medium and resuspended in 10 mmol/L MgSO4 at an optimal density at 600 nm (OD600) of 0.1 (1.4×108 CFU/mL). For root-cutting inoculation, bacterial suspension was poured onto the root wounded plants to achieve a final concentration of 107 CFU/mL. Plants were cultivated in a temperature-controlled culture room at 25 ℃ under 10000 lux (16 h light/8 h dark). Virulence assays were tested on four-week old eggplant (Solanum melongena cv. Senryo-nigou) and tobacco (Nicotiana tabacum cv. Bright Yellow) respectively. Eggplant plants were cultivated in rock-wool (Nittobo) and tobacco plants were cultivated in pots containing a mixture of vermiculite/peat moss (3:1). Each bacterial inoculation was tested on at least 4 plants and was carried out multi-replicates. Disease symptoms were scored daily for 16 days. Plants were rated according to a scale ranging: 0=no wilting; 1=1% to 25% wilting; 2=26% to 50% wilting; 3=51% to 75% wilting; and 4=76% to 100% wilting or dead.
1.4 Bacterial growth in infiltrated leaf tissue Internal bacterial populations were determined at selected time intervals after inoculation. Four-week old eggplant (Solanum melongena cv. Senryo-nigou) was chosen as host for leaf-infiltration. Bacterial cell suspensions (at 106 CFU/mL) in 10 mmol/L MgSO4 were infiltrated into plant leaves. Plants were grown at 25 ℃ (16 h light/8 h dark). Leaves were sampled every day. Leaf disks (an area of 0.38 cm2) were cut from infiltrated area by a sterile borer, transferred by a sterile forceps into a sterile tube containing 500 μL of sterile 10 mmol/L MgSO4, and crushed at 3000 r/min for 60 s with a 5 mm-diameter zirconia bead using Micro smash MS-100 (TOMY SEIKO). Standard 10-fold dilution plating onto BG medium supplemented with PB was carried out. Colonies were counted after 2-day incubation at 28 ℃, and the bacterial populations were calculated as CFU/cm2 of leaf area.
1.5 CI assays by leaf-infiltration Growth attenuation was identified by CI assay. Mutant and WT suspension (106 CFU/mL) were mixed at a ratio of 1:1 and then were injected on leaf by a blunt syringe[12]. Leaf tissue was smashed as mentioned above. Serial dilution of the smashed sample was spread onto BG medium with and without the corresponding antibiotic. Both of RK7197 (OE1-1 derivative, Gmr) and mutant could grow on medium with PB but only RK7197 survived on medium with PB and Gm (Figure 3). The CI was defined as the mutant/wild-type ratio within the output sample divided by the corresponding ratio in the inoculum. CI was calculated as following equation (Eq. 1).
 | 公式(Eq. 1) |
 |
| Figure 3 The flow diagram of CI assay on host plant by leaf-infiltration. Mutant and RK7197 (OE1-1 derivative, Gmr) are separated on BG medium supplemented with different antibiotics gentamicin (Gm) and polymyxin B (PB). |
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2 Results 2.1 Characteristic of SKWP effectors in Ralstonia solanacearum strain OE1-1 Plant pathogen R. solanacearum OE1-1 encodes a large repertoire (up to 70 candidates) of putative T3Es. These effectors are characterized with various internal repeats which are presumably involved in protein-protein interaction[13]. These effectors are shown in a list of GALA (seven members), AWR (five members), PopP (three members), SKWP (six members) and HLK (three members). SKWP family members contain a novel amino acid repeat domain from its conserved motif which is a type of α-helices structure known to mediate protein-protein interactions. SKWP4 contains 18 tandem SKWP repeats of a 42 aa motif and other members contain 12-15 SKWP repeats[14]. Genomic analysis of the skwp genes in strain OE1-1 showed some interesting features: three genes skwp4, skwp1, skwp6 (RSc1839, RSc3401, RSc2130) locate on the chromosome whereas skwp5, skwp3, skwp2 (RSp0296, RSp0930, RSp1374) live on the megaplasmid (Table 3).
Table 3. Characteristics of SKWP effectors
| Gene in OE1-1 | SKWP | Size/aa | Features | Distribution in Rs speciesa |
| RSc3401 | SKWP1 | 2574 | Heat/armadillo-related repeats | Variable |
| RSp1374 | SKWP2 | 2467 | Conserved | |
| RSp0930 | SKWP3 | 2208 | Conserved | |
| RSc1839 | SKWP4 | 2574 | Conserved | |
| RSp0296 | SKWP5 | 2298 | Variable | |
| RSc2130 | SKWP6 | 817 | Variable | |
| aDistribution is considered "conserved" when the gene is found in > 90% of a set of 45 R. solanacearum strains through comparative genomic hybridization, otherwise it is considered "variable". | ||||
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2.2 A competition assay of R. solanacearum in tobacco and eggplant leaves Macho et al. has developed a sensitive and reliable CI assay that allows quantifying the R. solanacearum bacterial fitness in planta by leaf-infiltration. Although stem injection and soil-drenching infection are main infection modes for pathogenicity assay with R. solanacearum, there is a complementation effect appears between wild type strain and mutant strain in mixed infection during the inoculation procedures, which indicated those two modes were not appropriate for CI assay. We infiltrated tobacco and eggplant leaves with wild type OE1-1 and its derivative strain RK7197. 500 μL bacterial suspension of RK7197 strain (106 CFU/mL) and 500 μL bacterial suspension of wild type strain OE1-1 (106 CFU/mL) were performed for single and mixed inoculation. CI was calculated as above Eq. 1. CI=1 would reflect that the mutant strain is able to grow as efficiently as the wild type, and CI < 1 would indicate that growth of the mutant strain is attenuated.
A control experiment was carried out to test whether mixed inoculation affects the growth of two equally virulent bacteria towards tobacco and eggplant (Figure 4). Bacterial population were calculated since day post inoculation and differentiated using antibiotic selection. Both WT and RK7197 strains grew to the same level in single and mixed inoculations in eggplant and tobacco (Figure 4-A, C). The CI value in eggplant was close to 1 which indicated that the bacterial multiplication was not affected by limiting thresholds or genetic drift (Figure 4-B). The mutant strain cannot be complemented by the wild-type or interference between co-inoculated strains barely appeared during CI analysis. However, the CI value in tobacco was less than 1 indicated that RK7197 bacterial growth was attenuated (Figure 4-D). These observation correlated to Macho's speculation that bacterial fitness could vary from one plant to another. Therefore, we chose eggplant as host to analyze the contribution of T3Es to bacterial fitness.
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| Figure 4 Bacterial growth and competitive index (CI) values of OE1-1 versus RK7197 individually or mixed inoculated in eggplant and tobacco leaves. RK7197 is a derivative of the wild type OE1-1 carrying a gentamicin resistance cassette that does not affect virulence. A: bacterial growth of OE1-1 and RK7197 at 3 days post inoculation (DPI), either individually inoculated or mixed-inoculated in eggplant by leaf infiltration; B: CI mean value generated from mixed inoculation of OE1-1 and RK7197 in eggplant leaf; C: bacterial growth of OE1-1 and RK7197 at 3 days post inoculation (DPI), either individually inoculated or mixed-inoculated in tobacco by leaf infiltration; D: CI mean value generated from mixed inoculation of OE1-1 and RK7197 in tobacco leaf. Each experiment is repeated in triplicate and error bars represent the standard error. |
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2.3 Contribution of SKWP effectors to bacterial fitness in eggplant Previous research suggests that most T3Es are collectively essential but individually dispensable for the ability to produce symptoms in plants. The contribution of single T3Es is hard to be identified during the pathogenicity process. Solé identified AWR2 as the major contributor of its family to virulence in tomato by a series of complementation experiments[8]. Chen found HLK2 played more important role than other two members by construction of multiple deletion mutant[15]. In this study, all six mutants with single deletion of skwp gene were pathogenicity on tobacco and eggplant by root-cutting (Table 4).
Table 4. Pathogenicity tests of single skwp deletion mutant on host plants by root-cutting
| Strain | Gene | Virulence | |||||||
| skwp1 | skwp2 | skwp3 | skwp4 | skwp5 | skwp6 | Eggplant | Tobacco | ||
| OE1-1 | + | + | + | + | + | + | Virulence | Virulence | |
| RK7029 | - | + | + | + | + | + | Virulence | Virulence | |
| RK7058 | + | - | + | + | + | + | Virulence | Virulence | |
| RK7028 | + | + | - | + | + | + | Virulence | Virulence | |
| RK7033 | + | + | + | - | + | + | Virulence | Virulence | |
| RK7050 | + | + | + | + | - | + | Virulence | Virulence | |
| RK7032 | + | + | + | + | + | - | Virulence | Virulence | |
| Tobacco (N. tabacum cv. Bright Yellow) and eggplant (Solanum melongena cv. Senryo-nigou) plants were inoculated with R. solanacearum cells to achieve the final concentration at 107 CFU/mL by root-cutting. "-" means the effector gene was deleted while "+" means the effector gene existed in the mutant strain. | |||||||||
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Take eggplant for instance, the contribution of each member was hard to clarify during the pathogenicity process. Although there were slight differences of wilting symptoms among six deletion mutants, the function of each SKWP member remains unclear (Figure 5). This observation suggested that plant pathogenicity assay was not sensitive enough to reveal subtle phenotypic differences.
 |
| Figure 5 Pathogenicity test of skwp single deletion mutants on eggplant (Solanum melongena cv. Senryo-nigou). Bacterial suspension was poured onto the root wounded plants to achieve a final concentration of 107 CFU/mL. Disease symptoms were scored daily for 16 days. Plants were rated according to a scale ranging of 0 to 4 (0, no wilting; 1, 1%-25% wilting; 2, 26%-50% wilting; 3, 51%-75% wilting; 4, 76%-100% wilting). Each bacterial inoculation was tested on at least 4 plants and was repeated in triplicate. The average and standard error were calculated. |
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To unravel the role of each SKWP effector in contributing to bacterial multiplication inside host plant, we used competition assay in mixed infection to analysis the single skwp gene behavior in eggplant. All deletion mutants were found to multiply as efficient as RK7197 except RK7033 which was the mutant of skwp4 deficiency. The bacterial population of all five mutants varied from 6-log to 7-log while the bacterial population of RK7033 was below this range (Figure 6-A). Interestingly, we found that RK7033 strain exhibited CI values significantly less than 1 which indicated that its fitness in eggplant tissues was reduced (Figure 6-B). Other five mutants showed the CI value slightly higher than 1 or close to 1. This suggested that SKWP effectors affected the bacterial proliferation on some extent while SKWP4 was most significant. This speculation co-related with previous study that SKWP4 of R. solanacearum GMI1000 provided a competitive advantage only on eggplant but not tomato and pea.
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| Figure 6 Bacterial growth and CI assay. A: bacterial growth of RK7197 and skwp deletion mutant at 3 days post inoculation in a mixed infection in eggplant leaves. B: CI mean value generated from mixed inoculation of RK7197 and single skwp deletion mutant in eggplant leaves. Each experiment is repeated in triplicate and error bars represent the standard error. |
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3 Discussion The plant pathogen R. solanacearum typically enters hosts through root wound and colonizes the water-transporting xylem tissue, spreading up into plant stem. It encodes a large repertoire (up to 70 candidates) of putative T3SS effectors. These effectors are major determinant controlling the virulence of bacterial pathogens on both mammal and plant hosts[16-17]. Generally, these effectors are collectively essential but are individually dispensable for wilting symptoms in plant. GALA effectors jointly contribute to pathogenicity much more on Arabidopsis than on tomato[18]. AWR gene family collectively contributes to bacterial virulence on tomato, although AWR2 is the major contributor to virulence. HLK2 (HLK family, three members) played an important role in bacterial fitness in planta. All these symptoms are explained to a functional accumulation among effectors. Since the normal plant pathogenicity assays was not sensitive enough to reveal subtle phenotypic differences[19-20]. Macho developed a CI assay to detect individual contribution of T3E to bacterial fitness in a more sensitive manner than monitoring symptom development in pathogenicity assay.
In present study, the CI assay on R. solanacearum OE1-1 bacterial fitness in different host plants was investigated and eggplant was proved to be appropriate for this novel assay. Furthermore, we identified the contribution of six skwp genes to their bacterial proliferation through CI assay. All six SKWP effectors were found to be required for bacterial growth in eggplant tissues but affected the bacterial fitness differently in planta. The mutant with the deletion of skwp4 exhibited a reduced bacterial fitness in eggplant while others not, which indicated SKWP4 effector play a important role than other members. Macho et al. identified several T3Es among tomato, eggplant and bean, and confirmed that the contribution to fitness of T3Es can differ quite significantly on different host plants[9]. Wild type strain and mutant strains carry kanamycin and gentamicin antibiotics respectively which evaluate the individual contribution among effectors sensitively and efficiently in his method. Our study distinguished the wild type strain and mutant strains by adding gentamicin and polymyxin B antibiotic. The identification was quantitative but not as sensitive as Macho's method. Therefore, the identification of SKWP to bacterial fitness required the mutants more representative characteristic. Further study on consecutive deletion mutant and complemented mutants of SKWP would encourage us characterizing the contribution of these effectors to bacterial fitness and broaden our knowledge of the virulence strategies developed by R. solanacearum.
However, we should be clear that the competition experiment in plant leaf tissues is a way to monitor bacterial fitness in specific environment which is not appropriate to be considered as a representative assay to replace traditional pathogenicity assay. Because R. solanacearum invasion stage was complex and multistage. The bacteria has to attach to the root, find nutrients, multiply, migrate into plant tissues, and then penetrate the xylem in order to cause wilting disease[21-23]. The current leaf-infiltration injection mode ignores several key infectious processes. Nevertheless, the CI assay provides a new sight to discriminate 6 skwp members' contribution on bacterial fitness in specific host plant. This knowledge will be helpful in the future to demonstrate functional involvement or functional independence of the whole SKWP family.
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