Isolation of an insertion sequence from Ralstonia solanacearum race 1 and its potential use for strain characterization and detection

A new insertion sequence (IS), IS1405, was isolated and characterized from a Ralstonia solanacearum race 1 strain by the method of insertional inactivation of the sacB gene. Sequence analysis indicated that the IS is closely related to the members of IS5 family, but the extent of nucleotide sequence identity in 5' and 3' noncoding regions between IS1405 and other members of IS5 family is only 23 to 31%. Nucleotide sequences of these regions were used to design specific oligonucleotide primers for detection of race 1 strains by PCR. The PCR amplified a specific DNA fragment for all R. solanacearum race 1 strains tested, and no amplification was observed with some other plant-pathogenic bacteria. Analysis of nucleotide sequences flanking IS1405 and additional five endogenous IS1405s that reside in the chromosome of R. solanacearum race 1 strains indicated that IS1405 prefers a target site of CTAR and has two different insertional orientations with respect to this target site. Restriction fragment length polymorphism (RFLP) pattern analysis using IS1405 as a probe revealed extensive genetic variation among strains of R. solanacearum race 1 isolated from eight different host plants in Taiwan. The RFLP patterns were then used to subdivide the race 1 strains into two groups and several subgroups, which allowed for tracking different subgroup strains of R. solanacearum through a host plant community. Furthermore, specific insertion sites of IS1405 in certain subgroups were used as a genetic marker to develop subgroup-specific primers for detection of R. solanacearum, and thus, the subgroup strains can be easily identified through a rapid PCR assay rather than RFLP analysis.     

The phenotype and pathogenicity of Ralstonia solanacearum transformed under prolonged stress of excessive exogenous nitrogen

Bacterial wilt, caused by soil-borne pathogen Ralstonia solanacearum, is a serious disease in many plants such as Solanaceae. To investigate the effects of accumulated nitrogen in soil on the phenotype and pathogenicity of the R. solanacearum, a serial passage experiment (SPE) was designed. Specifically speaking, minimal medium supplied with a slight excess of ammonium sulphate (AS) or ammonium nitrate (AN) was used to simulate the nutrition of soil containing excess nitrogen. During the period of 30 SPE, the phenotype, pathogenicity and relative expression of nitrogen metabolism genes in R. solanacearum were monitored. Phenotypic analysis results illustrated that the colony morphology of R. solanacearum changed after long-term culture, from high virulence colonies with strong fluidity to small, round non-mucoid colonies; The strain after prolonged stress of excessive exogenous nitrogen was a no-virulence phenotype conversion type (PC-type). The time for a change in colony morphology to occur after exposure to exogenous AS or AN was significantly less than the untreated samples, which treated without exogenous nitrogen. The results of pathogenicity also demonstrated that the cultures treated with exogenous AN or AS reduced virulence more quickly than the control. The disease index of 10 SPE with AN treatment or AS treatment was 89% or 68% lower than that of the control, respectively. In addition, as the incubation time increased, the swimming motility and the number of biofilms formation of the cultures were significantly changed under both treatments in comparison to the untreated samples. Furthermore, the relative expression of the nitric oxide reductase norB gene in the cultures treated with AN was 1.51-fold higher compared with the control after 30 SPE. These results indicated that excessive nitrogen supply in the environment could accelerate the transformation of R. solanacearum from high virulence wild-type into a PC-type, probably for the purpose of adapting to the adverse environment.     

Spontaneous mutations in a regulatory gene induce phenotypic heterogeneity and adaptation of Ralstonia solanacearum to changing environments

An evolution experiment with the bacterial plant pathogen Ralstonia solanacearum revealed that several adaptive mutations conferring enhanced fitness in plants arose in the efpR gene encoding a regulator of virulence and metabolic functions. In this study, we found that an efpR mutant systematically displays colonies with two morphotypes: the type S (‘smooth’, similar to the wild type) and the type EV (‘efpR variant’). We demonstrated that the efpH gene, a homologue of efpR, plays a key role in the control of phenotypic heterogeneity, the ΔefpR-ΔefpH double mutant being stably locked into the EV type. Using mixed infection assays, we demonstrated that the type EV is metabolically more proficient than the type S and displays fitness gain in specific environments, whereas the type S has a better fitness into the plant environment. We provide evidence that this efpR-dependent phenotypic heterogeneity is a general feature of strains of the R. solanacearum species complex and could occur in natural conditions. This study highlights the potential role of phenotypic heterogeneity in this plant pathogen as an adaptive trait to changing environments.     

Genetic rearrangement associated with in vivo mucoid conversion of Pseudomonas aeruginosa PAO is due to insertion elements.

The conversion of Pseudomonas aeruginosa PAO to the mucoid phenotype has been reported for a chronic pulmonary infection model in rats (D. E. Woods, P. A. Sokol, L. E. Bryan, D. G. Storey, S. J. Mattingly, H. J. Vogel, and H. Ceri, J. Infect. Dis. 163:143-149, 1991). This conversion was associated with a genetic rearrangement upstream of the exotoxin A gene. To characterize the genetic rearrangement, the region upstream of the toxA gene was cloned from PAO, PAO-muc (a mucoid strain), and PAO-rev (a nonmucoid revertant strain). The nucleotide sequence of a 4.8-kb fragment from PAO-muc was determined. A+T-rich regions of approximately 2 kb (IS-PA-4) and 0.4 kb (IS-PA-5) were identified in this fragment. DNA probes constructed internal to these regions hybridized to PAO-muc but not to PAO or PAO-rev, suggesting that PAO-muc contains an insertion element. Sequence analysis of the nonmucoid clones indicated that a 2,561-bp fragment corresponding to IS-PA-4 and a 992-bp fragment corresponding to IS-PA-5 were not present in PAO or PAO-rev. Both nonmucoid clones, however, contained in the same location as IS-PA-4, a 1,313-bp region which was not present in PAO-muc. DNA probes complementary to this sequence, designated IS-PA-6, did not hybridize with PAO-muc, indicating that this sequence had been replaced upon conversion to the mucoid phenotype. Between IS-PA-4 and IS-PA-5 there was a 500-bp sequence which was 94% identical to the 500-bp sequence downstream of IS-PA-6. These insertion elements had some DNA sequence similarity to plasmid and transposon sequences, suggesting that they may be of plasmid origin. IS-PA-4 and IS-PA-5 were shown also to be present in two mucoid isolates from cystic fibrosis patients. The insertions occurred in the same location upstream of the toxA gene, suggesting that this type of genetic recombination may also be associated with mucoid conversion in some P. aeruginosa clinical isolates.     

Conserved sequence elements associated with exon skipping

One of the major forms of alternative splicing, which generates multiple mRNA isoforms differing in the precise combinations of their exon sequences, is exon skipping. While in constitutive splicing all exons are included, in the skipped pattern(s) one or more exons are skipped. The regulation of this process is still not well understood; so far, cis- regulatory elements (such as exonic splicing enhancers) were identified in individual cases. We therefore set to investigate the possibility that exon skipping is controlled by sequences in the adjacent introns. We employed a computer analysis on 54 sequences documented as undergoing exon skipping, and identified two motifs both in the upstream and downstream introns of the skipped exons. One motif is highly enriched in pyrimidines (mostly C residues), and the other motif is highly enriched in purines (mostly G residues). The two motifs differ from the known cis-elements present at the 5′ and 3′ splice site. Interestingly, the two motifs are complementary, and their relative positional order is conserved in the flanking introns. These suggest that base pairing interactions can underlie a mechanism that involves secondary structure to regulate exon skipping. Remarkably, the two motifs are conserved in mouse orthologous genes that undergo exon skipping.     

Distribution and sequence analysis of a family of type ill-dependent effectors correlate with the phylogeny of Ralstonia solanacearum strains

In Ralstonia solanacearum, we previously have reported on the characterization of popP1 and popP2 genes. These genes encode type III-dependent pathogenicity effectors related to the large family of AvrRxv/YopJ cysteine proteases that are shared among pathogens of plants and animals. In this study, we identify a third gene, named popP3, that is inactivated in the genome sequence of strain GMI1000 by insertion of a copy of the insertion sequence ISRso13. The three popP genes are localized on two large chromosomal pathogenicity islands, with popP1 and popP2 being present on the same island. Phylogenic analysis demonstrated that the PopP2 and PopP3 proteins are clearly distinct from other effectors of this family previously characterized in plant and animal pathogens. Analysis of the distribution and allelic variations of the three genes in 30 strains representative of the biodiversity of R. solanacearum established that popP genes are distributed widely among strains from two of the three phyla previously defined on the basis of the structure of the core genome. Sequencing of the popP genes from the different strains revealed limited allelic variations at the three loci but did not show evidence of recombination between the popP genes. Limited allelic variation together with occurrence of insertion sequences within or in the close vicinity of popP genes and the presence of gene duplications in these pathogenicity islands suggest that genomic rearrangements might be a major evolutionary driving force controlling evolution of the genes encoded in these regions. The implications of these observations in terms of bacterial evolution, gene acquisition, and horizontal gene transfers are discussed.     

A non-parametric approach to translating gene region heterogeneity associated with phenotype into location heterogeneity

MOTIVATION: The analysis of genetic data poses statistical problems in the form of high dimensionality with small sample sizes. The construction of a composite gene region (sequence pair) heterogeneity measure is one technique for reducing the dimensionality of the problem. This approach however is not without cost, since the contribution of locations to observed gene region differences between groups becomes entangled in this summary measure. This is problematic since it is of scientific interest to identify locations that together depict phenotype. RESULTS: A method is proposed for relating observed gene region heterogeneity back to the location level. In the spirit of a factor analysis-type setting, the approach focuses on identifying a latent variable structure among locations to explain within and between group genetic differences associated with phenotype. The method is flexible for identifying either the additive contribution from individual locations or the additive contribution from a group of locations, to observed gene region heterogeneity, depending upon the weighting scheme used in constructing a gene region heterogeneity measure. The approach is illustrated with clinical trial data, where the problem of altered HIV drug susceptibility is examined through characterizing location contributions to HIV protease gene region differences associated with a phenotypic treatment response. AVAILABILITY: The Splus (MathSoft, Inc. S-Plus 2000, Seattle, WA, 1999) developed menu-driven functions for obtaining results, GENE_ S (J.Kowalski, Harvard School of Public Health, Boston, MA 2001), is available from the author upon request.     

Interactions of Ralstonia solanacearum and Pectobacterium carotovorum with Meloidogyne incognita on potato

Effect of interactions of Meloidogyne incognita with Ralstonia solanacearum and interaction of M. incognita with Pectobacterium carotovorum were studied in sequential and simultaneous inoculations on potato (Solanum tuberosum). Inoculation of M. incognita caused a lesser reduction in plant growth than caused by R. solanacearum. Inoculation of M. incognita plus R. solanacearum caused a greater reduction in plant growth than the damage caused by either pathogen. Inoculation of M. incognita prior to R. solanacearum resulted in a greater reduction in plant growth than R. solanacearum was inoculated prior to M. incognita. However, inoculation of M. incognita or P. carotovorum caused similar reduction in plant growth. Inoculation of P. carotovorum prior to M. incognita caused lesser reduction in plant growth than simultaneous inoculation of both pathogens. Inoculation of M. incognita caused galling in potato roots but the size of galls was small. Inoculation of P. carotovorum or R. solanacearum with M. incognita had adverse effect on galling and nematode multiplication. Wilting or soft rot index was 3 when R. solanacearum or P. carotovorum was inoculated alone. In other treatments, where R. solanacearum or P. carotovorum was inoculated with M. incognita, wilting or soft rot indices were 5.     

Histopathology of Susceptible and Resistant Capsicum annuum Cultivars Infected with Ralstonia solanacearum

The vascular colonisation of resistant and susceptible hot chilli (Capsicum annuum) cultivars by Ralstonia solanacearum was examined using transmission electron microscopy. Tap roots of artificially-inoculated plants, grown in sterilised soil were investigated to observe the morphological barriers involved in the restriction of bacterial spread. In the resistant cultivar, several responses induced in response to bacterial infection, were observed. First, a cell wall coating material developed together with swelling of the primary wall of the xylem vessels, limiting the bacterial spread. Second, formation of various types of vesicles in the vascular parenchyma cells, which enveloped the bacterial mass and also partly restricted the pathogen spread. Third, induction of hypersensitive reaction in the xylem vessels resulted in the distortion and lysis of the bacteria. In the susceptible cultivar, vascular coating, production of vesicle and induction of hypersensitive reaction were not observed and bacterial spread was not limited. Rapid vascular colonisation of the susceptible cultivar seemed to be generalised which resulted in the rapid wilting of affected plants. Other reactions involved in both resistant and susceptible cultivars include disorganisation of cytoplasm of parenchyma cells, disintegration of nuclei, and rupturing of xylem vessel walls. The restriction of pathogen spread associated with the resistance in C. annuum to bacterial wilt was mainly attributed to some induced, morphological and physical barriers.     

The population genetic test Tajima's D identifies genes encoding pathogen‐associated molecular patterns and other virulence‐related genes in Ralstonia solanacearum

The detection of pathogen‐associated molecular patterns (PAMPs) by plant pattern recognition receptors (PRRs) is an essential part of plant immunity. Until recently, elf18, an epitope of elongation factor‐Tu (EF‐Tu), was the sole confirmed PAMP of Ralstonia solanacearum, the causal agent of bacterial wilt disease, limiting our understanding of R. solanacearum–plant interactions. Therefore, we set out to identify additional R. solanacearum PAMPs based on the hypothesis that genes encoding PAMPs are under selection to avoid recognition by plant PRRs. We calculated Tajima's D, a population genetic test statistic which identifies genes that do not evolve neutrally, for 3003 genes conserved in 37 R. solanacearum genomes. The screen flagged 49 non‐neutrally evolving genes, including not only EF‐Tu but also the gene for Cold Shock Protein C, which encodes the PAMP csp22. Importantly, an R. solanacearum allele of this PAMP was recently identified in a parallel independent study. Genes coding for efflux pumps, some with known roles in virulence, were also flagged by Tajima's D. We conclude that Tajima's D is a straightforward test to identify genes encoding PAMPs and other virulence‐related genes in plant pathogen genomes.     

Measurement of Horizontal and Vertical Movement of Ralstonia solanacearum in Soil

Two model systems were constructed to measure horizontal and vertical movement of bacteria in soil. These systems were applied to measuring movement of Ralstonia solanacearum (race 1, biovar 3), a causal agent of bacterial wilt of tomato, in andosol and sand at 28°C. The first system was used to measure horizontal movement of the bacteria in soil packed in a narrow horizontal frame. Suspension of the pathogen was applied to soil at one end of the frame, and bacterial number per gram of soil was measured over distance from the inoculation point after 4 days. Horizontal movement of R. solanacearum in supersaturated soil, but without flow, was possibly due to diffusion and the front advanced at 2.2 cm/day in andosol, and at 8.1 cm/day in sand. Using the same experimental system, but applying water inflow to one end of the frame only, the bacterium was detected at the front of water in andosol and sand. The front of the distribution advanced at 20.4 cm/h in andosol and 66.3 cm/h in sand. In the second experimental system, a cylinder of soil packed in a short tube was soaked with water, and soil at the top of the tube was inoculated with bacterial suspension. Immediately, soil cylinders were turned upward, and the bacterial number per gram of soil was measured along vertical distance from the inoculation point after 7 days. Using the system with andosol, the capillary water front rose to 32.5 cm over 7 days after inoculation, and R. solanacearum reached to 18.8 cm height. In sand, capillary water rose to 20.0 cm and the bacteria reached to 16.3 cm height.     

Changes in mumps virus gene sequence associated with variability in neurovirulent phenotype

Mumps virus is highly neurotropic and, prior to widespread vaccination programs, was the major cause of viral meningitis in the United States. Nonetheless, the genetic basis of mumps virus neurotropism and neurovirulence was until recently not understood, largely due to the lack of an animal model. Here, nonneurovirulent (Jeryl Lynn vaccine) and highly neurovirulent (88-1961 wild type) mumps virus strains were passaged in human neural cells or in chicken fibroblast cells with the goal of neuroadapting or neuroattenuating the viruses, respectively. When tested in our rat neurovirulence assay against the respective parental strains, a Jeryl Lynn virus variant with an enhanced propensity for replication (neurotropism) and damage (neurovirulence) in the brain and an 88-1961 wild-type virus variant with decreased neurotropic and neurovirulent properties were recovered. To determine the molecular basis for the observed differences in neurovirulence and neuroattenuation, the complete genomes of the parental strains and their variants were fully sequenced. A comparison at the nucleotide level associated three amino acid changes with enhanced neurovirulence of the neuroadapted vaccine strain: one each in the nucleoprotein, matrix protein, and polymerase and three amino acid changes with reduced neurovirulence of the neuroattenuated wild-type strain: one each in the fusion protein, hemagglutinin-neuraminidase protein, and polymerase. The potential role of these amino acid changes in neurotropism, neurovirulence, and neuroattenuation is discussed.     

Specific and sensitive detection of Ralstonia solanacearum in soil with quantitative, real-time PCR assays

Aims:  The aim of this study was to develop a sensitive and an effective method suitable for large-scale detection and quantification of Ralstonia solanacearum in soil.
Methods and Results:  Based on the specific sequence of R. solanacearum strain G1000, the primer pair R.sol1-R.sol2 and the TaqMan probe Rs-pro were designed, and specific and sensitive PCR detection methods were successfully established. The detection limit was 100 fg μl⁻¹ DNA in conventional PCR and 1·2 fg μl⁻¹ in real-time PCR. By combining real-time PCR with the modified protocols to extract DNA from soil, it was possible to achieve real-time detection of R. solanacearum in soil, and the degree of sensitivity was 100 fg μl⁻¹. To detect inhibition in soil samples, an exogenous internal positive control (IPC) was included preventing false negative results, and IPC was successfully amplified from all samples tested. The methodology developed was used to detect the presence of R. solanacearum in tobacco fields in China.
Conclusions:  The real-time PCR combined with the protocol to extract DNA from soil led to the development of a specific, sensitive and rapid detection method for R. solanacearum in soil.
Significance and Impact of the Study:  The real-time PCR improves the detection sensitivity and specificity and provides an important tool for routine detection of R. solanacearum in soil samples and for epidemiological and ecological studies.     

Induction of resistance and expression of defense-related genes in tobacco leaves infiltrated with Ralstonia solanacearum

Ralstonia solanacearum 8107 (8107) is non-pathogenic to tobacco and elicits the hypersensitive response (HR). In Nicotiana tabacum cv. Samsun NN leaves infiltrated with 8107, acquired resistance to challenging tobacco mosaic virus (TMV) was induced 2-6 d after 8107-infiltration. hsr203J and hin1 genes were expressed only in the 8107-infiltrated area. On the other hand, the expression of PR-1a and PR-1b genes was not detected in the 8107-infiltrated area, but in areas other than that developing the HR. Expression of these PR-1 genes was regulated simultaneously and the kinetics of the expression was dependent on the distance from the infiltration area. Therefore, diffusible signal(s) might be produced in HR-causing cells and transmitted to peripheral cells resulting in expression of PR genes. In NahG10 tobacco infiltrated with 8107, the HR was induced but resistance to TMV was not. Analysis using NahG10 tobacco also showed that the salicylic acid (SA)-dependent signal regulated the expression of hsr203J and PR-1a, but not that of hin1 and PR-1b. These results suggest that resistance of tobacco to 8107 is SA-independent and involves a quite different mechanism from acquired resistance to TMV induced by 8107-infiltration which is SA-dependent.     

茄科雷尔氏菌脂酰-CoA合成酶的功能鉴定

【目的】茄科雷尔氏菌是一种常见的农作物致病菌,引起植物青枯病。研究其脂肪酸代谢途径将有助于寻找新的抗菌药物靶点,为防治青枯病害提供新的思路。【方法】利用大肠杆菌FadD序列,进行同源比对发现茄科雷尔氏菌GMI1000中RSc2857(RsFadD)具有较高的相似性,推测其具有脂酰-CoA合成酶活性。采用PCR扩增方法获得RsfadD基因,连入表达载体pBAD24M后互补大肠杆菌fadD突变株,并检测转化子的生长情况。RsfadD与pET-28b连接后,在大肠杆菌BL(DE3)中表达,并利用Ni-NTA纯化获得带有组氨酸标签的RsFadD,体外测定RsFadD的活性。利用同源重组方法,获得RsfadD敲除突变株,分析突变株的生长性状。【结果】RsfadD异体互补大肠杆菌fadD突变株,恢复突变株在以脂肪酸为碳源的基础培养基上生长。体外活性测定RsFadD具有脂酰-CoA合成酶活性,对不同链长的脂肪酸都具有活性,但活性低于大肠杆菌FadD。RsfadD突变株在添加不同链长脂肪酸的基础培养上仅能微弱生长,而在丰富培养基上生长无差异。【结论】茄科雷尔氏菌中RsfadD编码脂酰-CoA合成酶,在脂肪酸利用过程中发挥重要作用。但RsfadD突变株在基础培养基上微弱生长,说明茄科雷尔氏菌基因组中还有其他的脂酰-CoA合成酶基因。以上研究结果为进一步研究茄科雷尔氏菌中脂酰-CoA合成酶以及脂肪酸利用机制奠定了基础。     

青枯菌致病机理及作物抗青枯病研究进展

青枯菌(Rdstonia solancearum)是引起植物青枯病的病原细菌.青枯菌通过T3S(Ⅲ型分泌系统)、T2S(Ⅱ型分泌系统)等分泌系统将多种毒性因子输送到胞外使寄主植物致病.转基因抗病、培育抗性品种和生物防治是防治青枯病的主要途径.