| Ralstonia solanacearum,a soil borne gram-negative plant pathogenic bacterium,is a causal agent of bacterial wilt disease on more than 450 plant species worldwide.It is currently ranked as one of the 10 most destructive plant-pathogenic bacteria due to its caused severe losses in many economically important plants worldwide,such as tomato,potato and tobacco.To date,the bacterial wilt diseases have been reported in nearly thirty provinces in China,which is particularly severe in tomato and tobacco plants in the Yangtze River Basin and some southern provinces of china,and seriously threatens our agricultural production.To invade host plants,R.solanacearum produces abundant pathogenicity factors,including virulence factors(so-called type Ⅲ effectors,T3Es),extracellular proteases(such as cellulase,pectinase and endoglucanase),extracellular polysaccharides(EPS)and small molecule toxins(such as ferritin),and the bacteria uses a complex network to globally regulate production of these pathogenicity factors.Among them,a syringe-like type Ⅲ secretion system(T3SS)plays a key role on infection of R.solanacearum toward host plants and it is especially produced in the early stage of infection.The T3 SS is conserved in many other gram-negative pathogenic bacteria of animals and plants,and bacteria use the T3 SS to inject T3 Es into host cytosol to subvert host defense.Expression of the T3 SS and T3 E genes is directly controlled by Hrp B,an Ara C-family of transcriptional regulator,and expression of hrp B is positively regulated by two close paralogs Hrp G and Prh G,which are two-component system response regulators and can respond to host signals or some mimic signals by phosphorylation.Expression of hrp G is activated by a Prh A-Prh I/R-Prh J signaling cascade but negatively regulated by a global regulator Phc A.Expression of prh G is independent from the Prh A signaling cascade but positively regulated by Phc A.Phc A is well known to be quorum sensing-dependent,and R.solanacearum might switch from using Hrp G to Prh G for hrp B activation in a cell density-dependent manner.To further elucidate the complex regulation on the R.solanaceartum T3 SS,we previously screened a subset of T3SS-regulating candidates with transposon mutagenesis.RSc2427 and RSc0454 were identified among the transposon mutants of interest,and we here focus on these two novel T3SS-regulating candidates to investigate their functional roles,regulation of the T3 SS,and contribution to pathogenicity of R.solanacearum.(1)A CysB regulator positively regulates cysteine synthesis,expression of type Ⅲ secretion system genes,and pathogenicity in Ralstonia solanacearumR.solanacearum RSc2427 is annotated as a Lys R-family of CysB-like transcriptional regulator,which is responsible for cysteine synthesis in many bacteria.The cys B mutants were indeed cysteine auxotrophs that failed to grow in minimal medium.Supplementary cysteine substantially restored impaired growth of cys B mutants in the minimal medium.Genes of cys U and cys I regulons are annotated for cysteine synthesis,and CysB positively regulated these expressions.Supplementary cysteine in the diluted Hoagland medium at 0.25 m M or higher was extremely pernicious that withered and dried tomato cuttings quickly within 1-2 days,while that at 0.12 m M was competent for growth of tomato cuttings.After treatment with supplementary cysteine(0.1 m M)for 2 days,tomato cuttings were subjected to inoculation by the petiole-inoculation method.The cys B mutants grew slightly in host plants but failed to wilt tomato plants.Supplementary cysteine substantially restored impaired growth of cys B mutants in inside host plants,indicating that the impaired in planta proliferation of cys B mutants is partially due to insufficient cysteine inside host plants.Moreover,CysB positively regulated expression of the T3 SS genes both in vitro and in planta through the Prh G to Hrp B pathway,whilst impaired expression of the T3 SS genes in cys B mutants was independent of growth deficiency under nutrient-limited conditions.CysB was also demonstrated to be required for production of the exopolysaccharide and swimming motility,which contribute jointly to host colonization and infection process of R.solanacearum.All these results characterized CysB as a novel regulator on the T3 SS expression in R.solanacearum.These results can also provide novel insights into understanding of various biological functions of CysB regulators and complex regulatory networks on the T3 SS in R.solanacearum.(2)Involvement of a FAD-Linked Oxidase RSc0454 for Expression of the Type Ⅲ Secretion System genes and Pathogenicity in Ralstonia solanacearumR.solanacearum RSc0454 is predicted as a FAD-linked oxidase,which contains distinct domains of lactate dehydrogenase(LDH)and succinate dehydrogenase(SDH).The rsc0454 mutant grew normally in rich medium but grew faintly in host plants,and failed to grow in minimal medium.Supplementary succinate but not lactate could substantially restore some phenotypes of rsc0454 mutants,including faint growth in host plants,diminished growth in the minimal medium,and lost pathogenicity toward host plants,indicating that diminished pathogenicity of the rsc0454 mutant is possibly due to insufficient succinate in host plants.Rsc0454 is involved in expression of the T3 SS genes both in vitro and in planta is not because of growth deficiency.Expression of T3 SS genes is directly controlled by a master regulator,Hrp B,and hrp B expression is positively regulated by Hrp G and Prh G in parallel ways.Deletion of rsc0454 substantially reduced expression levels of hrp B and T3 SS both in vitro and in planta.Moreover,RSc0454 is revealed to be required for the T3 SS expression via Hrp G and Prh G through some novel pathway.Supplementary succinate(20 m M)at low concentrations did not restore growth of rsc0454 mutant in minimal medium,the addition of 40 m M succiniate acid significantly restored the growth of mutant in minimal medium,while high concentration of succiniate acid(100 mm)completely inhibited the growth of R.solanacearum.Supplementary NADH at concentrations of 0.1 to 5 m M or NAD+ at 0.1 m M did not alter growth of the wild type strain,whereas supplementary NAD+ at 5 m M diminished growth of the wild-type strain in minimal medium,indicating that excess NAD+ might disturb redox status inside cells.It is striking that supplementary.Supplementary NADH or NAD+ at 0.1 m M did not restore growth of the RSc0454 mutant in the minimal medium,whereas they began to partially restore growth of RSc0454 mutants only in the presence of succinate at 10 m M in the minimal medium at 36 hpi.Supplementary succinate and NADH and NAD+ together restored redox balance inside RSc0454 mutant cells.The unbalanced redox in the RSc0454 mutant might be responsible for its lack of growth in the minimal medium.All these results provide novel insights into understanding of various biological functions of this FAD-linked oxidase RSc0454 and involvement of the redox balance on the T3 SS expression in R.solanacearum.In conclusion,CysB transcription factor indirectly affects T3 SS and infection process while regulating cysteine synthesis,while RSc0454(FAD-linked oxidase)indirectly affects T3 SS and infection process while affecting intracellular redox balance.These results show that R.solanacearum uses extremely complex regulatory network to regulate T3 SS to complete the infection of host plants.The results of this study help to deeply understand the multiple biological functions of CysB transcription factor and RSc0454 oxidase and the complex network of Ralstonia solanacearum regulating T3 SS,and finally provide new insights for comprehensive understanding of the pathogenic mechanism of R.solanacearum and the prevention and control of plant bacterial diseases. |
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