Characterization And Evaluation Of Biological And Disease Resistant Function Of Popw From Ralstonia Solanacearum

Ralstonia solanacearum is a major soil-borne plant pathogen in subtropical and tropical areas that naturally infects roots and multiplies in xylem vessels which has an unusually wide host range since plant species susceptible to the pathogen have been observed to occur in more than 200 plant species belonging to approximately 50 botanical families. Huge economic losses are caused annually by this pathogen in China and many other countries R. solanacearum is a very complex community with dishtingushed physiological characterization. It is difficult to control because strains from different regions and host have significant difference in pathogenicty, biovar and serology. Many scientists have done some researchs about R. solanacearum using many methods from different aspects.Harpins are extracellular proteins encoded by Gram-negative bacterial hrp (hypersensitive response and pathogenity) gene cluster. They are a major class of proteins that travel the pathway, elicit the HR (hypersensitive reaction) when infiltrated into the apoplast of leaf tissue. They are heat stable, rich in Gly and/or Ser, lack Cys, and differ in their primary sequences.popW(1143 bp in length) and its dervivates,popW(1-159)and popW(160-366) were cloned from Ralstonia solanacearum ZJ3721. The three genes were liagased to expression vector pET30a (+) and expressed successfully in Escherichia coli BL21 (DE3) induced by 1mM IPTG. The optimum concentration of IPTG for the expression of PopW is 0.01 mM. The fused PopW protein was purified from supernatant of its host by Ni column. Finally, the popW-deficient mutant of Ralstonia solanacearum was construced by homolougous recombination.This study identified a new extracellular bacterial protein, PopW, from R. solanacearum ZJ3721. PopW, like other known harpins, is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into plants, PopW induced rapid tissue collapse on tobacco (non-host). This hypersensitive reaction (HR)-eliciting activity of PopW was heat stable and protease sensitive. PopW of R. solanacearum consists of two domains connected by a Pro-and Ser-rich sequence. The N-terminal domain (amino acids 1 to 159) alone was able to elicit the HR, while its C-terminal region (amino acids 160 to 366) is homologous to pectate lyases (PLs). Neither this C-terminal fragment nor the full-length PopW showed PL activity. PopW was widely distributed in other R. solanacearum strains from different genetic diversity by PCR amplification identification. The subcellular location analysis via transfering popW and GFP fused gene to onion epimdermal meideated by Agrobacterium tumefaciens revealed that PopW targeted plant cell wall. PopW did not affect the virulence of R. solanacearum because popW-deficient mutants retained the wild-type ability to elicit HR on non-host plants and to cause disease on tomato host plants. Thus, we concluded that PopW is a cell-wall associated,hrpB-dependent,39.79 kilodaltons new harpin which is widely distributed in different R. solanacearum strains and has no influence on the pathogenicity of this pathogen.We have investigated the tobacco systemic acquire resistance induced by PopW protein from R. solanacearum. Nicotiana tobacum cv. Sam sun NN tobacco leaves were treated by 25μg/mL PopW followed by the infection of TMV onto these leaves, and the upper and lower two leaves of each one. The Lesion number infected by TMV is significantly less than blank control treated with sterile water and TMV. H2O2 bursted in the leaves sprayed by PopW, which reached the maximum of 1.972μM/g FW (fresh weight) 24h after the tobacco was treated. The activities of defence enzyme (PAL, POD and PPO) were also changed. The activity of PAL reached the climax of 1215.33 U/g 12h after treatment. POD and PPO achieved the peak activities 10h and 24h after treatment, which were 863.67 U/g and 1110.5 U/g, separately. The PR-1 gene in tobacco and Arabidopsis were up-expressed after PopW treatment, while the PR-1 did not express in transgenic nahG tobacco and Arabidopsis. In conclusion, plant resistance induced by PopW is systemic acquired resistance meditaed by salicylic acid.Verticillium wilt on eggplant is a devastating soilbrone disease. PopW was found effectively inhibiting Verticillium daliae infection by inducing eggplant acquired resistance. A chitinase-secreting strain CH2 was selectef from 353 strains isolated from rhizosphere of eggplant. This strain was identified to be of Bacillus cereus based on 16S rRNA gene sequence alignment and biochemical and physiological characteristics. The strain can live on chitin-Ayers (CA) medium which only has chitin as the carbon resource, the evaluation of its activity, combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), showed it can secreted a 15.0 KDa chitinase. On glass slides, germination of the fungal spores was effectively suppressed by the bacterial suspension, supernatant from the suspension, and 0.005%solution of chitinase extracted from the strain CH2. The optimum pH for chitinase was 7.1 and optimum temperature was 40℃. At that temperature, high-level chitinolytic activity was retained for 10 days. In greenhouse experiments, suspension of the cells of the CH2 strain reduced the severity of Verticillium wilt on eggplant by 69.69%,25μg/mL PopW solution, CH2 supernatant by 57.46 and 54.04%, and the enzyme diluted to 0.01% strength by 53.13% in 14 days. Strain CH2 and its chitinase have good commercial potential in controlling Verticillium wilt.