The Physiological Effect Of Two Bacteriophytochromes From Agrobacterium Fabrum C58 In Horizontal Gene Transfer

Phytochrome is of photoreceptor which makes respond to red and far-red light.It is widely distributed in plant,bacterium and fungi.Normally,it has two stable spectral forms,i.e.Pr(red light absorbing state)and Pfr state(far-red light absorbing state).Numerous evidence revealed that plant phytochrome is involved in the regulation of plant photo-morphogenesis.However,up to date,little useful information is available concerning on the physiological function of bacteriophytochrome(BphP)from photosynthetic and non-photosynthetic bacterium,especially in non-photosynthetic bacterium.Instead,people payed more attention on their structure and photochemical characteristic.Agrobacterium fabrum C58,one non-photosynthetic bacterium,had been widely used as one common transgenic vector mainly due to its capability of horizontal gene transfer(HGT),including transformation and conjugation.Meanwhile,Two BphPs named Agp1 and Agp2 were identified via DNA sequencing.Although Tilman's group found that both Agp1 and Agp2 exerted some effects on the transformation of A.fabrum C58 against Arabidopsis thaliana and cucumber.However,no systematic studies have been done on the relationship between these BphPs and transormation.In addition,given the conjugation in A.fabrum C58,it is also unclear if the conjugation of A.fabrum C58 would be affected by Agp1 or Agp2.Because there is no work reported in such a field.Therefore,in this thesis,we focused on the roles of BphPs from A.fabrum C58 in transformation and conjugation.The results will not only broaden our knowledge on the physiological function of BphPs,but also provide the therotical support for the mechanism of both transformation and conjugation.In order to explore their physiological function in HGT,we firstly constructed the Agp1 and Agp2 single or double knockout mutants through homologue recombinant.Meanwhile,we also obtained the complementation and over-expression strains by using pBBR122 as expresssion vector.Based on above strains,we explored the effects of Agp1 and Agp2 in conjugation through double-resistance selection.It was observed that the effects of Agp1 and Agp2 in conjugation were dependent on light irradiation and the cells where they were located.In the donor strain,the Pr state of Agp1 or Agp2 inhibited the conjugation and their Pfr state promoted the conjugation;in the recipient strain,Agp2 present the weak effect in conjugation.The Agp1 from recipient cell played a key role in conjugation.Its Pr form up-regulated the conjugation and Pfr form down-regulated the reaction.In comparison with previous results,the Agp1 and Agp2 in donor cell with pTiC58 exhibited more significant effects in conjugation.Moreover,both Agp1 and Agp2 could promote the conjugation.In order to discuss the mechanism of Agp1 in conjugation,we targeted the phosphorylation site in Agp1 and constructed H519 R mutant.The loss of phosphorylation capability gave rise to the complete inhibition on the conjugation.It indicated that Agp1 regulated the conjugation via its phosphorylation level.Additionally,we also conducted some studies about the role of both PhrA and PhrB on the conjugation.These results would provide the therotical support for the further mechanism researches.Subsequently,we studied the roles of Agp1 and Agp2 in transformation.Firsly,GUS staining and GUS enzymatic activity were measured following the infection of both wt and the mutated A.fabrum C58 into BY-2 cell.The results showed that the presence of both Agp1 and Agp2 helped to enhance the transformation capability.Afterwards,we also studied the effects of Agp1 and Agp2 on the motility of A.fabrum C58.The results revealed that,the Pr state of Agp1 and Agp2 produced under far-red light condition played the negative effects in motiliy of A.fabrum C58 when pH varied from 6.0 to 8.0;in contrast,their Pfr state played a positive role on the motility.In this work,we also carried out some spectroscopic studies to explore if some structural changes happened in different domain of full-length Agp1 when Pr state was transformed to Pfr state.Six mutants were single-labelled or double-labelled by using FRET pair,i.e.Atto495 and Atto565.FRET happened in the hetero-labelled proteins,indicating that two Agp1 monomers were arranged in the parallel form in the dimer.In addition,fluorescence emission spectra experienced some small changes or no changes in various mutants.It showed that the red light irradiation induced some structual changes in PHY domain,while no significant changes were observed in Histidine Kinase domain during the photoconversion.More work needs to be done in the future to gain deeper understanding on the signal conduction mechanism in Agp1 and Agp2.In conclusion,the results revealed that the bacteriophytochromes from A.fabrum C58 as a non-photosynthetic bacterium played the important roles in transformation and conjugation.Meanwhile,Agp1 and Agp2 were involved in conjugation and transformation by their own phosphorylation level.These results could provide an insight into the study of the physiological function of other bacteriophytochromes.And it also could provide the theoretical support for the cognition of bacterial HGT regulation pathway.