The Discovery And Genetic Modification Of Ralstonia Solanacearum Filamentous Phages RSBg

Bacterial wilt is a plant soil-borne disease caused by Ralstonia solanacearum and is regarded as the plant cancer.It can infect more than hundreds of plants in 50 families,including tobacco,peanut,tomato,potato,eggplant and other important cash crops.Bacterial wilt is one of the most destructive plant bacterial disease in south China.R.solanacearum has strong adaptability and high diversity.In order to find safe and green control measures,phage biocontrol has been paid more and more attention.Phages are viruses that infect bacteria.They are common in nature and occur wherever bacteria are present.Filamentous phage is a kind of phage classified according to morphology,and has been neglected because of its inability to kill bacteria directly.Filamentous phages can continuously infect bacteria and secrete from the infected bacteria.Many filamentous phages can integrate genome DNA into the host bacteria genome forming lysogenic prophages.In this paper,an intact filamentous phage,named RSBg,was found in the genome of R.solanacearum Bg07 by bioinformatics prediction.Phage RSBg was isolated from the supernatant of Bg07 culture.Phage RSBg significantly delayed the growth of the host R.solanacearum GMI1000,but did not lyse the infected R.solanacearum,and did not affect the final growth of the host R.solanacearum.Transmission electron microscopy showed that RSBg virion is a typical filamentous structure,with a length of about 4200 nm and a diameter of about 30 nm.The genomic DNA extracting,PCR amplification and sequencing were performed to obtain flanking sequence of the junction site of phage RSBg.Combining the partial DNA sequence and RSBg prophage sequence,it was found that the RSBg genome size of phage is 7.5 Kb,encoding 11 genes in total.Moreover,phage RSBg was genetically modified because of its weak biocontrol effect on tomato bacterial wilt.Segments of the full-length phage RSBg was amplified and cloned to pUC19 by seamless cloning,generating pUCRSBg.Then,Insert "gene scissors" cas9 was cloned into plasmid pUCRSBg,resulting p UCRSBg cas9.The sgRNA sequence targeting hrpB,the key regulatory factor of the pathogenic gene of R.solanacearum was inserted into plasmid pUCRSBgcas9,generating pUCRSBgcas9 hrpB.The recombinant plasmid is expected to be transformed into R.solanacearum,and produce recombinant infective phage.The recombinant phage may break hrpB gene of R.solanacearum and play a better effect in bacterial wilt biocontrol.