| Bacterial wilt of plants caused by Ralstonia solanacearum is an important soil-borne disease worldwide.Ralstonia solanacearum can adapt to the complex and changing natural environment through its internal regulation mechanisms.Then ppGpp is a signaling alarmone,relA gene is responsible for the synthesis of ppGpp and spo T is the bifunctional gene which is responsible for the synthesis and degradation of pp Gpp.The quantity of ppGpp in cells changes to regulate the biological functions of bacterials such as motility and biofilm formation,so that the bacteria can better adapt the adversity conditions.In this study,Z-Aq-1,the 4 race of Ralstonia solanacearum,was used as the research object.By means of reverse genetics relA and spoT gene deletion strains and complementary strains were constructed.To analyze the connection between relA,spoT genes and biological characteristics such as pathogenicity,motility and biofilm.The eutrophic medium is a commonly used medium for the isolation and cultivation of Ralstonia solanacearum and the oligotrophic medium is closer to the growth environment of Ralstonia solanacearum in host plants.Transcriptome sequencing technology was used to determine the transcript differences between the Z-Aq-1 strain and the relA and spoT gene mutant strains under eutrophic and oligotrophic culture conditions.Explored the function of relA and spoT genes and discovered genes related to motility will lay a theoretical foundation for further clarifying the regulatory mechanism of Ralstonia solanacearum under adversity.1.Functional verification of pp Gpp-related genes relA and spoT geneThe pathogenicity determination showed that the pathogenicity of Z-Aq-1△relA and Z-Aq-1△spoT strains was not significantly different compared with the wild-type strains.The growth curve showed no significant difference in the growth rates of mutant strain and wild-type strain in NB medium.While in hrp medium the growth rates of both relA and spo T mutant strain increased significantly compared with the wild-type strain in logarithmic period and spoT mutant strain was faster than relA mutant strain.The biofilm test showed that the biofilm formation ability of both relA and the spoT mutant strain was significantly increased compared with the wild-type strain,and the spo T mutant strain was stronger than the relA mutant strain.The metabolic activity assay showed that the utilization metabolism of 96 substrates in spo T and relA mutant strains was not significantly different from that in wild-type strains.The motility measurement showed that compared with wild-type strains,the motility of both relA and spoT mutant strains significantly decreased and the motility of spoT mutant strains decreased more significantly than that of relA mutant strains.The culture experiment of adversity induction showed that Z-Aq-1△relA was more tolerance to higher temperature and osmotic pressure than Z-Aq-1△spoT strain and the mutant strains were more tolerance to adversity than the wild-type strain.For this reason,it is speculated that the relA and spoT genes are not involved in the pathogenic regulatory network of Ralstonia solanacearum.But it can regulate the phenotype of Ralstonia solanacearum motility,biofilm and growth rate.2.Transcriptome studies of wild-type and mutant strainsThe study identified genes that were differentially expressed between mutant and wild-type strainsunder eutrophic and oligotrophic culture conditions.Under oligotrophic culture conditions,compared with wild-type strains the expression levels of three motA,motB and flgK genes related to motility were significantly down-regulated in relA mutant strains and expression levels of motility-related genes were significantly down-regulated in spoT mutant strains.TetR,LysR,AsnC,MarR and other regulatory transcription factors were differentially expressed in both mutant strains.Therefore,it is speculated that relA and spoT genes are involved in the regulation of Ralstonia solanacearum motility.MotA,motB and flgK are the key genes that regulate Ralstonia solanacearum motility.Transcriptome sequencing results further clarified the ppGpp regulatory network in Ralstonia solanacearum and discovered new genes related to motility. |
