Investigation Of The Mechanism Of Biosynthesis Of The Non-coding RNA SR220 In Xanthomonas Campestris Pathovar Campestris

Xanthomonas campestris pv.campestris（Xcc）is the casual agent of black rot in many important crops such as rape,cabbage,radish,cabbage and the model plant Arabidopsis thaliana.It is also a model bacteria for studying the interaction between bacterial pathogen and host plants.Our lab previously found that,the non-coding RNA（nc RNA）SR220 of Xcc is an RsmA-binding RNA,which can specifically bind to and inhibit RsmA protein,the core component of the Rsm（Repression of secondary metabolism）post-transcriptional regulatory system,and thereby negatively regulates Xcc virulence.More interestingly,unlike other RsmA-binding nc RNA（such as Rsm B of Erwinia carotovora）,SR220 is derived from the 3’untranslated region（3’UTR）of the m RNA of a protein-coding gene（XC1332）,but not transcribed from an independent nc RNA gene,indicating that it has a unique biosynthetic pathway.The purpose of this study is:（1）clarify whether the biosynthesis of SR220 is positively regulated by RsmA at the transcriptional level,just like other RsmA-binding nc RNAs;（2）determine the 5’upstream flanking sequence essential for SR220 cleavage;（3）identify the ribonuclease that involved in SR220 cleavage.To clarify whether the synthesis of SR220 is positively regulated by RsmA,the expression levels of SR220 in the wild-type strain and the rsm A deletion mutant was detected and compared by Northern blotting.The result showed that SR220 highly expressed in the wild-type strain,but no expression signal was detected in the rsm A deletion mutant,indicating that the production of SR220requires RsmA.More importantly,further experiments revealed that SR220production requires direct binding of SR220 with RsmA,suggesting that RsmA regulates SR220 synthesis at the post-transcriptional level in Xcc,unlike the situation in other bacteria in which RsmA activates the synthesis of its RNA antagonists at the transcriptional level.Next,the"shorten-fragment complementation"method was used to identify the 5’upstream flanking sequences essential for SR220 cleavage.The results showed that the 5’flanking sequence（G₅G₄T₃T₂C₁）at positions 1^th to 5^thupstream of SR220 is necessary for the correct cleavage of SR220.Furthermore,"single base mutation and complementation"experiments showed that the G₅,G₄ and C₁ are critical for the correct cleavage of SR220.To identify the ribonuclease involved in SR220 cleavage,the RNA interactome capture（RIC）experiment was firstly performed,but unfortunately the experiment is unsuccessful.Then,this study tried to identify the ribonuclease involved in SR220 cleavage by detecting the expression of SR220 in the mutant of the 20 ribonuclease genes in Xcc genome respectively.The results showed that deletion of the rn D,a gene encoding RNase D,resulted in abnormal production of SR220,indicating that RNase D is involved in the cleavage of SR220.In vitro cleavage experiments showed that the purified RNase D can cleave SR220 precursor,further support the conclusion that RNase D is involved in the cleavage of SR220.In conclusion,in this study,the mechanism of the biosynthesis of SR220,the only virulence-related nc RNA in Xcc,was investigated,and get the following conclusions:（1）SR220 is positively regulated by RsmA at the post-transcriptional level;（2）The 5’upstream flanking sequence and key bases necessary for the correct cleavage of SR220 were determined;（3）RNase D was found to be involved in the cleavage of SR220.These findings increase our understanding of the mechanisms of bacterial nc RNA biosynthesis as well as the virulence regulation of Xcc.