Mechanism Revealed By RNA-Seq For The Attentated Virulence By Inhibitors In Rice Pathogen Acidovorax Oryzae

Acidovorax oryzae that widely distributed in rice planting area can cause serious losses of rice production by contaminated seeds. Based on the resistance of strain RS-1to Ampicillin (Amp), this study examined the effect of Amp on the virulence related phenotype of strain RS-1, such as pathogenicity, motility, biofilm formation, exopolysaccharides (EPS) and bacterial colonization; then, the gene expression was analysised by RNA-Seq and validated by qRT-PCR and gene knockout; next, effector secretion was tested by ELISA as well as protein interaction was measured by bacterial two-hybrid and GST-pull down assays. Restults indicated that Amp did not influence the growth and biofilm formation of strain RS-1, but significantly reduced the virulence and colonization of strain RS-1. Furthermore, transcriptome analysis by RNA-Seq showed that638genes were uniquely expressed under Amp (+) conditions, while89genes were specificly expressed under Amp (-) conditions. Among differentially expressed genes under Amp (+) vs. Amp (-) conditions, in particular,526genes involved in bacterial virulence and resistance. The reliability of the transcriptomic data in this study was first evidenced by the high correlation between two biological replicates, and validated by qRT-PCR of21type six secretion system (T6SS) related genes. The down-regulated expression of hcp by RNA-Seq analysis and the reduced secretion of Hcp protein by ELISA assays showed Amp significantly influenced the transcription and expression of hcp gene which encodes a very important effector protein of T6SS. Similar phenotypes between wild type and△hcp mutant further confirmed that hcp plays a very important role in Amp response and the attenuated virulence in strain RS-1. Interaction of Hcp with other T6SS components, such as VgrG or Lipo, highlights the importance of Hcp in T6SS.In summary, this study found that the virulence of strain RS-1was able to be attenuated by Amp exposure, which will be considered in disease prediction due to the increasing contamination of antibiotics in agricultural ecosystem, and provided a new insight to elucidate the pathogenic mechanmism by attenuation of virulence.