New insights into virulence mechanisms of rice pathogen Acidovorax avenae subsp. avenae strain RS-1 following exposure to ß-lactam antibiotics.

Acidovorax avenae subsp. avenae (Aaa) strain RS-1 has been reported to have strong virulence to rice plants1,2,3,4,5. One of the fundamental goals in the study of this rice pathogen is to identify virulence determinants during its interaction with the host6,7,8,9. Whilst the genome-wide transcriptomic response of strain RS-1 to in vivo infection of this rice pathogen has been examined with RNA-Seq previously10, in general, it is not easy to collect in vivo plant pathogenic bacteria, due to the contamination of plant tissues and low cell numbers in water. Therefore, an alternate method should be developed for genome-wide identification of virulence-associated genes in strain RS-1. Recent studies have found that in addition to direct killing effect, exposure to antibiotics at sub-inhibitory concentrations could cause an increase or reduction in virulence of human and animal bacterial pathogens, while the pathogenic consequence of antibiotic resistance depends on the kind and concentration of antibiotics, as well as the tested bacterial strains11,12,13,14,15,16. Interestingly, a preliminary study revealed the naturally occurring s-lactam-resistance of strain RS-1, which makes it necessary to examine the pathogenic consequence of β-lactam antibiotics exposure in this rice bacterial pathogen. A number of studies have been performed to explore the underlying causes for the changes in virulence of human and animal bacterial pathogens when exposure to antibiotics17,18,19. In particular, differential expression of several genes encoding known virulence determinants has recently been proposed to be involved in bacterial virulence and antibiotic resistance20,21,22,23,24. However, little information was available about genome-wide analysis of gene expression under exposure to antibiotics, which will provide a comprehensive insight into molecular mechanisms of virulence in bacterial pathogen. In this study, phenotypic changes and the genome-wide transcriptomic response of strain RS-1 were examined following exposure to s-lactam antibiotics. The transcriptome was measured with RNA-Seq and validated by qRT-PCR using the primers listed in Table S1 and knockout mutations of the type VI secretion system (T6SS) genes. Secreted levels of the T6SS effector, Hcp, were measured by western blot analysis and ELISA, and its interaction with other T6SS components was investigated by using both a bacterial two-hybrid assay and a GST pull-down assay.