Functional Analysis Of Sigma 54 Factors In Xanthomonas Oryzae Pv.oryzae

Bacterial leaf blight of rice caused by Xanthomonas oryzae pv.oryzae(Xoo)is one of the most severe bacterial diseases in rice production in the world,especially in Asia and Africa.The transcription of genes related to bacterial virulence ph enotypes,such as motility,chemotaxis,exopolysaccharide(EPS)production,biofilm formation,and metabolisms of cyclic dimeric guanosine monophosphate(c-di-GMP),are controlled by diverse transcriptional factors.?⁵⁴ factor(Rpo N)plays a crucial role in bacterial motility,virulence,growth,and other biological functions.Two homologous?⁵⁴factors,Rpo N1 and Rpo N2,were previously identified from Xoo the biological functions that remain fully elucidated.In this study,RNA-seq analysis was performed to analyze the transcriptome-wide regulomes of Rpo N1 and Rpo N2,and assess their specific and co-regulatory pathways in Xoo.A total of 127 overlapping differentially-expressed genes(DEGs)regulated by Rpo N1 and Rpo N2 were identified.These DEGs were significantly enriched in flagellar assembly,bacterial chemotaxis,and two-component system groups.In addition,35 genes encoding flagellar structural components and regulatory factors were down-regulated by Rpo N1 and Rpo N2,and their expression levels were significantly reduced in the?rpo N2 mutant compared to that in the?rpo N1 mutant,suggesting that Rpo N2 has a more important regulatory role in flagellar assembly than Rpo N1.Interestingly,the expression level of rpo N2 decreased in?rpo N1,but that of Rpo N1 did not change significantly in?rpo N2.Furthermore,no direct binding of Rpo N1 to the promoter of rpo N2 was observed,indicating that Rpo N1 indirectly regulates the rpo N2 expression in Xoo.In addition,remarkably reduced growth of?rpo N1 but not?rpo N2 was found compared to the wild-type strain,suggesting that Rpo N1 played a more crucial role in regulating bacterial growth and basal metabolism than Rpo N2 in Xoo.The type ? pilus(T4P),a special class of bacterial surface filament,plays crucial roles in surface adhesion,motility,biofilm formation,and virulence in pathogenic bacteria.Direct regulation of T4P genes by Rpo N2 and Pil RX in Xoo was found in this study.Furthermore,the interaction of Pil RX with Rpo N2 was identified,and the role of Rpo N2-Pil RX in regulating the T4P genes expression,motility,virulence,and biofilm formation in Xoo was demonstrated.These findings suggested that the Rpo N2-Pil RX regulatory system played an essential role in pilus-dependent biological functions by directly regulating T4P genes in Xoo.