Functional Analysis Of Flagellin And Its Glycosylation In Rice-xanthomonas Oryzae Pv.Oryzae Interaction

Plants have evolved a sensitive defense response system that detects and recognizes various pathogen-associated molecular patterns(PAMPs)(e.g.flagellin)and induces immune responses to protect againstinvasion.Transcriptional responses in rice to PAMPs produced by Xanthomonas oryzae pv.oryzae(Xoo),the bacterial blight pathogen of rice,have not yet been defined.In this study,we characterized transcriptomic responses in rice inoculated with the wildtype(WT)Xoo and flagellin-deficient mutant ?fliC through RNA-seq analysis.Digital gene expression(DGE)analysis based on Solexa/Illumina sequencing was used to investigate transcriptomic responses in 30 day-old seedlings of rice(Oryza sativa L.cv.Nipponbare).1,680 genes were differentially-expressed(DEGs)in rice inoculated with WT relative to ?fliC;among which 1,159 genes were upregulated and 521 were down-regulated.Expression patterns of 12 randomly-selected DEGs assayed by quantitative real time PCR(qRT-PCR)were similar to those detected by DGE analyses,confirming reliability of the DGE data.Functional annotations revealed the up-regulated DEGs are involved in the cell wall,lipid and secondary metabolism,defense response and hormone signaling,whereas the down-regulated ones are associated with photosynthesis.Moreover,57 and 21 specifically expressed genes were found afterWT and ?fliC treatments,respectively.This study provided additional insights into molecular basis of rice response to bacterial infection and revealed potential functions of bacterial flagellin in the rice-Xoo interactions.Flagellin glycosylation plays crucial roles in flagellar assembly,motility and virulence in several pathogenic bacteria.However,little is known about the genetic determinants and biological functions of flagellin glycosylation in Xoo.Here,the structure,regulation and functions of a ten-gene cluster gigX(glycosylation island genes of Xoo),which was embedded in a flagellar regulon were characterized.gigXl through gigX10,encoded putative enzymes or proteins involved in glycan biosynthesis and transfer,including a nucleotide sugar transaminase,a carrier protein(ACP),a 3-oxoacyl-ACP synthase,a 3-oxoacyl-ACP reductase,a dehydrogenase,an acetyltransferase,a ring hydroxylating dioxygenase,a hypothetical protein,a methyltransferanse,and a glycosyltransferase,respectively.The gigX genes were co-transcribed in an operon and up-regulated by the upstream ?54 factor RpoN2 and transcriptional activator FleQ.In frame deletion of each gigX gene resulted in abolishment of flagellin glycosylation,leading to the mutant unglycosylated flagellin smaller than the wildtype glycosylated one.No changes in flagellar filament and motility were observed in the ?gigX mutants.Importantly,all mutants except ?gigX9 showed significantly increased virulence and bacterial growth in the susceptible rice cultivar IR24,and AgigXl and ?gigX10 showed enhanced type ? secretion system(T3SS)-releted gene expression.Moreover,the glycosylated flagellin of wildtype induced higher H2O2 levels in rice leaves than the unglycosylated ones of AgigXl or?gigX10.Taken together,this study reveals that gigX cluster determines flagellin glycosylation,and implicates glycosylation of flagellin in regulation of motility and virulence of Xoo.