Transcriptional And Functional Analysis Of The Intergenic Region Between HRPX And HRPG Of Xanthomonas Campestris Pvathovar Campestris

Xanthomonas campestris pv. campestris（Xcc） is the causal agent of cruciferous plant including cabbage, cauliflower, radish, mustard, and arabidopsis thaliana. Xcc is one of the model bacteria for studying the mechanism of plant pathogenic bacteria-host plants interactions. Xcc is also an important industrial bacteria used for producing the industrial raw material xanthan gum. Up to date, the whole genomes of three Xcc strains,8004, ATCC33913and B100, have been sequenced.In the genome of Xcc8004,84.79%of the sequences are protein-coding sequences and15.21%are locating in intergenic regions （IGRs）. At present, the functional study of IGR has become one of hotspots in the field of functional genomics. Recently, it has been shown that some IGRs are transcribed and have important functions. In this study, we focus on:whether the IGR of hrpX and hrpG, the two key virulence regulatory genes of Xcc8004, is transcribed and has any biological function.In Xcc8004genome, hrpX （gene ID:XC₃076） and hrpG （gene ID: XC₃077） are closely adjacent but with opposite transcriptional orientations: hrpX is encoded on the minus strand and hrpG is encoded on the plus strand of the genome. The spacer between the start codons of the two genes is823bp （base pair）.5’-Race analysis demonstrated that the transcriptional start site of hrpX is located in79bp upstream of its start codon, and the transcriptional start site of hrpG is located in129bp upstream of its start codon. Promoter analyses reveal that the core promoter regions of hrpX and hrpG are68bp and70bp upstream of their transcriptional start sites, respectively. Thus, the476bp region between the hrpX and hrpG promoters is the area without any known function; we define this region as the IGR of hrpX and hrpG, named IGRhrpXG·Our previous RNA deep sequencing results showed that transcripts from IGRhrpXG are detected in RNA samples isolated from Xcc8004grown in the minimal medium MMX and the rich medium NYG, indicating that IGRhrpXG is transcribed. RT-PCR analysis show that specific products are detectable using different IGRhrpXG-specific primer sets, demonstrating that IGRhrpXG is indeed transcribed. We try to clarify the transcriptional orientation of the transcripts from IGRhrpXG by using Strand-Specific RT-PCR, but failed for an unknown reason.To identify the biology function of IGRhrpXG, the deletion mutant of named8004⊿GR476hrpX-G, was constructed. Phenotype analysis showed that,8004⊿IGR476hrpX-G displayed a reduced virulence on host plant and lost hypersensitive reaction （HR） on non-host plant. Semi-quantitative RT-PCR assays showed that the transcriptional level of hrpX and hrpG was significantly reduced compared to that in the wild type strain, suggesting that the reduction in virulence and lost HR of the IGRhrpXG deletion mutant are due to the reduction in hrpX and hrpG transcription.The trans-complementary strains8004⊿IGR476hrpX-G/pL6-476and8004⊿hrpG/pL6-828were constructed, can’t recover pathogenicity and HR response of wild type strain. Guess hrpX and hrpG core promoter is not enough to their normal transcription.The trans-complementary strain8004⊿hrpG/pL6-70hrpG and8004⊿IGR476hrpX-G/pL6-70hrppG were constructed. Phenotypes of the assay results demonstrated that the virulence and hypersensitive reaction of complementary compensated. By means of semiquantitative RT-PCR assays, transcriptional level of hrpX and hrpG recover in two. The core promoter of hrpX and hrpG is enough their normal transcription.In order to explore the function of the476bp sequence, we constructed cis-complementary strain8004⊿IGR476hrpX-G/476,476bp of sequence in which with two extra Xba I enzyme loci. The phenotypes of the assay results shewed that the virulence and hypersensitive reaction werere covered. Transcription level of hrpG in8004⊿IGR476hrpX-G/476increased to a certain degree, as a result, HrpG concentration increased, which enough to activate the hrpX transcription and hrpX transcription returned to normal levels.We harbor the expection that hrpX-hrpG gene spacer in Xcc8004is functional, which might contain upstream components outside the core promoter or upstream regulation sequence of hrpG. The hrpX-hrpG spacer regulates the transcription of hrpX, via regulating transcription of hrpG, thereby regulates the hrp gene cluster downstream. However, the transcription regulation mechanism remains to be further study.