Functional Analysis Of MiaA?hflx And SRNA In Acidovorax Citrulli

Bacterial fruit blotch(BFB)is a worldwide quarantine bacterial disease.It can seriously damage watermelon,melon and other cucurbit crops through seed transmission,which is common in melon producing regions of the world,and has taken serious economic losses.The pathogen of BFB is Acidovorax citrulli,gram-negative bacteria,single root flagella,which optimal growth temperature is 24~28 ?.The type III secretion system(T3SS)is a mainly pathogenic factor of A.citrulli which can secrete effectors in the host cells.At present,there is still no commercial disease-resistant seed for BFB.The regulation of post-transcriptional levels is important for gene expression,normal assembly of proteins,and maintenance of life activities.Hfq is an RNA chaperone protein that can combine with sRNA to regulate the post-transcriptional level of genes(small non-coding RNA)and promote or inhibit the translation of target mRNA.MiaA is a tRNA isopentenyl pyrophosphate transferase that plays a key role in promoting translation fidelity.In addition,HflX is one of the additional ribosome-associated proteins required for the synthesis of effective peptides during the translation phase.The gene encoding HflX is located downstream of the hfq gene and encodes a GTP-binding protein.The hydrolysis of GTP can drive the functional cycle of other related proteins.In this study,the hfq,miaA,hflX genes were analyzed by bioinformatics,and the gene information in the A.citrulli was studied.The mutant strains and complementary strains of miaA and hflX genes were constructed.The function of ?miaA and ?hflX was studied through phenotypic determination,and by qRT-PCR,we studied the possible regulatory relationship between hfq,miaA and hflX.In addition,the candidate Hfq-dependent sRNA was detected by bioinformatics prediction and construction of hfq overexpressing strains.Through determinate the expression of the sRNAs in the pressure environment,the candidate sRNA NC-27 was selected.NC-27 was knocked out and replenished in the genome,and its function in the A.citrulli was studied.Then we predicted target mRNAs of NC-27.The following results were obtained from this study:(1)Through bioinformatics analysis,the Hfq protein is encoded by hfq gene in A.citrulli which contains 83 amino acids,no special domain,and can function as an RNA chaperone protein;MiaA protein consists of 350 amino acids with two low complexity domains and an IPPT domain,which catalyze the modification of adenosine near the anticodon;HflX consists of 390 amino acids with two GTP binding domains and one Feo_B domain.Through the prediction of protein interaction,it is found that there may be an interaction relationship between Hfq,MiaA and HflX.(2)The presences of miaA and hflX have positive contribution to the virulence of A.citrulli.The deletion of miaA significantly reduced the hrpX,which a key gene of the type III secretory system,and significantly decreased the pathogenicity and biofilm formation ability,and increased brown pigment secretion,but didn't affect its ability to cause the hypersensitive response.The deletion of the hflX gene also didn't affect the ability of A.citrulli Aac5 strain to cause the hypersensitive response.Although it showed stronger biofilm formation ability,its exercise ability decreased significantly.At the same time,the key genes hrpG and hrpE of the type III secretion system were significantly down-regulated,and its pathogenic ability was also significantly decreased.(3)There is a regulatory relationship between miaA,hflX and hfq.The results of qRTPCR revealed,the hfq gene was positivetly regulated by hflX and miaA genes.The hflX gene was positivetly regulated by the miaA gene,and the miaA gene was negatively regulated by the hflX gene.(4)We has predicted 31 sRNAs in A.citrulli,and the stability of the 6 candidate sRNAs was dependent on the Hfq protein.Using SIPHT,31 potential sRNAs were predicted in A.citrulli AAC00-1.Real-time PCR was performed using the cDNA of hfq overexpressing strains.It was found that the expression levels of 6 sRNAs were significantly up-regulated,that is,the stability of these sRNAs may be dependence on Hfq protein.The expression levels of candidate sRNAs vary under different pressure conditions.(5)The deletion of the candidate sRNA NC-27 increased the virulence of A.citrulli Aac5 and increased the accumulation of hrpG mRNA,resulted an increase in the T3 SS protein HrpG.As an upstream regulator of hrpX,the increase also led to the up-regulation of hrpX mRNA and HrpX,and the device protein HrcJ was also increased.Its virulence is enhanced.(6)By Target RNA2,we predicted 29 target mRNAs of candidate sRNA NC-27 in A.citrulli.By analyzing the secondary structure of NC-27,it was found to have a uracil-rich at the 3' end.The sequence of U can contact the amino acid of the inner circle of the Hfq protein,and the binding site of the candidate target mRNA is mainly located between the 102~117 nucleic acids.