Functional Analysis Of A Rice DEAD-box RNA Helicase Gene OsBIRH1 And Preliminary Study On The YTH Protein Family In Arabidopsis And Rice

Recent studies have demonstrated that DEAD-box RNA helicases and RNA-binding proteins play important roles in plant growth and development as well as in plant responses to hormones and environmental stresses.In our previous studies aimed at elucidating molecular mechanisms of induced immunity in rice,two differentially expressed genes,which encode a putative DEAD-box RNA helicase and a YTH domain-containing unknown protein,were identified to be associated with induced immunity phenotype induced by benzothaidiazole,a chemical inducer of immunity in plants.In present studies,I performed a functional analysis of a rice DEAD-box RNA helicase gene,OsBIRH1,by combined biochemical and genomics approaches.Moreover,I also identified through bioinformatics methods the YTH (YT521-B homology)domain-containing protein family from Arabidopsis thaliana and rice and performed a preliminary study on the biochemical activity and gene expression patterns of the family members in Arabidopsis.The full-length cDNA of the OsBIRH1 gene is 2108 bp in size with a predicted 1926 bp open reading frame.OsBIRH1 encodes a protein of 641 amino acids and the deduced OsBIRH1 protein contains all conserved domains characteristic of the DEAD-box RNA helicase proteins.The OsBIRH1 gene locates on chromosome 3 of the rice genome as a single copy gene and consists of eight exons and seven introns. Recombinant OsBIRH1 protein was purified from E.coli and was shown to have RNA-dependent ATPase and RNA helicase activities,which are common biochemical characteristic of DEAD-box RNA helicases.Northern blotting analysis showed that the OsBIRH1 gene has a relatively high level of basal expression in rice leaf tissue. However,expression of OsBIRH1 was activated in rice seedling leaf tissues after treatment with BTH,salicylic acid(SA),l-aminocyclopropane-1-carboxylic acid (ACC)and jasmonic acid(JA).Expression of OsBIRH1 was up-regulated in incompatible interaction between M.grisea and a resistant genotype during the first 6 h after inoculation,but its expression remained unchanged in compatible interaction.A functional analysis of OsBIRH1 was performed in transgenic arabidopsis plants.The OsBIRH1 coding region was cloned into a plant transformation binary vector pCAMBIA99-1 under control of the CaMV 35S promoter.A total of 15 independent transgenic Arabidopsis lines were obtained through floral dip transformation method.Southern analysis of the transgenic lines showed that the OsBIRH1 gene was integrated into the arabidopsis genome with 1～2 copies;and RT-PCR indicated that the OsBIRH1 gene is expressed normally in most of the transgenic tobacco lines.Disease resistance phenotype assays demonstrated that the OsBIRH1-overexpressing transgenic Arabidopsis plants showed an enhanced disease resistance against Alternaria brassicicola and Pseudomonas syringae pv tomato DC3000.Interestingly,the defense-related PR-1,PR-2,PR-5 and PDF1.2 genes showed an enhanced constitutive expression in the transgenic lines.Moreover,the transgenic arabidopsis plants overexpressing OsBIRH1 also showed an enhanced tolerance to drought and oxidative stress.The results suggest that OsBIRH1 encodes a functional DEAD-box RNA helicase and plays important roles in disease resistance responses and abiotic stress tolerance.YTH domain is recently identified conserved domain in a set of protein with unknown biological function.The YTH domain-containing proteins are considered to have a putative RNA binding activity.To understand the biological function of YTH domain-containing proteins,I identified 13 and 12 genes that encode putative YTH proteins in the genomes of Arabidopsis thaliana and rice and named AtYTHO1～13 and OsYTH01～12,respectively.This suggests that YTH domain-containing proteins consist of a small gene family in higher plants.All members of Arabidopsis and rice YTH protein family contain a conserved YTH domain,but no additional known domain or conserved region was identified outside the YTH domain in protein sequences.Recombinant AtYTH05 and AtYTH07 proteins were purified from E.coli expressing plasmids carrying coding regions of these two genes.Results from electrophoretic mobility shift assays demonstrated that recombinant AtYTH05 protein could bind to double stranded RNA duplex in vitro,suggesting that the YTH protein have RNA binding activity.Expression patterns of all members of the Arabidopsis YTH family were analyzed by reverse transcriptase(RT)-PCR.Results showed that transcriptional expression of Arabidopsis YTH family genes was differentially regulated in different organs and at different developmental stages.Promoters of AtYTH05 and AtYTH07 genes were cloned and AtYTH05/07 promoter::GUS constructs were made to transform into Arabidopsis.GUS staining demonstrated that expression of AtYTH05 and AtYTH07 genes was significantly induced during flowering stage.The expression patterns of AtYTH05 and AtYTH07 genes as revealed by promoter::GUS staining were similar to those from RT-PCR analysis.Results obtained from this preliminary study indicate that YTH family proteins are a novel grop of RNA binding proteins and may play important roles in plant growth and development.