| Regulation of defense responses in plants requires a complicated signal pathway network,in which a number of regulatory components are involved.During the course of our studies aimed at elucidating the molecular biology of rice defense response induced by benzothiadiazole,two defense-related differentially expressed cDNA clones,BIHN-w1 and BNHN-w3,were identified.BIHN-w1 seems to be a part of a gene encoding a putative RING-finger(RING-H2)protein,while BNHN-w3 appears as a gene coding for a serine carboxypeptidase-like protein.In this study,we cloned and identified these two genes,OsBIRF1 and OsBISCPL1,and performed functional analysis for their biological role in plant defense responses against biotic and abiotic stress.The full-length cDNA of OsBIRF1 is 1873 bp with an open reading frame(ORF)of 1191 bp,which encodes a 396 amino acid protein containing a conserved RING-H2-type RING finger domain,and thus OsBIRF1 is a rice RING-H2-type RING finger protein.OsBIRF1 is located on chromosome 2 of the rice genome and consists of a single exon without intron.RT-PCR analysis showed that expression of OsBIRF1 was up-regulated in rice leaf tissues after treatment with benzothaidiazole(BTH), salicylic acid(SA),1-aminocyclopropane-1-carboxylic acid(ACC)and jasmonic acid (JA),which are known defense-related signal molecules in plants.Most importantly, expression of OsBIRF1 was up-regulated in an incompatible interaction between Magnaporthe grisea and a resistant genotype,and no significant induced expression was observed in a compatible interaction.To further study the function of OsBIRF1 in disease resistance response,a functional analysis of OsBIRF1 in transgenic tobacco plants was performed.OsBIRF1 was introduced in tobacco,and 10 individual transgenic plants(T0 progeney)were obtained.Five independent transgenic lines with single copy of transgene were obtained through screening based on segregation of antiobitoc resistance marker.RT-PCR analysis indicated that OsBIRF1 is expressed normally in transgenic tobacco lines.OsBIRF1 transgenic plants showed better in growth as compared that of wild-type plants.Disease resistance assays revealed that overexpression of OsBIRF1 in transgenic tobacco plants enhanced disease resistance against tobacco mosaic virus(TMV)and Pseudomonas syringae pv.tabaci,and up-regulated expression levels of defense-related PR gene,e.g.PR-1,PR-2,PR-3 and PR-5.Moreover,OsBIRF1-overexpressing transgenic tobacco plants showed a reduced ABA sensitivity,and enhanced tolerance to oxidative stress and drought stress.These results suggest that OsBIRF1 is a rice RING-finger protein,and plays important roles in defense responses against biotic and abiotic stress.The full-length of OsBISCPL1 is 1747 bp with an ORF of 1407 bp.OsBISCPL1 is located on chromosome 8 of the rice genome and consists of 12 exons and 11 introns. OsBISCPL1 encodes a 468 aa protein containing conserved peptidase S10 domain, serine active site and a signal peptide at N-terminus.OsBISCPL1 is expressed ubiquitously in rice,including roots,stems,leaves and spikes.Expression of OsBISCPL1 in leaves was significantly up-regulated after treatments with BTH,SA, JA and ACC,and also up-regulated in an incompatible interaction between rice and the blast fungus,M.grsiea.Transgenic Arabidopsis plants with constitutive expression of OsBISCPL1 were generated and disease resistance assays indicated that the OsBISCPL1-overexprressing plants showed an enhanced disease resistance against Pseudomonas syringae pv.tomato and Alternaria brassicicola.Expression levels of some defense-related genes,e.g.PR1,PR2,PR5 and PDF1.2,were constitutively up-regulated in transgenic plants as compared with those in wild-type plants. Furthermore,the OsBISCPL1-overexprressing plants also showed an increased tolerance to oxidative stress and up-regulated expression of oxidative stress-related genes.The results suggested that the OsBISCPL1 may be involved in regulation of defense responses against pathogen infection and oxidative stress. |
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