Isolation And Characterization Of Rice Genes Interacting With Spl11

Spl11 is a negative regulator of cell death and defense response in rice.It encodes a U-box E3 ligase that is involved in ubiquitination-mediated protein degradation.To identify putative substrates of the SPL11 protein,we performed a yeast two-hybrid(Y2H)screening using the SPL11 ARM repeat domain as the bait. Eight different SPL11 interacting proteins or SPINs(SPIN1-SPIN8)were identified. Except for SPIN6,all proteins have intermediate to strong interactions with SPL11's ARM domain.SPIN1 encodes a K homolog(KH)domain-containing RNA-binding protein.SPIN2 encodes a homolog of plant myosin heavy chain protein.SPIN3 encodes a MOM-like protein.SPIN4 encodes an RNAse P Rpr2/Rpp21(RPR) domain-containing protein with putative pre-mRNA cleavage function.SPIN5 encodes an acetylglutamate kinase-like protein.SPIN6 encodes a Rho-GTPase activating protein.Two of the SPINs,SPIN7 and SPIN8,show no hits to known functional groups using the BLAST algorithm search.Among the SPIN genes,SPIN3,SPIN4,SPIN6 were selected for functional analysis.RNAi and over-expression constructs were generated for the three genes. Forty-one lines of SPIN3-RNAi,six lines of SPIN3-overexpression,one line of SPIN4-RNAi,two lines of SPIN4-overexpression,forty-six lines of SPIN6-RNAi and nine lines of SPIN6-overexpression were obtained and confirmed to carry the transgenes.Silencing and over-expression of the target genes were verified using PCR. Disease resistance of these lines to the blast fungus Magnaporthe oryzae and bacterial blight pathogen Xanthomonas oryzae pv.oryzae(Xoo)were evaluated.Among all the RNAi lines,only SPIN4 RNAi lines showed enhanced resistance to Xoo.In contrary, over-expression of the SPIN3 and SPIN6 genes in transgenic rice conferred enhanced resistance to Xoo.In addition,we observed that the PAL activity in the SPIN3 over-expression plants was significantly higher than that in the wild type and RNAi plants,consistent with their enhanced resistance to Xoo.These results suggest that SPIN3,SPIN4 and SPIN6 might be involved in the Spl11-mediated cell death and defense pathway.More detailed analysis of the transgenic lines generated from this study is being carried out to identify the exact function of these genes in rice.