Researches On The RF5 Which Forming An Endonuclease Complex In Fertility Restoration Of Honglian-CMS Rice

Cytoplasmic male sterility (CMS) is a widespread phenomenon well known in higher plants. Synergistically, a kind of genes encoded by nucleus can rescue this phenomenon and make cytoplasmic male sterility line produce functional pollens. These genes were named fertility restorer genes (Rf genes). Honglian CMS (HL-CMS) do not produce functional pollens which aborted in bicellular microspore stage and belonged to gametophytic male sterility. To date, we have cloned two restorer genes Rf5 and Rf6; all two genes are belonging to PPR gene family according to bioinformatics analysis. Based on primary research of RF5 restoration mechanism, we found that RF5 can not directly process the HL-CMS transcript, atp6-orfH79. We uncovered that RF5 and others factor could for an about 500KD protein complex which responsible for transcript atp6-orfH79 cleavage. Furthermore, previous research showed that GRP162 and RFC2 were identified and characterized, which belonging to restoration of fertility complex (RFC). But, the rest of RFC and complex assembling remain to unclear, especially, the cleavage factor of RFC is still unknown in HL-CMS transcripts processing. Taking these questions based on previous studies, we started the study of deeply mechanism of RF5 RFC which processing the male sterility transcripts. According to new subunits identification, functional characterization, complex assembling and cleavage assays, we have obtained some new clues and the specific mechanism of restoration for RF5 RFC in HL-CMS fertility restoration pathways. The main results are summarized as follows:1. According to yeast hybrid assay, BIFC, CO-IP, Pull-down assays, we identified and characterized candidates RFC3 (encoded WD40 and DUF1620 domain) and RFC4 (encoded ERCC4 domain). Furthermore, we exhibited the relationship of RF5, GRP162, RFC2, RFC3 and RFC4 according to interaction assays. Subsequently, sub-cellular localization showed RFC3 is located to mitochondria whatever in sterile line and fertile line. Interestingly, RFC4 target to mitochondria only in fertile line (Rf5 NIL) and is diffused in cell for sterile line (YTA). Protein sequence alignment showed RFC3 and RFC4 are conserved in higher plants, furthermore, rice RFC3 and RFC4 are closet to homologous in Zeamays and Sorghum compared with Arabidopsis.2. Using RNA interference methods, selecting specific gene fragment for RFC3 and RFC4 to construct RNAi transgenic vector, which were transformed into callus of Rf5 NIL. According to qRT-PCR and Northern blots analysis, we found that HL-CMS associated transcripts, atp6-orfH79, and toxic protein ORFH79 accumulation in RFC3 and RFC4 extreme down regulation lines comparatively. Furthermore, pollen staining assay exhibited about 50% abortive pollen grains in RNAi lines, by self-cross and genetics analysis, the individuals of RFC3 and RFC4 transgenic lines exhibited gametophytic segregation ratio (1:1) which is consistent with our expectation. These results uncovered the important of RFC3 and RFC4 in HL-CMS RF5 restoration pathways.3. To find out the specific function of RFC3 based on previous bioinformatics analysis, BN-PAGE technology was employed to analysis the RFC stability firstly. Mitochondria isolated from sterile line YTA, fertile line Rf5 NIL and RFC3 RNAi lines were analyzed by BN-PAGE and continual Western blots, results showed that RFC in mitochondria of RFC3 RNAi lines is diffused compare with fertile line Rf5 NIL which exhibiting stable RFC located in same position at BN gel. For sterile line YTA, we only detected the RFC3 signal in a low molecule size position; inversely, we have not find RF5 and GRP162 accumulation in mitochondria. These conclusions suggest that RFC3 play an irreplaceable role in RFC assembling and is responsible for normal function and stability of RFC. Furthermore, we separated the membrane fractions from matrix using purified mitochondria from Rfi NILs, Western blot result showed RFC3 is anchored in mitochondria membrane fractions using antibody against RFC3 and COXII respectively. These results implied RFC3 is a membrane protein and the Rf5 molecular restoration complex is a membrane bound complex.4. To verify the candidate protein RFC4 whether involving in HL-CMS transcript atp6-orfH79 cleavage, we expressed and purified RFC4 from E.coli firstly. In vitro incubation experiments suggested that rRFC4 can directly cleavage atp6-orfH79 probes specifically. These results implied we have found out the direct executor which acting on HL-CMS associated transcripts. Furthermore, because of the differential location of RFC4 in sterile line YTA and fertile line Rf5 NIL respectively, we constructed fused Ubi-MTS-RFC4 expression vectors firstly. Subsequently, this vector was transformed in callus from sterile line YTA and the fused with MTS (mitochondria target sequence) peptide RFC4 rescued the sterile phenotype. These interesting results implied that location of RFC4 is very important for HL-CMS fertility restoration and uncover the important relationship between the nucleus and cytoplasm. Summarily, we have uncovered the specific function of RFC3 and RFC4 in restoration pathways; more importantly, we have identified HL-CMS associated RNA cleavage factor RFC4 specifically and uncovered its function in RNA processing in fertility restoration pathways. All of these promote us to knowing the mechanism of HL-CMS fertility restoration deeply and give us a new sight for CMS/Rfs pathways, also providing a clue for new type hybrid rice breeding.