Xianfeng Zhao

Identification of a New Rice Blast Resistance Gene, Pid3, by Genomewide Comparison of Paired Nucleotide-Binding Site–Leucine-Rich Repeat Genes and Their Pseudogene Alleles Between the Two Sequenced Rice Genomes

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. The two major subspecies of Asian cultivated rice (Oryza sativa L.), indica and japonica, have shown obvious differences in rice blast resistance, but the genomic basis that underlies the difference is not clear. We performed a genomewide comparison of the major class of resistant gene family, the nucleotide-binding site–leucine-rich repeat (NBS–LRR) gene family, between 93-11 (indica) and Nipponbare (japonica) with a focus on their pseudogene members. We found great differences in either constitution or distribution of pseudogenes between the two genomes. According to this comparison, we designed the PCR-based molecular markers specific to the Nipponbare NBS–LRR pseudogene alleles and used them as cosegregation markers for blast susceptibility in a segregation population from a cross between a rice blast-resistant indica variety and a susceptible japonica variety. Through this approach, we identified a new blast resistance gene, Pid3, in the indica variety, Digu. The allelic Pid3 loci in most of the tested japonica varieties were identified as pseudogenes due to a nonsense mutation at the nucleotide position 2208 starting from the translation initiation site. However, this mutation was not found in any of the tested indica varieties, African cultivated rice varieties, or AA genome-containing wild rice species. These results suggest that the pseudogenization of Pid3 in japonica occurred after the divergence of indica and japonica.

Oryza sativa Dicer-like4 Reveals a Key Role for Small Interfering RNA Silencing in Plant Development

MicroRNAs and small interfering RNAs (siRNAs) are two classes of small regulatory RNAs derived from different types of precursors and processed by distinct Dicer or Dicer-like (DCL) proteins. During evolution, four Arabidopsis thaliana DCLs and six rice (Oryza sativa) DCLs (Os DCLs) appear to have acquired specialized functions. The Arabidopsis DCLs are well characterized, but those in rice remain largely unstudied. Here, we show that both knockdown and loss of function of rice DCL4, the homolog of Arabidopsis DCL4, lead to vegetative growth abnormalities and severe developmental defects in spikelet identity. These phenotypic alterations appear to be distinct from those observed in Arabidopsis dcl4 mutants, which exhibit accelerated vegetative phase change. The difference in phenotype between rice and Arabidopsis dcl4 mutants suggests that siRNA processing by DCL4 has a broader role in rice development than in Arabidopsis. Biochemical and genetic analyses indicate that Os DCL4 is the major Dicer responsible for the 21-nucleotide siRNAs associated with inverted repeat transgenes and for trans-acting siRNA (ta-siRNA) from the endogenous TRANS-ACTING siRNA3 (TAS3) gene. We show that the biogenesis mechanism of TAS3 ta-siRNA is conserved but that putative direct targets of Os DCL4 appear to be differentially regulated between monocots and dicots. Our results reveal a critical role of Os DCL4-mediated ta-siRNA biogenesis in rice development.

Overexpression of microRNA319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.).

MicroRNA319 (miR319) family is one of the conserved microRNA (miRNA) families among diverse plant species. It has been reported that miR319 regulates plant development in dicotyledons, but little is known at present about its functions in monocotyledons. In rice (Oryza sativa L.), the MIR319 gene family comprises two members, Osa-MIR319a and Osa-MIR319b. Here, we report an expression pattern analysis and a functional characterization of the two Osa-MIR319 genes in rice. We found that overexpressing Osa-MIR319a and Osa-MIR319b in rice both resulted in wider leaf blades. Leaves of osa-miR319 overexpression transgenic plants showed an increased number of longitudinal small veins, which probably accounted for the increased leaf blade width. In addition, we observed that overexpressing osa-miR319 led to enhanced cold tolerance (4 °C) after chilling acclimation (12 °C) in transgenic rice seedlings. Notably, under both 4 and 12 °C low temperatures, Osa-MIR319a and Osa-MIR319b were down-regulated while the expression of miR319-targeted genes was induced. Furthermore, genetically down-regulating the expression of either of the two miR319-targeted genes, OsPCF5 and OsPCF8, in RNA interference (RNAi) plants also resulted in enhanced cold tolerance after chilling acclimation. Our findings in this study demonstrate that miR319 plays important roles in leaf morphogenesis and cold tolerance in rice.

Characterizations and fine mapping of a mutant gene for high tillering and dwarf in rice (Oryza sativa L.)

A rice htd-1 mutant, related to tillering and dwarfing, was characterized. We show that the htd-1 mutant increases its tiller number by releasing axillary buds from dormant stage rather than by initiating more axillary buds. The dwarf is caused by averagely reducing each internode and panicle. Based on this dwarfing pattern, the htd-1 mutant could be grouped into dn-type dwarf defined by Takeda (Gamma Field Symp 16:1, 1977). In addition, the dwarfing of the htd-1 mutant was found independent of GA based on the analyses of two GA-mediated processes. Based on the quantitative determination of IAA and ABA and application of the two hormones exogenously to the seedlings, we inferred that the high tillering capacity of the htd-1 mutant should not be attributed to a defect in the synthesis of IAA or ABA. The genetic analysis of the htd-1 mutant indicated that the phenotypes of high tillering and dwarf were controlled by a recessive gene, termed htd1. By map-based cloning, the htd1 gene was fine mapped in a 30-kb DNA region on chromosome 4. Sequencing the target DNA region and comparing the counterpart DNA sequences between the htd-1 mutant and other rice varieties revealed a nucleotide substitution corresponding to an amino acid substitution from prolin to leucine in a predicted rice gene, OsCCD7, the rice orthologous gene of AtMAX3/CCD7. With the evidence of the association between the presence of one amino acid change in OsCCD7 and the abnormal phenotypes of the htd-1 mutant, OsCCD7 was identified as the candidate of the HTD1 gene.

Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference

The rice OsMADS16 gene is phylogenetically related to the angiosperm B-function MADS-box genes. To investigate if OsMADS16 functions as an AP3/DEF orthologue to regulate the development of lodicules and stamens in rice, we isolated its genomic sequences and characterized its functions in planta by RNA interference. The genomic sequence of the OsMADS16 gene shows that it shares high similarity in genomic structure and the deduced amino acid sequence with the maize B-class gene, Si1. Transgenic lines from the introduced gene expressing double-stranded RNA with the OsMADS16 cDNA fragment were male-sterile and displayed alternations of lodicules and stamens, occasionally depressed palea and overgrown glume. The two lodicules were converted into four palea/lemma-like organs and some stamens into carpels. Further investigations of the transcription of OsMADS16 gene in these transgenic lines by RT-PCR revealed that its transcript was significantly reduced. Transcription of a rice PI homologous gene, OsMADS4, was also reduced remarkably in the transgenic plants. Our results demonstrate that OsMADS16 is an AP3/DEF orthologue to specify the identities of lodicules and stamens in rice flower and also support that OsMADS4 is a PI orthologue. In addition, these results suggest that RNA interference is a useful tool for functional genomics in rice.

Functional characterization of rice OsDof12.

DNA-binding with one finger (Dof) proteins are a large family of transcription factors involved in a variety of biological processes in plants. In rice, 30 different Dof genes have been identified through genome analysis. Here we report the functional characteristics of a rice Dof gene, OsDof12, which encodes a predicted Dof protein. The nuclear localization of OsDof12 was investigated by the transient expression assays of the OsDof12–GFP fusion protein in onion epidermal cells. Trans-activation assays in a yeast one-hybrid system indicated that OsDof12 had transcriptional activity. RNA expression analyses showed that the expression of OsDof12 was not tissue-specific in general and fluctuated at different development stages in rice. In addition, OsDof12 was strongly inhibited by dark treatments. The transgenic lines overexpressing OsDof12 showed early flowering under long-day (LD) conditions, whereas OsDof12 overexpression had no effect on flowering time under short-day (SD) conditions. In transgenic lines overexpressing OsDof12, the transcription levels of Hd3a and OsMADS14 were up-regulated under LD conditions but not SD conditions, whereas the expression of Hd1, OsMADS51, Ehd1 and OsGI did not change under LD and SD conditions. These results suggested that OsDof12 might regulate flowering by controlling the expression of Hd3a and OsMADS14.

STAMENLESS 1, encoding a single C2H2 zinc finger protein, regulates floral organ identity in rice

Floral organ identity is defined by organ homoetic genes whose coordinated expression is crucial with respect to the time and place of floral organ formation. Here, we report molecular cloning and characterization of the rice STAMENLESS 1 (SL1) gene that is involved in floral development. The sl1 mutant largely resembles the rice B-class gene mutant spw1; both exhibit homeotic conversions of lodicules and stamens to palea/lemma-like organs and carpels. Additionally, sl1 produces flowers with varied numbers of inner floral organs, and amorphous tissues without floral organ identity were frequently formed in whorls 3 and 4. We also show that SL1 specifies lodicule and stamen identities through positive transcriptional regulation of SPW1/OsMADS16 expression. SL1 encodes a member of the C2H2 family of zinc finger proteins, closely related to JAG of Arabidopsis. The functional divergence between SL1 and JAG implies that SL1 was co-opted for its distinctive roles in specification of floral organ identity in rice after the lineage split from Arabidopsis.

An AT-hook gene is required for palea formation and floral organ number control in rice

Grasses have highly specialized flowers and their outer floral organ identity remains unclear. In this study, we identified and characterized rice mutants that specifically disrupted the development of palea, one of the outer whorl floral organs. The depressed palea1 (dp1) mutants show a primary defect in the main structure of palea, implying that palea is a fusion between the main structure and marginal tissues on both sides. The sterile lemma at the palea side is occasionally elongated in dp1 mutants. In addition, we found a floral organ number increase in dp1 mutants at low penetration. Both the sterile lemma elongation and the floral organ number increase phenotype are enhanced by the mutation of an independent gene SMALL DEGENERATIVE PALEA1 (SDP1), whose single mutation causes reduced palea size. E function and presumable A function floral homeotic genes were found suppressed in the dp1-2 mutant. We identified the DP1 gene by map-based cloning and found it encodes a nuclear-localized AT-hook DNA binding protein, suggesting a grass-specific role of chromatin architecture modification in flower development. The DP1 enhancer SDP1 was also positional cloned, and was found identical to the recently reported RETARDED PALEA1 (REP1) gene encoding a TCP family transcription factor. We further found that SDP1/REP1 is downstreamly regulated by DP1.

The interactions among DWARF10, auxin and cytokinin underlie lateral bud outgrowth in rice.

Previous studies have shown that DWARF10 (D10) is a rice ortholog of MAX4/RMS1/DAD1, encoding a carotenoid cleavage dioxygenase and functioning in strigolactones/strigolactone-derivatives (SL) biosynthesis. Here we use D10- RNA interference (RNAi) transgenic plants similar to d10 mutant in phenotypes to investigate the interactions among D10, auxin and cytokinin in regulating rice shoot branching. Auxin levels in node 1 of both decapitated D10-RNAi and wild type plants decreased significantly, showing that decapitation does reduce endogenous auxin concentration, but decapitation has no clear effects on auxin levels in node 2 of the same plants. This implies that node 1 may be the location where a possible interaction between auxin and D10 gene would be detected. D10 expression in node 1 is inhibited by decapitation, and this inhibition can be restored by exogenous auxin application, indicating that D10 may play an important role in auxin regulation of SL. The decreased expression of most OsPINs in shoot nodes of D10-RNAi plants may cause a reduced auxin transport capacity. Furthermore, effects of auxin treatment of decapitated plants on the expression of cytokinin biosynthetic genes suggest that D10 promotes cytokinin biosynthesis by reducing auxin levels. Besides, in D10-RNAi plants, decreased storage cytokinin levels in the shoot node may partly account for the increased active cytokinin contents, resulting in more tillering phenotypes.

Sequencing and de novo assembly of a near complete indica rice genome

A high-quality reference genome is critical for understanding genome structure, genetic variation and evolution of an organism. Here we report the de novo assembly of an indica rice genome Shuhui498 (R498) through the integration of single-molecule sequencing and mapping data, genetic map and fosmid sequence tags. The 390.3 Mb assembly is estimated to cover more than 99% of the R498 genome and is more continuous than the current reference genomes of japonica rice Nipponbare (MSU7) and Arabidopsis thaliana (TAIR10). We annotate high-quality protein-coding genes in R498 and identify genetic variations between R498 and Nipponbare and presence/absence variations by comparing them to 17 draft genomes in cultivated rice and its closest wild relatives. Our results demonstrate how to de novo assemble a highly contiguous and near-complete plant genome through an integrative strategy. The R498 genome will serve as a reference for the discovery of genes and structural variations in rice.