OsSYP121 Accumulates at Fungal Penetration Sites and Mediates Host Resistance to Rice Blast.

Magnaporthe oryzae is a fungal pathogen that causes rice (Oryza sativa) blast. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are key components in vesicle trafficking in eukaryotic cells and are known to contribute to fungal pathogen resistance. Syntaxin of Plants121 (SYP121), a Qa-SNARE, has been reported to function in nonhost resistance in Arabidopsis (Arabidopsis thaliana). However, the functions of SYP121 in host resistance to rice blast are largely unknown. Here, we report that the rice SYP121 protein, OsSYP121, accumulates at fungal penetration sites and mediates host resistance to rice blast. OsSYP121 is plasma membrane localized and its expression was obviously induced by the rice blast in both the blast-resistant rice landrace Heikezijing and the blast-susceptible landrace Suyunuo (Su). Overexpression of OsSYP121 in Su resulted in enhanced resistance to blast. Knockdown of OsSYP121 expression in Su resulted in a more susceptible phenotype. However, knockdown of OsSYP121 expression in the resistant landrace Heikezijing resulted in susceptibility to the blast fungus. The POsSYP121 ::GFP-OsSYP121 accumulated at rice blast penetration sites in transgenic rice, as observed by confocal microscopy. Yeast two-hybrid results showed that OsSYP121 can interact with OsSNAP32 (Synaptosome-associated protein of 32 kD) and Vesicle-associated membrane protein714/724. The interaction between OsSYP121 and OsSNAP32 may contribute to host resistance to rice blast. Our study reveals that OsSYP121 plays an important role in rice blast resistance as it is a key component in vesicle trafficking.

Characterization of a vacuolar zinc transporter OZT1 in rice (Oryza sativa L.).

The CDF family is a ubiquitous family that has been identified in prokaryotes, eukaryotes, and archaea. Members of this family are important heavy metal transporters that transport metal ions out of the cytoplasm. In this research, a full length cDNA named Oryza sativa Zn Transporter 1 (OZT1) that closely related to rat ZnT-2 (Zn Transporter 2) gene was isolated from rice. The OZT1 encoding a CDF family protein shares 28.2 % ~ 84.3 % of identities and 49.3 % ~ 90.9 % of similarities with other zinc transporters such as RnZnT-2, HsZnT-8, RnZnT-8 and AtMTP1. OZT1 was constitutively expressed in various rice tissues. The OZT1 expression was significantly induced both in the seedlings of japonica rice Nipponbare and indica rice IR26 in response to Zn(2+) and Cd(2+) treatments. Besides, OZT1 expression was also increased when exposed to other excess metals, such as Cu(2+), Fe(2+) and Mg(2+). Subcellular localization analysis indicated that OZT1 localized to vacuole. Heterologous expression of OZT1 in yeast increased tolerance to Zn(2+) and Cd(2+) stress but not the Mg(2+) stress. Together, OZT1 is a CDF family vacuolar zinc transporter conferring tolerance to Zn(2+) and Cd(2+) stress, which is important to transporting and homeostasis of Zn, Cd or other heavy metals in plants.

Functional analysis of a novel Cys2/His2-type zinc finger protein involved in salt tolerance in rice.

The Cys2/His2-type zinc finger proteins have been implicated in different cellular processes involved in plant development and stress responses. Through microarray analysis, a salt-responsive zinc finger protein gene ZFP179 was identified and subsequently cloned from rice seedlings. ZFP179 encodes a 17.95 kDa protein with two C2H2-type zinc finger motifs having transcriptional activation activity. The real-time RT-PCR analysis showed that ZFP179 was highly expressed in immature spikes, and markedly induced in the seedlings by NaCl, PEG 6000, and ABA treatments. Overexpression of ZFP179 in rice increased salt tolerance and the transgenic seedlings showed hypersensitivity to exogenous ABA. The increased levels of free proline and soluble sugars were observed in transgenic plants compared to wild-type plants under salt stress. The ZFP179 transgenic rice exhibited significantly increased tolerance to oxidative stress, the reactive oxygen species (ROS)-scavenging ability, and expression levels of a number of stress-related genes, including OsDREB2A, OsP5CS OsProT, and OsLea3 under salt stress. Our studies suggest that ZFP179 plays a crucial role in the plant response to salt stress, and is useful in developing transgenic crops with enhanced tolerance to salt stress.

[Cloning and expression analysis of ZFP207 encoding a TFA-type zinc finger protein in rice].

The zinc finer proteins consist of a large transcription factor family involved in plant development and responses to environmental stresses. In this paper, a TFA-type zinc finger protein gene ZFP207 (GenBank assession number AK063147.1) was cloned from rice variety Jiucaiqing by RT-PCR approach. This gene contains an open reading frame (ORF) of 567 bp, which encodes a peptide of 188 amino acid residues. The isoelectric point (pI) of the protein is 9.67, and its molecular weight is 20.72 kDa. Bioinformatic analysis showed that the ZFP207 protein comprises a typical TFA-type zinc finger domain and an EAR-motif at its C-terminus. However, nuclear localization signals (NLS) commonly existing in TFA-type zinc finger proteins was not found in the ZFP207 amino acid sequence. In addition, based on the alignments of the whole amino acid sequences of some known TFA-type zinc finger proteins in plants, a phylogenetic tree was con-structed by the neighbour joining method. The phylogenetic tree showed that ZFP207 and other TFA-type zinc finger proteins with single zinc finger domain were grouped into the same branch. The expression pattern of ZFP207 gene was also investigated in various rice tissues at adult stage by RT-PCR and the results showed that ZFP207 was expressed with high levels in culms and leaves, but lower in roots and spikes. Finally, the trans-activation assay in yeast cells revealed that ZFP207 lacked the trans-activation activity.

[Preparation, characterization and application of rice Qb-SNARE protein OsNPSN11 polyclonal antibody].

Membrane fusion in vesicle trafficking in the cells of eukaryotic organisms is mediated by soluble-N-ethyl- maleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins. OsNPSN11 is a member of Qb-SNARE gene family isolate from rice. The cDNA of OsNPSN11 was subcloned into pET-30a and fusion to the 6 × His tag. Induced by 0.5 mmol/L IPTG for four hours, the recombinant protein was highly expressed in Escherichia coli, which was purified by Ni2+ -NTA His-bind resin affinity chromatography column to be used as an antigen to raise the antibody in New Zealand rabbits. Western blotting analysis showed that the antibody can specifically recognize the expressed antigen and the OsNPSN11 in plasma membrane protein from various rice tissues. This indicated that the antibody can be used for expres-sion analysis in transgenic rice.

Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245.

ZFP245 is a cold- and drought-responsive gene that encodes a zinc finger protein in rice. The ZFP245 protein localizes in the nucleus and exhibits trans-activation activity. Transgenic rice plants overexpressing ZFP245 were generated and found to display high tolerance to cold and drought stresses. The transgenic plants did not exhibit growth retardation, but showed growth sensitivity against exogenous abscisic acid, increased free proline levels and elevated expression of rice pyrroline-5-carboxylatesynthetase and proline transporter genes under stress conditions. Overproduction of ZFP245 enhanced the activities of reactive oxygen species-scavenging enzymes under stress conditions and increased the tolerance of rice seedlings to oxidative stress. Our data suggest that ZFP245 may contribute to the tolerance of rice plants to cold and drought stresses by regulating proline levels and reactive oxygen species-scavenging activities, and therefore may be useful for developing transgenic crops with enhanced tolerance to abiotic stress.

SRWD: a novel WD40 protein subfamily regulated by salt stress in rice (OryzasativaL.).

By analysis with microarray data, we found that a gene encoding a novel protein containing five WD40 repeats, was regulated by salt stress in rice and named as SRWD1 (Salt responsive WD40 protein 1). By database searching, additional four SRWD1-like genes (SRWD2-SRWD5) were found in rice genome, and these five SRWD genes formed a novel WD40 subfamily. Phylogenetic analysis showed that plant SRWD proteins divided into four groups. The significant functional divergences during SRWD evolution were found. The tissue-specific and salt responsive expression profiling for SRWD genes was investigated based on microarray data. It was found that all five SRWD genes in rice were regulated by salt stress. Further, we found that SRWD1 was regulated with different patterns by salt stress in two rice cultivars responding differently to salt stress. Our study correlates WD40 proteins with salt stress in plants and provides fundamental information for the further investigation of plant SRWD proteins.

Expression analysis of rice A20/AN1-type zinc finger genes and characterization of ZFP177 that contributes to temperature stress tolerance.

The A20/AN1-type zinc finger protein family is conserved in animals and plants. Using human AWP1 protein as a query, we identified twelve A20/AN1-type zinc finger proteins in japonica rice. Most of these genes were constitutively expressed in leaves, roots, culms and spikes. Through microarray analysis, it was found that four genes (ZFP177, ZFP181, ZFP176, ZFP173), two genes (ZFP181 and ZFP176) and one gene (ZFP157) were significantly induced by cold, drought and H(2)O(2) treatments, respectively. Further expression analysis showed that ZFP177 was responsive to both cold and heat stresses, but down-regulated by salt. The subcellular localization assay indicated that ZFP177 was localized in cytoplasm in tobacco leaf and root cells. Yeast-one hybrid assay showed that ZFP177 lacked trans-activation potential in yeast cells. Overexpression of ZFP177 in tobacco conferred tolerance of transgenic plants to both low and high temperature stresses, but increased sensitivity to salt and drought stresses. Further we found expression levels of some stress-related genes were inhibited in ZFP177 transgenic plants. These results suggested that ZFP177 might play crucial but differential roles in plant responses to various abiotic stresses.

Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.).

We previously identified a salt and drought stress-responsive TFIIIA-type zinc finger protein gene ZFP252 from rice. Here we report the functional analysis of ZFP252 using gain- and loss-of-function strategies. We found that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes and enhanced rice tolerance to salt and drought stresses, as compared with ZFP252 antisense and non-transgenic plants. Our findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced tolerance to salt and drought stresses.

Overexpression of the Qc-SNARE gene OsSYP71 enhances tolerance to oxidative stress and resistance to rice blast in rice (Oryza sativa L.).

OsSYP71 is an oxidative stress and rice blast response gene that encodes a Qc-SNARE protein in rice. Qc-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), which function as important components of the vesicle trafficking machinery in eukaryotic cells. In this paper, 12 Qc-SNARE genes were isolated from rice, and expression patterns of 9 genes were detected in various tissues and in seedlings challenged with oxidative stresses and inoculated with rice blast. The expression of OsSYP71 was clearly up-regulated under these stresses. Overexpression of OsSYP71 in rice showed more tolerance to oxidative stress and resistance to rice blast than wild-type plants. These results indicate that Qc-SNAREs play an important role in rice response to environmental stresses, and OsSYP71 is useful in engineering crop plants with enhanced tolerance to oxidative stress and resistance to rice blast.

Quantitative trait loci analysis for rice seed vigor during the germination stage.

Seed vigor is an important characteristic of seed quality, and rice cultivars with strong seed vigor are desirable in direct-sowing rice production for optimum stand establishment. In the present study, the quantitative trait loci (QTLs) of three traits for rice seed vigor during the germination stage, including germination rate, final germination percentage, and germination index, were investigated using one recombinant inbred line (RIL) population derived from a cross between japonica Daguandao and indica IR28, and using the multiple interval mapping (MIM) approach. The results show that indica rice presented stronger seed vigor during the germination stage than japonica rice. A total of ten QTLs, and at least five novel alleles, were detected to control rice seed vigor, and the amount of variation (R(2)) explained by an individual QTL ranged from 7.5% to 68.5%, with three major QTLs with R(2)>20%. Most of the QTLs detected here are likely to coincide with QTLs for seed weight, seed size, or seed dormancy, suggesting that the rice seed vigor might be correlated with seed weight, seed size, and seed dormancy. At least five QTLs are novel alleles with no previous reports of seed vigor genes in rice, and those major or minor QTLs could be used to significantly improve the seed vigor by marker-assisted selection (MAS) in rice.

Cloning and characterization of three genes encoding Qb-SNARE proteins in rice.

Qb-SNARE proteins belong to the superfamily of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and function as important components of the vesicle trafficking machinery in eukaryotic cells. Here, we report three novel plant SNARE (NPSN) genes isolated from rice and named OsNPSN11, OsNPSN12 and OsNPSN13. They have about 70% nucleotide identity over their entire coding regions and similar genomic organization with ten exons and nine introns in each gene. Multiple alignment of deduced amino acid sequences indicate that the OsNPSNs proteins are homologous to AtNPSNs from Arabidopsis, containing a Qb-SNARE domain and a membrane-spanning domain in the C-terminal region. Semi-quantitative RT-PCR assays showed that the OsNPSNs were ubiquitously and differentially expressed in roots, culms, leaves, immature spikes and flowering spikes. The expression of OsNPSNs was significantly activated in rice seedlings treated with H(2)O(2), but down-regulated under NaCl and PEG6000 stresses. Transient expression method in onion epidermal cells revealed that OsNPSNs were located in the plasma membrane. Transformed yeast cells with OsNPSNs had better growth rates than empty-vector transformants when cultured on either solid or liquid selective media containing various concentrations of H(2)O(2), but more sensitive to NaCl and mannitol stresses. The 35S:OsNPSN11 transgenic tobacco also showed more tolerance to H(2)O(2) and sensitivity to NaCl and mannitol than non-transgenic tobacco. These results indicate that OsNPSNs may be involved in different aspects of the signal transduction in plant and yeast responses to abiotic stresses.

Molecular cloning and characterization of a novel SNAP25-type protein gene OsSNAP32 in rice (Oryza sativa L.).

The SNAP25-type proteins belong to the superfamily of the SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), and function as important components of the vesical trafficking machinery in eukaryotic cells. In this paper, we report the cloning and expression characterization of OsSNAP32 gene, and the subcellular localization of its encoded protein. The OsSNAP32 gene contains five exons and four introns, and is located between RFLP markers C12276S and S1917 on chromosome 2 in rice. The OsSNAP32 has a molecular weight of 31.3 kD, comprises 283 amino acid residues, and contains Qb-SNARE and Qc-SNARE domains in the N- and C-terminal, respectively. Multiple sequence alignment of the SNARE domains indicates that OsSNAP32 protein is homologous to HvSNAP34 and HvSNAP28 (63% and 55% of amino acid identity respectively) from barley. The transient expression method in onion epidermal cells, revealed that OsSNAP32 is located in the plasma membrane, like other SNAP25-type proteins. Semi-quantitative RT-PCR assay showed that the OsSNAP32 is highly expressed in leaves and culms, and low in roots of rice, while hardly detected in immature spikes and flowering spikes. The expression of OsSNAP32 was significantly activated in rice seedlings treated with H2O2, PEG6000, and low temperature or after inoculation with rice blast (Magnaporthe grisea strain Hoku 1). The results suggest that this gene belongs to a novel member of this gene family encoding SNAP25-type proteins, involved in the rice responses to biotic and abiotic stresses.