A genome-wide survey of interaction between rice and Magnaporthe oryzae via microarray analysis.

The main aim of the work is to study the regulation of gene expression in the interaction between rice and Magnaporthe oryzae by gene chip technology. In this study, we mainly focused on changes of gene expression at 24, 48, and 72 hours post-inoculation (hpi), through which we could conduct a more comprehensive analysis of rice blast-related genes in the process of infection. The results showed that the experimental groups contained 460, 1227, and 3937 significant differentially expressed genes at 24, 48, and 72 hpi, respectively. Furthermore, 115 significantly differentially expressed genes were identified in response to rice blast infection at all three time points. By annotating these 115 genes, they were divided into three categories: metabolic pathways, proteins or enzymes, and organelle components. As expected, many of these genes were known rice blast-related genes; however, we discovered new genes with high fold changes. Most of them encoded conserved hypothetical proteins, and some were hypothetically conserved genes. Our study may contribute to finding new resistance genes and understanding the mechanism of rice blast development.

Rice OsAAA-ATPase1 is Induced during Blast Infection in a Salicylic Acid-Dependent Manner, and Promotes Blast Fungus Resistance.

Fatty acids (FAs) have been implicated in signaling roles in plant defense responses. We previously reported that mutation or RNAi-knockdown (OsSSI2-kd) of the rice OsSSI2 gene, encoding a stearoyl acyl carrier protein FA desaturase (SACPD), remarkably enhanced resistance to blast fungus Magnaporthe oryzae and the leaf-blight bacterium Xanthomonas oryzae pv. oryzae (Xoo). Transcriptomic analysis identified six AAA-ATPase family genes (hereafter OsAAA-ATPase1-6) upregulated in the OsSSI2-kd plants, in addition to other well-known defense-related genes. Here, we report the functional analysis of OsAAA-ATPase1 in rice's defense response to M. oryzae. Recombinant OsAAA-ATPase1 synthesized in Escherichia coli showed ATPase activity. OsAAA-ATPase1 transcription was induced by exogenous treatment with a functional analogue of salicylic acid (SA), benzothiadiazole (BTH), but not by other plant hormones tested. The transcription of OsAAA-ATPase1 was also highly induced in response to M. oryzae infection in an SA-dependent manner, as gene induction was significantly attenuated in a transgenic rice line expressing a bacterial gene (nahG) encoding salicylate hydroxylase. Overexpression of OsAAA-ATPase1 significantly enhanced pathogenesis-related gene expression and the resistance to M. oryzae; conversely, RNAi-mediated suppression of this gene compromised this resistance. These results suggest that OsAAA-APTase1 plays an important role in SA-mediated defense responses against blast fungus M. oryzae.

Molecular characterization and application of a novel cytoplasmic male sterility-associated mitochondrial sequence in rice.

BACKGROUND: Cytoplasmic male sterility (CMS) is a maternally inherited inability to produce functional pollen found in numerous flowering plant species. CMS is associated with mitochondrial DNA mutation, novel chimeric open reading frames (ORFs), and rearrangement of coding and noncoding regions of the mitochondrial genome. RESULTS: BLAST (Basic Local Alignment Search Tool) analysis indicated that L-sp1, a new sequence-characterized amplified region, is non-homologous to atp6-orfH79 (or atp6-orf79) and WA352 cloned CMS-associated genes. L-sp1 was found in 11 of 102 wild rice accessions belonging to four AA genome species: Oryza rufipogon, Oryza nivara, Oryza glumaepatula, and Oryza meridionalis. Using L-sp1, two new CMS lines were developed, from either low natural fertility plants or sterile plants, by backcrossing BC1F1 with Yuetai B. Northern blot and RT-PCR revealed that L-sp1 was only expressed in the anthers of w1/YTB, w2/YTB, w1/YTB//YTB, and w2/YTB//YTB when in the same cytoplasm background. CONCLUSIONS: L-sp1 is a single-copy chimeric CMS-associated gene found in the mitochondrial genome. It can be expressed in anthers with the same specific cytoplasm background, and will be a useful molecular marker for the development and marker-assisted selection of new CMS lines.

Expression analysis of innate immunity related genes in the true/field blast resistance gene-mediated defence response.

Rice blast resistance (R) genes-mediated resistance response depends on various resistance-related genes involved in incompatible interactions. In this work, the expression profiles of innate rice immunity related genes were examined in the mediated resistance response of true/field resistance genes. Three sets of rice near-isogenic lines (NILs) were used: the resistant NILs carrying true resistance genes in the genetic background of the susceptible cultivar Nipponbare (NB), NB-Pib, NB-Pizt, NB-Pik and NB-Pita2; NILs bearing field resistance genes pi21 in the susceptible cultivar Aichiasahi (AA) AA-pi21, Kahei (KHR). The marker gene OsWRKY45 of salicylic acid (SA) signalling was upregulated in all tested cultivars. And, JAmyb (marker gene of jasmonic acid signalling) showed higher upregulation in the resistance lines with nucleotide-binding sites and leucine-rich repeat (NBS-LRR) R genes Pib, Pizt, Pik, Pita2 and Pikahei than in NB and KHS. SalT of abscisic acid (ABA) signalling may be involved in the R/Avr interaction, including Pizt, Pik, pi21 and Pikahei. However, SalT was shown to negatively regulate Pib/AvrPib interaction. OsPR1b and PBZ1 were differentially expressed and strongly activated at a later stage by 48 h post-inoculation. Interestingly, there was evidence that OsPR1b and PBZ1 played an important role in the pi21-mediated response. It was shown that OsRAR1 could be upregulated in the true resistance line NB-Pita2 and the field resistance line KHR, while OsSGT1 and OsHSP90 could be upregulated in all tested lines. The involvement of these genes illustrated the complexity of the downstream signalling pathways in the mediated resistance response of true/field resistance genes.

An amperometric biosensor based on rat cytochrome p450 1A1 for benzo[a]pyrene determination.

Using the plasmid pCW, high-level expression of rat cytochrome p4501A1 (CYP1A1) has been achieved by making NH(2)-terminal translational fusions to bacterial leader sequences ompA (ompA-1A1/pCW). The construct ompA-1A1 was compared with an expression construct in which the Ala codon GCT was placed in the second position and 5'-terminal codons were maximized for A T content (1A1/pCW). Both constructs produced spectrally active, functional protein. However, the ompA-1A1 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with the construct 1A1/pCW. The expressed 1A1 from the construct ompA-1A1/pCW in bacterial membrane fractions were collected and immobilized in nano-Na-montmorillonite (nano-SWy-2) and dihexadecylphosphate (DHP) composite film. The direct electrochemistry of CYP1A1 in a nano-SWy-2-DHP film on an edge-plane pyrolytic graphite electrode (EPG) has been obtained and the catalytic activity of the enzyme to benzo[a]pyrene has been investigated by the cyclic voltammetry. The immobilized CYP1A1 displayed a pair of redox peaks with a formal potential of -0.36 mV in pH 7.0 O(2)-free phosphate buffers at scan rate of 1 V s(-1). The CYP1A1 in the nano-SWy-2-DHP film retained its bioactivity and could catalyze the reduction of dissolved oxygen. Upon the addition of its substrate benzo[a]pyrene (B[a]P) to the air-saturated solution, the reduction peak current of dissolved oxygen increased, which indicates the catalytic behavior of CYP1A1 to B[a]P. By amperometry a calibration linear range for B[a]P was obtained to be 3.31-16.56 µM with a sensitivity of 58.57 µA mM(-1). And the apparent Michaelis-Menten constant for the electrocatalytic activity of CYP1A1 was estimated to be 46.27 µM for B[a]P.

Expression, purification and direct eletrochemistry of cytochrome P450 6A1 from the house fly, Musca domestica.

A plasmid (pCW) was modified to code for the complete sequence of house fly (Musca domestica) cytochrome P450 6A1 (CYP6A1) with only the second amino acid changed in the N-terminal portion and this plasmid was used to express the enzyme CYP6A1 in Escherichia coli cells. With the addition of delta-aminolevulinic acid and FeCl(3) to the culture, the enzyme was produced at a level about 0.25 micromol L(-1) (15mgL(-1)) of culture with approximately 50% of the P450 being associated with the membrane fraction. The CYP6A1 protein was characterized and the content of CYP6A1 in each fraction was determined by the spectroscopic method. A nearly homogenous CYP6A1 was obtained by purification with a combination of DEAE Sepharose fast flow and hydroxyapatite chromatography. Direct electrochemistry of CYP6A1 in a didodecyldimethylammonium bromide (DSAB) film on an edge-plane pyrolytic graphite electrode (EPG) has been obtained and the catalytic activity of the enzyme to aldrin has been demonstrated by the cyclic voltammetry.

Identification and fine mapping of Pi39(t), a major gene conferring the broad-spectrum resistance to Magnaporthe oryzae.

Blast, caused by the ascomycete fungus Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. The Chinese native cultivar (cv.) Q15 expresses the broad-spectrum resistance to most of the isolates collected from China. To effectively utilize the resistance, three rounds of linkage analysis were performed in an F(2) population derived from a cross of Q15 and a susceptible cv. Tsuyuake, which segregated into 3:1 (resistant/susceptible) ratio. The first round of linkage analysis employing simple sequence repeat (SSR) markers was carried out in the F(2) population through bulked-segregant assay. A total of 180 SSR markers selected from each chromosome equally were surveyed. The results revealed that only two polymorphic markers, RM247 and RM463, located on chromosome 12, were linked to the resistance (R) gene. To further define the chromosomal location of the R gene locus, the second round of linkage analysis was performed using additional five SSR markers, which located in the region anchored by markers RM247 and RM463. The locus was further mapped to a 0.27 cM region bounded by markers RM27933 and RM27940 in the pericentromeric region towards the short arm. For fine mapping of the R locus, seven new markers were developed in the smaller region for the third round of linkage analysis, based on the reference sequences. The R locus was further mapped to a 0.18 cM region flanked by marker clusters 39M11 and 39M22, which is closest to, but away from the Pita/Pita(2) locus by 0.09 cM. To physically map the locus, all the linked markers were landed on the respective bacterial artificial chromosome clones of the reference cv. Nipponbare. Sequence information of these clones was used to construct a physical map of the locus, in silico, by bioinformatics analysis. The locus was physically defined to an interval of approximately 37 kb. To further characterize the R gene, five R genes mapped near the locus, as well as 10 main R genes those might be exploited in the resistance breeding programs, were selected for differential tests with 475 Chinese isolates. The R gene carrier Q15 conveys resistances distinct from those conditioned by the carriers of the 15 R genes. Together, this valuable R gene was, therefore, designated as Pi39(t). The sequence information of the R gene locus could be used for further marker-based selection and cloning.

Quantitative analysis of cytochrome C released from rice mitochondria using the adsorptive polarographic wave of guanidine modified Co(II)-cytochrome C complex.

A new, simple and sensitive method for the quantitative analysis of cytochrome C (Cyt C) based on the reduction wave of guanidine modified Co(II)-Cyt C complex at about -1.74 V (vs. SCE) by single sweep polarography in the solution containing 8 x 10(-6) mol L(-1) CoCl2, 0.04 mol L(-1) guanidine hydrochloride, 0.2 mol L(-1) NaOH and 0.5% Na2SO3. The peak height is linearly proportional to the concentration of Cyt C in the range of 0.005 approximately 1.500 mg L(-1) (correlation coefficient 0.999). Common amino acids, saccharide, organic acid and metal ions of appropriate concentrations have no interference on the Cyt C determination. The released Cyt C in the process of mitochondrial permeability transition of Hong-Lian cytoplasmic male sterile line of rice has been measured by the method, and the result is satisfactory.

[RAPD Identification of Three Types of Three-lines Hybrid Rice with Defferent Male Sterile Cytoplasms and Their Parent Lines.].

The RAPD analysis was conducted on genome DNA from 21 sets of rice, including 6 three-line hybrid rice combinations, separately derived from three different kinds of cytoplasmic male sterile (CMS) lines and their related parents. Out of 264 random primers screened first, 25 primers displayed well in polymorphisms. It was shown that only 7 bands amplified respectively from 7 primers were enough to discriminate the different types of CMS, the hybrid combinations and their parents.

Localizing the Bacterial Blight Resistance Gene, Xa22(t), to a 100-Kilobase Bacterial Artificial Chromosome.

ABSTRACT The rice bacterial blight resistance gene, Xa22(t), provides resistance to a broad spectrum of Xanthomonas oryzae pv. oryzae isolates. Here, we localize the gene to a small 100-kb fragment of chromosome 11 by a combination of genetic recombination analysis and physical mapping. Mapping was done with two F(2) populations from the cross between Zhachanglong and Zhenzhuai. The first population consisted of 248 random individuals and 404 highly susceptible individuals selected from an F(2) population of more than 2,000 individuals and was used to construct a linkage map around the Xa22(t) locus. For the second F(2) population, 7,680 plants were examined with simple sequence repeat markers flanking the Xa22(t) locus to identify recombinants useful for fine-genetic mapping. Two large-insert bacterial artificial chromasome (BAC) libraries (from cvs. Teqing and Minghui63) were screened with a marker (R1506) which cosegregated perfectly with Xa22(t) in the first population. Restriction mapping of the resulting BAC clones enabled a physical map of the area to be constructed, and subclones from the BAC clones provided additional restriction fragment length polymorphism probes which could be placed on the fine-structure genetic map using the recombinants from the second mapping population. The Xa22(t) locus was mapped to a approximately 100-kb interval delimited by the R1506 marker and a subclone from the M3H8 BAC clone.