A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

A Gγ subunit promoter T-DNA insertion mutant――A1-412 of Magnaporthe grisea is defective in appressorium formation, penetration and pathogenicity

Heterotrimeric G-proteins consisting of α, β and γ-subunits are essential for the transduction of ex- tracellular signals to various downstream intracellular effectors in eukaryotes. Previous studies showed that Gα and Gβ were involved in regulating     

MoFLP1, encoding a novel fungal fasciclin-like protein, is involved in conidiation and pathogenicity in Magnaporthe oryzae

Fasciclin family proteins have been identified as cell adhesion molecules in various organisms. In this study, a novel Magnaporthe oryzae fasciclin-like protein encoding gene, named MoFLP1, was isolated from a subtractive suppressive cDNA library and functionally analyzed. Sequence analysis showed that the MoFLP1 gene contains an open reading frame (ORF) of 1050 nucleotides encoding 349 amino acids with a calculated molecular weight of 35.85 kDa and a pI of 7.76. The deduced MoFLP1 protein contains a 17-amino acid secretion signal sequence and an 18-amino acid sequence with the characteristics of a glycosylphosphotidylinositol (GPI) anchor additional signal at its N- and C-terminuses, respectively. Potential N-glycosylation sites and domains involving cell adhesion were also identified in MoFLP1. Sequence analysis and subcellular localization by the expression of MoFLP1-GFP fusion construct in M. oryzae indicated that the MoFLP1 protein is probably localized on the vacuole membrane. Two MoFLP1 null mutants generated by targeted gene disruption exhibited marked reduction of conidiation, conidial adhesion, appressorium turgor, and pathogenicity. Our results indicate that fasciclin proteins play important roles in fungal development and pathogenicity in M. oryzae.     

Genetic analysis and gene cloning of a triangular hull 1 (tri1) mutant in rice (Oryza sativa L.)

Grain shape and size are two key factors that determine rice yield and quality. In the present study, a rice triangular hull mutant (tri1) was obtained from the progeny of japonica rice variety Taipei 309 treated with 60Co γ-rays. Compared to the wild type, the tri1 mutant presents a triangular hull, and exhibits an increase in grain thickness and protein content, but with a slight decrease in plant height and grain weight. Genetic analysis indicated that the mutant phenotype was controlled by a recessive nuclear gene which is stably inherited. Using a map-based cloning strategy, we fine-mapped tri1 to a 47-kb region between the molecular markers CHR0122 and CHR0127 on the long arm of chromosome 1, and showed that it co-segregates with the molecular marker CHR0119. According to the rice genome sequence annotation there are six predicated genes within the mapped region. Sequencing analysis of the mutant and the wild type indicated that there was a deletion of an A nucleotide in exon 3 of the OsMADS32 gene, which could result in a downstream frameshift mutation and premature termination of the predicted polypeptide. Both semi-quantitative and real-time RT-PCR analyses showed that this gene expressed highly in young inflorescences, while expressed at very low levels in other tissues. These results implied that the OsMADS32 gene could be a candidate of TRI1. Taken together, the results of this study lay the foundation for further investigation into the molecular mechanisms regulating rice caryopsis development.     

Identification and gene mapping of a narrow and upper-albino leaf mutant in rice (Oryza sativa L.)

The leaf blade consists of color and shape traits. Studies of leaf-blade development are important for improvement of rice yield and quality because it is an essential organ for photosynthesis. A narrow and upper-albino leaf mutant (nul1) was identified from among progeny of the indica restorer line Jinhui10 raised from seeds treated with ethyl methane sulfonate. Under field conditions, the mutant displayed narrow and upper-albino leaf blades with significantly decreased photosynthetic pigment contents throughout their development. The narrow-leaf trait is caused by a decreased number of small veins. In contrast to the wild type, the growth period was extended by approximately 8 d and agronomic traits, such as effective panicle number, percentage seed set and 1000-grain weight, declined significantly in the nul1 mutant. Genetic analysis suggested that the narrow and upper-albino leaf characteristics showed coseparation and were controlled by one recessive gene. The Nul1 gene was mapped onto chromosome 7 between the Indel marker Ind07-1 and the Simple Sequence Repeat marker RM21637. The physical distance between the markers was 75 kb and eight genes were annotated in this region based on the rice Nipponbare genome sequence. These results provide a foundation for cloning and function analysis of Nul1.     

Analysis of type II toxin-antitoxin genes <Emphasis Type="Italic">all</Emphasis> 3211-<Emphasis Type="Italic">asl</Emphasis> 3212 in <Emphasis Type="Italic">Anabaena</Emphasis> PCC 7120

The type II toxin-antitoxin genes are responsible for the phenotypic switch to a quasi-dormant state that enables cell survival under stresses, a similar function to heterocyst of cyanobacteria. In this paper, we particularly study the role of gene pair all3211-asl3212 under Spectinomycin stress to reveal how the type II toxin-antitoxin involved in environmental stress responses. Bioinformatics prediction shows that toxin protein gene All3211 is homologous to MazF, a member of mazEF family that encoding nucleases. We clone gene all3211-asl3212 into expression vectors to identify its molecular characteristics. Deletion mutant strains of all3211-asl3212 are selected in a tri-parental mating screen. Phenotype comparisons of mutant and wild type reveals no difference of single-deletion-mutants in pigment integrity, the sensitivity to antibiotics, and heterocyst formation. The results show that deletion mutation of single TAS gene pair all3211-asl3212 results in limited effects on the cellular growth of PCC 7120. Thus, we suggest that dosage compensating might be provided from redundant genes or bypass pathways to offset obvious phenotypic differences.     

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.     

Morphological and physiological analysis of narrow and striped leaf 1 （nsl1） mutant of rice （Oryza sativa L.） and the gene mapping

The shape and color of rice leaves are impor- tant agronomic traits that directly influence the proportion of sunlight energy utilization and ultimately affect the yield and quality. A new mutant exhibiting stable inheritance was identified as derived from ethyl methane sulfonate （EMS）-treated restorer Jinhui 10, tentatively named as narrow and striped leaf 1 （nsll）. The nsll displayed pale white leaves at the seeding stage and then white striped leaves in parallel to the main vein at the jointing stage. Meanwhile, its leaf blades are significantly narrower than the control group of Jinhui 10. The chloroplast structures of cells in the white striped area of the nsll mutant break down, and the photosynthetic pigments are significantly lower than that of the wild type. Moreover, fluorescence parameters, such as Fo, Fv/Fm, ФpsⅡ, qP, and ETR, in the nsll mutant are significantly lower than those of the wild type, and the photosynthetic efficiency is also significantly decreased. These changes in leaf color and shape, together with physiological changes in the nsll, result in smaller plant height and a decrease in the most important agro- nomic traits, such as the number of grains per panicle, grain weight, etc. Genetic analysis shows that the narrow and striped traits of the nsll mutant are controlled by a single recessive nuclear gene, which is located between InDel 16 and InDel 12 in chromosome 3. The physical distance is 204 kb. So far, no similar genes of such leaf color and shape in this area have been reported, This study has laid asolid foundation for the gene cloning and function analysis of NSL 1.     

Genetic analysis and fine mapping of an enclosed panicle mutant esp2 in rice （Oryza sativa L.）

The phenomenon of panicle enclosure in rice is mainly caused by the shortening of uppermost internode.Elucidating the molecular mechanism of panicle enclosure will be helpful for solving the problem of panicle enclosure in male sterile lines and creating new germplasms in rice.We acquired a monogenic recessive enclosed panicle mutant,named as esp2 (enclosed shorter panicle 2),from the tissue culture progeny of indica rice cultivar Minghui-86.In the mutant,panicles were entirely enclosed by flag leaf sheaths and the uppermost internode was almost completely degenerated,but the other internodes did not have obvious changes in length.Genetic analysis indicated that the mutant phenotype was controlled by a recessive gene,which could be steadily inherited and was not affected by genetic background.Apparently,ESP2 is a key gene for the development of uppermost internode in rice.Using an F 2 population of a cross between esp2 and a japonica rice cultivar Xiushui-13 as well as SSR and InDel markers,we fine mapped ESP2 to a 14-kb region on the end of the short arm of chromosome 1.According to the rice genome sequence annotation,only one intact gene exists in this region,namely,a putative phosphatidylserine synthase gene.Sequencing analysis on the mutant and the wild type indicated that this gene was inserted by a 5287-bp retrotransposon sequence.Hence,we took this gene as a candidate of ESP2.The results of this study will facilitate the cloning and functional analysis of ESP2 gene.     

SRO1 regulates heavy metal mercury stress response in Arabidopsis thaliana

Heavy metals in the environment are harmful limiting factors for the normal growth and development of plants. Here, we isolated and identified an Arabidopsis thaliana T-DNA insertion mutant, named srol-1, which showed a hyper-sensitive response to HgCl2. The SRO1 protein contains a WWE domain that mediates proteinprotein interactions. Under HgCl2 treatment, when compared with the wild-type plants, the growth of srol-1 was repressed dramatically and the number of true leaves was reduced and etiolated. The electrolyte leakage rates showed that cell membrane integrity in srol-1 was damaged more severely than in the wild type. DAB （3,5-diaminobenzidine） staining and confocal microscopy showed that Hg2＋ stress induced more hydrogen peroxide accumulation in srol-1 than in the wild type. The qRT-PCR results indicated that the expression of some abiotic stress-induced genes, such as L-ascorbate peroxidase （APX1）, was reduced under oxidative or Hg2＋ stress. Transgenic plants containing a GFP：：SRO1 fusion protein showed that SRO1 was localized in the nucleus of the cells. SRO1 was shown to be expressed in various tissues, and was most highly expressed in the vigorous tissues. Our results suggest thatSRO1 may play an important role in the stress response of A. thaliana to heavy metals.     

Fluorescent co-localization of PTS1 and PTS2 and its application in analysis of the gene function and the peroxisomal dynamic in Magnaporthe oryzae

The peroxisomal matrix proteins involved in many important biological metabolism pathways in eukaryotic cells are encoded by nucleal genes, synthesized in the cytoplasm and then transported into the organelles. Targeting and import of these proteins depend on their two peroxisomal targeting signals （PTS 1 and PTS2） in sequence as we have known so far. The vectors of the fluorescent fusions with PTS, i.e., green fluorescence protein （GFP）-PTS1, GFP-PTS2 and red fluorescence protein （RFP）-PTS1, were constructed and introduced into Magnaporthe oryzae Guy ll cells. Transformants containing these fusions emitted fluorescence in a punctate pattern, and the locations of the red and green fluorescence overlapped exactly in RFP-PTS 1 and GFP-PTS2 co-transformed strains. These data indicated that both PTS1 and PTS2 fusions were imported into peroxisomes. A probable higher efficiency of PTS1 machinery was revealed by comparing the fluorescence backgrotmds in GFP-PTS1 and GFP-PTS2 transformants. By introducing both RFP-PTS1 and GFP-PTS2 into Amgpex6 mutants, the involvement of MGPEX6 gene in both PTS1 and PTS2 pathways was proved. In addition, using these transformants, the inducement ofperoxisomes and the dynamic of peroxisomal number during the pre-penetration processes were investigated as well. In summary, by the localization and co-localization of PTS1 and PTS2, we provided a useful tool to evaluate the biological roles of the peroxisomes and the related genes.     

Functional analysis of the ABA-responsive protein family in ABA and stress signal transduction in Arabidopsis

The phytohormone abscisic acid (ABA) plays an important role in plant growth and development, for example in seed dormancy and germination, as well as in plant responses to environmental stresses, such as drought and high salinity. Previous studies have shown that ABA regulates the expression of genes with an ABA-responsive element (ABRE) and their corresponding physiological responses. Bioinformatics analysis identified a GRAM domain-containing gene family that has a multiple ABRE cis-element, which was termed the ABA-responsive protein (ABR) family. To analyze the function of the ABR family, we identified homozygous T-DNA insertion mutants and constructed abr1, 2, 3 double mutants and triple mutant. The abr1, abr2 and abr3 single mutants showed a normal phenotype; however, the germination of seeds of the double mutants and triple mutant were insensitive to ABA, NaCl, mannitol and glucose. ABR1-GFP was distributed as a punctate structure in the cytosol and may be localized in the endomembrane system. The ABR2-GFP and ABR3-GFP proteins localized in the cytoplasm. In addition, ABR1, ABR2 and ABR3 were expressed in various tissues, and could be induced by several abiotic stresses, especially by ABA. The expressions of these genes were significantly suppressed in aba2, abi1 and abi2 null mutants. These results suggested that the ABR family may act downstream of ABI1 and ABI2 in the ABA signal transduction process in plants.     

稻瘟菌致病性变异突变体的筛选及共分离分析

通过筛选稻瘟菌(Magnaporthe grisea)P131小种的REMI(Restriction Enzyme MediatedIntegration)转化体库获得对水稻品种梅雨明致病性变异的突变体,命名为PX1.与野生型菌株P131相比,该突变体对水稻品种梅雨明致病性丧失,在洋葱表皮上侵染钉形成率显著降低,而孢子萌发率和附着胞形成率差异不显著.遗传分析表明,该突变体的突变表型和潮霉素抗性标记共分离,说明突变是由于外源质粒插入引起的,因此,可以此为标记克隆控制该表型的基因.     

Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29

An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel® P-100. The protein was absorbed on DEAE-cellulose and Bio-Gel® P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited inhibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia sclerotiorum. The 50% inhibitory concentrations (IC₅₀) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B29I also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germinated spores.     

Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae

Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentiation in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.     

Role of two phosphohexomutase genes in glycogen synthesis in Synechocystis sp. PCC6803

Phosphohexomutases catalyze the interconversion between hexose-6-phosphate and hexose-l-phosphate and play important roles in polysaccharide synthesis. In Synechocystis sp. PCC 6803, sl10726 is predicted to encode PGM （phosphoglucomutase）, slr1334 is predicted to encode a PGM/PMM （phosphomannomutase） bifunction enzyme. In comparison to the wild type, a sllO726-null mutant showed 3.4% PGM activity but 45%-69% glycogen content. Down-regulation of slr1334, an essential gene, by using a copper regulated promoter further decreased the PGM activity in the sllO726：：Kmr PpetE-slr1334 double mutant to 0.3% of the wild type level. However, the glycogen content was not further decreased in parallel. In vitro, recombinant Sl10726 or S1r1334 showed predicted enzyme activities. Our results indicate that a relatively high level of glycogen can be maintained in Synechocystis mutants with low levels of PGM activity. The high PGM activity in the cyanobacterium may be required for turnover of glycogen or synthesis of other polysaccharides or oligosaccharides.     

Sinorhizobium meliloti lsrB is involved in alfalfa root nodule development and nitrogen-fixing bacteroid differentiation

Rhizobia interact with host legumes to induce the formation of nitrogen-fixing nodules, which is very important in agriculture and ecology. The development of nitrogen-fixing nodules is stringently regulated by host plants and rhizobial symbionts. In our previous work, a new Sinorhizobium meliloti LysR regulator gene (lsrB) was identified to be essential for alfalfa nodulation. However, how this gene is involved in alfalfa nodulation was not yet understood. Here, we found that this gene was associated with prevention of premature nodule senescence and abortive bacteroid formation. Heterogeneous deficient alfalfa root nodules were induced by the in-frame deletion mutant of lsrB (lsrB1-2), which was similar to the plasmid-insertion mutant, lsrB1. Irregular senescence zones earlier appeared in these nodules where bacteroid differentiation was blocked at different stages from microscopy observations. Interestingly, oxidative bursts were observed in these nodules by DAB staining. The decreased expression of lipopolysaccharide core genes (lpsCDE) was correspondingly determined in these nodules. S. meliloti lipopolysaccharide is required for suppression of oxidative bursts or host cell defense. These findings demonstrate that the S. meliloti lsrB gene is involved in alfalfa root nodule development and bacteroid differentiation by suppressing oxidative bursts or defense responses in host cells.