Comparative Proteomic Analysis of Plasma Membrane Proteins in Rice Leaves Reveals a Vesicle Trafficking Network in Plant Immunity That Is Provoked by Blast Fungi.

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases in rice and can affect rice production worldwide. Rice plasma membrane (PM) proteins are crucial for rapidly and precisely establishing a defense response in plant immunity when rice and blast fungi interact. However, the plant-immunity-associated vesicle trafficking network mediated by PM proteins is poorly understood. In this study, to explore changes in PM proteins during M. oryzae infection, the PM proteome was analyzed via iTRAQ in the resistant rice landrace Heikezijing. A total of 831 differentially expressed proteins (DEPs) were identified, including 434 upregulated and 397 downregulated DEPs. In functional analyses, DEPs associated with vesicle trafficking were significantly enriched, including the "transport" term in a Gene Ontology enrichment analysis, the endocytosis and phagosome pathways in a Encyclopedia of Genes and Genomes analysis, and vesicle-associated proteins identified via a protein-protein interaction network analysis. OsNPSN13, a novel plant-specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 13 protein, was identified as an upregulated DEP, and transgenic plants overexpressing this gene showed enhanced blast resistance, while transgenic knockdown plants were more susceptible than wild-type plants. The changes in abundance and putative functions of 20 DEPs revealed a possible vesicle trafficking network in the M. oryzae-rice interaction. A comparative proteomic analysis of plasma membrane proteins in rice leaves revealed a plant-immunity-associated vesicle trafficking network that is provoked by blast fungi; these results provide new insights into rice resistance responses against rice blast fungi.

Identification and fine mapping of qGR6.2, a novel locus controlling rice seed germination under salt stress.

BACKGROUND: Rice growth is frequently affected by salinity. When exposed to high salinity, rice seed germination and seedling establishment are significantly inhibited. With the promotion of direct-seeding in Asia, improving rice seed germination under salt stress is crucial for breeding. RESULTS: In this study, an indica landrace Wujiaozhan (WJZ) was identified with high germinability under salt stress. A BC1F2 population derived from the crossing WJZ/Nip (japonica, Nipponbare)//Nip, was used to quantitative trait loci (QTL) mapping for the seed germination rate (GR) and germination index (GI) under H2O and 300 mM NaCl conditions. A total of 13 QTLs were identified, i.e. ten QTLs under H2O conditions and nine QTLs under salt conditions. Six QTLs, qGR6.1, qGR8.1, qGR8.2, qGR10.1, qGR10.2 and qGI10.1 were simultaneously identified under two conditions. Under salt conditions, three QTLs, qGR6.2, qGR10.1 and qGR10.2 for GR were identified at different time points during seed germination, which shared the same chromosomal region with qGI6.2, qGI10.1 and qGI10.2 for GI respectively. The qGR6.2 accounted for more than 20% of phenotypic variation under salt stress, as the major effective QTL. Furthermore, qGR6.2 was verified via the BC2F2 population and narrowed to a 65.9-kb region with eleven candidate genes predicted. Based on the microarray database, five candidate genes were found with high transcript abundances at the seed germination stage, of which LOC_Os06g10650 and LOC_Os06g10710 were differentially expressed after seed imbibition. RT-qPCR results showed the expression of LOC_Os06g10650 was significantly up-regulated in two parents with higher levels in WJZ than Nip during seed germination under salt conditions. Taken together, it suggests that LOC_Os06g10650, encoding tyrosine phosphatase family protein, might be the causal candidate gene for qGR6.2. CONCLUSIONS: In this study, we identified 13 QTLs from a landrace WJZ that confer seed germination traits under H2O and salt conditions. A major salt-tolerance-specific QTL qGR6.2 was fine mapped to a 65.9-kb region. Our results provide information on the genetic basis of improving rice seed germination under salt stress by marker-assisted selection (MAS).

Functional analysis of OsHSBP1 and OsHSBP2 revealed their involvement in the heat shock response in rice (Oryza sativa L.).

The heat shock response (HSR) induces the production of heat shock proteins (HSPs) through the activation of heat shock factors (HSF). HSF binding protein (HSBP) is reported to modulate the function of HSF by binding to their trimer and hence to regulate HSR. This report describes the role of OsHSBP1 and OsHSBP2 in the regulation of the HSR and seed development of rice. Both genes expressed ubiquitously in all tissues under normal growth conditions while their expression levels were significantly increased during recovery after heat shock treatment. Subcellular localization revealed the cytosol-nuclear localization of both OsHSBP1 and OsHSBP2 in onion epidermal cells. The yeast two-hybrid assay depicted the self-binding ability of both genes. Both genes were also important for seed development, as their knock-down lines were associated with significant seed abortion. The thermotolerance assay revealed that OsHSBP1 and OsHSBP2 are negative regulators of HSR and involved in acquired thermotolerance but not in basal thermotolerance since their over-expression transgenic lines pre-heated at sublethal temperature, showed significantly decreased seedling survival after heat shock treatment. Furthermore, antioxidant activity and gene expression of catalase and peroxidase was significantly increased in knock-down transgenic seedlings of OsHSBP1 and OsHSBP2 after heat stress compared with the wild type. The expression of heat specific HSPs was also increased significantly in knockdown line of both genes but in a specific manner, suggesting the involvement of HSBP genes in different pathways. Overall, the present study reveals the role of OsHSBP1 and OsHSBP2 in the regulation of the HSR and seed development of rice.

Phylogenetic characterization of a microsporidium (Nosema sp.) isolated from the mulberry pest, Hemerophila atrilineata.

Microsporidia are a group of obligate intracellular unicellular eukaryotes that can parasitize a wide variety of other eukaryotes ranging from protists to invertebrates and vertebrates. In this study, we examined the microsporidium Nosema sp. isolated from the mulberry pest, Hemerophila atrilineata Butler, 1881, named herein "Nosema sp. HA". The fresh spores were long oval in shape, 3.8 +/- 0.4 microm in length and 1.9 +/- 0.3 microm in width. Analysis of tissue infection of silkworm, Bombyx mori Linnaeus, 1758, indicated that the midgut, Malpighian tubules, muscle, fat body, silk glands, hemocytes, nerve tissue and gonads of silkworm were infected with Nosema sp. HA. The complete rRNA gene sequence of this microsporidium contained 4 305 base pairs (GenBank Accession JN882299), including the large subunit rRNA (2492 bp), the internal transcribed spacer (187 bp), the small subunit rRNA (1232 bp), the intergenic spacer (279 bp) and the 5S region (115 bp). The organization of the rRNA gene is 5'-LSU-ITS-SSU-IGS-5S-3'. Phylogenetic analysis, comparison of sequence identities and the arrangement in the rRNA gene subunits suggested that this isolate is separate from other Nosema species.

Complete sequence and gene organization of the Nosema heliothidis ribosomal RNA gene region.

By sequencing the entire ribosomal RNA (rRNA) gene region of Nosema heliothidis isolated from cotton bollworm (Helicoverpa armigera), we showed that its gene organization is similar to the type species, Nosema bombycis: the 5'-large subunit rRNA (2,490 bp)-internal transcribed spacer (192 bp)-small subunit rRNA (1,232 bp)-intergenic spacer (274 bp)-5S rRNA (115 bp)-3'. We constructed two phylogenetic trees, analyzed phylogenetic relationships, examined rRNA organization of microsporidia, and compared the secondary structure of small subunit rRNA with closely related microsporidia. The latter two features may provide important information for the classification and phylogenetic analysis of microsporidia.

Phylogenetic analysis of complete rRNA gene sequence of Nosema philosamiae isolated from the lepidopteran Philosamia cynthia ricini.

ABSTRACT. The microsporidian Nosema philosamiae is a pathogen that infects the eri-silkworm Philosamia cynthia ricini. The complete sequence of rRNA gene (4,314 bp) was obtained by polymerase chain reaction amplification with specific primers and sequencing. The sequence analysis showed that the organization of the rRNA of N. philosamiae was similar to the pattern of Nosema bombycis. Phylogenetic analysis of rRNA gene sequences revealed that N. philosamiae had a close relationship with other Nosema species, confirming that N. philosamiae is correctly assigned to the genus Nosema.

Sequence and phylogenetic analysis of SSU rRNA gene of five microsporidia.

The complete small subunit rRNA (SSU rRNA) gene sequences of five microsporidia including Nosema heliothidis, and four novel microsporidia isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori, respectively, were obtained by PCR amplification, cloning, and sequencing. Two phylogenetic trees based on SSU rRNA sequences had been constructed by using Neighbor-Joining of Phylip software and UPGMA of MEGA4.0 software. The taxonomic status of four novel microsporidia was determined by analysis of phylogenetic relationship, length, G+C content, identity, and divergence of the SSU rRNA sequences. The results showed that the microsporidia isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata have close phylogenetic relationship with the Nosema, while another microsporidium isolated from Bombyx mori is closely related to the Endoreticulatus. So, we temporarily classify three novel species of microsporidia to genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA. Another is temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The result indicated as well that it is feasible and valuable to elucidate phylogenetic relationships and taxonomic status of microsporidian species by analyzing information from SSU rRNA sequences of microsporidia.