The Secreted Protein MoHrip1 Is Necessary for the Virulence of Magnaporthe oryzae.

Secreted effectors from Magnaporthe oryzae play critical roles in the interaction with rice to facilitate fungal infection and disease development. M. oryzae-secreted protein MoHrip1 can improve plant defense as an elicitor in vitro, however, its biological function in fungal infection is not clear. In this study, we found that the expression of mohrip1 was significantly induced in the stages of fungal penetration and colonization. Although dispensable for the growth and conidiation, MoHrip1 was necessary for the full virulence of M. oryzae. Deletion of mohrip1 remarkably compromised fungal virulence on rice seedlings and even on rice leaves with wounds. Rice sheath inoculation assay further demonstrated the defects of mohrip1-deleted mutants on penetration and proliferation in rice cells. Additionally, compared with WT and complementation strain, the inoculation of mohrip1-deleted mutants induced a higher expression of specific defense related genes and a higher production of specific defensive compounds in rice leaves. These data collectively indicated that MoHrip1 is necessary for fungal penetration and invasive expansion, and further full virulence of rice blast fungus.

Draft genome sequence, disease-resistance genes, and phenotype of a Paenibacillus terrae strain (NK3-4) with the potential to control plant diseases.

Paenibacillus terrae NK3-4 is a plant growth-promoting rhizobacterium that may be useful for controlling plant diseases. We conducted a genomic analysis and identified the genes mediating antimicrobial functions. Additionally, an extracellular antifungal protein component was isolated and identified. The draft genome sequence was assembled into 54 contigs, with 5 458 568 bp and a G+C content of 47%. Moreover, 4 690 015 bp encoded 5090 proteins, 7 rRNAs, and 54 tRNAs. Forty-four genes involved in antimicrobial functions were detected. They mainly encode 19 non-ribosomal peptide synthetases (NRPSs); one polyketide synthase/NRPSs hybrid enzyme; four Zn-dependent metalloproteases; three antilisterial bacteriocin subtilosin biosynthesis proteins (AlbA); four serine proteases; five pectate lyases; three beta-glucanases; and four 1,4-beta-xylanases. These include four novel NRPSs that have not been found in any species of Paenibacillus. Furthermore, five proteins exhibiting antifungal activity were identified from the antifungal extracellular protein component based on MS/MS and the strain NK3-4 predicted protein library. On the basis of these features, we propose that strain NK3-4 represents a promising biocontrol agent for protecting plant from diseases. The draft genome sequence described herein may provide the genetic basis for the characterization of the molecular mechanisms underlying the biocontrol functions. It may also facilitate the development of rational strategies for improving the strain.

Inducible and constitutive expression of an elicitor gene Hrip1 from Alternaria tenuissima enhances stress tolerance in Arabidopsis.

Hrip1 is a novel hypersensitive response-inducing protein secreted by Alternaria tenuissima that activates defense responses and systemic acquired resistance in tobacco. This study investigates the role that Hrip1 plays in responses to abiotic and biotic stress using transgenic Arabidopsis thaliana expressing the Hrip1 gene under the control of the stress-inducible rd29A promoter or constitutive cauliflower mosaic virus 35S promoter. Bioassays showed that inducible Hrip1 expression in rd29A∷Hrip1 transgenic lines had a significantly higher effect on plant height, silique length, plant dry weight, seed germination and root length under salt and drought stress compared to expression in 35S∷Hrip1 lines and wild type plants. The level of enhancement of resistance to Botrytis cinerea by the 35S∷Hrip1 lines was higher than in the rd29A∷Hrip1 lines. Moreover, stress-related gene expression in the transgenic Arabidopsis lines was significantly increased by 200 mM NaCl and 200 mM mannitol treatments, and defense genes in the jasmonic acid and ethylene signaling pathway were significantly up-regulated after Botrytis inoculation in the Hrip1 transgenic plants. Furthermore, the activity of some antioxidant enzymes, such as peroxidase and catalase increased after salt and drought stress and Botrytis infection. These results suggested that the Hrip1 protein contributes to abiotic and biotic resistance in transgenic Arabidopsis and may be used as a useful gene for resistance breeding in crops. Although the constitutive expression of Hrip1 is suitable for biotic resistance, inducible Hrip1 expression is more responsive for abiotic resistance.

An insecticidal protein from Xenorhabdus budapestensis that results in prophenoloxidase activation in the wax moth, Galleria mellonella.

Xenorhabdus budapestensis can produce a variety of proteins that help this bacterium and its mutualistic nematode vector kill the host insect. In this report, we purified one protein fraction from the intracellular extract of X. budapestensis D43, which was designated HIP57. By injection, HIP57 caused Galleria mellonella larval bodies to blacken and die with an LD(50) of 206.81 ng/larva. Analyzes of HIP57 by two-dimensional gel electrophoresis showed that this protein was a single spot on the gel with a molecular weight of 57 kDa and a pI of ∼5. Sequencing and bioinformatic analysis suggested that the HIP57 toxin was homologous to GroEL. GroEL has been accepted as molecule chaperon; however, our research revealed that HIP57 (GroEL) possesses another novel function as an insecticide. A GroEL phylogenetic tree defined the relationship among the related species of mutualistic bacteria (Xenorhabdus and Photorhabdus) from the entomopathogenic nematodes and the evolution within the family Enterobacteriaceae. Thus, GroEL could be a complement to 16S rDNA for studying the molecular phylogenies of the family Enterobacteriaceae. Phenoloxidase (PO) activity analysis of G. mellonella larvae injected with HIP57 suggested that the toxin activates the PO cascade, which provides an extensive defense reaction that potentially responsible for G. mellonella larval death.

Protein elicitor PemG1 from Magnaporthe grisea induces systemic acquired resistance (SAR) in plants.

Elicitors can stimulate defense responses in plants and have become a popular strategy in plant disease control. Previously, we isolated a novel protein elicitor, PemG1, from Magnaporthe grisea. In the present study, PemG1 protein expressed in and purified from Escherichia coli improved resistance of rice and Arabidopsis to bacterial infection, induced transient expression of pathogenesis-related (PR) genes, and increased accumulation of hydrogen peroxide in rice. The effects of PemG1 on disease resistance and PR gene expression were mobilized systemically throughout the rice plant and persisted for more than 28 days. PemG1-induced accumulation of OsPR-1a in rice was prevented by the calcium channel blockers LaCl3, BAPTA, EGTA, W7, and TFP. Arabidopsis mutants that are insensitive to jasmonic acid (JA) and ethylene showed increased resistance to bacterial infection after PemG1 treatment but PemG1 did not affect resistance of mutants with an impaired salicylic acid (SA) transduction pathway. In rice, PemG1 induced overexpressions of the SA signal-related genes (OsEDS1, OsPAL1, and OsNH1) but not the JA pathway-related genes (OsLOX2 and OsAOS2). Our findings reveal that PemG1 protein can function as an activator of plant disease resistance, and the PemG1-mediated systemic acquired resistance is modulated by SA- and Ca(2+)-related signaling pathways.

The protein elicitor Hrip1 enhances resistance to insects and early bolting and flowering in Arabidopsis thaliana.

The elicitor Hrip1 isolated from necrotrophic fungus Alternaria tenuissima, could induce systemic acquired resistance in tobacco to enhance resistance to tobacco mosaic virus. In the present study, we found that the transgenic lines of Hrip1-overexpression in wild type (WT) Arabidopsis thaliana were more resistant to Spodoptera exigua and were early bolting and flowering than the WT. A profiling of transcription assay using digital gene expression profiling was used for transgenic and WT Arabidopsis thaliana. Differentially expressed genes including 40 upregulated and three downregulated genes were identified. In transgenic lines of Hrip1-overexpression, three genes related to jasmonate (JA) biosynthesis were significantly upregulated, and the JA level was found to be higher than WT. Two GDSL family members (GLIP1 and GLIP4) and pathogen-related gene, which participated in pathogen defense action, were upregulated in the transgenic line of Hrip1-overexpression. Thus, Hrip1 is involved in affecting the flower bolting time and regulating endogenous JA biosynthesis and regulatory network to enhance resistance to insect.

Transcriptional profiling of wheat (Triticum aestivum L.) during a compatible interaction with the cereal cyst nematode Heterodera avenae.

Cereal cyst nematode (CCN, Heterodera avenae) presents severe challenges to wheat (Triticum aestivum L.) production worldwide. An investigation of the interaction between wheat and CCN can greatly improve our understanding of how nematodes alter wheat root metabolic pathways for their development and could contribute to new control strategies against CCN. In this study, we conducted transcriptome analyses of wheat cv. Wen 19 (Wen19) by using RNA-Seq during the compatible interaction with CCN at 1, 3 and 8 days past inoculation (dpi). In total, 71,569 transcripts were identified, and 10,929 of them were examined as differentially expressed genes (DEGs) in response to CCN infection. Based on the functional annotation and orthologous findings, the protein phosphorylation, oxidation-reduction process, regulation of transcription, metabolic process, transport, and response process as well as many other pathways previously reported were enriched at the transcriptional level. Plant cell wall hydrolysis and modifying proteins, auxin biosynthesis, signalling and transporter genes were up-regulated by CCN infection to facilitate penetration, migration and syncytium establishment. Genes responding to wounding and jasmonic acid stimuli were enriched at 1 dpi. We found 16 NBS-LRR genes, 12 of which were down-regulated, indicating the repression of resistance. The expression of genes encoding antioxidant enzymes, glutathione S-transferases and UDP-glucosyltransferase was significantly up-regulated during CCN infection, indicating that they may play key roles in the compatible interaction of wheat with CCN. Taken together, the results obtained from the transcriptome analyses indicate that the genes involved in oxidation-reduction processes, induction and suppression of resistance, metabolism, transport and syncytium establishment may be involved in the compatible interaction of Wen 19 with CCN. This study provides new insights into the responses of wheat to CCN infection. These insights could facilitate the elucidation of the potential mechanisms of wheat responses to CCN.

[Research on optimization of forming techniques for Compound Tall Gastrodia Tuber drop pills by uniform design method].

OBJECTIVE: To select the best froming techniques for Compound Tall Gastrodia Tuber drop pills. METHODS: According to the factors that affect the quality of drop pill, we selected four factors as temperature of drop solution, the variety of matrix, the ratio between drug and matrix, the dropping distance, and then we selected the RSD of weight of dripping pills and the roundness as index examination. RESULTS: The optimum techniques as follows: the ratio between drug and matrix was 3 : 7, the temperature of drug solution was 80 degrees C, the dropping distance was 1 cm, the composition of matrix was PEG4000: PEG6000 = 1 : 1. The method adopted was steady and feasible. CONCLUSION: It's feasible to seek the optimum techniques making Compound Tall Gastrodia Tuber drop pills by uniform design method.

[Expression of Alternaria tenuissima peaT2 gene in Pichia pastoris and its function].

A positive clone was screened from Alternaria tenuissima expression library. The result of sequencing and gene analysis indicated that the cloned DNA fragment has a complete ORF, which was named peaT2 (Protein Elicitor from Alternaria tenuissima 2) with the GenBank accession number EF212880. The gene was amplified by PCR and subcloned into the pPIC9K of Pichia pastoris expression system. The resulting recombinant plasmid pPIC9K/peaT2 was verified by sequencing and digested by Sac I. The linearized DNA was transformed into P. pastoris GS115 by electroporation. By means of MD and G418-YPD plates and PCR, the recombinant P. pastoris strains (his- mut+) were obtained. A recombinant clone cultivated on YPD plate with high concentration of G418 was randomly selected as expression strain. The protein expression was induced by methanol and analyzed by 12% SDS-PAGE. The results of SDS-PAGE and Western blot indicated that this gene was expressed successfully in P. pastoris with the induction of methanol. In BMMY culture medium, the expressed protein reached its maximum amount at 72h, whereas no corresponding protein was detected in the negative control. Bioassay was performed with the expressed protein. After soaking with the expressed protein for 8h, wheat seeds were cultured, the height of seedlings was measured after 24h, 36h, 48h, 7d, respectively, and the root length was measured after 7 days. The results showed that the expressed protein can promote seeding growth and root length obviously in appropriate concentration. It is revealed that PeaT2 can act as protein elicitor.

[Effects and mechanism of total saponins of Psammosilene tunicoids against rheumatoid arthritis].

OBJECTIVE: To observe the antiarthritic effects and the possible mechanism of total saponins of Psammruosilene tunicoids (TSPT) against rheumatoid arthritis (RA). METHOD: After establishing AA rat model, the TSPT'S antiarthritic effects and mechanism against RA were studied through observing the changes of ankle swelling, arthritis index and levels of IL-1beta and TNF-alpha after medication. RESULT: TSPT could effectively inhibits articular swelling, decrease arthritis index and regulate down the content of IL-1beta and TNF-alpha in the inflammatory tissue soak of AA rats. CONCLUSION: TSPT has good antiarthritic effects and the possible mechanism may be related to its down-regulation of IL-1beta and TNF-alpha.

[Construction of a cDNA library of Alternaria sp. strain JH505 using Gateway technology].

A cDNA library was constructed based on the Gateway system that is a method to construct a high-quality cDNA library without the use of restriction enzyme for cloning. This is the first report that the Gateway system is used to construct a cDNA library for Alternaria sp.. The entry cDNA library was constructed in this report has a high titer of 1 x 10(7) cfu/mL and contain a total clones of 9 x 10(7) cfu, with an average inserts size of about 1510bp. In order to screen for a gene encoding the plant activator protein from library using an antiserum, the entry cDNA library was transferred into Gateway destination vector to create an expression library. The expression library show a high titer of 1.58 x 10(6) cfu/mL and contains a total clones of 6.32 x 10(6) cfu, with an average inserts size of 1680bp.

[Method for studying traditional Chinese compound prescription].

Treatment with traditional Chinese medicinal composite is one of the most important characteristics of traditional Chinese medicine (TCM). Studying material base of TCM composite and its mechanism is a key to modernizing the industry of Chinese medicinal herbs. The research for TCM composite can be carried out from many different angles, including multiple components, multiple actions, multiple levels and multiple targets. Such a way to study TCM composite will be beneficial to improving the theory of TCM composite, guiding clinical administration and developing new products.