Yonghua Zhu

Isolation of a rice endophytic bacterium, Pantoea sp. Sd-1, with ligninolytic activity and characterization of its rice straw degradation ability.

This study focused on an endophytic bacterial strain, Pantoea sp. Sd‐1, which can be used to degrade lignin and rice straw. This strain was isolated from rice seeds by an optimized surface sterilization method. Pantoea sp. Sd‐1 showed exceptional ability to degrade rice straw and lignin. In rice straw or kraft lignin‐containing medium supplemented with 1% glucose and 0·5% peptone, Pantoea sp. Sd‐1 effectively reduced the rice straw mass weight by 54·5% after 6 days of treatment. The strain was also capable of reducing the lignin colour (52·4%) and content (69·1%) after 4 days of incubation. The findings suggested that the rice endophytic bacterium Pantoea sp. Sd‐1 could be applied for the degradation of lignocellulose biomass, such as rice straw.

Over-expression of a glutamate dehydrogenase gene, MgGDH, from Magnaporthe grisea confers tolerance to dehydration stress in transgenic rice

AbstractMain conclusionHeterologous expression of a fungal NADP(H)-GDH gene (MgGDH) fromMagnaporthe griseacan improve dehydration stress tolerance in rice by preventing toxic accumulation of ammonium.n Glutamate dehydrogenase (GDH; EC 1.4.1.2 and EC 1.4.1.4) may act as a stress-responsive enzyme in detoxification of high intracellular ammonia and production of glutamate for proline synthesis under stress conditions. In present study, a fungal NADP(H)-GDH gene (MgGDH) from Magnaporthe grisea was over-expressed in rice (Oryza sativa L. cv. ‘kitaake’), and the transgenic plants showed the improvement of tolerance to dehydration stress. The kinetic analysis showed that His-TF-MgGDH preferentially utilizes ammonium to produce l-glutamate. Moreover, the affinity of His-TF-MgGDH for ammonium was dramatically higher than that of His-TF-OsGDH for ammonium. Over-expressing MgGDH transgenic rice plants showed lower water-loss rate and higher completely close stomata than the wild-type plants under dehydration stress conditions. In transgenic plants, the NADP(H)-GDH activities were markedly higher than those in wild-type plants and the amination activity was significantly higher than the deamination activity. Compared with wild-type plants, the transgenic plants accumulated much less NH4+ but higher amounts of glutamate, proline and soluble sugar under dehydration stress conditions. These results indicate that heterologous expression of MgGDH can prevent toxic accumulation of ammonium and in return improve dehydration stress tolerance in rice.

Over-expression of a fungal NADP(H)-dependent glutamate dehydrogenase PcGDH improves nitrogen assimilation and growth quality in rice

Glutamate dehydrogenase (GDH) tends to have a lower affinity for ammonium than glutamine synthetase (GS) in higher plants. Consequently, nitrogen is mostly assimilated as ammonium by the GS/glutamate synthase pathway which requires 2-oxoglutarate (2-OG) as carbon skeletons. In contrast, the NADP(H)-dependent GDH in fungi has a higher affinity for ammonium than that in higher plants and plays a more significant part in ammonium assimilation. We isolated an NADP(H)-GDH gene (PcGDH) from the fungus Pleurotus cystidiosus and heterologously expressed it in rice (Oryza sativa L.). Alterations in nitrogen assimilation, growth, metabolism, and grain yield were observed in the transgenic plants. An investigation of the kinetic properties of the purified recombinant protein demonstrated that the amination activity (7.05xa0±xa00.78xa0μmoLxa0min−1xa0mg soluble protein−1) of PcGDH was higher than the deamination activity (3.36xa0±xa00.42xa0μmoLxa0min−1xa0mg soluble protein−1) and that the Km value for ammonium (Kmxa0=xa03.73xa0±xa00.23xa0mM) was lower than that for the glutamate (Kmxa0=xa015.97xa0±xa00.31xa0mM), indicating that the PcGDH tends to interconvert 2-OG and glutamate. Examination of the activity of NADP(H)-GDH in control and transgenic lines demonstrated that NADP(H)-GDH activity in the transgenic lines was markedly higher than that in the control lines; in particular, the amination activity was significantly higher than the deamination activity in shoots of the transgenic lines. The results of the hydroponics experiment revealed that shoot and root length, fresh weight, chlorophyll content, nitrogen content, and amino acid levels (glutamate, glutamine, and total amino acids) were elevated in transgenic lines in comparison with those of the control line under different nitrogen conditions at seedling stage. The 1,000-grain weight and the panicle number in transgenic lines were considerably augmented in the field condition, yet the filled grain rate dropped slightly and there was no apparent change in the grain yield. The levels of glutelin and prolamine in the transgenic seeds were considerably higher than those in control seeds. In conclusion, these results demonstrate that heterologous expression of P. cystidiosus GDH (PcGDH) could improve nitrogen assimilation and growth in rice.

Molecular cloning, characterization and function analysis of a GDH gene from Sclerotinia sclerotiorum in rice

The full-length cDNA encoding a glutamate dehydrogenase (GDH) which catalyzes the reaction of reductive amination of α-oxoglutarate (α-OG) to glutamate (the anabolic activity) and the reverse reaction of oxidative deamination of glutamate (the catabolic activity) was isolated from Sclerotinia sclerotiorum, we designated it as SsGDH. Bioinformatics analysis revealed that SsGDH had a typical GDH spatial structure and extensive homology with other fungal or bacteria GDHs. To evaluate its function in rice, rice (Oryza sativa L. cv. ‘kitaake’) was transformed with SsGDH in a binary vector construct by Agrobacterium-mediated transformation. Transgenic rice plants showed that transcripts and proteins of SsGDH accumulated at higher levels and GDH enzymatic activity was obviously higher in transgenic rice plants compared with the non-transformant rice plants (CK), though phenotype including plant height, fresh weight and dry weight became slightly weaker compared with CK under 50, 500 and 5,000xa0μM nitrogen gradient nutrient solution treatment (NH4NO3 as a nitrogen source) after introducing SsGDH into rice. For enzymatic activity assay in vitro, recombinant His6-SsGDH protein was expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA agarose. Results suggested that recombinant His6-SsGDH protein had GDH activity using ammonium, α-OG, and l-glutamate separately as a substrate at two different concentrations, especially the affinity for ammonium was very high, and its Km value was only 0.28xa0±xa00.03xa0mM, indicating that SsGDH can assimilate more ammonium into rice. According to previous reports, transgenic plants expressing fungal or bacteria GDHs might show improved herbicide resistance. Basta resistance test showed that SsGDH expression in rice can significantly enhanced their tolerance to Basta than CK. In conclusion, our results may provide some clues for further investigation on nitrogen utilization via introducing exogenous GDHs from lower organisms into rice.

Isolation and evaluation of endophytic Streptomyces endus OsiSh-2 with potential application for biocontrol of rice blast disease

BACKGROUNDnBiocontrol is a promising strategy in the control of rice blast disease. In the present study, we isolated and characterized a novel antagonist to the pathogen Magnaporthe oryzae from rice endophytic actinomycetes.nnnRESULTSnOut of 482 endophytic actinomycetes isolated from rice blast infected and healthy rice, Streptomyces endus OsiSh-2 exhibited remarkable in vitro antagonistic activity. Scanning electron microscopy observations of M.u2009oryzae treated by OsiSh-2 revealed significant morphological alterations in hyphae. In 2-year field tests, the spraying of OsiSh-2 spore solution (107 u2009sporesu2009mL-1 ) is capable of reducing rice blast disease severity by 59.64%. In addition, a fermentation broth of OsiSh-2 and its cell-free filtrates could inhibit the growth of M.u2009oryzae, suggesting the presence of active enzymes and secondary metabolites. OsiSh-2 tested positive for polyketide synthase-I and nonribosomal peptide synthetase genes and can produce cellulase, protease, gelatinase, siderophore, indole-3-acetic acid and 1-amino-cyclopropane-1-carboxylate deaminase. A preliminary separation indicated that the methanol extract of OsiSh-2 could suppress the growth of pathogens. The major active component was identified as nigericin.nnnCONCLUSIONnEndophytic S.u2009endus OsiSh-2 has potential as a biocontrol agent against rice blast in agriculture.

The Receptor-like Cytoplasmic Kinase STRK1 Phosphorylates and Activates CatC, thereby Regulating H2O2 Homeostasis and Improving Salt Tolerance in Rice

The receptor-like cytoplasmic kinase STRK1 positively regulates rice salt and oxidative stress tolerance through maintaining H2O2 homeostasis by phosphorylating CatC mainly at Tyr-210. Salt stress can significantly affect plant growth and agricultural productivity. Receptor-like kinases (RLKs) are believed to play essential roles in plant growth, development, and responses to abiotic stresses. Here, we identify a receptor-like cytoplasmic kinase, salt tolerance receptor-like cytoplasmic kinase 1 (STRK1), from rice (Oryza sativa) that positively regulates salt and oxidative stress tolerance. Our results show that STRK1 anchors and interacts with CatC at the plasma membrane via palmitoylation. CatC is phosphorylated mainly at Tyr-210 and is activated by STRK1. The phosphorylation mimic form CatCY210D exhibits higher catalase activity both in vitro and in planta, and salt stress enhances STRK1-mediated tyrosine phosphorylation on CatC. Compared with wild-type plants, STRK1-overexpressing plants exhibited higher catalase activity and lower accumulation of H2O2 as well as higher tolerance to salt and oxidative stress. Our findings demonstrate that STRK1 improves salt and oxidative tolerance by phosphorylating and activating CatC and thereby regulating H2O2 homeostasis. Moreover, overexpression of STRK1 in rice not only improved growth at the seedling stage but also markedly limited the grain yield loss under salt stress conditions. Together, these results offer an opportunity to improve rice grain yield under salt stress.

LRRK1, a receptor-like cytoplasmic kinase, regulates leaf rolling through modulating bulliform cell development in rice

Moderate leaf rolling is useful in improving photosynthetic efficiency and grain yields. Receptor-like cytoplasmic kinases (RLCKs) play important roles in plant growth and development. However, little is known about their functions in rice leaf morphogenesis. Here, we report the isolation and characterization of LRRK1 (leaf rolling receptor-like cytoplasmic kinase 1), an RLCK gene involved in the regulation of leaf rolling. LRRK1 was mainly localized at the plasma membrane and was phosphorylated in vivo. Overexpression of LRRK1 in rice reduced the size of bulliform cells at the adaxial cell layers, which caused in turn adaxially rolled leaves. However, deficiency of LRRK1 in the lrrk1 mutant did not result in a detectable visual phenotype. LRRK1 could upregulate the expression of negative regulators but downregulate the expression of positive regulators of bulliform cell development. These results indicate that LRRK1 is a negative regulator involved in the bulliform cell development. Furthermore, the panicle numbers in LRRK1-overexpressing plants increased significantly compared with the wild-type plants under a rational close planting condition. Taken together, these findings suggest that LRRK1 plays an important role in regulating leaf rolling and is a promising candidate gene for breeding rice with ideal plant architecture and improved grain yield.

The Role of Iron Competition in the Antagonistic Action of the Rice Endophyte Streptomyces sporocinereus OsiSh-2 Against the Pathogen Magnaporthe oryzae

Rice blast caused by Magnaporthe oryzae severely impacts global rice yield stability. The rice endophyte Streptomyces sporocinereus OsiSh-2, with strong antagonistic activity towards M. oryzae, has been reported in our previous study. To decipher the model of the antagonistic action of OsiSh-2 towards M. oryzae, we compared the iron-capturing abilities of these two strains. The cultivation of OsiSh-2 and a M. oryzae strain under iron-rich and iron-starved conditions showed that M. oryzae depended more on iron supplementation for growth and development than did OsiSh-2. Genomic analysis of the S. sporocinereus and M. oryzae species strains revealed that they might possess different iron acquisition strategies. The actinobacterium OsiSh-2 is likely to favor siderophore utilization compared to the fungus M. oryzae. In addition, protein annotations found that OsiSh-2 contains the highest number of the siderophore biosynthetic gene clusters among the 13 endophytic actinomycete strains and 13 antifungal actinomycete strains that we compared, indicating the prominent siderophore production potential of OsiSh-2. Additionally, we verified that OsiSh-2 could excrete considerably more siderophores than Guy11 under iron-restricted conditions and displayed greater Fe3+-reducing activity during iron-supplemental conditions. Measurements of the iron mobilization between the antagonistic OsiSh-2 and Guy11 showed that the iron concentration is higher around OsiSh-2 than around Guy11. In addition, adding iron near OsiSh-2 could decrease the antagonism of OsiSh-2 towards Guy11. Our study revealed that the antagonistic capacity displayed by OsiSh-2 towards M. oryzae was related to the competition for iron. The highly efficient iron acquisition system of OsiSh-2 may offer valuable insight for the biocontrol of rice blast.

Identification and characterization of a novel bacterial pyranose 2-oxidase from the lignocellulolytic bacterium Pantoea ananatis Sd-1

ObjectiveTo identify and characterize a novel bacterial pyranose 2-oxidase (P2Ox) and investigate its potential use in lignin degradation applications.ResultsA new bacterial P2Ox (PaP2Ox) enzyme was identified in the lignocellulolytic bacterium Pantoea ananatis Sd-1. The PaP2Ox open reading frame was cloned, and the encoded protein was heterologously expressed in an Escherichia coli expression system. Unlike another reported bacterial P2Ox enzyme, the purified PaP2Ox exhibits a homotetrameric spatial conformation that is similar to fungal P2Oxs, with each subunit having a molecular mass of 65xa0kDa. The recombinant PaP2Ox exhibits maximum activity at 50xa0°C and pH 6.5 with d-glucose as its preferred substrate. In addition, this enzyme was shown to work in combination with bacterial laccase in lignin degradation.ConclusionsThe bacterial enzyme PaP2Ox has potential use in ligninolytic systems and shows promising value in industrial biotechnological applications.