Putative Phosphatase UvPsr1 Is Required for Mycelial Growth,Conidiation, Stress Response and Pathogenicity in Ustilaginonidea virens

Ustilaginoidea virens is the causal agent of rice false smut, which can be a highly destructive disease of rice. The plasma membrane phosphatase Psr1 proteins, which act as a regulator of the salinity stress response in yeast, are widely distributed across fungi, but their functional characterization is sketchy. In this study, we characterized the functions of Psr1 protein, UvPsr1, in U. virens. Analyses of the ΔUvpsr1 and its complementation strain showed that UvPsr1 is required for normal mycelial growth, conidiation and tolerance to oxidative, osmotic and cell wall stresses. When rice panicles were inoculated with the ΔUvpsr1 strains, no symptoms of false smut disease developed, showing that UvPSR1 also contributes to the pathogenicity of the fungus. The deletion mutant of UvPSR1 also appeared to produce a smaller titer of toxic compounds able to inhibit elongation of the germinated seeds. In conclusion, our results indicated that UvPsr1 is a new pathogenic factor of U. virens.     

UvWhi2 Is Required for Stress Response and Pathogenicity in Ustilaginoidea virens

Some stress response-related genes have been identified in Ustilaginoidea virens, but it is not clear whether and how defects of stress responses affect the pathogenesis processes of U. virens. In this study, we identified a general stress response factor UvWHI2 as a homolog of Saccharomyces cerevisiae Whi2 in U. virens. The relative expression level of UvWhi2 was significantly up-regulated during infection, suggesting that UvWHI2 may be involved in pathogenesis. Furthermore, knockout of UvWhi2 showed decreased mycelial growth, increased conidiation in the potato sucrose medium and a defect in pathogenicity. In addition, the RNA-Seq and phenotypic analysis showed that UvWHI2 was involved in response to oxidative, hyperosmotic, cell wall stress and nutrient limitation. Further studies revealed that the defects of stress responses of the ΔUvwhi2 mutant affected the formation of secondary spores on the nutrient limited surface and the rice surface, resulting in a significant reduction of pathogenicity of U. virens. Our results suggest that UvWHI2 is necessary for fungal growth, stress responses and the formation of secondary spores in U. virens. In addition, the defects of stress responses can affect the formation of secondary spores on the rice surface, and then compromise the pathogenicity of U. virens.     

Effect of water stress at different development stages on vegetative and reproductive growth of corn

A field study was carried out from 1995 to 1997 in order to determine the effect of irrigation and water stress imposed at different development stages on vegetative growth, grain yield and other yield components of corn (Zea mays L.). The field trials were conducted on a silty loam Entisol soil, with Pioneer 3377 corn hybrid. A randomised complete block design with three replications was used. Four known growth stages of the plant were considered and a total of 16 (including rain fed) irrigation treatments were applied. The effect of irrigation or water stress at any stage of development on plant height, leaf area index, grain yield per hectare, as well number of ears per plant, grain yield per cob and 1000 kernels weight, were evaluated. Results of this 3-year study show that all vegetative and yield parameters were significantly affected by water shortage in the soil profile due to omitted irrigation during the sensitive tasselling and cob formation stages. Water stress occurring during vegetative and tasselling stages reduced plant height, as well as leaf area development. Short-duration water deficits during the rapid vegetative growth period caused 28–32% loss of final dry matter weight. Highest yields were observed in the fully irrigated control (VTCM) and the treatment which allowed water stress during the vegetative growth stage (TCM). Even a single irrigation omission during one of the sensitive growth stages, caused up to 40% grain yield losses during dry years such as 1996. Much greater losses of 66–93% could be expected as a result of prolonged water stress during tasselling and ear formation stages. Seasonal irrigation water amounts required for non-stressed production varied by year from 390 to 575 mm. Yield response factor (k_y) values (unitless parameter) relating yield loss to water deficits) obtained for the first, second and third experimental years were determined to be 1.22, 1.36 and 0.81, respectively.     

UvSMEK1, a Suppressor of MEK Null,Regulates Pathogenicity,Conidiation and Conidial Germination in Rice False Smut Fungus Ustilaginoidea virens

Rice false smut, which is caused by Ustilaginoidea virens, is an emerging disease of rice spikelets in rice-growing areas worldwide. However, the infection mechanism of U. virens on rice spikelets is still unclear. Here, we characterized a suppressor of mitogen-activated protein kinase kinase or ERK kinase(MEK) null(UvSMEK1) in U. virens that is conserved among filamentous fungi. Compared with wild type U. virens strain P-1, Uv SMEK1 deletion mutants were defective in pathogenicity and conidial germination. In addition, conidiation of UvSMEK1 deletion mutants was significantly reduced on yeast extract tryptone(YT) plates, but increased in YT broth compared with the wild type. Compared with Uv SMEK1 expression level during the vegetative mycelia and conidiation stages, UvSMEK1 dramatically increased during infection of rice florets. Surprisingly, the Uv SMEK1 deletion mutants exhibited higher tolerance to H2 O2 and NaCl. In summary, presented evidence suggested that UvSMEK1 positively regulated pathogenicity, conidial germination and conidiation in YT broth, and negatively regulated conidiation on YT medium and tolerance to oxidative and osmotic stresses. The results enhance our understanding of the regulatory mechanism of pathogenicity of U. virens, and present a potential molecular target for blocking rice infection by U. virens.     

Cytosolic streaming in vegetative mycelium and aerial structures of Aspergillus niger

Aspergillus niger forms aerial hyphae and conidiophores after a period of vegetative growth. The hyphae within the mycelium of A. niger are divided by septa. The central pore in these septa allows for cytoplasmic streaming. Here, we studied inter- and intra-compartmental streaming of the reporter protein GFP in A. niger. Expression of the gene encoding nuclear targeted GFP from the gpdA or glaA promoter resulted in strong fluorescence of nuclei within the vegetative hyphae and weak fluorescence in nuclei within the aerial structures. These data and nuclear run on experiments showed that gpdA and glaA are higher expressed in the vegetative mycelium when compared to aerial hyphae, conidiophores and conidia. Notably, gpdA or glaA driven expression of the gene encoding cytosolic GFP resulted in strongly fluorescent vegetative hyphae and aerial structures. Apparently, GFP streams from vegetative hyphae into aerial structures. This was confirmed by monitoring fluorescence of photo-activatable GFP (PA-GFP). In contrast, PA-GFP did not stream from aerial structures to vegetative hyphae. Streaming of PA-GFP within vegetative hyphae or within aerial structures of A. niger occurred at a rate of 10–15 μm s^-1. Taken together, these results not only show that GFP streams from the vegetative mycelium to aerial structures but it also indicates that its encoding RNA is not streaming. Absence of RNA streaming would explain why distinct RNA profiles were found in aerial structures and the vegetative mycelium by nuclear run on analysis and micro-array analysis.Key words: aerial hypha, Aspergillus, conidia, conidiophore, cytoplasmic streaming, development, fungus, vegetative mycelium     

New Insights on the Terpenome of the Red Seaweed Laurencia dendroidea (Florideophyceae,Rhodophyta)

The red seaweeds belonging to the genus Laurencia are well known as halogenated secondary metabolites producers, mainly terpenoids and acetogennins. Several of these chemicals exhibit important ecological roles and biotechnological applications. However, knowledge regarding the genes involved in the biosynthesis of these compounds is still very limited. We detected 20 different genes involved in the biosynthesis of terpenoid precursors, and 21 different genes coding for terpene synthases that are responsible for the chemical modifications of the terpenoid precursors, resulting in a high diversity of carbon chemical skeletons. In addition, we demonstrate through molecular and cytochemical approaches the occurrence of the mevalonate pathway involved in the biosynthesis of terpenes in L. dendroidea. This is the first report on terpene synthase genes in seaweeds, enabling further studies on possible heterologous biosynthesis of terpenes from L. dendroidea exhibiting ecological or biotechnological interest.     

Identification of deletion mutants with improved performance under water-limited environments in rice (Oryza sativa L.)

Rice (Oryza sativa L.) is a semi-aquatic member of the grass family that is poorly adapted to dry environments and has greater sensitivity to water-deficits than other important cereals in this family. To increase productivity in aerobic or water-limited environments rice must overcome its adaptations to flooded environments. Deletion mutants offer an alternative genetic resource for improving drought tolerance. Almost 3500 IR64 deletion mutants were screened under vegetative and reproductive stage drought stress in the field and evaluated for leaf drying and/or grain yield. Seven novel conditional mutants of rice which showed gain of function through continued growth as drought stress developed compared to the wild type were identified. Mutant recovery rate was 0.1%. Further evaluation of putative drought mutants revealed that their average shoot biomass at maturity and grain yield per plant under stress exceeded those of the wild type by two-fold. Studies under controlled conditions confirmed mutants to have continued growth of both roots and shoots as drought developed compared to the wild type, and a tendency for greater water extraction. We propose that deletions in these mutants have affected a regulator of the highly conservative growth response common to irrigated lowland rice cultivars. Our results suggest that screening deletion mutants for performance under managed drought stress in the field could be a highly effective way to identify valuable genetic resources for improved drought response and aerobic adaptation in rice.     

玉米大斑病菌StSte12基因对H_2O_2氧化胁迫的应激调节

通过比较野生型菌株Wt01-23与StSte12基因RNAi沉默突变体菌株StRNAi9-10和StRNAi3-6在H_2O_2胁迫下生长和发育方面的差异,分析转录因子基因StSte12对玉米大斑病菌氧化胁迫的调节能力。在不同浓度H_2O_2胁迫条件下,测定野生型菌株和突变体菌株的菌落生长速度、菌丝形态、产孢量和菌丝萌发率。结果表明,随着H_2O_2浓度的增加,玉米大斑病菌野生型菌株和突变体菌株的菌落生长速度、产孢量和菌丝萌发率均显著降低,但突变体菌株的降低程度显著高于野生型菌株,表明StSte12基因对玉米大斑病菌的氧化应激调节具有重要的调控功能。     

Water relations and growth of the weed,goosegrass (Eleusine indica), under drought stress

The relative sensitivity to water stress at different growth stages of goosegrass (Eleusine indica (L.) Gaertner) was investigated by measuring water status and growth of groups of plants stressed during the vegetative stage, the reproductive stage, and during both stages. Plants were grown from seed in large pots in a controlled-environment chamber at 29/23°C and 14-h photoperiod. In all treatments, decreasing leaf water-potential was correlated with decreasing osmotic and pressure potentials. Plants stressed during the flowering stage maintained greater pressure potentials at any leaf water-potential than plants stressed during vegetative growth or stressed twice. Prestressing the plants did not induce lower leaf osmotic potentials at full turgor. However, dehydration was the main cause of low osmotic potentials measured in the leaves of the plants stressed twice. Stomatal closure occurred over a relatively large range of leaf water-potentials. The sensitivity of stomata to water stress in this species was fairly similar in the two growth stages studied. Leaves of plants stressed during the flowering stage had a smaller decline in total biomass during the stress period, and a higher rate of growth after rewatering, compared to the plants that received a stress during vegetative growth and flowering or were stressed during vegetative growth.     

Development in Aspergillus

The genus Aspergillus represents a diverse group of fungi that are among the most abundant fungi in the world. Germination of a spore can lead to a vegetative mycelium that colonizes a substrate. The hyphae within the mycelium are highly heterogeneous with respect to gene expression, growth, and secretion. Aspergilli can reproduce both asexually and sexually. To this end, conidiophores and ascocarps are produced that form conidia and ascospores, respectively. This review describes the molecular mechanisms underlying growth and development of Aspergillus.Key words: Aspergillus, fungi, asexual reproduction, sexual reproduction, development, conidium, conidiophore, vegetative mycelium, heterogeneity, ascocarp, ascospore, fruiting body     

偃麦草ErABF1基因克隆及功能分析

抗逆相关bZIP (Basic leucine zipper) 转录因子家族基因主要参与ABA、干旱、高盐等胁迫应答反应,其过表达能够显著增强植物的抗逆性。本研究从偃麦草（Elytrigria repens L.）中分离到一个抗逆相关 ErABF1（E. repens ABA Binding Factor 1）基因,氨基酸序列比对分析发现,该基因与小麦、玉米、拟南芥等bZIP转录因子基因同源性较高,亲缘关系较近;ErABF1基因的表达受到ABA、干旱、高盐、低温的强烈诱导;在2% PEG、200 mmol·L^-1 NaCl胁迫培养基上初步功能分析表明, ErABF1过表达提高了转基因烟草对干旱、高盐的胁迫耐性。     

小麦木瓜类半胱氨酸蛋白酶基因TaRD21的克隆及赤霉病菌诱导下的表达分析

木瓜类半胱氨酸蛋白酶（PLCPs）作为一类重要的蛋白水解酶,在植物生长发育以及胁迫应答过程中都发挥着重要作用。本研究从抗、感赤霉病小麦品种差异表达基因谱中获得1个注释为RD21 Cysteine proteases的EST（表达序列标签）,以此序列检索小麦最新基因组数据库并设计引物,从小麦中克隆到3个基因,分别命名为TaRD21-2A、TaRD21-2B和TaRD21-2D,属于PLCPs RD21家族。序列分析表明,3个基因的开放阅读框长度分别为1 410、1 428和1 419 bp,分别编码469、475和472个氨基酸。序列比对发现,3个基因的序列相似性为89.3％,所编码蛋白的氨基酸序列相似性为95.6％。系统进化分析表明,TaRD21-2A、TaRD21-2B和TaRD21-2D蛋白的同源性较高,且与乌拉尔图小麦TuRD21A蛋白聚为一类。qRT-PCR分析表明,3个TaRD21基因均受水杨酸(SA)、乙烯利(ETH)以及赤霉病菌诱导表达;感病品种中,TaRD21-2A对SA和赤霉病菌的响应更迅速,且表达量较高;抗病品种中,TaRD21-2B和TaRD21-2D基因对ETH的响应更迅速。     

Construction of Escherichia coli Mutant with Decreased Endotoxic Activity by Modifying Lipid A Structure

Escherichia coli BL21 (DE3) and its derivatives are widely used for the production of recombinant proteins, but these purified proteins are always contaminated with lipopolysaccharide (LPS). LPS is recognized by the toll-like receptor 4 and myeloid differentiation factor 2 complex of mammalian immune cells and leads to release of pro-inflammatory cytokines. It is a vital step to remove LPS from the proteins before use for therapeutic purpose. In this study, we constructed BL21 (DE3) ∆msbB28 ∆pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity. The plasmids harboring pagL and/or lpxE were then introduced into this mutant to further modify the LPS. The new strain (S004) carrying plasmid pQK004 (pagL and lpxE) produced mono-phosphoryated tetra-acylated lipid A, which induces markedly less production of tumor necrosis factor-α in the RAW264.7 and IL-12 in the THP1, but still retains ability to produce recombinant proteins. This study provides a strategy to decrease endotoxic activity of recombinant proteins purified from E. coli BL21 backgrounds and a feasible approach to modify lipid A structure for alternative purposes such as mono-phosphoryl lipid A (MPL) as vaccine adjuvants.     

Identification and Characterization of a New Type III Polyketide Synthase from a Marine Yeast,Naganishia uzbekistanensis

A putative Type III Polyketide synthase (PKSIII) encoding gene was identified from a marine yeast, Naganishia uzbekistanensis strain Mo29 (UBOCC-A-208024) (formerly named as Cryptococcus sp.) isolated from deep-sea hydrothermal vents. This gene is part of a distinct phylogenetic branch compared to all known terrestrial fungal sequences. This new gene encodes a C-terminus extension of 74 amino acids compared to other known PKSIII proteins like Neurospora crassa. Full-length and reduced versions of this PKSIII were successfully cloned and overexpressed in a bacterial host, Escherichia coli BL21 (DE3). Both proteins showed the same activity, suggesting that additional amino acid residues at the C-terminus are probably not required for biochemical functions. We demonstrated by LC-ESI-MS/MS that these two recombinant PKSIII proteins could only produce tri- and tetraketide pyrones and alkylresorcinols using only long fatty acid chain from C8 to C16 acyl-CoAs as starter units, in presence of malonyl-CoA. In addition, we showed that some of these molecules exhibit cytotoxic activities against several cancer cell lines.     

Tasco(®), a product of Ascophyllum nodosum, imparts thermal stress tolerance in Caenorhabditis elegans

Tasco^®, a commercial product manufactured from the brown alga Ascophyllum nodosum, has been shown to impart thermal stress tolerance in animals. We investigated the physiological, biochemical and molecular bases of this induced thermal stress tolerance using the invertebrate animal model, Caenorhabiditis elegans. Tasco^® water extract (TWE) at 300 μg/mL significantly enhanced thermal stress tolerance as well as extended the life span of C. elegans. The mean survival rate of the model animals under thermal stress (35 °C) treated with 300 μg/mL and 600 μg/mL TWE, respectively, was 68% and 71% higher than the control animals. However, the TWE treatments did not affect the nematode body length, fertility or the cellular localization of daf-16. On the contrary, TWE under thermal stress significantly increased the pharyngeal pumping rate in treated animals compared to the control. Treatment with TWE also showed differential protein expression profiles over control following 2D gel-electrophoresis analysis. Furthermore, TWE significantly altered the expression of at least 40 proteins under thermal stress; among these proteins 34 were up-regulated while six were down-regulated. Mass spectroscopy analysis of the proteins altered by TWE treatment revealed that these proteins were related to heat stress tolerance, energy metabolism and a muscle structure related protein. Among them heat shock proteins, superoxide dismutase, glutathione peroxidase, aldehyde dehydrogenase, saposin-like proteins 20, myosin regulatory light chain 1, cytochrome c oxidase RAS-like, GTP-binding protein RHO A, OS were significantly up-regulated, while eukaryotic translation initiation factor 5A-1 OS, 60S ribosomal protein L18 OS, peroxiredoxin protein 2 were down regulated by TWE treatment. These results were further validated by gene expression and reporter gene expression analyses. Overall results indicate that the water soluble components of Tasco^® imparted thermal stress tolerance in the C. elegans by altering stress related biochemical pathways.     

Growth,stomatal resistance,and transpiration of Aloe vera under different soil water potentials

Aloe vera (Sábila) is used in folklore medicine and commercial cosmetology products in many countries. Little is known about the plant's physiological, growth, and yield responses under different irrigation regimes. The plant has a crassulacean acid metabolism (CAM) that allows water conservation within the tissue, and therefore, resistance to high water stress. A. vera plants were submitted to different irrigation regimes in a greenhouse experiment to evaluate the response of the physiologic processes such as stomatal resistance and transpiration as well as leaf growth and yield. The experiment consisted of three irrigation regimes under a completely randomized design. No initial effect on stomatal resistance or transpiration was exhibited, but as time elapsed changes in these variables were noted. We suggest that the high water content in the parenchyma maintains stomatal opening despite water stress. In a subsequent period, the leaves that were submitted to water stress at the beginning, showed stomatal opening reduction related to low soil water potential. The low soil water potential reduced leaf weight, plant growth rate, and leaf number, mainly in leaf growth during the experiment confirming the sensitivity of new leaves to water stress. The results suggest that the low leaf temperature increases stomatal resistance, decreases plant and leaf growth rates. This behavior is opposite to other CAM species in semiarid condition.     

Integrated management of major diseases of mungbean by seed treatment and foliar application of insecticide,fungicides and bioagent

Field experiments were conducted during the rainy seasons of 2009 and 2010 for the management of the major diseases of mungbean, namely, wet root rot (Rhizoctonia solani), cercospora leaf spots (Cercospora canescens and Pseudocercospora cruenta) and yellow mosaic (Mungbean Yellow Mosaic Virus) by using different combinations of an insecticide, fungicide, and bio-formulation as seed treatment, with or without foliar sprays. A combination of seed treatment with thiamethoxam (Cruiser™) at 4 g kg⁻¹, carboxin (Vitavax™) at 2 g kg⁻¹ and Pusa 5SD (Trichoderma virens) at 4 g kg⁻¹ followed by simultaneous foliar sprays of thiamethoxam (Actara™) 0.02% and carbendazim (Bavistin™) 0.05% at 21 and 35 days after sowing resulted in the highest seed germination and grain yield in mungbean with the lowest intensities of cercospora leaf spots and mungbean yellow mosaic, and moderate incidence of wet root rot. The lowest whitefly population was also observed in this treatment during all stages of the crop. The treatment combinations having Pusa 5SD as seed treatment provided the lowest wet root rot incidence. Two sprays were superior to single spray for all variables recorded, but in combination with seed treatment, single spray was found to be more cost effective as it obtained the highest return per rupee of input. Use of T. virens based bio-formulation Pusa 5SD with insecticide thiamethoxam has been effectively demonstrated for the first time along with fungicides Bavistin and Vitavax for the management of the major diseases of mungbean.