Search

Destruction of citrus fruits by fungal pathogens during preharvest and postharvest stages can result in severe losses for the citrus industry. Antagonistic microorganisms used as biological agents to control citrus pathogens are considered alternatives to synthetic fungicides. In this study, we aimed to identify fungal pathogens causing dominant diseases on citrus fruits in a specialized citrus cultivation region of Vietnam and inspect soilborne Bacillus isolates with antifungal activity against these pathogens. Two fungal pathogens were characterized as Colletotrichum gloeosporioides and Penicillium digitatum based on morphological characteristics and ribosomal DNA internal transcribed spacer sequence analyses. Reinfection assays of orange fruits confirmed that C. gloeosporioides causes stem-end rot, and P. digitatum triggers green mold disease. By the heterologous expression of the green fluorescent protein (GFP) in C. gloeosporioides using Agrobacterium tumefaciens-mediated transformation, we could observe the fungal infection process of the citrus fruit stem-end rot caused by C. gloeosporioides for the first time. Furthermore, we isolated and selected two soilborne Bacillus strains with strong antagonistic activity for preventing the decay of citrus fruits by these pathogens. Molecular analyses of 16 S rRNA and gyrB genes showed that both isolates belong to B. velezensis. Antifungal activity assays indicated that bacterial culture suspensions could strongly inhibit C. gloeosporioides and P. digitatum, and shield orange fruits from the invasion of the pathogens. Our work provides a highly effective Bacillus-based preservative solution for combating the fungal pathogens C. gloeosporioides and P. digitatum to protect citrus fruits at the postharvest stages.

The 2,4-diacetylphloroglucinol (2,4-DAPG) biosynthetic gene clusters were amplified from Pseudomonas protegens Pf-5 and Pseudomonas sp. G22 (lab isolate) and cloned into pUCPM18Gm vector for constitutive expression in diazotrophic endophytic Pseudomonas sp. WS5 isolated from wheat plant. 2,4-DAPG production in recombinant strains as quantitated by HPLC analysis was 12–14 μg ml −1 while the native 2,4-DAPG producing strains showed <2 μg ml −1 . The antifungal extract of the recombinant strains showed antagonistic effect against Magnaporthe oryzae B157 and Rhizoctonia solani . Endophytic colonization and plant protection ability of recombinant strains was observed in rice, sorghum, and wheat after 60 d of growth in plants challenged with the fungal pathogens, indicating the endophytic recombinant strain was effective in promoting the plant growth during infection. Pathogenesis-related genes expression in rice plants as analyzed by qPCR showed that the rice plants colonized by 2,4-DAPG producing recombinant strain when challenged with M. oryzae B157 had elevated levels of NPR1 and PR10a gene expression. [ABSTRACT FROM AUTHOR]

Neurospora crassa BGT-1 (NCU06381) and BGT-2 (NCU09175) are two putative glycoside hydrolases (GHs) with additional predicted glycosyltransferase activity and binding sites for a glycosyl phosphatidyl inositol (GPI) anchor that would facilitate their attachment to the plasma membrane (PM). To discern their role in key morphogenetic events during vegetative development of N . crassa , BGT-1 and BGT-2 were labeled with the green fluorescent protein (GFP). The gfp was inserted immediately after the signal peptide sequence, within the bgt - 1 encoding sequence, or directly before the GPI-binding site in the case of bgt - 2 . Both BGT-1-GFP and BGT-2-GFP were observed at the PM of the hyphal apical dome, excluding the foremost apical region and the Spitzenkörper (Spk), where chitin and β-1,3-glucan synthases have been previously found. These and previous studies suggest a division of labor of the cell wall synthesizing machinery at the hyphal dome: at the very tip, glucans are synthesized by enzymes that accumulate at the Spk, before getting incorporated into the PM, whereas at the subtending zone below the apex, glucans are presumably hydrolyzed, producing amenable ends for further branching and crosslinking with other cell wall polymers. Additionally, BGT-1-GFP and BGT-2-GFP were observed at the leading edge of new developing septa, at unreleased interconidial junctions, at conidial poles, at germling and hyphal fusion sites, and at sites of branch emergence, all of them processes that seemingly involve cell wall remodeling. Even though single and double mutant strains for the corresponding genes did not show a drastic reduction of growth rate, bgt - 2 Δ and bgt - 1 Δ:: bgt - 2 Δ strains exhibited an increased resistance to the cell wall stressors calcofluor white (CW) and congo red (CR) than the reference strain, which suggests they present significant architectural changes in their cell wall. Furthermore, the conidiation defects observed in the mutants indicate a significant role of BGT-1 and BGT-2 on the re-arrangement of glucans needed at the conidiophore cell wall to allow conidial separation. [ABSTRACT FROM AUTHOR]

Green fluorescent protein (GFP) is a powerful tool for studying gene expression, protein localization, protein-protein interactions, calcium concentrations, and redox potentials owing to its intrinsic fluorescence. However, GFP not only contains a chromophore but is also tightly folded in a temperature-dependent manner. The latter property of GFP has recently been exploited (1) to characterize the translocase of the outer mitochondrial membrane and (2) to discriminate between protein transport across and into biomembranes in vivo. I therefore suggest that GFP could be a valuable tool for the general analysis of protein transport machineries and pathways in a variety of organisms. Moreover, results from such studies could be important for the interpretation and optimization of classical experiments using GFP tagging. [ABSTRACT FROM AUTHOR]

The article presents a study which aims to help gain understanding concerning all the protein functions in the fission yeast as a eukaryotic model. It examines seven gene products localized in the nucleus or chromatin, mitosis-specific spindle pole body component and human homologue. Results show that three other proteins were found in the medial ring upon cytokinsesis and another was on the cell periphery while remaining 38 proteins were detected in the cytoplasm.

β-1,6- N-Acetylglucosaminidase (dispersin B), which cleaves poly-ß-(1,6)-linked N-acetylglucosamine, is encoded by dspB of Aggregatibacter actinomycetemcomitans. To enhance the production of dispersin B, we engineered dspB to transcribe mRNAs devoid of the trinucleotide ACA. Transcription and translation levels of ACA-less and wild-type dspB expressed in Escherichia coli ( E. coli) under T5 and T7 promoters were analyzed by real-time RT-PCR and protein quantification, respectively. The ACA-less dspB mRNA level was significantly higher ( P < 0.01) and produced 77.6 and 34.9% more dispersin B than wild-type dspB expressed under T7 and T5 promoters, respectively. Dispersin B expression under T7 promoter caused a 98–99.5% drop in the glyceraldehyde-3-phosphate dehydrogenase ( gapA) mRNA level, which was not observed with T5 promoter. Fusion of green fluorescent protein (GFP) with dispersin B allowed rapid quantification of dispersin B production by measuring fluorescence intensity in culture broth. Although the cultures containing 0.1% glucose showed sustained increase in dispersin B-GFP production until 12 h, no significant increase in dispersin B activity was observed beyond 4 and 6 h after induction when expressed under T7 and T5 promoters, respectively. This study demonstrates the effectiveness of ACA-less mRNA and the advantage of GFP tagging for enhanced dispersin B production and quantification, which could be adapted for improving the production of other commercially important proteins in E. coli. [ABSTRACT FROM AUTHOR]

ABSTRACT Rhizobia and associative bacteria promote growth in rice plants (Oryza sativa L.) through a series of mechanisms, but most studies on inoculation have been performed based on inoculation with these bacteria in a separate or singular manner. The objective of this study was to assess the efficiency of single/isolated inoculation and inoculation combined with symbiotic rhizobia from forage legume and with Azospirillum brasilense on promoting growth and the root colonization process in wetland rice. Two rhizobia among four isolates from a greenhouse and a laboratory experiment were selected that efficiently promoted seed germination and rice plant growth in a sterilized substrate and in soil. The two most efficient isolates (UFRGS Vp16 and UFRGS Lc348) were inoculated alone or in combination with a commercial product containing A. brasilense in two field experiments using two wetland rice cultivars over two growing seasons. In the field experiments, these isolates coinoculated with A. brasilense promoted larger increases in the agronomic variables of wetland rice compared to the control without inoculation. Confocal laser microscopy confirmed the presence of inoculated bacteria tagged with gfp (UFRGS Vp16, UFRGS Lc348, and A. brasilense) colonizing the root surface of the rice seedlings, mainly in the root hairs and lateral roots.

At first glance, mitosis in plants looks quite different from that in animals. In fact, terrestrial plants have lost the centrosome during evolution, and the mitotic spindle is assembled independently of a strong microtubule organizing center. The phragmoplast is a plant-specific mitotic apparatus formed after anaphase, which expands centrifugally towards the cell cortex. However, the extent to which plant mitosis differs from that of animals at the level of the protein repertoire is uncertain, largely because of the difficulty in the identification and in vivo characterization of mitotic genes of plants. Here, we discuss protocols for mitosis imaging that can be combined with endogenous green fluorescent protein (GFP) tagging or conditional RNA interference (RNAi) in the moss Physcomitrella patens, which is an emergent model plant for cell and developmental biology. This system has potential for use in the high-throughput study of mitosis and other intracellular processes, as is being done with various animal cell lines.

We have defined a network of interacting Drosophila cell surface proteins in which a 21-member IgSF subfamily, the Dprs, binds to a nine-member subfamily, the DIPs. The structural basis of the Dpr-DIP interaction code appears to be dictated by shape complementarity within the Dpr-DIP binding interface. Each of the six dpr and DIP genes examined here is expressed by a unique subset of larval and pupal neurons. In the neuromuscular system, interactions between Dpr11 and DIP-γ affect presynaptic terminal development, trophic factor responses, and neurotransmission. In the visual system, dpr11 is selectively expressed by R7 photoreceptors that use Rh4 opsin (yR7s). Their primary synaptic targets, Dm8 amacrine neurons, express DIP-γ. In dpr11 or DIP-γ mutants, yR7 terminals extend beyond their normal termination zones in layer M6 of the medulla. DIP-γ is also required for Dm8 survival or differentiation. Our findings suggest that Dpr-DIP interactions are important determinants of synaptic connectivity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Summary Following differential screening of gene expression during tomato fruit development, we isolated developmentally regulated (DR) clones, including several putative transcription factors. Based on sequence homology, DR1, DR3, DR4 and DR8 are members of the Aux/IAA family, and DR12 belongs to the auxin response factor (ARF) family of transcription factors. Importantly, mRNA accumulation for the Aux/IAA -like genes was regulated by ethylene in tomato fruit but not in the leaves, indicating that these putative auxin response components also participate to the ethylene-dependent regulation of gene expression in a tissue-specific manner. The functional significance of DR12 , the ARF -like gene, was investigated by cellular biology and reverse genetics approaches. Heterologous protein targeting studies, carried out using a DR12–GFP gene fusion construct, revealed specific nuclear localization of the DR12-encoded protein, in accordance with its putative function as a transcriptional regulator. Transgenic plants over- and under-expressing DR12 were generated in order to explore the physiological role of the gene. Both antisense and sense co-suppressed DR12-inhibited lines displayed a pleiotropic phenotype that included dark-green immature fruit, unusual cell division in the fruit pericarp, blotchy ripening, enhanced fruit firmness, upward curling leaves and increased hypocotyl and cotyledon growth. While a perturbation of the response to auxin may explain some of the phenotypes, surprisingly, the expression of members of four classes of early auxin-regulated genes was unaffected in the DR12-inhibited plants. The involvement of this ARF-like encoded protein in mediating the auxin response is discussed along with the possibility that it might affect responsiveness to other phytohormones in the tomato. [ABSTRACT FROM AUTHOR]

Exocytosis molecular mechanisms in plant cells are not fully understood. The full characterization of molecular determinants, such as SNAREs, for the specificity in vesicles delivery to the plasma membrane should shed some light on these mechanisms. Nicotiana tabacum Syntaxin 1 (NtSyr1 or SYP121) is a SNARE protein required for ABA control of ion channels and appears involved in the exocytosis of exogenous markers.NtSyr1 is mainly localized on the plasma membrane, but when over expressed the protein also appears on endomembranes. Since NtSyr1 is a tail-anchored protein inserted into the target membrane post-translationally, it is not clear whether its initial anchoring site is the ER or the plasma membrane.In this study, we investigated the sorting events of NtSyr1 in vivo using its full-length cDNA or its C-terminal domain, fused to a GFP tag and transiently expressed in protoplasts or in the leaves of Nicotiana tabacum cv. SR1. Five chimeras were produced of which two were useful to investigate the protein sorting within the endomembrane system. One (GFP-H3M) had a dominant negative effect on exocytosis; the other one (SP1-GFP) resulted in a slow targeting to the same localization of the full-length chimera (GFP-SP1). The insertion of signal peptides on SP1-GFP further characterized the insertion site for this protein. Our data indicates that NtSyr1 is firstly anchored on ER membrane and then sorted to plasma membrane.