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Tomato chlorosis virus (ToCV) is an emerging pathogen that cause severe yellow leaf disorder syndrome in tomato plants. In this study, we aimed to generate a recombinant ToCV tagged with green fluorescent protein (GFP) to enable real-time monitoring of viral infection in living plants. Transformation of the full-length cDNA construct of ToCV RNA1 into Escherichia coli resulted in instability issues, which were successfully overcome by inserting a plant intron into RNA1. Subsequently, a GFP tag was engineered into a cDNA construct of ToCV RNA2. The resulting recombinant ToCV-GFP could systemically infect Nicotiana benthamiana plants, and GFP expression was observed along the major veins. Utilizing ToCV-GFP, we also showed that ToCV engages in antagonistic relationships with two different tomato-infecting viruses in mixed infections in N. benthamiana. This study demonstrates the potential of ToCV-GFP as a valuable tool for the visual tracking of infection and movement of criniviruses in living plants.

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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.

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Morphogenesis of the intricate patterns of diatom silica cell walls is a protein-guided process, yet to date only very few such silica biomineralization proteins have been identified. Therefore, it is currently unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species-specific silica patterns. To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica, and Cyclotella cryptica) by complete demineralization of the cell walls. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the extracts identified 92 proteins that we name 'soluble silicome proteins' (SSPs). Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In-depth bioinformatics analyses revealed that SSPs could be grouped into distinct classes based on short unconventional sequence motifs whose functions are yet unknown. The results from the in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall.

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BACKGROUND: Sporisorium scitamineum is the causative agent of smut disease in sugarcane. The tricky life cycle of S. scitamineum consists of three distinct growth stages: diploid teliospores, haploid sporidia and dikaryotic mycelia. Compatible haploid sporidia representing opposite mating types (MAT-1 and MAT-2) of the fungus fuse to form infective dikaryotic mycelia in the host tissues, leading to the development of a characteristic whip shaped sorus. In this study, the transition of distinct stages of in vitro life cycle and in planta developmental stages of S. scitamineum are presented by generating stable GFP transformants of S. scitamineum. METHODS AND RESULTS: Haploid sporidia were isolated from the teliospores of Ss97009, and the opposite mating types (MAT-1 and MAT-2) were identified by random mating assay and mating type-specific PCR. Both haploid sporidia were individually transformed with pNIIST plasmid, harboring an enhanced green fluorescent protein (eGFP) gene and hygromycin gene by a modified protoplast-based PEG-mediated transformation method. Thereafter, the distinct in vitro developmental stages including fusion of haploid sporidia and formation of dikaryotic mycelia expressing GFP were demonstrated. To visualize in planta colonization, transformed haploids (MAT-1gfp and MAT-2gfp) were fused and inoculated onto the smut susceptible sugarcane cultivar, Co 97009 and examined microscopically at different stages of colonization. GFP fluorescence-based analysis presented an extensive fungal colonization of the bud surface as well as inter- and intracellular colonization of the transformed S. scitamineum in sugarcane tissues during initial stages of disease development. Noticeably, the GFP-tagged S. scitamineum led to the emergence of smut whips, which established their pathogenicity, and demonstrated initial colonization, active sporogenesis and teliospore maturation stages. CONCLUSION: Overall, for the first time, an efficient protoplast-based transformation method was employed to depict clear-cut developmental stages in vitro and in planta using GFP-tagged strains for better understanding of S. scitamineum life cycle development.

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Mastigonemes, tripartite tubular hairs on the anterior flagellum of Phytophthora zoospores, are instrumental for disease dissemination to new host plants. A previous study showed that PnMas2 was part of the tubular shaft of Phytophthora parasitica mastigonemes. In the current study, genes encoding two related proteins, PnMas1 and PnMas3, were identified in the genome of P. parasitica. PnMas1 interacts with PnMas2 and also occurs along the mastigoneme shaft. RNA-Seq analyses indicate that PnMas1 and PnMas2 genes have similar expression profiles both in vitro and in planta but that PnMas3 is expressed temporally prior to PnMas1 and PnMas2 during asexual development and plant infection. Immunocytochemistry and GFP-tagging document the occurrence of all three PnMas proteins within the specialised compartments of the ER during mastigoneme formation, but only PnMas1 and PnMas2 occur in mature mastigonemes on the flagellar surface. Anti-PnMas1 and anti-PnMas2 antibodies co-labelled two high-molecular-weight (~400 kDa) protein complexes in native gels but anti-PnMas3 antibodies labelled a 65 kDa protein complex. Liquid chromatography-mass spectrometry analysis identified PnMas1 and PnMas2 but not PnMas3 in flagellar extracts. These results suggest that PnMas3 associates with mastigonemes during their assembly within the ER but is not part of mature mastigonemes on the anterior flagellum. Phylogenetic analyses using homologues of Mas genes from the genomes of 28 species of Stramenopiles give evidence of three Mas sub-families, namely Mas1, Mas2 and Mas3. BLAST analyses showed that Mas genes only occur in flagellate species within the Stramenopile taxon.

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BACKGROUND: The interaction between arbuscular mycorrhizal fungi (AMF) and AMF spore associated bacteria (SAB) were previously found to improve mycorrhizal symbiotic efficiency under saline stress, however, the information about the molecular basis of this interaction remain unknown. Therefore, the present study aimed to investigate the response of maize plants to co-inoculation of AMF and SAB under salinity stress. RESULTS: The co-inoculation of AMF and SAB significantly improved plant dry weight, nutrient content of shoot and root tissues under 25 or 50 mM NaCl. Importantly, co-inoculation significantly reduced the accumulation of proline in shoots and Na+ in roots. Co-inoculated maize plants also exhibited high K+/Na+ ratios in roots at 25 mM NaCl concentration. Mycorrhizal colonization significantly positively altered the expression of ZmAKT2, ZmSOS1, and ZmSKOR genes, to maintain K+ and Na+ ion homeostasis. Confocal laser scanning microscope (CLSM) view showed that SAB were able to move and localize into inter- and intracellular spaces of maize roots and were closely associated with the spore outer hyaline layer. CONCLUSION: These new findings indicate that co-inoculation of AMF and SAB effectively alleviates the detrimental effects of salinity through regulation of SOS pathway gene expression and K+/Na+ homeostasis to improve maize plant growth.

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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.

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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.

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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 ß-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-2Δ and bgt-1Δ::bgt-2Δ 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.

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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.