Volume electron microscopy reconstruction uncovers a physical barrier that limits virus to phloem.

Most plant reoviruses are phloem-limited, but the mechanism has remained unknown for more than half a century. Southern rice black-streaked dwarf virus (Fijivirus, Reoviridae) causes phloem-derived tumors, where its virions, genomes, and proteins accumulate, and it was used as a model to explore how its host plant limits the virus within its phloem. High-throughput volume electron microscopy revealed that only sieve plate pores and flexible gateways rather than plasmodesmata had a sufficiently large size exclusion limit (SEL) to accommodate virions and potentially serve as pathways of virion movement. The large SEL gateways were enriched within the proliferated sieve element (SE) layers of tumors. The lack of such connections out of the SE-enriched regions of tumors defined a size-dependent physical barrier to high flux transportation of virions. A working model is proposed to demonstrate the mechanism underlying limitation of virus within phloem.

Transcriptomic and Metabolomic Investigation on Leaf Necrosis Induced by ZmWus2 Transient Overexpression in Nicotiana benthamiana.

WUSCHEL (WUS) is a crucial transcription factor in regulating plant stem cell development, and its expression can also improve genetic transformation. However, the ectopic expression of WUS always causes pleiotropic effects during genetic transformation, making it important to understand the regulatory mechanisms underlying these phenomena. In our study, we found that the transient expression of the maize WUS ortholog ZmWus2 caused severe leaf necrosis in Nicotiana benthamiana. We performed transcriptomic and non-target metabolomic analyses on tobacco leaves during healthy to wilted states after ZmWus2 transient overexpression. Transcriptomic analysis revealed that ZmWus2 transformation caused active metabolism of inositol trisphosphate and glycerol-3-phosphate, while also upregulating plant hormone signaling and downregulating photosystem and protein folding pathways. Metabolomic analysis mainly identified changes in the synthesis of phenylpropanoid compounds and various lipid classes, including steroid synthesis. In addition, transcription factors such as ethylene-responsive factors (ERFs), the basic helix-loop-helix (bHLH) factors, and MYBs were found to be regulated by ZmWus2. By integrating these findings, we developed a WUS regulatory model that includes plant hormone accumulation, stress responses, lipid remodeling, and leaf necrosis. Our study sheds light on the mechanisms underlying WUS ectopic expression causing leaf necrosis and may inform the development of future genetic transformation strategies.

Assessment of the mechanism of drug resistance in Trichophyton mentagrophytes in response to various substances.

BACKGROUND: Trichophyton mentagrophyte (TM), a zoonotic pathogen, has been endangering public health due to emerging drug resistance. Although increased attention is paid to this issue, there is very limited research available on drug resistance in TM. In this study, we studied the gene and proteomic changes, morphological changes, cellular fat localization, fat content changes, and biofilm of TM treated with different substances. RESULTS: The TM growth curve showed a positive correlation with the concentration of Fenarimol (FE), genistein (GE), clotrimazole (KM), and Miconazole nitrate salt (MK). The morphology of TM cells changed in different degrees after treatment with different substances as observed by TEM and SEM. The results showed that under KM and berberine hydrochloride (BB) treatment, a total of 3305 differentially expressed genes were detected, with the highest number in the KM-treated group (578 up-regulated and 615 down-regulated). A total of 847 proteins and 1850 peptides were identified in TM proteomics. Nile red staining showed that the fat content of TM was significantly higher in the BB-, ethidium bromide- (EB), FE-, KM-, Adriamycin hydrochloride- (YA), and MK-treated group compared to the control group. Results of the biofilm thickness showed that it gradually increased under treatment with specific concentrations of KM or BB, which may be related to the up-regulation of ERG25 and CYP related gene proteins. CONCLUSIONS: It is suggested that in order to effectively deal with dermatomycosis caused by TM, it is necessary to inhibit the expression of ERG25 and CYP related genes and fat metabolism, which can result in the inhibition of the production of biofilm by the fungus and solve the problem of fungal drug resistance in clinical settings.

Structural and functional organization of the MYC transcriptional factors in Camellia sinensis.

MAIN CONCLUSION: Genome-wide identification, expression analysis of the MYC family in Camellia sinensis, and potential functional characterization of CsMYC2.1 have laid a solid foundation for further research on CsMYC2.1 in jasmonate (JA)-mediated response. Myelocytomatosis (MYC) of basic helix-loop-helix (bHLH) plays a major role in JA-mediated plant growth and developmental processes through specifically binding to the G-box in the promoters of their target genes. In Camellia sinensis, studies on the MYC gene family are limited. Here, we identified 14 C. sinensis MYC (CsMYC) genes, and further analyzed the evolutionary relationship, gene structure, and motif pattern among them. The expression patterns of these CsMYC genes in different tissues suggested their important roles in diverse function in tea plant. Four MYC transcription factors with the highest homology to MYC2 in Arabidopsis were localized in the nucleus. Two of them, named CsMYC2.1 and CsMYC2.2, exhibited transcriptional self-activating activity, and, therefore, could significantly activate the promoter containing G-box motif, whereas CsJAM1.1 and CsJAM1.2 lack the transcriptional self-activating activity, indirectly mediating the JA pathway through interacting with CsMYC2.1 and CsMYC2.2. Furthermore, Yeast Two-Hybrid (Y2H) and Bimolecular Fluorescent Complimentary (BiFC) assays showed that CsMYC2.1 could interact with CsJAZ3/7/8 proteins. Genetically, the complementation of CsMYC2.1 in myc2 mutants conferred the ability to restore the sensitivity to JA signals. The results provide a comprehensive characterization of the 14 CsMYCs in C. sinensis, establishing a solid foundation for further research on CsMYCs in JA-mediated response.

A novel pathogenicity determinant hijacks maize catalase 1 to enhance viral multiplication and infection.

Pathogens have evolved various strategies to overcome host immunity for successful infection. Maize chlorotic mottle virus (MCMV) can cause lethal necrosis in maize (Zea mays) when it coinfects with a virus in the Potyviridae family. However, the MCMV pathogenicity determinant remains largely unknown. Here we show that the P31 protein of MCMV is important for viral accumulation and essential for symptom development. Ectopic expression of P31 using foxtail mosaic virus or potato virus X induced necrosis in systemically infected maize or Nicotiana benthamiana leaves. Maize catalases (CATs) were shown to interact with P31 in yeast and in planta. P31 accumulation was elevated through its interaction with ZmCAT1. P31 attenuated the expression of salicylic acid (SA)-responsive pathogenesis-related (PR) genes by inhibiting catalase activity during MCMV infection. In addition, silencing of ZmCATs using a brome mosaic virus-based gene silencing vector facilitated MCMV RNA and coat protein accumulation. This study reveals an important role for MCMV P31 in counteracting host defence and inducing systemic chlorosis and necrosis. Our results have implications for understanding the mechanisms in defence and counter-defence during infection of plants by various pathogens.

Horizontal Transfer of a Retrotransposon from the Rice Planthopper to the Genome of an Insect DNA Virus.

Horizontal transfer of genetic materials between virus and host has been frequently identified. Three rice planthoppers, Laodelphax striatellus, Nilaparvata lugens, and Sogatella furcifera, are agriculturally important insects because they are destructive rice pests and also the vector of a number of phytopathogenic viruses. In this study, we discovered that a small region (∼300 nucleotides [nt]) of the genome of invertebrate iridescent virus 6 (IIV-6; genus Iridovirus, family Iridoviridae), a giant DNA virus that infects invertebrates but is not known to infect planthoppers, is highly homologous to the sequences present in high copy numbers in these three planthopper genomes. These sequences are related to the short interspersed nuclear elements (SINEs), a class of non-long terminal repeat (LTR) retrotransposons (retroposons), suggesting a horizontal transfer event of a transposable element from the rice planthopper genome to the IIV-6 genome. In addition, a number of planthopper transcripts mapped to these rice planthopper SINE-like sequences (RPSlSs) were identified and appear to be transcriptionally regulated along the different developmental stages of planthoppers. Small RNAs derived from these RPSlSs are predominantly 26 to 28 nt long, which is a typical characteristic of PIWI-interacting RNAs. Phylogenetic analysis suggests that IIV-6 acquires a SINE-like retrotransposon from S. furcifera after the evolutionary divergence of the three rice planthoppers. This study provides further examples of the horizontal transfer of an insect transposon to virus and suggests the association of rice planthoppers with iridoviruses in the past or present.IMPORTANCE This study provides an example of the horizontal transfer event from a rice planthopper genome to an IIV-6 genome. A small region of the IIV-6 genome (∼300 nt) is highly homologous to the sequences presented in high copy numbers of three rice planthopper genomes that are related to the SINEs, a class of retroposons. The expression of these planthopper SINE-like sequences was confirmed, and corresponding Piwi-interacting RNA-like small RNAs were identified and comprehensively characterized. Phylogenetic analysis suggests that the giant invertebrate iridovirus IIV-6 obtains this SINE-related sequence from Sogatella furcifera through a horizontal transfer event in the past. To the best of our knowledge, this is the first report of a horizontal transfer event between a planthopper and a giant DNA virus and also is the first evidence for the eukaryotic origin of genetic material in iridoviruses.

Filamentous Structures Induced by a Phytoreovirus Mediate Viral Release from Salivary Glands in Its Insect Vector.

Numerous viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. These viruses circulate in the vector body, enter the salivary gland, and then are released into the apical plasmalemma-lined cavities, where saliva is stored. The cavity plasmalemma of vector salivary glands thus represents the last membrane barrier for viral transmission. Here, we report a novel mechanism used by a persistent virus to overcome this essential barrier. We observed that the infection by rice gall dwarf virus (RGDV), a species of the genus Phytoreovirus in the family Reoviridae, induced the formation of virus-associated filaments constructed by viral nonstructural protein Pns11 within the salivary glands of its leafhopper vector, Recilia dorsalis Such filaments attached to actin-based apical plasmalemma and induced an exocytosis-like process for viral release into vector salivary gland cavities, through a direct interaction of Pns11 of RGDV and actin of R. dorsalis Failure of virus-induced filaments assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene inhibited the dissemination of RGDV into salivary cavities, preventing viral transmission by R. dorsalis For the first time, we show that a virus can exploit virus-induced inclusion as a vehicle to pass through the apical plasmalemma into vector salivary gland cavities, thus overcoming the last membrane barrier for viral transmission by insect vectors.IMPORTANCE Understanding how persistent viruses overcome multiple tissue and membrane barriers within the insect vectors until final transmission is the key for viral disease control. The apical plasmalemma of the cavities where saliva is stored in the salivary glands is the last barrier for viral transmission by insect vectors; however, the mechanism is still poorly understood. Here we show that a virus has evolved to exploit virus-induced filaments to perform an exocytosis-like process that enables viral passage through the apical plasmalemma into salivary cavities. This mechanism could be extensively exploited by other persistent viruses to overcome salivary gland release barriers in insect vectors, opening new perspectives for viral control.

Generalized Pseudo-Unit-Cell model for long-wavelength optical phonons of multinary mixed crystals: application to A(x)B(1-x)C(y)D(1-y) type mixed crystals.

Long-wavelength optical phonons in multinary mixed crystals are studied based on the Pseudo-Unit-Cell model. A unitary matrix method is developed to calculate the eigenfrequencies of optical phonons in multinary mixed crystals. The analytical expressions of oscillator strengths and dielectric constants of the multinary mixed crystals are obtained as a function of the phonon frequencies. The results indicate that the composition dependence of oscillator strengths shows clearly the phonon-mode behaviors of the mixed crystals. The theory and calculation method can be applied to any type of multinary mixed crystals. It is found that there is a composition independent point for the dielectric constant of quaternary mixed crystals.

Improved quantification of immune-gold labeling and its use to compare the distribution of cellular factors among sub-chloroplast compartments.

Quantification of immuno-gold labeling can provide valuable information on the quantity and localization of a target within a region of interest (ROI). Background subtraction usually requires preparation of material with a deliberately reduced amount of target component often by gene knockout/knockdown. This paper reports a modified method without the need for gene knockout/knockdown, by using a region outside the ROI as a background and non-immune serum to verify the reliability of the data. An optimized parameter for use in image processing was also developed to improve semi-automatic segmentation of gold particles, by using the standard deviation of pixel intensity together with default parameters (size and intensity) to improve specificity. The modified methods were used to quantify the gold labeling of various components within chloroplasts and their 3 sub-organelle compartments (thylakoid, stroma and starch). Rubisco, actin, myosin, ß-tubulin, Endoplasmic reticulum-retention signal HDEL, Sterol methyltransferase 1, and double stranded RNA were all effectively and consistently quantified at the level of the different sub-chloroplast compartments. The approach should be applicable more widely for high resolution labelling of samples in which a background requiring gene knockout/knockdown is not a realistic option.

Viral pathogens hitchhike with insect sperm for paternal transmission.

Arthropod-borne viruses (arboviruses) can be maternally transmitted by female insects to their offspring, however, it is unknown whether male sperm can directly interact with the arbovirus and mediate its paternal transmission. Here we report that an important rice arbovirus is paternally transmitted by the male leafhoppers by hitchhiking with the sperm. The virus-sperm binding is mediated by the interaction of viral capsid protein and heparan sulfate proteoglycan on the sperm head surfaces. Mating experiments reveal that paternal virus transmission is more efficient than maternal transmission. Such paternal virus transmission scarcely affects the fitness of adult males or their offspring, and plays a pivotal role in maintenance of viral population during seasons unfavorable for rice hosts in the field. Our findings reveal that a preferred mode of vertical arbovirus transmission has been evolved by hitchhiking with insect sperm without disturbing sperm functioning, facilitating the long-term viral epidemic and persistence in nature.

Endoplasmic reticulum remodeling induced by Wheat yellow mosaic virus infection studied by transmission electron microscopy.

Plant virus was a kind of organism lived depending on infecting viable host cell and propagated their posterity by replicating its hereditary nucleotide, transcripting into protein, assembling protein and nucleotide into virion (Ortín and Parra, 2006; Sanfaçon, 2005). Viral infection usually induces remodeling of host cell, especially endoplasmic reticulum (ER) for generating membrane packed viral factory. During the infection of Bymovirus, a kind of membranous body (MB) was generated in host cells, which is thought as an ER aggregate. In present study we performed a study on Wheat yellow mosaic virus (WYMV) induced MB by several transmission electron microscopy (TEM) based methods, including cytological observation, component analysis by immuno-gold labeling and structural analysis by electron tomography (ET). WYMV infection induced at least two morphologies of MB, including the lamella dominated morphology (lamella-MB) looked like sprawling cirrus, and the tubule dominated morphology (tubule-MB) looked like latticed network. MB was verified composing of ER as revealed by immuno-gold labeling by antibody against endoplasmic reticulum (ER) retention signal as well as by detailed observation of MB construction modules as double layer membrane. By immuno-gold labeling, both two MB morphologies (lamella-MB and tubule-MB) had same components in viral derived protein and membrane origination (from ER). Structural analysis by ET reconstruction revealed the organization of ER in MB. Lamella-MB was composed of cesER like structures arranged irregularly whereas tubule-MB was composed of tubER like structures arranged regularly. This study provided insights into the structural details in how Bymovirus utilizing host membrane system.

Virus-Induced Tubules: A Vehicle for Spread of Virions into Ovary Oocyte Cells of an Insect Vector.

Many arthropod-borne viruses are persistently propagated and transovarially transmitted by female insect vectors through eggs, but the mechanism remains poorly understood. Insect oocytes are surrounded by a layer of follicular cells, which are connected to the oocyte through actin-based microvilli. Here, we demonstrate that a plant reovirus, rice gall dwarf virus (RGDV), exploits virus-containing tubules composed of viral non-structural protein Pns11 to pass through actin-based junctions between follicular cells or through actin-based microvilli from follicular cells into oocyte of its leafhopper vector Recilia dorsalis, thus overcoming transovarial transmission barriers. We further determine that the association of Pns11 tubules with actin-based cellular junctions or microvilli of the ovary is mediated by a specific interaction between Pns11 and actin. Interestingly, RGDV can replicate and assemble progeny virions in the oocyte cytoplasm. The destruction of the tubule assembly by RNA interference with synthesized double-stranded RNA targeting the Pns11 gene strongly inhibits transovarial transmission of RGDV by its vectors. For the first time, we show that a virus can exploit virus-induced tubule as a vehicle to overcome the transovarial transmission barrier by insect vectors.