Restriction-ligation-independent production of a TVCV infectious clone and a TVCV-based gene expression vector.

Transgenic expression of proteins in plants is central to research and biotechnology, and, often, it is desirable to obtain this expression without altering the nuclear or plastid genomes. Thus, expression vectors based on plant viruses that infect multiple cells are useful; furthermore, they are also advantageous for studies of the life cycle of the virus itself. Here, we report the development of an expression vector based on a Turnip vein-clearing virus (TVCV), a tobamovirus known to easily infect two model plants, Nicotiana benthamiana, and Arabidopsis thaliana. Avoiding restriction digestion, we utilized a restriction-ligation-independent cloning approach to construct an infectious cDNA clone of TVCV from the viral RNA and then to convert this clone to a gene expression vector adapted for Gateway-based recombination cloning for transgene insertion. The functionality of the resulting vector, designated pTVCV-DEST, was validated by the expression of an autofluorescent reporter transgene following agroinoculation of the target plant.

Arabidopsis LSH10 transcription factor and OTLD1 histone deubiquitinase interact and transcriptionally regulate the same target genes.

Histone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene expression. In plants, OTLD1, a member of the ovarian tumor (OTU) deubiquitinase family, deubiquitylates histone 2B and represses the expression of genes involved in growth, cell expansion, and hormone signaling. OTLD1 lacks the intrinsic ability to bind DNA. How OTLD1, as well as most other known plant histone deubiquitinases, recognizes its target genes remains unknown. Here, we show that Arabidopsis transcription factor LSH10, a member of the ALOG protein family, interacts with OTLD1 in living plant cells. Loss-of-function LSH10 mutations relieve the OTLD1-promoted transcriptional repression of the target genes, resulting in their elevated expression, whereas recovery of the LSH10 function results in down-regulated transcription of the same genes. We show that LSH10 associates with the target gene chromatin as well as with DNA sequences in the promoter regions of the target genes. Furthermore, without LSH10, the degree of H2B monoubiquitylation in the target promoter chromatin increases. Hence, our data suggest that OTLD1-LSH10 acts as a co-repressor complex potentially representing a general mechanism for the specific function of plant histone deubiquitinases at their target chromatin.

Gain-of-function mutant of movement protein allows systemic transport of a defective tobacco mosaic virus.

Functional compensation in response to gene dysfunction is a fascinating phenomenon that allows mutated viruses to regain the capabilities of their wild-type parental strains. In this study, we isolated mutants of tobacco mosaic virus capable of CP-independent systemic movement. These gain-of-function mutants lacked the 16 C-terminal amino acids of the movement protein (MP). Whereas this deletion did not affect the cell-to-cell movement of MP, it dramatically enhanced the viral genomic RNA levels and MP accumulation within the infected cells and altered the subcellular localization of MP from exclusively plasmodesmata (PD) to both PD and plasma membrane. The adapted defective virus suppressed the expression of the ethylene pathway and phloem-associated resistance factors in the inoculated leaves. These findings demonstrate the potential for plant viral MPs to gain a new function that allows viral genomes to move systemically in the absence of the natural viral factor that mediates this spread.

Investigating Interactions Between Histone Modifying Enzymes and Transcription Factors in vivo by Fluorescence Resonance Energy Transfer.

Epigenetic regulation of gene expression is commonly affected by histone modifying enzymes (HMEs) that generate heterochromatic or euchromatic histone marks for transcriptional repression or activation, respectively. HMEs are recruited to their target chromatin by transcription factors (TFs). Thus, detecting and characterizing direct interactions between HMEs and TFs are critical for understanding their function and specificity better. These studies would be more biologically relevant if performed in vivo within living tissues. Here, a protocol is described for visualizing interactions in plant leaves between a plant histone deubiquitinase and a plant transcription factor using fluorescence resonance energy transfer (FRET), which allows the detection of complexes between protein molecules that are within <10 nm from each other. Two variations of the FRET technique are presented: SE-FRET (sensitized emission) and AB-FRET (acceptor bleaching), in which the energy is transferred non-radiatively from the donor to the acceptor or emitted radiatively by the donor upon photobleaching of the acceptor. Both SE-FRET and AB-FRET approaches can be adapted easily to discover other interactions between other proteins in planta.

Overexpression of purple acid phosphatase GmPAP2.1 confers resistance to Soybean mosaic virus in a susceptible soybean cultivar.

A purple acid phosphatase, GmPAP2.1, from the soybean (Glycine max) cultivar L29 may function as a resistance factor acting against specific strains of Soybean mosaic virus (SMV). In this study, we found that overexpression of GmPAP2.1 from L29 conferred SMV resistance to a susceptible cultivar, Lee 74. We determined that GmPAP2.1 interacted with the SMV protein P1 in the chloroplasts, resulting in the up-regulation of the ICS1 gene, which in turn promoted the pathogen-induced salicylic acid (SA) pathway. SA accumulation was elevated in response to the co-expression of GmPAP2.1 and SMV, while transient knockdown of endogenous SA-related genes resulted in systemic infection by SMV strain G5H, suggesting that GmPAP2.1-derived resistance depended on the SA-pathway for the activation of a defense response. Our findings thus suggest that GmPAP2.1 purple acid phosphatase of soybean cultivar L29 functions as an SA-pathway-dependent resistance factor acting against SMV.

Receptor-like kinase BAM1 facilitates early movement of the Tobacco mosaic virus.

Cell-to-cell movement is an important step for initiation and spreading of virus infection in plants. This process occurs through the intercellular connections, termed plasmodesmata (PD), and is usually mediated by one or more virus-encoded movement proteins (MP) which interact with multiple cellular factors, among them protein kinases that usually have negative effects on MP function and virus movement. In this study, we report physical and functional interaction between MP of Tobacco mosaic virus (TMV), the paradigm of PD-moving proteins, and a receptor-like kinase BAM1 from Arabidopsis and its homolog from Nicotiana benthamiana. The interacting proteins accumulated in the PD regions, colocalizing with a PD marker. Reversed genetics experiments, using BAM1 gain-of-function and loss-of-function plants, indicated that BAM1 is required for efficient spread and accumulation the virus during initial stages of infection of both plant species by TMV. Furthermore, BAM1 was also required for the efficient cell-to-cell movement of TMV MP, suggesting that BAM1 interacts with TMV MP to support early movement of the virus. Interestingly, this role of BAM1 in viral movement did not require its protein kinase activity. Thus, we propose that association of BAM1 with TMV MP at PD facilitates the MP transport through PD, which, in turn, enhances the spread of the viral infection.

Rapid generation of inoculum of a plant RNA virus using overlap PCR.

We have developed an efficient method to rapidly generate infectious inoculum of a plant RNA virus and confirmed its infectivity by mechanical inoculation. The method takes advantage of overlap PCR to bypass the cloning steps, which makes it relatively simple, rapid, and inexpensive compared to the traditional methods. Using this approach, inoculum of a tobamovirus, Turnip vein clearing virus (TVCV), was generated. PCR products specific for the 35S promoter and TVCV genome were used as templates for overlap PCR to form a single product containing the full-length TVCV cDNA under the control of the double 35S promoter, and the entire process took only 8 h. This inoculum was infectious in Nicotiana benthamiana, and its infectivity was ca. 67% compared to 0% and 100% with negative and positive controls, respectively. Thus, this rapid method generates efficient infectious inoculum for a plant RNA virus.

Self-Assembled Amphiphilic Starch Based Drug Delivery Platform: Synthesis, Preparation, and Interactions with Biological Barriers.

Core-shell structured nanoparticles (NPs) render the simultaneous coloading capacity of both hydrophobic and hydrophilic drugs and may eventually enhance therapeutic efficacy. In this study, we employed a facile squalenoylation technology to synthesize a new amphiphilic starch derivative from partially oxidized starch, which self-assembled into core-shell starch NPs (StNPs) only at a squalenyl degree of substitution (DoS) of ∼1%. The StNPs characteristics could be tuned as the functions of the polymer molecular weight, DoS, and NPs concentration. The biopharmaceutical features of the StNPs, including colloidal stability, carrier properties, and biocompatibility, were carefully investigated. The interaction study between StNPs and mucin glycoproteins, the main organic component of mucus, revealed a moderate mucin interacting profile. Furthermore, the StNPs also showed good penetration through Pseudomonas aeruginosa biofilms. These results nominate StNPs as a versatile drug delivery platform with potential applications for mucosal drug delivery and the treatment of persistent infections.

Identification of Viruses and Viroids Infecting Tomato and Pepper Plants in Vietnam by Metatranscriptomics.

Tomato (Lycopersicum esculentum L.) and pepper (Capsicum annuum L.) plants belonging to the family Solanaceae are cultivated worldwide. The rapid development of next-generation sequencing (NGS) technology facilitates the identification of viruses and viroids infecting plants. In this study, we carried out metatranscriptomics using RNA sequencing followed by bioinformatics analyses to identify viruses and viroids infecting tomato and pepper plants in Vietnam. We prepared a total of 16 libraries, including eight tomato and eight pepper libraries derived from different geographical regions in Vietnam. We identified a total of 602 virus-associated contigs, which were assigned to 18 different virus species belonging to nine different viral genera. We identified 13 different viruses and two viroids infecting tomato plants and 12 viruses and two viroids infecting pepper plants with viruses as dominantly observed pathogens. Our results showed that multiple infection of different viral pathogens was common in both plants. Moreover, geographical region and host plant were two major factors to determine viral populations. Taken together, our results provide the comprehensive overview of viral pathogens infecting two important plants in the family Solanaceae grown in Vietnam.

A plant intron enhances the performance of an infectious clone in planta.

Although infectious clones are fundamental tools in virology and plant pathology, their efficacy is often reduced by the instability of viral sequences in Escherichia coli. In this study, we constructed an infectious clone of PepMoV (pPepMoV) in a bacterial binary vector (pSNU1); the clone induces symptoms of PepMoV in agroinfiltrated plants. During its modification and maintenance in E. coli, however, the pPepMoV infectious clone was instable in the bacteria. Manipulation of this unstable clone in the bacterial strain DH10B led to the spontaneous formation of a recombined clone with high stability in the bacteria but with reduced infectivity due to an unwanted insertion of an E. coli sequence in the NIa-protease coding region. Replacement of this sequence with a plant intron restored infectivity and maintained plasmid stability. In addition to restoring plasmid growth in both E. coli and Agrobacterium, the presence of the intron in the PepMoV sequence enhanced the accumulation of PepMoV in agroinfiltrated leaves and resulted in symptom induction in upper systemic leaves that was nearly as strong as with PepMoV sap-inoculation. Plant introns have been previously used to stabilize plasmids in E. coli without any effect or with an unexpected lag in symptom development. In contrast, the current results demonstrated the in vivo enhancement of an infectious clone by a plant intron.

Isolation and validation of a candidate Rsv3 gene from a soybean genotype that confers strain-specific resistance to soybean mosaic virus.

Soybean mosaic virus (SMV), a member of the genus Potyvirus, significantly reduces soybean production worldwide. Rsv3, which confers strain-specific resistance to SMV, was previously mapped between the markers A519F/R and M3Satt in chromosome 14 of the soybean [Glycine max (L.) Merr.] genotype L29. Analysis of the soybean genome database revealed that five different NBS-LRR sequences exist between the flanking markers. Among these candidate Rsv3 genes, the full-length cDNA of the Glyma.14g204700 was successfully cloned from L29. Over-expression of Glyma.14g204700 in leaves inoculated with SMV inhibited viral infection in a soybean genotype lacking Rsv3. In addition, the transient silencing of the candidate gene caused a high accumulation of an avirulent strain in L29 carrying Rsv3. Our results therefore provide additional line of evidence to support that Glyma.14g204700 is likely Rsv3 gene that confers strain-specific resistance to SMV.

Engineering an auto-activated R protein that is in vivo activated by a viral protease.

Autonomous hypersensitive responses (self-HRs) are caused by constitutively active R proteins. In this study, we identified an auto-activated form of the R gene Pvr9 (autoPvr9); the auto-activation results from an amino acid substitution between its NBS and LRR domains. Self-HR was strongly reduced or completely inhibited by fusion of an extra peptide to the autoPvr9 N-terminal or C-terminal, respectively. When an NIa recognition site was placed between autoPvr9 and the extra peptide, the fusion construct could trigger an NIa-dependent HR. Several C-terminal fusions were tested, but only those that maintained detectable protein expression were capable of an NIa-dependent HR. Our results suggest the potential for transforming malfunctioning and auto-activated R proteins into a new construct targeting potyviral NIa proteases.

Virus-induced gene silencing reveals signal transduction components required for the Pvr9-mediated hypersensitive response in Nicotiana benthamiana.

Resistance to pathogens mediated by plant resistance (R) proteins requires different signaling transduction components and pathways. Our previous studies revealed that a potyvirus resistance gene in pepper, Pvr9, confers a hypersensitive response (HR) to pepper mottle virus in Nicotiana benthamiana. Our results show that the Pvr9-mediated HR against pepper mottle virus infection requires HSP90, SGT1, NDR1, but not EDS1. These results suggest that the Pvr9-mediated HR is possibly related to the SA pathway but not the ET, JA, ROS or NO pathways.

Molecular characterization of Pvr9 that confers a hypersensitive response to Pepper mottle virus (a potyvirus) in Nicotiana benthamiana.

There are some R genes against potyviruses which were mapped in pepper. However, none of them has been characterized at the molecular level. In this study, we characterized Pvr9 which is an Rpi-blb2 ortholog from pepper and confers a hypersensitive response to Pepper mottle virus (PepMoV) in a transient expression system in Nicotiana benthamiana. This gene putatively encoded for 1298 amino acids and is located on pepper chromosome 6. PepMoV NIb was the elicitor of the Pvr9-mediated hypersensitive response. NIb from several other potyviruses also elicited the hypersensitive response. Inoculation of pepper with PepMoV resulted in a minor increase in Pvr9 transcription in the resistant cultivar CM334 and a slight down-regulation in the susceptible cultivar Floral Gem. The 5' upstream region of Pvr9 from cultivar CM334 had higher transcription activity than the region from cultivar Floral Gem. The cultivars CM334 and Floral Gem had non-functional Pvr9 homologs with loss-of-function mutations.

A simple method for screening of plant NBS-LRR genes that confer a hypersensitive response to plant viruses and its application for screening candidate pepper genes against Pepper mottle virus.

Plant NBS-LRR genes are abundant and have been increasingly cloned from plant genomes. In this study, a method based on agroinfiltration and virus inoculation was developed for the simple and inexpensive screening of candidate R genes that confer a hypersensitive response to plant viruses. The well-characterized resistance genes Rx and N, which confer resistance to Potato virus X (PVX) and tobamovirus, respectively, were used to optimize a transient expression assay for detection of hypersensitive response in Nicotiana benthamiana. Infectious sap of PVX and Tobacco mosaic virus were used to induce hypersensitive response in Rx- and N-infiltrated leaves, respectively. The transient expression of the N gene induced local hypersensitive response upon infection of another tobamovirus, Pepper mild mottle virus, through both sap and transcript inoculation. When this method was used to screen 99 candidate R genes from pepper, an R gene that confers hypersensitive response to the potyvirus Pepper mottle virus was identified. The method will be useful for the identification of plant R genes that confer resistance to viruses.