CRISPR Screen Reveals PACT as a Pro-Viral Factor for Dengue Viral Replication.

The dengue virus is a single-stranded, positive-sense RNA virus that infects ~400 million people worldwide. Currently, there are no approved antivirals available. CRISPR-based screening methods have greatly accelerated the discovery of host factors that are essential for DENV infection and that can be targeted in host-directed antiviral interventions. In the present study, we performed a focused CRISPR (Clustered Regularly Interspaced Palindromic Repeats) library screen to discover the key host factors that are essential for DENV infection in human Huh7 cells and identified the Protein Activator of Interferon-Induced Protein Kinase (PACT) as a novel pro-viral factor for DENV. PACT is a double-stranded RNA-binding protein generally known to activate antiviral responses in virus-infected cells and block viral replication. However, in our studies, we observed that PACT plays a pro-viral role in DENV infection and specifically promotes viral RNA replication. Knockout of PACT resulted in a significant decrease in DENV RNA and protein abundances in infected cells, which was rescued upon ectopic expression of full-length PACT. An analysis of global gene expression changes indicated that several ER-associated pro-viral genes such as ERN1, DDIT3, HERPUD1, and EIF2AK3 are not upregulated in DENV-infected PACT knockout cells as compared to infected wildtype cells. Thus, our study demonstrates a novel role for PACT in promoting DENV replication, possibly through modulating the expression of ER-associated pro-viral genes.

MYADM binds human parechovirus 1 and is essential for viral entry.

Human parechoviruses (PeV-A) are increasingly being recognized as a cause of infection in neonates and young infants, leading to a spectrum of clinical manifestations ranging from mild gastrointestinal and respiratory illnesses to severe sepsis and meningitis. However, the host factors required for parechovirus entry and infection remain poorly characterized. Here, using genome-wide CRISPR/Cas9 loss-of-function screens, we identify myeloid-associated differentiation marker (MYADM) as a host factor essential for the entry of several human parechovirus genotypes including PeV-A1, PeV-A2 and PeV-A3. Genetic knockout of MYADM confers resistance to PeV-A infection in cell lines and in human gastrointestinal epithelial organoids. Using immunoprecipitation, we show that MYADM binds to PeV-A1 particles via its fourth extracellular loop, and we identify critical amino acid residues within the loop that mediate binding and infection. The demonstrated interaction between MYADM and PeV-A1, and its importance specifically for viral entry, suggest that MYADM is a virus receptor. Knockout of MYADM does not reduce PeV-A1 attachment to cells pointing to a role at the post-attachment stage. Our study suggests that MYADM is a multi-genotype receptor for human parechoviruses with potential as an antiviral target to combat disease associated with emerging parechoviruses.

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.

The transporter associated with antigen processing (TAP) is a key player in the major histocompatibility class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and triggers its proteasomal degradation. How UL49.5 promotes TAP degradation has, so far, remained unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal. We propose that the C terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the cullin-RING E3 ligase in endoplasmic reticulum-associated degradation.

The activated plant NRC4 immune receptor forms a hexameric resistosome.

Innate immune responses against microbial pathogens in both plants and animals are regulated by intracellular receptors known as Nucleotide-binding Leucine-rich Repeats (NLR) proteins. In plants, these NLRs play a crucial role in recognizing pathogen effectors, thereby initiating the activation of immune defense mechanisms. Notably, certain NLRs serve as "helper" NLR immune receptors (hNLR), working in tandem with "sensor" NLR immune receptors (sNLR) counterparts to orchestrate downstream signaling events to express disease resistance. In this study, we reconstituted and determined the cryo-EM structure of the hNLR required for cell death 4 (NRC4) resistosome. The auto-active NRC4 formed a previously unanticipated hexameric configuration, triggering immune responses associated with Ca 2+ influx into the cytosol. Furthermore, we uncovered a dodecameric state of NRC4, where the coil-coil (CC) domain is embedded within the complex, suggesting an inactive state, and expanding our understanding of the regulation of plant immune responses. One Sentence Summary: The hexameric NRC4 resistosome mediates cell death associated with cytosolic Ca 2+ influx.

The herpesvirus UL49.5 protein hijacks a cellular C-degron pathway to drive TAP transporter degradation.

The transporter associated with antigen processing (TAP) is a key player in the MHC class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 (BoHV-1) impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and promotes its proteasomal degradation. How UL49.5 promotes TAP degradation is unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal in human cells. We propose that the C-terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the CRL2 E3 in ER-associated degradation.

LYSET/TMEM251- a novel key component of the mannose 6-phosphate pathway.

Degradation of macromolecules delivered to lysosomes by processes such as autophagy or endocytosis is crucial for cellular function. Lysosomes require more than 60 soluble hydrolases in order to catabolize such macromolecules. These soluble hydrolases are tagged with mannose6-phosphate (M6P) moieties in sequential reactions by the Golgi-resident GlcNAc-1-phosphotransferase complex and NAGPA/UCE/uncovering enzyme (N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase), which allows their delivery to endosomal/lysosomal compartments through trafficking mediated by cation-dependent and -independent mannose 6-phosphate receptors (MPRs). We and others recently identified TMEM251 as a novel regulator of the M6P pathway via independent genome-wide genetic screening strategies. We renamed TMEM251 to LYSET (lysosomal enzyme trafficking factor) to establish nomenclature reflective to this gene's function. LYSET is a Golgi-localized transmembrane protein important for the retention of the GlcNAc-1-phosphotransferase complex in the Golgi-apparatus. The current understanding of LYSET's importance regarding human biology is 3-fold: 1) highly pathogenic viruses that depend on lysosomal hydrolase activity require LYSET for infection. 2) The presence of LYSET is critical for cancer cell proliferation in nutrient-deprived environments in which extracellular proteins must be catabolized. 3) Inherited pathogenic alleles of LYSET can cause a severe inherited disease which resembles GlcNAc-1-phosphotransferase deficiency (i.e., mucolipidosis type II).Abbreviations: GlcNAc-1-PT: GlcNAc-1-phosphotransferase; KO: knockout; LSD: lysosomal storage disorder; LYSET: lysosomal enzyme trafficking factor; M6P: mannose 6-phosphate; MPRs: mannose-6-phosphate receptors, cation-dependent or -independent; MBTPS1/site-1 protease: membrane bound transcription factor peptidase, site 1; MLII: mucolipidosis type II; WT: wild-type.

Establishment and validation of the survival prediction risk model for appendiceal cancer.

Objective: Establishing a risk model of the survival situation of appendix cancer for accurately identifying high-risk patients and developing individualized treatment plans. Methods: A total of 4,691 patients who were diagnosed with primary appendix cancer from 2010 to 2016 were extracted using Surveillance, Epidemiology, and End Results (SEER) * Stat software. The total sample size was divided into 3,283 cases in the modeling set and 1,408 cases in the validation set at a ratio of 7:3. A nomogram model based on independent risk factors that affect the prognosis of appendix cancer was established. Single-factor Cox risk regression, Lasso regression, and multifactor Cox risk regression were used for analyzing the risk factors that affect overall survival (OS) in appendectomy patients. A nomogram model was established based on the independent risk factors that affect appendix cancer prognosis, and the receiver operating characteristic curve (ROC) curve and calibration curve were used for evaluating the model. Survival differences between the high- and low-risk groups were analyzed through Kaplan-Meier survival analysis and the log-rank test. Single-factor Cox risk regression analysis found age, ethnicity, pathological type, pathological stage, surgery, radiotherapy, chemotherapy, number of lymph nodes removed, T stage, N stage, M stage, tumor size, and CEA all to be risk factors for appendiceal OS. At the same time, multifactor Cox risk regression analysis found age, tumor stage, surgery, lymph node removal, T stage, N stage, M stage, and CEA to be independent risk factors for appendiceal OS. A nomogram model was established for the multifactor statistically significant indicators. Further stratified with corresponding probability values based on multifactorial Cox risk regression, Kaplan-Meier survival analysis found the low-risk group of the modeling and validation sets to have a significantly better prognosis than the high-risk group (p < 0.001). Conclusion: The established appendix cancer survival model can be used for the prediction of 1-, 3-, and 5-year OS and for the development of personalized treatment options through the identification of high-risk patients.

Comparison of incidence and prognosis between young and old gastric cancer patient in North-Western China: A retrospective cohort study.

Gastric cancer (GC) is the fourth most common cancer in the world and the second most common cancer in China. In this study, we compared the clinicopathological features and prognosis of GC between young and old patients after curative resection. Six hundred and eighty-six patients with GC resected were divided two groups according to patient age: Younger GC patients ≤40 years of age (YGC, n = 52) and older GC patients >40 years of age (OGC, n = 634). The YGC group had 52 (7.6%) patients in total 686 GC patients. YGC patients was predominant in women (53.8% vs 26.5%) compared with OGC patients. 5-year overall survival exhibited differences in tumor sites, tumor sizes, macroscopic types, T staging, N staging, rate of N staging (rN), tumor node metastasis staging, scope of gastrectomy, radical degree, and lymphatic vascular invasion within each of YGC and OGC group. Univariate analysis of the clinical factors affecting overall survival in YGC group revealed the significant differences in tumor size, macroscopic types (except Borrmann), T staging (except T2), N staging (N3a and N3b), rN, tumor node metastasis staging (III), scope of gastrectomy, radical degree, and lymphatic vascular invasion. Gender, N staging, rN, radical degrees were the independent prognostic factors of younger patients with GC. Similar results were found in the OGC groups. The significant differences in radical degree and lymphatic vascular invasion were found between male and female patients in YGC group. Similar results were found in the OGC groups. Our results showed that YGC patients differ from OGC patients in predominance of women. Gender, N staging, rN, radical degrees were independent risk factors for the prognosis in YGC patients.

Development of an innovative nomogram of risk factors to predict postoperative recurrence of gastrointestinal stromal tumors.

BACKGROUND: There are many staging systems for gastrointestinal stromal tumors (GISTs), and the risk indicators selected are also different; thus, it is not possible to quantify the risk of recurrence among individual patients. AIM: To develop and internally validate a model to identify the risk factors for GIST recurrence after surgery. METHODS: The least absolute shrinkage and selection operator (LASSO) regression model was performed to identify the optimum clinical features for the GIST recurrence risk model. Multivariable logistic regression analysis was used to develop a prediction model that incorporated the possible factors selected by the LASSO regression model. The index of concordance (C-index), calibration curve, receiver operating characteristic curve (ROC), and decision curve analysis were used to assess the discrimination, calibration, and clinical usefulness of the predictive model. Internal validation of the clinical predictive capability was also evaluated by bootstrapping validation. RESULTS: The nomogram included tumor site, lesion size, mitotic rate/50 high power fields, Ki-67 index, intracranial necrosis, and age as predictors. The model presented perfect discrimination with a reliable C-index of 0.836 (95%CI: 0.712-0.960), and a high C-index value of 0.714 was also confirmed by interval validation. The area under the curve value of this prediction nomogram was 0.704, and the ROC result indicated good predictive value. Decision curve analysis showed that the predicting recurrence nomogram was clinically feasible when the recurrence rate exceeded 5% after surgery. CONCLUSION: This recurrence nomogram combines tumor site, lesion size, mitotic rate, Ki-67 index, intracranial necrosis, and age and can easily predict patient prognosis.

The human disease gene LYSET is essential for lysosomal enzyme transport and viral infection.

Lysosomes are key degradative compartments of the cell. Transport to lysosomes relies on GlcNAc-1-phosphotransferase-mediated tagging of soluble enzymes with mannose 6-phosphate (M6P). GlcNAc-1-phosphotransferase deficiency leads to the severe lysosomal storage disorder mucolipidosis II (MLII). Several viruses require lysosomal cathepsins to cleave structural proteins and thus depend on functional GlcNAc-1-phosphotransferase. We used genome-scale CRISPR screens to identify lysosomal enzyme trafficking factor (LYSET, also named TMEM251) as essential for infection by cathepsin-dependent viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). LYSET deficiency resulted in global loss of M6P tagging and mislocalization of GlcNAc-1-phosphotransferase from the Golgi complex to lysosomes. Lyset knockout mice exhibited MLII-like phenotypes, and human pathogenic LYSET alleles failed to restore lysosomal sorting defects. Thus, LYSET is required for correct functioning of the M6P trafficking machinery and mutations in LYSET can explain the phenotype of the associated disorder.

TMEM41B and VMP1 modulate cellular lipid and energy metabolism for facilitating dengue virus infection.

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses.

Comparison of clinicopathologic profiles and prognosis of gastric cancer in the upper, middle and lower third of the stomach: A retrospective cohort study.

Gastric cancer (GC) is the fourth most common cancer in the world and the second most common cancer in China. The aim of this study was to investigate the clinicopathologic profiles and prognosis of GC in the upper third (UT), middle third (MT) and low third (LT) of the stomach.Five hundred and forty-two patients with GC resected between January 2010 and January 2014 were retrospectively studied and divided in 3 groups according to cancer location: upper third gastric cancer (UTGC) (n = 62); MTGC (n = 131) and LTGC (n = 349). Clinical and pathological parameters including gender, age, tumor size, macroscopic types, histological types, depth of invasion, lymph node metastasis, venous infiltration and lymph embolism were compared among groups. Overall survival (OS) was calculated based on the aforementioned parameters. Univariate and multivariate survival was analyzed and Cox regression was conducted for each location. The prognostic accuracy was determined using receiver operating characteristic curve analysis.Patients with UTGC was similar to those with MTGC and both were distinct from those with LTGC based on the tumor size, macroscopic types, depth of invasion and 5-year OS. Patients with MTGC were similar to those with LTGC and distinct from UTGC patients based on gender. 5-year OS were lower for patients with UTGC than those with LTGC (P = .001) and were comparable between MTGC and LTGC. No significant differences in 5-year OS were observed between UTGC and MTGC. Cox regression revealed that macroscopic types, depth of invasion and lymph node metastasis were the independent prognostic factors for GC patients regardless of locations. Receiver operating characteristic curve analysis revealed that macroscopic types, depth of invasion and lymph node metastasis were the significantly effective prognosis for the 5-year OS in GC patients regardless of locations.Our results showed that UTGC is distinct from LTGC whereas MTGC shares some characteristics from both UTGC and LTGC.

Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism.

Genome-wide analysis of the mode of action of GSK983, a potent antiviral agent, led to the identification of dihydroorotate dehydrogenase as its target along with the discovery that genetic knockdown of pyrimidine salvage sensitized cells to GSK983. Because GSK983 is an ineffective antiviral in the presence of physiological uridine concentrations, we explored combining GSK983 with pyrimidine salvage inhibitors. We synthesized and evaluated analogs of cyclopentenyl uracil (CPU), an inhibitor of uridine salvage. We found that CPU was converted into its triphosphate in cells. When combined with GSK983, CPU resulted in large drops in cellular UTP and CTP pools. Consequently, CPU-GSK983 suppressed dengue virus replication in the presence of physiological concentrations of uridine. In addition, the CPU-GSK983 combination markedly enhanced the effect of RNA-dependent RNA polymerase (RdRp) inhibition on viral infection. Our findings highlight a new host-targeting strategy for potentiating the antiviral activity of RdRp inhibitors.

A memory of eS25 loss drives resistance phenotypes.

In order to maintain cellular protein homeostasis, ribosomes are safeguarded against dysregulation by myriad processes. Remarkably, many cell types can withstand genetic lesions of certain ribosomal protein genes, some of which are linked to diverse cellular phenotypes and human disease. Yet the direct and indirect consequences from these lesions are poorly understood. To address this knowledge gap, we studied in vitro and cellular consequences that follow genetic knockout of the ribosomal proteins RPS25 or RACK1 in a human cell line, as both proteins are implicated in direct translational control. Prompted by the unexpected detection of an off-target ribosome alteration in the RPS25 knockout, we closely interrogated cellular phenotypes. We found that multiple RPS25 knockout clones display viral- and toxin-resistance phenotypes that cannot be rescued by functional cDNA expression, suggesting that RPS25 loss elicits a cell state transition. We characterized this state and found that it underlies pleiotropic phenotypes and has a common rewiring of gene expression. Rescuing RPS25 expression by genomic locus repair failed to correct for the phenotypic and expression hysteresis. Our findings illustrate how the elasticity of cells to a ribosome perturbation can drive specific phenotypic outcomes that are indirectly linked to translation and suggests caution in the interpretation of ribosomal protein gene mutation data.

A Distinct, Non-Virion Plant Virus Movement Protein Encoded by a Crinivirus Essential for Systemic Infection.

Plant-infecting viruses utilize various strategies involving multiple viral and host factors to achieve successful systemic infections of their compatible hosts. Lettuce infectious yellows virus (LIYV), genus Crinivirus, family Closteroviridae, has long, filamentous flexuous virions and causes phloem-limited infections in its plant hosts. The LIYV-encoded P26 is a distinct non-virion protein that shows no similarities to proteins in current databases: it induces plasmalemma deposits over plasmadesmata (PD) pit fields and is speculated to have roles in LIYV virion transport within infected plants. In this study, P26 was demonstrated to be a PD-localized protein, and its biological significance was tested in planta by mutagenesis analysis. An LIYV P26 knockout mutant (P26X) showed viral RNA replication and virion formation in inoculated leaves of Nicotiana benthamiana plants, but failed to give systemic infection. Confirmation by using a modified green fluorescent protein (GFP)-tagged LIYV P26X showed GFP accumulation only in infiltrated leaf tissues, while wild-type LIYV GFP readily spread systemically in the phloem. Attempts to rescue P26X by complementation in trans were negative. However a translocated LIYV P26 gene in the LIYV genome rescued systemic infection, but P26 orthologs from other criniviruses did not. Mutagenesis in planta assays showed that deletions in P26, as well as 2 of 11 specific alanine-scanning mutants, abolished the ability to systemically infect N. benthamianaIMPORTANCE Plant viruses encode specific proteins that facilitate their ability to establish multicellular/systemic infections in their host plants. Relatively little is known of the transport mechanisms for plant viruses whose infections are phloem limited, including those of the family Closteroviridae. These viruses have complex, long filamentous virions that spread through the phloem. Lettuce infectious yellows virus (LIYV) encodes a non-virion protein, P26, which forms plasmalemma deposits over plasmodesmata pit fields, and LIYV virions are consistently found attached to those deposits. Here we demonstrate that P26 is a unique movement protein required for LIYV systemic infection in plants. LIYV P26 shows no sequence similarities to other proteins, but other criniviruses encode P26 orthologs. However, these failed to complement movement of LIYV P26 mutants.

Two Crinivirus-Conserved Small Proteins, P5 and P9, Are Indispensable for Efficient Lettuce infectious yellows virus Infectivity in Plants.

Genomic analysis of Lettuce infectious yellows virus (LIYV) has revealed two short open reading frames (ORFs) on LIYV RNA2, that are predicted to encode a 5-kDa (P5) and a 9-kDa (P9) protein. The P5 ORF is part of the conserved quintuple gene block in the family Closteroviridae, while P9 orthologs are found in all Criniviruses. In this study, the expression of LIYV P5 and P9 proteins was confirmed; P5 is further characterized as an endoplasmic reticulum (ER)-localized integral transmembrane protein and P9 is a soluble protein. The knockout LIYV mutants presented reduced symptom severity and virus accumulation in Nicotiana benthamiana or lettuce plants, indicating their importance in efficient virus infection. The P5 mutant was successfully complemented by a dislocated P5 in the LIYV genome. The structural regions of P5 were tested and all were found to be required for the appropriate functions of P5. In addition, P5, as well as its ortholog P6, encoded by Citrus tristeza virus (CTV) and another ER-localized protein encoded by LIYV RNA1, were found to cause cell death when expressed in N. benthamiana plants from a TMV vector, and induce ER stress and the unfolded protein response (UPR).

Efficient Protein Expression and Virus-Induced Gene Silencing in Plants Using a Crinivirus-Derived Vector.

Plant virus-based vectors are valuable tools for recombinant gene expression and functional genomics for both basic and applied research. In this study, Lettuce infectious yellows virus (LIYV) of the genus Crinivirus was engineered into a virus vector that is applicable for efficient protein expression and virus-induced gene silencing (VIGS) in plants. We examined gene replacement and “add a gene” strategies to develop LIYV-derived vectors for transient expression of the green fluorescent protein (GFP) reporter in Nicotiana benthamiana plants. The latter yielded higher GFP expression and was further examined by testing the effects of heterologous controller elements (CEs). A series of five vector constructs with progressively extended LIYV CP sgRNA CEs were tested, the longest CE gave the highest GFP expression but lower virus accumulation. The whitefly transmissibility of the optimized vector construct to other host plants, and the capability to accommodate and express a larger gene, a 1.8 kb β-glucuronidase (GUS) gene, were confirmed. Furthermore, the LIYV vector was also validated VIGS by silencing the endogenous gene, phytoene desaturase (PDS) in N. benthamiana plants, and the transgene GFP in N. benthamiana line 16c plants. Therefore, LIYV-derived vectors could provide a technical reference for developing vectors of other economically important criniviruses.

Accumulation of 24 nucleotide transgene-derived siRNAs is associated with crinivirus immunity in transgenic plants.

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing-mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA-dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus-derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non-transgenic N. benthamiana plants as well as transgene-derived siRNAs (t-siRNAs) derived from the immune-transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t-siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t-siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t-siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24-nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24-nucleotide t-siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24-nucleotide t-siRNAs is associated with crinivirus immunity in transgenic plants.

Inspirations on Virus Replication and Cell-to-Cell Movement from Studies Examining the Cytopathology Induced by Lettuce infectious yellows virus in Plant Cells.

Lettuce infectious yellows virus (LIYV) is the type member of the genus Crinivirus in the family Closteroviridae. Like many other positive-strand RNA viruses, LIYV infections induce a number of cytopathic changes in plant cells, of which the two most characteristic are: Beet yellows virus-type inclusion bodies composed of vesicles derived from cytoplasmic membranes; and conical plasmalemma deposits (PLDs) located at the plasmalemma over plasmodesmata pit fields. The former are not only found in various closterovirus infections, but similar structures are known as 'viral factories' or viroplasms in cells infected with diverse types of animal and plant viruses. These are generally sites of virus replication, virion assembly and in some cases are involved in cell-to-cell transport. By contrast, PLDs induced by the LIYV-encoded P26 non-virion protein are not involved in replication but are speculated to have roles in virus intercellular movement. These deposits often harbor LIYV virions arranged to be perpendicular to the plasma membrane over plasmodesmata, and our recent studies show that P26 is required for LIYV systemic plant infection. The functional mechanism of how LIYV P26 facilitates intercellular movement remains unclear, however, research on other plant viruses provides some insights on the possible ways of viral intercellular movement through targeting and modifying plasmodesmata via interactions between plant cellular components and viral-encoded factors. In summary, beginning with LIYV, we review the studies that have uncovered the biological determinants giving rise to these cytopathological effects and their importance in viral replication, virion assembly and intercellular movement during the plant infection by closteroviruses, and compare these findings with those for other positive-strand RNA viruses.

A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability.

Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to support protoplasts in the form of uniform thin layer, which has three unique properties. •The tissue culture-treated membrane has a good affinity for the plant cell; thus, protoplasts can spread evenly and form a very thin layer.•There are more choices for membrane pore size, depending on the application.•It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol-gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.