The function of chloroplast ferredoxin-NADP+ oxidoreductase positively regulates the accumulation of bamboo mosaic virus in Nicotiana benthamiana.

A gene down-regulated in Nicotiana benthamiana after bamboo mosaic virus (BaMV) infection had high identity to the nuclear-encoded chloroplast ferredoxin NADP+ oxidoreductase gene (NbFNR). NbFNR is a flavoenzyme involved in the photosynthesis electron transport chain, catalysing the conversion of NADP+ into NADPH. To investigate whether NbFNR is involved in BaMV infection, we used virus-induced gene silencing to reduce the expression of NbFNR in leaves and protoplasts. After BaMV inoculation, the accumulation of BaMV coat protein and RNA was significantly reduced. The transient expression of NbFNR fused with orange fluorescent protein (OFP) localized in the chloroplasts and elevated the level of BaMV coat protein. These results suggest that NbFNR could play a positive role in regulating BaMV accumulation. Expressing a mutant that failed to translocate to the chloroplast did not assist in BaMV accumulation. Another mutant with a catalytic site mutation could support BaMV accumulation to some extent, but accumulation was significantly lower than that of the wild type. In an in vitro replication assay, the replicase complex with FNR inhibitor, heparin, the RdRp activity was reduced. Furthermore, BaMV replicase was revealed to interact with NbFNR in yeast two-hybrid and co-immunoprecipitation experiments. Overall, these results suggest that NbFNR localized in the chloroplast with functional activity could efficiently assist BaMV accumulation.

Exploration of wheat yellow mosaic virus-responsive miRNAs and their targets in wheat by miRNA and degradome sequencing.

Wheat (Triticum aestivum) is one of the most important food crops around the world. China is the largest wheat production country and wheat yellow mosaic virus (WYMV) is a non-negligible threat to wheat production. This study aimed to explore miRNAs and their corresponding target genes responsive to WYMV in wheat. Linmai and Jimai were used for miRNA and degradome high-throughput sequencing. After comparison and analysis, differentially expressed miRNAs and their target genes between normal wheat and WYMV-infected wheat were identified. GO and KEGG pathway enrichment analysis were then performed on target genes. A total of 530 miRNAs were identified in all samples, including 106 known miRNAs and 424 novel miRNAs. Among them, 131 miRNAs, corresponding to 85 target genes, were differentially expressed between normal wheat and WYMV-infected wheat. 85 target genes were significantly enriched in 21 GO terms and two KEGG pathways, Plant hormone signal transduction and Monobactam biosynthesis. In conclusion, 131 differentially expressed miRNAs, corresponding to 85 target genes, were identified between normal wheat and WYMVinfected wheat. Our findings provide more evidence on the roles of miRNAs and their target genes in wheat- WYMV interactions.

Dissection and cytological mapping of chromosome arm 4VS by the development of wheat-Haynaldia villosa structural aberration library.

KEY MESSAGE: A cytological map of Haynaldia villosa chromosome arm 4VS was constructed to facilitate the identification and utilization of beneficial genes on 4VS. Induction of wheat-alien chromosomal structure aberrations not only provides new germplasm for wheat improvement, but also allows assignment of favorable genes to define physical regions. Especially, the translocation or introgression lines carrying alien chromosomal fragments with different sizes are useful for breeding and alien gene mapping. Chromosome arm 4VS of Haynaldia villosa (L.) Schur (syn. Dasypyrum villosum (L.) P. Candargy) confers resistances to eyespot and wheat yellow mosaic virus (WYMV). In this research, we used both irradiation and the pairing homoeologous gene (Ph) mutant to induce chromosomal aberrations or translocations. By using the two approaches, a structural aberration library of chromosome arm 4VS was constructed. In this library, there are 57 homozygous structural aberrations, in which, 39 were induced by the Triticum aestivum cv. Chinese Spring (CS) ph1b mutant (CS ph1b) and 18 were induced by irradiation. The aberrations included four types, i.e., terminal translocation, interstitial translocation, deletion and complex structural aberration. The 4VS cytological map was constructed by amplification in the developed homozygous aberrations using 199 4VS-specific markers, which could be allocated into 39 bins on 4VS. These bins were further assigned to their corresponding physical regions of chromosome arm 4DS based on BLASTn search of the marker sequences against the reference sequence of Aegilops tauschii Cosson. The developed genetic stocks and cytological map provide genetic stocks for wheat breeding as well as alien gene tagging.

Variable 3'polyadenylation of Wheat yellow mosaic virus and its novel effects on translation and replication.

BACKGROUND: Polyadenylation influences many aspects of mRNA as well as viral RNA. variable polyadenylation at the 3' end have been reported in RNA viruses. It is interesting to identify the characteristic and potential role of 3' polyadenylation of Wheat yellow mosaic virus (WYMV), which has been reported to contain two genomic RNAs with 3' poly(A) tails and caused severe disease on wheat in East Asia region. METHODS: 3' RACE was used to identify sequences of the 3' end in WYMV RNAs from naturally infected wheat by WYMV. In vitro translation assay was performed to analyze effect of UTRs of WYMV with or without 3'polyadenylation on translation. In vitro replication mediated by WYMV NIb protein were performed to evaluate effect of variable polyadenylation on replication. RESULTS: Variable polyadenylation in WYMV RNAs was identified via 3' RACE. WYMV RNAs in naturally infected wheat in China simultaneously present with regions of long, short, or no adenylation at the 3' ends. The effects of variable polyadenylation on translation and replication of WYMV RNAs were evaluated. 5'UTR and 3'UTR of WYMV RNA1 or RNA2 synergistically enhanced the translation of the firefly luciferase (Fluc) gene in in vitro WGE system, whereas additional adenylates had an oppositive effect on this enhancement on translation mediated by UTRs of WYMV. Additional adenylates remarkably inhibited the synthesis of complementary strand from viral genome RNA during the in vitro replication mediated by WYMV NIb protein. CONCLUSIONS: 3' end of WYMV RNAs present variable polyadenylation even no polyadenylation. 3' polyadenylation have opposite effect on translation mediated by UTRs of WYMV RNA1 or RNA2. 3' polyadenylation have negative effect on minus-strand synthesis of WYMV RNA in vitro. Variable polyadenylation of WYMV RNAs may provide sufficient selection on the template for translation and replication.

Detection and profiling of circular RNAs in uninfected and maize Iranian mosaic virus-infected maize.

Circular RNAs (circRNAs) are covalently closed non-coding RNAs that are usually derived from exonic regions of genes, but can also arise from intronic and intergenic regions. Studies of circRNAs in humans, animals and several plant species have shown an altered population of circRNAs in response to abiotic and biotic stress. Recently it was shown that circRNAs also occur in maize, but it is unknown if maize circRNAs are responsive to stress. Maize Iranian mosaic virus (MIMV, genus Nucleorhabdovirus, family Rhabdoviridae) causes an economically important disease in maize and other gramineous crops in Iran. In this study, we used data from RNA-Seq of MIMV-infected maize and uninfected controls to identify differentially expressed circRNAs. Such circRNAs were confirmed by two-dimensional polyacrylamide gel electrophoresis, northern blot, RT-qPCR and sequencing. A total of 1443 circRNAs were identified in MIMV-infected maize and 1165 circRNAs in uninfected maize. Two hundred and one circRNAs were in common between MIMV-infected and uninfected samples. Of these, 155 circRNAs were up-regulated and 5 down-regulated in MIMV infected plants, compared to the uninfected control. This study for the first time identified and profiled circRNA expression in maize in response to virus infection. Moreover, we predict that 33 circRNAs may bind 23 maize miRNAs, possibly affecting plant metabolism and development. Our data suggest a role for circRNAs in plant cell regulation and response to biotic stress such as virus infection, and give new insights into the complexity of plant-microbe interactions.

Chloroplast Proteome of Nicotiana benthamiana Infected by Tomato Blistering Mosaic Virus.

Tymovirus is a genus of plant pathogenic viruses that infects several dicotyledonous plants worldwide, causing serious diseases in economically important crops. The known cytopathic effect on the host cell organelles involves chloroplast membrane deformation and the induction of vesicles in its periphery. These vesicles are known to be the location where tymoviral genomic RNA replication occurs. Tomato blistering mosaic virus (ToBMV) is a tymovirus recently identified in tomato plants in Brazil, which is able to infect several other plants, including tobacco. In this work, we investigated the chloroplast proteomic profile of ToBMV-infected N. benthamiana using bidimensional electrophoresis (2-DE) and mass spectrometry, aiming to study the virus-host interaction related to the virus replication and infection. A total of approximately 200 spots were resolved, out of which 36 were differentially abundant. Differential spots were identified by mass spectrometry including photosynthesis-related and defense proteins. We identified proteins that may be targets of a direct interaction with viral proteins, such as ATP synthase ß subunit, RNA polymerase beta-subunit, 50S ribosomal protein L6 and Trigger factor-like protein. The identification of these candidate proteins gives support for future protein-protein interaction studies to confirm their roles in virus replication and disease development.

Over-expression of GmSN1 enhances virus resistance in Arabidopsis and soybean.

KEY MESSAGE: GmSN1 enhances virus resistance in plants most likely by affecting the expression of signal transduction and immune response genes. Soybean mosaic virus (SMV) infection causes severe symptom and leads to massive yield loss in soybean (Glycine max). By comparative analyzing gene expression in the SMV-resistant soybean cultivar Rsmv1 and the susceptible cultivar Ssmv1 at a transcriptome level, we found that a subgroup of Gibberellic Acid Stimulated Transcript (GAST) genes were down-regulated in SMV inoculated Ssmv1 plants, but not Rsmv1 plants. Sequence alignment and phylogenetic analysis indicated that one of the GAST genes, GmSN1, was closely related to Snakin-1, a well-characterized potato microbial disease resistance gene. When over-expressed in Arabidopsis and soybean, respectively, under the control of the 35S promoter, GmSN1 enhanced turnip mosaic virus resistance in the transgenic Arabidopsis plants, and SMV resistance in the transgenic soybean plants, respectively. Transcriptome analysis results showed that the up-regulated genes in the 35S:GmSN1 transgenic Arabidopsis plants were largely enriched in functional terms including "signal transduction" and "immune response". Real-time PCR assay indicated that the expression of GmAKT2, a potassium channel gene known to enhance SMV resistance when over-expressed in soybean, was elevated in the 35S:GmSN1 transgenic soybean plants. Taken together, our results suggest that GmSN1 enhances virus resistance in plants most likely by affecting the expression of signal transduction and immune response genes.

A sugarcane pathogenesis-related protein, ScPR10, plays a positive role in defense responses under Sporisorium scitamineum, SrMV, SA, and MeJA stresses.

KEY MESSAGE: A pathogenesis-related gene, ScPR10 , was isolated from sugarcane and its bio-function was characterized, demonstrating that ScPR10 was involved in plant defense responses to Sporisorium scitamineum , SrMV, SA, and MeJA stresses. Plant fungal and viral diseases are the major concerns in sugarcane industry. Many anti-fungal and antivirus components, including pathogenesis-related (PR) proteins, have been identified. The pathogenesis-related protein 10 (PR10) is the dominant group in PR families, involved in the plant defense mechanism. In this study, ScPR10 (GenBank Acc. No. KT887884), a 701-bp-length PR10 gene with a 483 bp-length open reading frame, was isolated from sugarcane. Its transient expression in the leaves of Nicotiana benthamiana indicated that the function role of ScPR10 is likely in the nucleus, and it increased the level of H2O2 accumulation in leaf cells. Moreover, ScPR10 could also enhance the resistance of N. benthamiana leaves to infection by Pseudomonas solanacearum and Fusarium solani var. coeruleum. Quantitative real-time PCR analysis revealed that ScPR10 was not constitutively expressed in sugarcane tissues due to its high expression in the buds and scant presence in root tips. In addition, the transcript of ScPR10 could be induced by a pathogenic fungus (Sporisorium scitamineum) and a virus (Sorghum mosaic virus, SrMV) in the resistant sugarcane cultivars, while it was down-regulated in the susceptible ones. After exposure to salicylic acid (SA) and methyl jasmonate (MeJA), ScPR10 peaked at 6 and 12 h, respectively. These results suggest that ScPR10 can play a positive role in sugarcane defense responses to S. scitamineum, SrMV, SA, and MeJA stresses.

Cassava Mosaic and Brown Streak Diseases: Current Perspectives and Beyond.

Cassava is the fourth largest source of calories in the world but is subject to economically important yield losses due to viral diseases, including cassava brown streak disease and cassava mosaic disease. Cassava mosaic disease occurs in sub-Saharan Africa and the Asian subcontinent and is associated with nine begomovirus species, whereas cassava brown streak disease has to date been reported only in sub-Saharan Africa and is caused by two distinct ipomovirus species. We present an overview of key milestones and their significance in the understanding and characterization of these two major diseases as well as their associated viruses and whitefly vector. New biotechnologies offer a wide range of opportunities to reduce virus-associated yield losses in cassava for farmers and can additionally enable the exploitation of this valuable crop for industrial purposes. This review explores established and new technologies for genetic manipulation to achieve desired traits such as virus resistance.

Drought increases cowpea (Vigna unguiculata [L.] Walp.) susceptibility to cowpea severe mosaic virus (CPSMV) at early stage of infection.

The physiological and biochemical responses of a drought tolerant, virus-susceptible cowpea genotype exposed to drought stress (D), infected by Cowpea severe mosaic virus (CPSMV) (V), and to these two combined stresses (DV), at 2 and 6 days post viral inoculation (DPI), were evaluated. Gas exchange parameters (net photosynthesis, transpiration rate, stomatal conductance, and internal CO2 partial pressure) were reduced in D and DV at 2 and 6 DPI compared to control plants (C). Photosynthesis was reduced by stomatal and biochemical limitations. Water use efficiency increased at 2 DPI in D, DV, and V, but at 6 DPI only in D and DV compared to C. Photochemical parameters (effective quantum efficiency of photosystem II and electron transport rate) decreased in D and DV compared to C, especially at 6 DPI. The potential quantum efficiency of photosystem II did not change, indicating reversible photoinhibition of photosystem II. In DV, catalase decreased at 2 and 6 DPI, ascorbate peroxidase increased at 2 DPI, but decreased at 6 DPI. Hydrogen peroxide increased at 2 and 6 DPI. Peroxidase increased at 6 DPI and chitinase at 2 and 6 DPI. ß-1,3-glucanase decreased in DV at 6 DPI compared to V. Drought increased cowpea susceptibility to CPSMV at 2 DPI, as verified by RT-PCR. However, at 6 DPI, the cowpea plants overcome this effect. Likewise, CPSMV increased the negative effects of drought at 2 DPI, but not at 6 DPI. It was concluded that the responses to combined stresses are not additive and cannot be extrapolated from the study of individual stresses.

Insight into virus encapsulation mechanism through in silico interaction study between coat protein and RNA operator loops of Sesbania mosaic virus.

Viruses are parasite by nature and they are responsible for many diseases. Inhibitor development is very difficult for viruses due to their rapid mutative nature. A common approach for treating virus infection is targeting them at the genomic level and an encapsulation mechanism can be one of the targets. Sesbania mosaic virus (SeMV) is a spherical virus and its capsid is formed by a coat protein, which contains the Arginine Rich Motif (ARM). This ARM interacts with RNA operator loops present in their genome and starts encapsulation. Though the structure of SeMV was already solved by crystallography, it lacks the critical ARM domain. We predicted the full-length three-dimensional structure of this protein by using crystal structure (lacking ARM) as a template along with tertiary structure of RNA operator loops. Docking studies were performed to discover the interacting residues of protein and RNA which are driving protein and RNA to interact with each other. We observed that these interactions lead to conformation changes in the coat protein structure, which starts genome encapsulation process. The ARM region is found to be crucial for these interactions. Molecular dynamics simulation studies were performed to check the conformational changes and free energy landscapes were generated to check the viability of these changes in terms of energy. In this work we proposed one RNA operator loop that is responsible for noticeable conformational changes in the SeMV structure and might be involved in the activation of the viral protein. The results of this in silico study can be tested further through in vitro studies and can be used to stop encapsulation.

Molecular Variability and Distribution of Sugarcane Mosaic Virus in Shanxi, China.

BACKGROUND: Sugarcane mosaic virus (SCMV) is responsible for large-scale economic losses in the global production of sugarcane, maize, sorghum, and some other graminaceous species. To understand the evolutionary mechanism of SCMV populations, this virus was studied in Shanxi, China. A total of 86 maize leaf samples (41 samples in 2012 and 45 samples in 2013) were collected from 4 regions of Shanxi. RESULTS: Double-antibody sandwich (DAS)-ELISA and RT-PCR showed 59 samples (30 samples in 2012 and 29 samples in 2013) to be positive for SCMV, from which 10 new isolates of SCMV were isolated and sequenced. The complete genomes of these isolates are 9610 nt long, including the 5' and 3' non-coding regions, and encode a 3063-amino acid polyprotein. Phylogenetic analyses revealed that 24 SCMV isolates could be divided on the basis of the whole genome into 2 divergent evolutionary groups, which were associated with the host species. Among the populations, 15 potential recombination events were identified. The selection pressure on the genes of these SCMV isolates was also calculated. The results confirmed that all the genes were under negative selection. CONCLUSIONS: Negative selection and recombination appear to be important evolutionary factors shaping the genetic structure of these SCMV isolates. SCMV is distributed widely in China and exists as numerous strains with distinct genetic diversity. Our findings will provide a foundation for evaluating the epidemiological characteristics of SCMV in China and will be useful in designing long-term, sustainable management strategies for SCMV.

NbRABG3f, a member of Rab GTPase, is involved in Bamboo mosaic virus infection in Nicotiana benthamiana.

The screening of differentially expressed genes in plants after pathogen infection can uncover the potential host factors required for the pathogens. In this study, an up-regulated gene was identified and cloned from Nicotiana benthamiana plants after Bamboo mosaic virus (BaMV) inoculation. The up-regulated gene was identified as a member of the Rab small guanosine triphosphatase (GTPase) family, and was designated as NbRABG3f according to its in silico translated product with high identity to that of RABG3f of tomato. Knocking down the expression of NbRABG3f using a virus-induced gene silencing technique in a protoplast inoculation assay significantly reduced the accumulation of BaMV. A transiently expressed NbRABG3f protein in N. benthamiana plants followed by BaMV inoculation enhanced the accumulation of BaMV to approximately 150%. Mutants that had the catalytic site mutation (NbRABG3f/T22N) or had lost their membrane-targeting capability (NbRABG3f/ΔC3) failed to facilitate the accumulation of BaMV in plants. Because the Rab GTPase is responsible for vesicle trafficking between organelles, a mutant with a fixed guanosine diphosphate form was used to identify the donor compartment. The use of green fluorescent protein (GFP) fusion revealed that GFP-NbRABG3f/T22N clearly co-localized with the Golgi marker. In conclusion, BaMV may use NbRABG3f to form vesicles derived from the Golgi membrane for intracellular trafficking to deliver unidentified factors to its replication site; thus, both GTPase activity and membrane-targeting ability are crucial for BaMV accumulation at the cell level.

Evidence for different, host-dependent functioning of Rx against both wild-type and recombinant Pepino mosaic virus.

The potato Rx gene provides resistance against Pepino mosaic virus (PepMV) in tomato; however, recent work has suggested that the resistance conferred may not be durable. Resistance breaking can probably be attributed to multiple mutations observed to accumulate in the capsid protein (CP) region of resistance-breaking isolates, but this has not been confirmed through directed manipulation of an infectious PepMV clone. The present work describes the introduction of two specific mutations, A-T78 and A-T114, into the coat protein minimal elicitor region of an Rx-controlled PepMV isolate of the EU genotype. Enzyme-linked immunosorbent assay (ELISA) and phenotypic evaluation were conducted in three Rx-expressing and wild-type solanaceous hosts: Nicotiana benthamiana, Nicotiana tabacum and Solanum lycopersicum. Mutation A-T78 alone was sufficient to confer Rx-breaking activity in N. benthamiana and S. lycopersicum, whereas mutation A-T114 was found to be associated, in most cases, with a secondary A-D100 mutation to break Rx-mediated resistance in S. lycopersicum. These results suggest that the need for a second, fitness-restoring mutation may be dependent on the PepMV mutant under consideration. Both mutations conferred Rx breaking in S. lycopersicum, whereas neither conferred Rx breaking in N. tabacum and only A-T78 allowed Rx breaking in N. benthamiana, suggesting that Rx may function in a different manner depending on the genetic background in which it is present.

Loss of CMD2-mediated resistance to cassava mosaic disease in plants regenerated through somatic embryogenesis.

Cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) are the two most important viral diseases affecting cassava production in Africa. Three sources of resistance are employed to combat CMD: polygenic recessive resistance, termed CMD1, the dominant monogenic type, named CMD2, and the recently characterized CMD3. The farmer-preferred cultivar TME 204 carries inherent resistance to CMD mediated by CMD2, but is highly susceptible to CBSD. Selected plants of TME 204 produced for RNA interference (RNAi)-mediated resistance to CBSD were regenerated via somatic embryogenesis and tested in confined field trials in East Africa. Although micropropagated, wild-type TME 204 plants exhibited the expected levels of resistance, all plants regenerated via somatic embryogenesis were found to be highly susceptible to CMD. Glasshouse studies using infectious clones of East African cassava mosaic virus conclusively demonstrated that the process of somatic embryogenesis used to regenerate cassava caused the resulting plants to become susceptible to CMD. This phenomenon could be replicated in the two additional CMD2-type varieties TME 3 and TME 7, but the CMD1-type cultivar TMS 30572 and the CMD3-type cultivar TMS 98/0505 maintained resistance to CMD after passage through somatic embryogenesis. Data are presented to define the specific tissue culture step at which the loss of CMD resistance occurs and to show that the loss of CMD2-mediated resistance is maintained across vegetative generations. These findings reveal new aspects of the widely used technique of somatic embryogenesis, and the stability of field-level resistance in CMD2-type cultivars presently grown by farmers in East Africa, where CMD pressure is high.

Viral Strain-Specific Differential Alterations in Arabidopsis Developmental Patterns.

Turnip mosaic virus (TuMV) infections affect many Arabidopsis developmental traits. This paper analyzes, at different levels, the development-related differential alterations induced by different strains of TuMV, represented by isolates UK 1 and JPN 1. The genomic sequence of JPN 1 TuMV isolate revealed highest divergence in the P1 and P3 viral cistrons, upon comparison with the UK 1 sequence. Infectious viral chimeras covering the whole viral genome uncovered the P3 cistron as a major viral determinant of development alterations, excluding the involvement of the PIPO open reading frame. However, constitutive transgenic expression of P3 in Arabidopsis did not induce developmental alterations nor modulate the strong effects induced by the transgenic RNA silencing suppressor HC-Pro from either strain. This highlights the importance of studying viral determinants within the context of actual viral infections. Transcriptomic and interactomic analyses at different stages of plant development revealed large differences in the number of genes affected by the different infections at medium infection times but no significant differences at very early times. Biological functions affected by UK 1 (the most severe strain) included mainly stress response and transport. Most cellular components affected cell-wall transport or metabolism. Hubs in the interactome were affected upon infection.

Resistance gene analogs involved in tolerant cassava--geminivirus interaction that shows a recovery phenotype.

The current literature describes recovery from virus-induced symptoms as a RNA silencing defense, but immunity-related genes, including the structurally specific resistance gene analogs (RGAs) that may play a key role in tolerance and recovery is not yet reported. In this study, the transcriptome data of tolerant cassava TME3 (which exhibits a recovery phenotype) and susceptible cassava T200 infected with South African cassava mosaic virus were explored for RGAs. Putative resistance protein analogs (RPAs) with amide-like indole-3-acetic acid-Ile-Leu-Arg (IAA-ILR) and leucine-rich repeat (LRR)-kinase conserved domains were unique to TME3. Common responsive RPAs in TME3 and T200 were the dirigent-like protein, coil-coil nucleotide-binding site (NBS) and toll-interleukin-resistance, disease resistance zinc finger chromosome condensation-like protein (DZC), and NBS-apoptosis repressor with caspase recruitment (ARC)-LRR domains. Mutations in RPAs in the MHD motif of the NBS-ARC2 subdomain associated with the recovery phase in TME3 were observed. Additionally, a cohort of 25 RGAs mined solely during the recovery process in TME3 was identified. Phylogenetic and expression analyses support that diverse RGAs are differentially expressed during tolerance and recovery. This study reveals that in cassava, a perennial crop, RGAs participate in tolerance and differentially accumulate during recovery as a complementary defense mechanism to natural occurring RNA silencing to impair viral replication.

Effects of Abiotic and Biotic Stresses on the Internalization and Dissemination of Human Norovirus Surrogates in Growing Romaine Lettuce.

Human norovirus (NoV) is the major causative agent of fresh-produce-related outbreaks of gastroenteritis; however, the ecology and persistence of human NoV in produce systems are poorly understood. In this study, the effects of abiotic and biotic stresses on the internalization and dissemination of two human NoV surrogates (murine norovirus 1 [MNV-1] and Tulane virus [TV]) in romaine lettuce were determined. To induce abiotic stress, romaine lettuce was grown under drought and flood conditions that mimic extreme weather events, followed by inoculation of soil with MNV-1 or TV. Independently, lettuce plants were infected with lettuce mosaic virus (LMV) to induce biotic stress, followed by inoculation with TV. Plants were grown for 14 days, and viral titers in harvested tissues were determined by plaque assays. It was found that drought stress significantly decreased the rates of both MNV-1 and TV internalization and dissemination. In contrast, neither flood stress nor biotic stress significantly impacted viral internalization or dissemination. Additionally, the rates of TV internalization and dissemination in soil-grown lettuce were significantly higher than those for MNV-1. Collectively, these results demonstrated that (i) human NoV surrogates can be internalized via roots and disseminated to shoots and leaves of romaine lettuce grown in soil, (ii) abiotic stress (drought) but not biotic stress (LMV infection) affects the rates of viral internalization and dissemination, and (iii) the type of virus affects the efficiency of internalization and dissemination. This study also highlights the need to develop effective measures to eliminate internalized viruses in fresh produce.

Barley Stripe Mosaic Virus (BSMV) Induced MicroRNA Silencing in Common Wheat (Triticum aestivum L.).

MicroRNAs (miRNAs) play important roles in growth, development, and response to environmental changes in plants. Based on the whole-genome shotgun sequencing strategy, more and more wheat miRNAs have been annotated. Now, there is a need for an effective technology to analyse endogenous miRNAs function in wheat. We report here that the modified barley stripe mosaic virus (BSMV)-induced miRNAs silencing system can be utilized to silence miRNAs in wheat. BSMV-based miRNA silencing system is performed through BSMV-based expression of miRNA target mimics to suppress miR159a and miR3134a. The relative expression levels of mature miR159a and miR3134a decrease with increasing transcript levels of their target genes in wheat plants. In summary, the developed approach is effective in silencing endogenous miRNAs, thereby providing a powerful tool for biological function analyses of miRNA molecules in common wheat.