Tomato brown rugose fruit virus: An emerging and rapidly spreading plant RNA virus that threatens tomato production worldwide.

Tomato brown rugose fruit virus (ToBRFV) is an emerging and rapidly spreading RNA virus that infects tomato and pepper, with tomato as the primary host. The virus causes severe crop losses and threatens tomato production worldwide. ToBRFV was discovered in greenhouse tomato plants grown in Jordan in spring 2015 and its first outbreak was traced back to 2014 in Israel. To date, the virus has been reported in at least 35 countries across four continents in the world. ToBRFV is transmitted mainly via contaminated seeds and mechanical contact (such as through standard horticultural practices). Given the global nature of the seed production and distribution chain, and ToBRFV's seed transmissibility, the extent of its spread is probably more severe than has been disclosed. ToBRFV can break down genetic resistance to tobamoviruses conferred by R genes Tm-1, Tm-2, and Tm-22 in tomato and L1 and L2 alleles in pepper. Currently, no commercial ToBRFV-resistant tomato cultivars are available. Integrated pest management-based measures such as rotation, eradication of infected plants, disinfection of seeds, and chemical treatment of contaminated greenhouses have achieved very limited success. The generation and application of attenuated variants may be a fast and effective approach to protect greenhouse tomato against ToBRFV. Long-term sustainable control will rely on the development of novel genetic resistance and resistant cultivars, which represents the most effective and environment-friendly strategy for pathogen control. TAXONOMY: Tomato brown rugose fruit virus belongs to the genus Tobamovirus, in the family Virgaviridae. The genus also includes several economically important viruses such as Tobacco mosaic virus and Tomato mosaic virus. GENOME AND VIRION: The ToBRFV genome is a single-stranded, positive-sense RNA of approximately 6.4 kb, encoding four open reading frames. The viral genomic RNA is encapsidated into virions that are rod-shaped and about 300 nm long and 18 nm in diameter. Tobamovirus virions are considered extremely stable and can survive in plant debris or on seed surfaces for long periods of time. DISEASE SYMPTOMS: Leaves, particularly young leaves, of tomato plants infected by ToBRFV exhibit mild to severe mosaic symptoms with dark green bulges, narrowness, and deformation. The peduncles and calyces often become necrotic and fail to produce fruit. Yellow blotches, brown or black spots, and rugose wrinkles appear on tomato fruits. In pepper plants, ToBRFV infection results in puckering and yellow mottling on leaves with stunted growth of young seedlings and small yellow to brown rugose dots and necrotic blotches on fruits.

ß-Cyanoalanine synthase protects mites against Arabidopsis defenses.

Glucosinolates are antiherbivory chemical defense compounds in Arabidopsis (Arabidopsis thaliana). Specialist herbivores that feed on brassicaceous plants have evolved various mechanisms aimed at preventing the formation of toxic isothiocyanates. In contrast, generalist herbivores typically detoxify isothiocyanates through glutathione conjugation upon exposure. Here, we examined the response of an extreme generalist herbivore, the two-spotted spider mite Tetranychus urticae (Koch), to indole glucosinolates. Tetranychus urticae is a composite generalist whose individual populations have a restricted host range but have an ability to rapidly adapt to initially unfavorable plant hosts. Through comparative transcriptomic analysis of mite populations that have differential susceptibilities to Arabidopsis defenses, we identified ß-cyanoalanine synthase of T. urticae (TuCAS), which encodes an enzyme with dual cysteine and ß-cyanoalanine synthase activities. We combined Arabidopsis genetics, chemical complementation and mite reverse genetics to show that TuCAS is required for mite adaptation to Arabidopsis through its ß-cyanoalanine synthase activity. Consistent with the ß-cyanoalanine synthase role in detoxification of hydrogen cyanide (HCN), we discovered that upon mite herbivory, Arabidopsis plants release HCN. We further demonstrated that indole glucosinolates are sufficient for cyanide formation. Overall, our study uncovered Arabidopsis defenses that rely on indole glucosinolate-dependent cyanide for protection against mite herbivory. In response, Arabidopsis-adapted mites utilize the ß-cyanoalanine synthase activity of TuCAS to counter cyanide toxicity, highlighting the mite's ability to activate resistant traits that enable this extreme polyphagous herbivore to exploit cyanogenic host plants.

Purification of Plasmodesmata-Enriched Fraction for Proteomic Analyses.

In plants, plasmodesmata (PD) are plasmamembrane-lined pores that traverse the cell wall to establish cytoplasmic and endomembrane continuity between neighboring cells. As intercellular channels, PD play pivotal roles in plant growth and development, defense responses, and are also co-opted by viruses to spread cell-to-cell to establish systemic infection. Proteomic analyses of PD-enriched fractions may provide critical insights on plasmodesmal biology and PD-mediated virus-host interactions. However, it is difficult to isolate PD from plant tissues as they are firmly embedded in the cell wall. Here, we describe a protocol for the purification of PD from Nicotiana benthamiana leaves for proteomic analysis.

The cis-expression of the coat protein of turnip mosaic virus is essential for viral intercellular movement in plants.

To establish infection, plant viruses are evolutionarily empowered with the ability to spread intercellularly. Potyviruses represent the largest group of known plant-infecting RNA viruses, including many agriculturally important viruses. To better understand intercellular movement of potyviruses, we used turnip mosaic virus (TuMV) as a model and constructed a double-fluorescent (green and mCherry) protein-tagged TuMV infectious clone, which allows distinct observation of primary and secondary infected cells. We conducted a series of deletion and mutation analyses to characterize the role of TuMV coat protein (CP) in viral intercellular movement. TuMV CP has 288 amino acids and is composed of three domains: the N-terminus (amino acids 1-97), the core (amino acids 98-245), and the C-terminus (amino acids 246-288). We found that deletion of CP or its segments amino acids 51-199, amino acids 200-283, or amino acids 265-274 abolished the ability of TuMV to spread intercellularly but did not affect virus replication. Interestingly, deletion of amino acids 6-50 in the N-terminus domain resulted in the formation of aberrant virions but did not significantly compromise TuMV cell-to-cell and systemic movement. We identified the charged residues R178 and D222 within the core domain that are essential for virion formation and TuMV local and systemic transport in plants. Moreover, we found that trans-expression of the wild-type CP either by TuMV or through genetic transformation-based stable expression could not rescue the movement defect of CP mutants. Taken together these results suggest that TuMV CP is not essential for viral genome replication but is indispensable for viral intercellular transport where only the cis-expressed CP is functional.

A plant RNA virus hijacks endocytic proteins to establish its infection in plants.

Endocytosis and endosomal trafficking play essential roles in diverse biological processes including responses to pathogen attack. It is well established that animal viruses enter host cells through receptor-mediated endocytosis for infection. However, the role of endocytosis in plant virus infection still largely remains unknown. Plant dynamin-related proteins 1 (DRP1) and 2 (DRP2) are the large, multidomain GTPases that participate together in endocytosis. Recently, we have discovered that DRP2 is co-opted by Turnip mosaic virus (TuMV) for infection in plants. We report here that DRP1 is also required for TuMV infection. We show that overexpression of DRP1 from Arabidopsis thaliana (AtDRP1A) promotes TuMV infection, and AtDRP1A interacts with several viral proteins including VPg and cylindrical inclusion (CI), which are the essential components of the virus replication complex (VRC). AtDRP1A colocalizes with the VRC in TuMV-infected cells. Transient expression of a dominant negative (DN) mutant of DRP1A disrupts DRP1-dependent endocytosis and supresses TuMV replication. As adaptor protein (AP) complexes mediate cargo selection for endocytosis, we further investigated the requirement of AP in TuMV infection. Our data suggest that the medium unit of the AP2 complex (AP2ß) is responsible for recognizing the viral proteins as cargoes for endocytosis, and knockout of AP2ß impairs intracellular endosomal trafficking of VPg and CI and inhibits TuMV replication. Collectively, our results demonstrate that DRP1 and AP2ß are two proviral host factors of TuMV and shed light into the involvement of endocytosis and endosomal trafficking in plant virus infection.

Development of a cucumber green mottle mosaic virus-based expression vector for the production in cucumber of neutralizing epitopes against a devastating animal virus.

Virus-based expression systems have been widely exploited for the production of recombinant proteins in plants during the last thirty years. Advances in technology have boosted scale-up manufacturing of plant-made pharmaceuticals to high levels, via the complementation of transient expression and viral vectors. This combination allows proteins of interest to be produced in plants within a matter of days and thus, is well suited for the development of plant-made vaccines or therapeutics against emerging infectious diseases and potential bioterrorism agents. Several plant-based products are currently in varying stages of clinical development. To investigate the viability of virus-based expression systems for plant-made vaccines against porcine reproductive and respiratory syndrome virus (PRRSV), the most devastating threat to the pork industry in Canada, we cloned the full-length genome of a cucumber green mottle mosaic virus (CGMMV) isolate and developed a CGMMV-based expression vector. We further employed this vector to express the neutralizing epitope (NE) of PRRSV glycoprotein 5 (GP5) in cucumber leaves via agroinfiltration. The coding region of the GP5 NE was inserted downstream of the open reading frame for coat protein (CP) and expressed by a readthrough mechanism. The chimeric virus particles were stable and the expression levels reached as high as 35.84 mg/kg of cucumber leaf fresh weight. This study offers a promising solution to the production of a low cost, versatile and robust vaccine for oral administration against PRRSV through a chimeric virus particle display system.

Wax Ester Composition of Songbird Preen Oil Varies Seasonally and Differs between Sexes, Ages, and Populations.

Chemical signaling has been well studied in invertebrates and mammals but less so in birds, due to the longstanding misconception that olfaction is unimportant or even non-existent in this taxon. However, recent findings suggest that olfaction plays an important role in avian mate choice and reproductive behavior, similar to other taxa. The leading candidate source for compounds involved in avian chemical communication is preen oil, a complex mixture secreted from the uropygial gland. Preen oil contains volatile compounds and their potential wax ester precursors, and may act as a reproductive chemosignal. Reproductive signals are generally sexually dimorphic, age-specific, seasonally variable, and may also vary geographically. We tested whether preen oil meets these expectations by using gas chromatography to examine the wax ester composition of preen oil in song sparrows (Melospiza melodia). We found that the wax ester composition of preen oil was significantly different between sexes, age classes, seasons, and populations. Collectively, our results suggest that song sparrow preen oil meets the criteria of a chemical cue that may influence mate choice and reproduction. Our findings in song sparrows, which are sexually monomorphic in plumage, also parallel patterns described for dark-eyed juncos (Junco hyemalis), a closely related songbird with sexually dimorphic plumage. Behavioral tests are needed to confirm that song sparrows attend to the cues present in preen oil, but our findings support the increasingly accepted idea that chemical communication is common and widespread in birds as it is in other taxa.

Glyoxylate cycle and metabolism of organic acids in the scutellum of barley seeds during germination.

During the developmental processes from dry seeds to seedling establishment, the glyoxylate cycle becomes active in the mobilization of stored oils in the scutellum of barley (Hordeum vulgare L.) seeds, as indicated by the activities of isocitrate lyase and malate synthase. The succinate produced is converted to carbohydrates via phosphoenolpyruvate carboxykinase and to amino acids via aminotransferases, while free organic acids may participate in acidifying the endosperm tissue, releasing stored starch into metabolism. The abundant organic acid in the scutellum was citrate, while malate concentration declined during the first three days of germination, and succinate concentration was low both in scutellum and endosperm. Malate was more abundant in endosperm tissue during the first three days of germination; before citrate became predominant, indicating that malate may be the main acid acidifying the endosperm. The operation of the glyoxylate cycle coincided with an increase in the ATP/ADP ratio, a buildup of H2O2 and changes in the redox state of ascorbate and glutathione. It is concluded that operation of the glyoxylate cycle in the scutellum of cereals may be important not only for conversion of fatty acids to carbohydrates, but also for the acidification of endosperm and amino acid synthesis.

Functional analysis of the DAT gene promoter using transient Catharanthus roseus and stable Nicotiana tabacum transformation systems.

The Catharanthus roseus DAT gene encodes the enzyme acetyl-CoA:deacetylvindoline-4-O-acetyltransferase involved in the last step of the indole alkaloid pathway leading to vindoline. This gene is characterized by specific cell type expression in idioblasts and laticifers. To understand the specific transcriptional regulation mechanism(s) of DAT, several DAT promoter GUS constructs were cloned into pCAMBIA1305.1. Agroinfiltration of different explant types of C. roseus resulted in organ-specific accumulation of GUS, albeit at various levels. Heterologous accumulation of GUS in transgenic tobacco revealed both general and non-specific expression with the exception of a stomata-specific expression when 2.3 kb of the DAT promoter was coupled with a portion of the DAT ORF. These results suggest that in addition to the 2.3 kb upstream of the DAT transcriptional start site, additional cis-acting elements may be responsible for the specific spatial expression of DAT in vivo. Furthermore, hairy roots transformed with DAT promoter GUS constructs demonstrated GUS expression in root tissues (visualized through GUS enzyme activity), even though DAT is repressed in non-transformed roots.

Hydrogen peroxide is required for poly(phenolic) domain formation during wound-induced suberization.

The requirement for hydrogen peroxide (H(2)O(2)) during suberization was demonstrated in wound-induced potato tubers by monitoring the extent of phenolic polymerization after the inhibition of H(2)O(2) production using diphenyleneiodonium (DPI). In DPI-treated tissues the extent of phenolic polymerization in suberized tissues, measured using DFRC (Derivatization Followed by Reductive Cleavage) and thioglycolic acid analyses, was greatly reduced relative to untreated controls. Concomitantly, a large quantity of new soluble phenolics accumulated in the DPI-treated tissue some of which were not present in the controls. We suggest that the inhibition of H(2)O(2) production prevented these phenolics from being oxidized by cell wall peroxidases. As a result, these phenolics were left unpolymerized and accumulated in the tissue. Several of the soluble phenolics were identified as hydroxycinnamic acid derivatives. From the data presented, it was concluded that H(2)O(2) is required for the polymerization step in the formation of the poly(phenolic) domain of suberized potato tubers.