Alcohol-Perturbed Self-Assembly of Tobacco Mosaic Virus Coat Protein

Self-assembly of Tobacco mosaic virus coat protein is significantly altered in alcohol-water mixtures. Alcohol cosolvents stabilize the disk aggregate and prevent formation of helical rods at low pH. High alcohol content favours stacked disk assemblies and large rafts, while low alcohol concentration favours individual disks and short stacks. These effects appear to be caused by the hydrophobicity of the alcohol additive, with isopropyl alcohol having the strongest effect, and methanol the weakest. We hypothesize that alcohols interact with the hydrophobic faces of TMV-cp disks, thereby disrupting the protein-protein interactions between disks that are necessary to form helical rods.

Kloning Gen Coat Protein (CP) Carnation Mottle Virus (CarMV) pada Vektor Ekspresi [Cloning of Carnation Mottle Virus (CarMV) Coat Protein Gene into Expression Vector]

<p>Carnation mottle virus (CarMV) termasuk anggota genus Carmovirus dalam famili Tombusviridae. Virus ini banyak ditemukan menginfeksi tanaman anyelir di Jawa Barat dan menyebabkan gejala mottle. Sebagai langkah awal untuk memproduksi antiserum melalui teknik ekspresi gen CP perlu diklon pada vektor yang sesuai. Penelitian ini bertujuan mendapatkan klon CarMV yang berfungsi melalui kloning dan subkloning gen CP CarMV ke dalam vektor ekspresi yang sesuai. Penelitian dilakukan dalam beberapa tahap, yaitu ekstraksi RNA total dan amplifikasi cDNA CarMV dengan RT-PCR, menggunakan primer spesifik CarMVF dan CarMVR yang mengandung situs enzim restriksi XhoI dan BamHI, kloning dan subkloning DNA sisipan, serta konfirmasi transforman. Rekombinan gen sisipan CP CarMV dalam bakteri dikonfirmasi dengan koloni PCR. Gen CP CarMV berhasil dikloning ke dalam TA vektor pTZ57R/T dan disubkloning ke vektor ekspresi pET28a. Sekuen rekombinan CP CarMV berhasil dikonfirmasi melalui perunutan DNA. Penelitian lebih lanjut diperlukan untuk mendapatkan produksi antigen rekombinan yang melimpah pada bakteri ekspresi dan kondisi yang sesuai.</p><p><strong>Keywords</strong></p><p>Dianthus caryophillus L.; Carmovirus; Kloning; Subkloning; Bakteri ekspresi</p><p><strong>Abstract</strong></p><p>Carnation mottle virus (CarMV) is a type member of Carmovirus genus in family of Tombusvirus. The virus infects carnation plants in the centre area production of West Java and it cause mottle symptoms. The research aimed to obtain functional clone(s) of CarMV CP gene in suitable expression kloning vector. The research was carried out through several steps, namely total RNA extraction and amplification of cDNA of CP CarMV by RT-PCR using specific primer pairs CarMVF and CarMVR containing restriction enzyme sites XhoI and BamHI, respectively, TA cloning, and subcloning into expression vector pET28a and confirmation of recombinant plasmids by colony PCR. CarMV CP gen was successfully cloned into TA cloning vector pTZ57R/T and subcloned into vector pET28a, alsowere confirmed by DNA sequencing. Future experiment is necessary to be conducted to obtain abundance recombinant antigen production of CarMV CP in suitable expression condition and bacterial host.</p>

Unconventional COPII-mediated secretory protein trafficking continues in the absence of the Sar1 GTPase

Coat protein complex II (COPII) plays an integral role in the packaging of secretory cargoes within membrane-enclosed transport carriers that leave the endoplasmic reticulum (ER) from discrete membrane subdomains. Lipid bilayer remodeling necessary for this process is driven initially by membrane penetration of the coat subunit Sar1 and further stabilized by assembly of a multi-layer complex of several COPII proteins. However, the relative contributions of these distinct factors to transport carrier formation and protein trafficking remain unclear. Here, we demonstrate that anterograde cargo transport from the ER continues in the absence of Sar1, although the unconventional carriers that form fail to efficiently deliver their contents to subsequent compartments in the secretory pathway. Instead, cargoes accumulate immediately adjacent to the perinuclear Golgi under these conditions, together with components of the COPII coat. Our findings highlight new mechanisms by which COPII promotes transport carrier biogenesis and strongly suggests that the Sar1 GTPase plays a critical role in transport carrier uncoating ahead of membrane fusion and secretory cargo delivery at acceptor compartments.

The Amino-Proximal Region of the Coat Protein of Cucumber Vein Yellowing Virus (Family Potyviridae) Affects the Infection Process and Whitefly Transmission

Most plant viruses rely on vector transmission for their spread and specific interactions between vector and virus have evolved to regulate this relationship. The whitefly Bemisia tabaci- transmitted cucumber vein yellowing virus (CVYV; genus Ipomovirus, family Potyviridae) is endemic in the Mediterranean Basin, where it causes significant losses in cucurbit crops. In this study, the role of the coat protein (CP) of CVYV for B. tabaci transmission and plant infection was investigated using a cloned and infectious CVYV cDNA and a collection of point and deletion mutants derived from this clone. Whitefly transmission of CVYV was abolished in a deletion mutant lacking amino acids in position 93–105 of the CP. This deletion mutant caused more severe disease symptoms compared to the cDNA clone representing the wild-type (wt) virus and movement efficiency was likewise affected. Two virus mutants carrying a partially restored CP were transmissible and showed symptoms comparable to the wt virus. Collectively, our data demonstrate that the N-terminus of the CVYV CP is a determinant for transmission by the whitefly vector and is involved in plant infection and symptom expression.

First Report of Ranunculus white mottle ophiovirus in Slovenia in Pepper with yellow leaf curling symptom and in Tomato

Pepper (Capsicum annuum) and Tomato (Solanum lycopersicum) plants showing virus-like disease symptoms were collected in 2017, 2019, and 2020, in different parts of Slovenia (Supplementary Figure 1). Total RNA was extracted from leaf tissue of individual samples using RNeasy Plant Mini kit (Qiagen) and pooled in four composite samples as follows: 2 pepper plants from 2017 (D2017), 5 pepper and 4 tomato plants from 2019 (D2019_P1), 7 tomato plants (D2020_P1), and 2 pepper and 4 tomato plants (D2020_P3) from 2020. The pooled RNA samples were sequenced using Illumina platforms, details of the sequencing experiments are in Supplementary Table 1. Reads were analyzed using CLC Genomics Workbench (v. 20.0, Qiagen) following the pipelines for plant virus discovery (Pecman et al., 2017). Reads and contigs mapping to Ranunculus white mottle ophiovirus (RWMV, GenBank accession no. AY542957 or NC_043389) were detected in all pools. The longest contig (1,255 bp) was obtained from the 2019 composite sample, mapping to the coat protein-coding RNA 3 segment of the RWMV genome (accession no. AY542957). Details of mapping, genome coverage, and other viruses detected in the pools are summarized in the Supplementary Table 1. To identify individual RWMV-infected plants from the pools, primers were designed for detection by reverse transcription-polymerase chain reaction (RT-PCR) targeting the coat protein gene (see Supplementary Table 2). Two pepper samples from two different farms, collected in 2017 and 2019 in southwest Slovenia, and four tomato samples from two different farms, collected in 2020 in central Slovenia tested positive for RWMV in RT-PCR assays. To assess the diversity of RWMV isolates, amplicons were purified using QIAquick PCR purification kit (Qiagen) and sent for Sanger sequencing. Based on maximum likelihood phylogenetic analysis, RWMV Italian and Slovenian isolates form a monophyletic clade within the genus (see Supplementary Figure 2). Pairwise nucleotide identities of the Slovenian isolates (accession no. MZ507604-MZ507609), relative to the original Italian isolate coat protein (accession no. AY542957) range from 92-97%, indicating a moderate level of diversity among isolates (see Supplementary Figure 2). Since only RWMV, bell pepper alphaendornavirus (BPEV), and pepper cryptic virus 2 (PepCV2), were present in a pepper sample from 2017, and BPEV and PepCV2 infection in pepper are not known to be associated with any of the disease symptoms (Okada et al., 2011; Saritha et al., 2016), the symptoms observed on this plant might be associated with RWMV infection. We observed mottling with interveinal chlorosis or yellowing, slight to full curling of leaves from lamina inward, as well as necrotic and aborted flowers on this plant (see Supplementary Figure 1). We cannot easily associate observed symptoms with RWMV in RWMV-positive tomatoes, since several viruses were detected in the pools containing these samples. Nevertheless, the prominent symptoms in tomato were mottling with interveinal chlorosis and leaf curling, similar to those observed in pepper. RWMV was discovered and characterized in buttercup (Ranunculus asiaticus), and detected in anemone (Anemone coronaria), from Italy (Vaira et al., 1996, 1997, 2000, 2003). It was recently detected in pepper from Australia showing veinal yellowing (Gambley et al., 2019). Here, we detected RWMV for the first time in Slovenia, and reported its first detection in tomato and pepper from Europe. These findings call for further studies on the effects of RWMV infection on tomato and pepper production, and its monitoring in neighboring European countries. Acknowledgment This study received funding from the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection, Slovenian Research Agency (ARRS) core financing (P4-0165), and the Horizon 2020 Marie Skłodowska-Curie Actions Innovative Training Network (H2020 MSCA-ITN) project “INEXTVIR” (GA 813542), under the management of the European Commission-Research Executive Agency. References Gambley, C., et al. 2019. New Dis. Rep. 40:13. doi:10.5197/j.2044-0588.2019.040.013. Okada, R., et al. 2011. J. Gen. Virol. 92:2664-2673. doi:10.1099/vir.0.034686-0. Pecman, A., et al. 2017. Front. Microbiol. 8:1-10. doi:10.3389/fmicb.2017.01998. Saritha, R. K., et al. 2016. VirusDisease 27:327-328. doi:10.1007/s13337-016-0327-7. Vaira, A. M., et al. 2003. Arch. Virol. 148:1037-1050. doi:10.1007/s00705-003-0016-x. Vaira, A. M., et al. 1996. Acta Hortic., 432:36-43. doi:10.17660/ActaHortic.1996.432.3. Vaira, A. M., et al. 1997. Arch. Virol. 142:2131-2146. doi:10.1007/s007050050231. Vaira, A. M., et al. 2000. Plant Dis. 84:1046-1046. doi:10.1094/PDIS.2000.84.9.1046B.

The VINE complex is a VPS9–domain GEF–containing SNX–BAR coat involved in endosomal sorting

Membrane trafficking pathways perform important roles in establishing and maintaining the endolysosomal network. Retrograde protein sorting from the endosome is promoted by conserved SNX–BAR–containing coat complexes including retromer which enrich cargo at tubular microdomains and generate transport carriers. In metazoans, retromer cooperates with VARP, a conserved VPS9–domain GEF, to direct an endosomal recycling pathway. The function of the yeast VARP homolog Vrl1 has been overlooked due an inactivating mutation in commonly studied strains. Here, we demonstrate that Vrl1 has features of a SNX–BAR coat protein and forms an obligate complex with Vin1, the paralog of the retromer SNX–BAR protein Vps5. Unique features in the Vin1 N–terminus allow Vrl1 to distinguish it from Vps5, thereby forming what we have named the VINE complex. VINE occupies endosomal tubules and promotes the delivery of a conserved mannose 6–phosphate receptor–like protein to the vacuolar membrane. In addition to sorting functions, membrane recruitment by Vin1 is essential for Vrl1 GEF activity, suggesting that VINE is a multifunctional coat complex that regulates trafficking and signaling events at the endosome.

Survey for Yellow Mosaic Disease Occurring on Blackgram [Vigna mungo (L.) Hepper] and Weed Hosts in Four Major Blackgram Growing Districts of Andhra Pradesh and Characterization of Associated Viruses using Coat Protein Gene

Background: Yellow mosaic disease (YMD) caused by Yellow mosaic virus is one of the major constraints in the pulse production in Andhra Pradesh (A.P.) due to fast evolution of strains, like Mungbean yellow mosaic India virus (MYMIV). Keeping this in view, a survey was undertaken in the major blackgram growing districts of A.P. to know the YMD incidence in blackgram and weed hosts and were characterized based on genetic features by comparing with other YMV isolates from different hosts and locations across the world. Methods: Roving survey was conducted during rabi 2019-20 in major blackgram growing districts of A.P. viz., Krishna, Guntur, West Godavari and Prakasam districts for YMD incidence. Blackgram plants showing characteristic symptoms were collected as representative samples from each mandal along with the suspected weed plants and were subjected to amplification using coat protein (CP) specific primers followed by molecular characterization. Phylogenetic tree for coat protein (CP) gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny. Result: Out of the four districts surveyed, the highest disease incidence was recorded at Machavaram village of Prakasam district (43.22%), whereas least disease incidence was recorded at Chinaganjam village of Praksam district (2.4%). Six weeds viz., Ageratum conyzoides, Amaranthus viridis, Parthenium hysterophporus, Vigna trilobata, Abelmoscus moschatus, Desmodium laxiflorum have showed positive result in PCR amplification with MYMIV specific coat protein primers. Four isolates from blackgram samples and two from weed plants shared 94.85 to 99.58% nucleotide identity among themselves.

Characterization and Establishment of a Recombinase Polymerase Amplification Assay for Rapid Detection of Tomato Spotted Wilt Virus in Pepper

Tomato spotted wilt virus (TSWV) is one of the most economically destructive and scientifically challenging plant viruses, which has seriously affected the production of commercial crops. At present, there is no effective strategy to control this virus. Therefore, there is an urgent need for a rapid and simple method to detect TSWV, which is of great significance to prevent its spread. In this study, an isolate of TSWV (TSWV-LNTL) infecting pepper from Liaoning Province of northeast China was obtained. A phylogenetic tree based on neighbor-joining using coat protein (CP) gene was established. A rapid method for detecting TSWV by recombinase polymerase amplification (RPA) was established. The phylogenetic tree based on the nucleotide sequences of coat protein (CP) genes of different TSWV isolates showed that the genetic relationship of TSWV-LNTL was most closely related to that of TSWV-LX-Lettuce-12 (Yunnan) and TSWV-TSHL (Shandong) isolates in China. It can be finished at 39 °C for 20 min and then purified by heating at 65 °C for 10 min. The RPA primers were highly specific and no cross-reactivity was detected with other selected viruses infecting pepper. The results of sensitivity test revealed that the detection limit of RPA is 1.0 × 103 copies/μL, which was tenfold lower than that of PCR method. In addition, the RPA method was successfully applied to detect TSWV in field samples. These results reported the occurrence of TSWV on crop in Liaoning Province of northeast China and demonstrated that the established RPA assay provided an effective molecular diagnostic tool for the accurate and rapid detection of TSWV to prevent its spread.