scholarly journals Transgenic Sugarcane Resistant toSorghum mosaic virusBased on Coat Protein Gene Silencing by RNA Interference

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Jinlong Guo ◽  
Shiwu Gao ◽  
Qinliang Lin ◽  
Hengbo Wang ◽  
Youxiong Que ◽  
...  
Keyword(s):  
Rna Interference ◽  
Coat Protein ◽  
Mosaic Virus ◽  
Resistance Rate ◽  
Mosaic Disease ◽  
Artificial Inoculation ◽  
Multiple Sequence ◽  
Sugarcane Cultivar ◽  
Resistant Lines ◽  
Transgenic Lines

As one of the critical diseases of sugarcane, sugarcane mosaic disease can lead to serious decline in stalk yield and sucrose content. It is mainly caused byPotyvirus sugarcane mosaic virus(SCMV) and/orSorghum mosaic virus(SrMV), with additional differences in viral strains. RNA interference (RNAi) is a novel strategy for producing viral resistant plants. In this study, based on multiple sequence alignment conducted on genomic sequences of different strains and isolates of SrMV, the conserved region of coat protein (CP) genes was selected as the target gene and the interference sequence with size of 423 bp in length was obtained through PCR amplification. The RNAi vector pGII00-HACP with an expression cassette containing both hairpin interference sequence andcp4-epspsherbicide-tolerant gene was transferred to sugarcane cultivar ROC22 viaAgrobacterium-mediated transformation. After herbicide screening, PCR molecular identification, and artificial inoculation challenge, anti-SrMV positive transgenic lines were successfully obtained. SrMV resistance rate of the transgenic lines with the interference sequence was 87.5% based on SrMV challenge by artificial inoculation. The genetically modified SrMV-resistant lines of cultivar ROC22 provide resistant germplasm for breeding lines and can also serve as resistant lines having the same genetic background for study of resistance mechanisms.

scholarly journals Recombinant Coat Protein of Banana Bract Mosaic Virus as a Potential Antigen for Serological Detection of the Virus

2020 ◽  
pp. 47-60
Author(s):  
Darsana Dilip ◽  
Vimi Louis ◽  
Pallavi Sabharwal ◽  
H. S. Savithri ◽  
P. M. Namitha ◽  
...  
Keyword(s):  
Coat Protein ◽  
Fluorescence Spectroscopy ◽  
Mosaic Virus ◽  
Gene Segment ◽  
Mosaic Disease ◽  
Polyclonal Antiserum ◽  
Expression Vectors ◽  
Affinity Column ◽  
Banana Bract Mosaic Virus

Banana bract mosaic disease caused by Banana bract mosaic potyvirus (BBrMV) is reported to instigate heavy loss in banana and plantain across Asia. Almost all the cultivars of banana succumb to the disease resulting in malformed bunches weighing less than half of normal ones. In the current study the coat protein (CP) gene segment present at the 3’ terminal region of the viral genome amplified by RT-PCR was cloned into expression vectors, pRSET-C and pGEX-4T-2 to use it for raising polyclonal antiserum which in turn will aid in developing assays to detect the virus. Recombinant BBrMV CP (rCP) in pRSET-C when expressed was insoluble whereas, it was in the soluble fraction when expressed from pGEX-4T-2. The GST-fusion protein was purified by GSH sepharose affinity column chromatography and western blot analysis was performed using anti GST antibodies. 360 µg/ml of protein was purified from 1 l of culture. The GST tag was cleaved from the purified protein by incubation with thrombin at 25°C overnight.  The rCP was characterized using ultracentrifugation, fluorescence spectroscopy and electron microscopy. The tagless monomer failed to assemble to virus like particles (VLPs) in vitro which was substantiated by fluorescence spectroscopy. This study will be first step towards deciphering structure and functions of Banana bract mosaic virus coat protein.

scholarly journals Homology-Dependent Virus Resistance in Transgenic Plants with the Coat Protein Gene of Sweet Potato Feathery Mottle Potyvirus: Target Specificity and Transgene Methylation

1999 ◽  
Vol 89 (5) ◽  
pp. 385-391 ◽  
Author(s):  
S. Sonoda ◽  
M. Mori ◽  
M. Nishiguchi
Keyword(s):  
Coat Protein ◽  
Sweet Potato ◽  
Virus Resistance ◽  
Potato Virus X ◽  
Transcriptional Gene Silencing ◽  
Target Specificity ◽  
Post Transcriptional Gene Silencing ◽  
Resistant Lines ◽  
Nontranslated Region ◽  
Transgenic Lines

Nicotiana benthamiana plants were transformed with the coat protein (CP) coding sequence and the 3′ nontranslated region (NTR) of the severe strain of sweet potato feathery mottle potyvirus (SPFMV-S). Regenerated lines were screened for virus resistance using recombinant potato virus X (PVX) engineered to contain the sequence homologous to the transgene. Out of 19 transgenic lines, 7 showed virus resistance after inoculation by the recombinant PVX. In most of the resistant lines, relatively low steady-state accumulation of the CP gene mRNA and little or no protein products were observed, suggesting that the resistance was manifested by a post-transcriptional gene-silencing mechanism. The resistant lines could be divided into two groups according to the target specificity of the silencing mechanism; one group recognizing the 3′ part of the transgene mRNA and the other not only the 3′ part, but also the 5′ and the central part of the transgene mRNA. Particular regions of the transgene corresponding to the RNA target in the resistant lines were differentially methylated compared with the transgene sequence in a susceptible line.

scholarly journals Generation of Transgenic Papaya with Double Resistance to Papaya ringspot virus and Papaya leaf-distortion mosaic virus

2009 ◽  
Vol 99 (11) ◽  
pp. 1312-1320 ◽  
Author(s):  
Yi-Jung Kung ◽  
Huey-Jiunn Bau ◽  
Yi-Ling Wu ◽  
Chiung-Huei Huang ◽  
Tsui-Miao Chen ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Field Tests ◽  
Ringspot Virus ◽  
Papaya Ringspot Virus ◽  
Coding Region ◽  
Transgenic Papaya ◽  
Resistant Lines ◽  
Transgenic Lines ◽  
Chimeric Construct ◽  
Leaf Distortion

During the field tests of coat protein (CP)-transgenic papaya lines resistant to Papaya ringspot virus (PRSV), another Potyvirus sp., Papaya leaf-distortion mosaic virus (PLDMV), appeared as an emerging threat to the transgenic papaya. In this investigation, an untranslatable chimeric construct containing the truncated CP coding region of the PLDMV P-TW-WF isolate and the truncated CP coding region with the complete 3′ untranslated region of PRSV YK isolate was transferred into papaya (Carica papaya cv. Thailand) via Agrobacterium-mediated transformation to generate transgenic plants with resistance to PLDMV and PRSV. Seventy-five transgenic lines were obtained and challenged with PRSV YK or PLDMV P-TW-WF by mechanical inoculation under greenhouse conditions. Thirty-eight transgenic lines showing no symptoms 1 month after inoculation were regarded as highly resistant lines. Southern and Northern analyses revealed that four weakly resistant lines have one or two inserts of the construct and accumulate detectable amounts of transgene transcript, whereas nine resistant lines contain two or three inserts without significant accumulation of transgene transcript. The results indicated that double virus resistance in transgenic lines resulted from double or more copies of the insert through the mechanism of RNA-mediated posttranscriptional gene silencing. Furthermore, three of nine resistant lines showed high levels of resistance to heterologous PRSV strains originating from Hawaii, Thailand, and Mexico. Our transgenic lines have great potential for controlling a number of PRSV strains and PLDMV in Taiwan and elsewhere.

scholarly journals Complete Genome Characterization and Coat Protein Genealogy of Isolates of Maize dwarf mosaic virus from Johnsongrass and Maize in Oklahoma and Missouri

Plant Disease ◽  
2020 ◽  
Vol 104 (4) ◽  
pp. 1214-1223
Author(s):  
Dulanjani Wijayasekara ◽  
Akhtar Ali
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Complete Genome ◽  
Purifying Selection ◽  
Aphid Transmission ◽  
The United States ◽  
Mosaic Disease ◽  
Maize Dwarf Mosaic Virus ◽  
Maize Cultivars ◽  
Nucleotide Insertion

Maize dwarf mosaic virus (MDMV) significantly affects maize production worldwide, including the United States. This study describes the distribution and biological and molecular characterization of MDMV isolates from Johnsongrass and maize. A total of 262 samples (symptomatic = 214, asymptomatic = 48) were collected in Oklahoma and Missouri during 2016, 2017, and 2019 growing seasons. Based on a dot-immunobinding assay (DIBA), the average incidence of maize dwarf mosaic disease varied from ∼71% (79/111) in 2016, ∼76% (81/106) in 2017, and 62% (28/45) in 2019. Sixty-five DIBA-positive samples for MDMV were further confirmed by RT-PCR, and the complete coat protein (CP) gene was cloned and sequenced. Phylogenetic analysis of 132 isolates (This study = 65; GenBank = 67) revealed two main groups (G1 and G2) of MDMV isolates. All 65 MDMV isolates contained a 39-nucleotide insertion in the N-terminal region of CP genes and clustered in G1 which were different from the isolates in G2, without 39-nucleotide insertion. The first complete genome (9,563 nucleotides) of a MDMV isolate (Bixby1) from Johnsongrass was sequenced, which was distantly related to eight previously reported MDMV isolates from maize. The dN/dS ratio showed mostly purifying selection on each of cistrons except 6K1 being subjected to the diversifying selection. Further analyses revealed three putative recombination events between MDMV-Bixby1 and MDMV isolates from other countries. The successful mechanical and aphid transmission of MDMV-Bixby1 onto maize cultivars was achieved. Altogether, this information showed that Johnsongrass harbors genetically diverse MDMV isolates, which could pose a threat to cultivated crops such as maize and sorghum.

scholarly journals Selective Interaction of Sugarcane eIF4E with VPgs from Sugarcane Mosaic Pathogens

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 518
Author(s):  
Zongtao Yang ◽  
Meng Dong ◽  
Guangyuan Cheng ◽  
Shuxian Liu ◽  
Hai Zhang ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Expression Profiles ◽  
Mosaic Disease ◽  
Translation Initiation Factor ◽  
Bimolecular Fluorescence Complementation ◽  
Initiation Factor ◽  
Pcr Analysis ◽  
Protein Coding ◽  
Sugarcane Cultivar ◽  
Real Time Quantitative Pcr

Eukaryotic translation initiation factor 4E (eIF4E) plays a key role in the infection of potyviruses in susceptible plants by interacting with viral genome-linked protein (VPg). Sugarcane (Saccharum spp.) production is threatened by mosaic disease caused by Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), and Sugarcane streak mosaic virus (SCSMV). In this study, two eIF4Es and their isoform eIF(iso)4E and 4E-binding protein coding genes were cloned from sugarcane cultivar ROC22 and designated SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP, respectively. Real-time quantitative PCR analysis showed different expression profiles of these four genes upon SCMV challenge. A subcellular localization assay showed that SceIF4Ea, SceIF4Eb, SceIF(iso)4E, and ScnCBP were distributed in the nucleus and cytoplasm. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SceIF4Ea/b and SceIF(iso)4E were selectively employed by different sugarcane mosaic pathogens, i.e., SCMV-VPg interacted with SceIF4Ea/b and SceIF(iso)4E, SrMV-VPg interacted with both SceIF4Eb and SceIF(iso)4E, and SCSMV-VPg interacted only with SceIF(iso)4E. Intriguingly, the BiFC assays, but not the Y2H assays, showed that ScnCBP interacted with the VPgs of SCMV, SrMV, and SCSMV. Competitive interaction assays showed that SCMV-VPg, SrMV-VPg, and SCMV-VPg did not compete with each other to interact with SceIF(iso)4E, and SceIF(iso)4E competed with SceIF4Eb to interact with SrMV-VPg but not SCMV-VPg. This study sheds light on the molecular mechanism of sugarcane mosaic pathogen infection of sugarcane plants and benefits sugarcane breeding against the sugarcane mosaic disease.

scholarly journals Distinguishing resistances of transgenic sugarcane generated from RNA interference and pathogen‐derived resistance approaches to combating sugarcane mosaic virus

2021 ◽  
Vol 26 (2) ◽  
pp. 107
Author(s):  
Weny Nailul Hidayati ◽  
Retnosari Apriasti ◽  
Hardian Susilo Addy ◽  
Bambang Sugiharto
Keyword(s):  
Rna Interference ◽  
Mosaic Virus ◽  
Plant Viruses ◽  
Sugarcane Mosaic Virus ◽  
Pcr Analysis ◽  
Post Inoculation ◽  
Gene Encoding ◽  
Transgenic Sugarcane ◽  
Transgenic Lines ◽  
Pathogen Derived Resistance

Sugarcane mosaic virus (SCMV) is a causative agent that reduces growth and productivity in sugarcane. Pathogen‐derived resistance (PDR) and RNA interference (RNAi) are the most common approaches to generating resis‐ tance against plant viruses. Two types of transgenic sugarcane have been obtained by PDR and RNAi methods using a gene‐encoding coat protein (CP) of SCMV (SCMVCp). This research aimed to distinguish resistance of the two transgenic sugarcanes in combating SCMV through artificial viral inoculation. The experiment was conducted using transgenic sugar‐ cane lines validated by PCR analysis. Insertion of gene‐encoding CP in the transgenic lines was confirmed by amplification of 702 bp of DNA fragment of SCMVCp. After viral inoculation, mosaic symptoms appeared earlier, at 21 days post inoculation (dpi) in PDR transgenic lines, but was at 26 dpi in RNAi transgenic lines. Symptom observation showed that 77.8% and 50% of the inoculated plants developed mosaic symptoms in PDR and RNAi transgenic lines, respectively. RT‐PCR analysis revealed that the nuclear inclusion protein b (Nib) gene of SCMV was amplified in the symptomatic leaves in plants classified as susceptible lines. Immunoblot analysis confirmed presence of viral CP with a molecular size of 37 kDa in the susceptible lines. Collectively, these results indicated that the RNAi approach targeting the gene for CP effectively produces more resistance against the SCMV infection in transgenic sugarcane compared to the PDR approach.

scholarly journals Maize resistance to Sugarcane mosaic virus

2017 ◽  
Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽  
pp. 542-544
Author(s):  
R. Pokorný ◽  
M. Porubová
Keyword(s):  
Mosaic Virus ◽  
Systemic Infection ◽  
Sugarcane Mosaic Virus ◽  
Resistant Line ◽  
Mechanisms Of Resistance ◽  
Long Distance ◽  
Maize Hybrids ◽  
Resistant Lines ◽  
Maize Resistance ◽  
Long Distance Movement

Under greenhouse conditions 12 maize hybrids derived from crosses of four resistant lines with several lines of different level of susceptibility were evaluated for resistance to Czech isolate of Sugarcane mosaic virus (SCMV). These hybrids were not fully resistant to isolate of SCMV, but the symptoms on their newly growing leaves usually developed 1 to 3 weeks later in comparison with particular susceptible line, the course of infection was significantly slower and rate of infection lower. As for mechanisms of resistance, the presence of SCMV was detected by ELISA in inoculated leaves both of resistant and susceptible lines, but virus was detected 7 days later in resistant line. Systemic infection developed only in susceptible lines. These results indicate restriction of viral long distance movement in the resistant line.

scholarly journals Multiple aromatic amino acids are involved in potyvirus movement by forming π-stackings to maintain coat protein accumulation

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Zhi-Yong Yan ◽  
Xiao-Jie Xu ◽  
Le Fang ◽  
Chao Geng ◽  
Yan-Ping Tian ◽  
...  
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Infectious Clone ◽  
Three Dimensional ◽  
Cell Movement ◽  
Systemic Infection ◽  
Watermelon Mosaic Virus ◽  
Protein Accumulation ◽  
Three Dimensional Model ◽  
Aromatic Residues

AbstractCoat protein (CP) is required for potyviruses to move and establish a systemic infection in plants. π-stackings formed by aromatic residues play critical roles in maintaining protein stability and functions. As we know, many aromatic residues located in the core region of potyvirus CPs are conserved. However, their roles in potyvirus infection remain largely unknown. Here, through analysis of the three-dimensional model of the tobacco vein banding mosaic virus (TVBMV; genus Potyvirus) CP, 16 aromatic residues were predicated to form π-stackings. The results of transient expression experiments demonstrated that deletion of any of these 16 aromatic residues reduced CP accumulation. Infectivity assays showed that deletion of any of these aromatic residues in the TVBMV infectious clone abolished cell-to-cell movement and reduced replication of the virus. Substitution of Y105 and Y147 individually with non-aromatic residues alanine or glycine reduced CP accumulation, virus replication, and abolished the ability of TVBMV to move intercellularly, while substitution of these two residues individually with aromatic residues phenylalanine or tryptophan, had no or little effect on CP accumulation and TVBMV systemic movement and replication. Similar results were obtained from the CP mutants of watermelon mosaic virus (WMV, genus Potyvirus). Taken together, our results demonstrate that multiple aromatic residues in CP are involved in potyvirus movement by forming π-stackings to maintain CP accumulation.

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