scholarly journals Engineering Resistance Against Cotton Leaf Curl Kokhran Virus-Burewala Strain Using CRISPR-Cas9 System in Nicotiana Benthamiana

2021 ◽  
Author(s):  
Muhammad Hamza ◽  
Muhammad Zuhaib Khan ◽  
Roma Mustafa ◽  
Hira Kamal ◽  
Aneela Hussain ◽  
...  
Keyword(s):  
Genome Editing ◽  
Nicotiana Benthamiana ◽  
Durable Resistance ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Pcr Primers ◽  
Cotton Leaf ◽  
A Genome ◽  
Intact Virus ◽  
Leaf Curl

Abstract Clustered regularly interspaced palindromic repeats (CRISPR) and associated Cas9 nuclease (CRISPR-Cas9) systems provide adaptive immunity to prokaryotes against infectious phage particles that can be engineered as a genome-editing tool. Guided by an RNA strand, the class II type II CRISPR-Cas9 system can be employed to provide resistance against plant DNA viruses. Here we describe an efficient CRISPR-Cas9 genome editing system based on simultaneous targeting of the highly conserved intergenic region (IR) of the virus that can provide resistance against Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bur) in Nicotiana benthamiana plants. The data revealed that upon infection, the transgenic plants harboring CRISPR-Cas9 and two gRNAs showed complete resistance against CLCuKoV-Bur/Cotton leaf curl Multan betasatellite (CLCuMB). All efforts failed to find the intact virus in CLCuKoV-Bur/CLCuMB challenged transgenic (OX:Cas9NB:IR) plants using either gene specific PCR primers or CLCuKoV-Bur as a probe in southern blot hybridization. Thus, our results have demonstrated an efficient CRISPR-Cas9 approach to engineer durable resistance against CLCuKoV-Bur in a model system. The implications of these findings are discussed.

scholarly journals In Planta Interaction and Transreplication of Distinct Begomoviruses and their Associated Components

2021 ◽  
Vol 26 (01) ◽  
pp. 45-51
Author(s):  
Muhammad Naeem Sattar
Keyword(s):  
Nicotiana Benthamiana ◽  
Blot Hybridization ◽  
Low Frequency ◽  
Southern Blot Hybridization ◽  
Bipartite Begomoviruses ◽  
In Planta ◽  
Monopartite Begomovirus ◽  
Structural Resemblance ◽  
Rep Proteins ◽  
Viral Components

The studies described here were intended to examine the transreplication and interactions abilities of a widespread ToLCNDV, and an emerging begomovirus PeLCV associated with its cognate betasatellite TbLCuB. PeLCV, a monopartite begomovirus, has been characterized from many important crops, vegetables and weeds along with its associated TbLCuB. The DNA-B of bipartite ToLCNDV genome has been successfully transreplicated by the DNA-A of different bipartite begomoviruses, albeit with low frequency. Whether PeLCV can transreplicate DNA-B of ToLCNDV is unknown. To unravel this notion, both these viruses were inoculated to the model Nicotiana benthamiana plants in all possible combinations and the in planta existence of viral components were verified by PCR and Southern blot hybridization. The results demonstrated that PeLCV transreplicated and maintained ToLCNDV DNA-B. Whereas, ToLCNDV DNA-A could not transreplicate TbLCuB. Analyses of Rep proteins structure of ToLCNDV and PeLCV revealed a structural resemblance, whereas putative iteron-binding sequences of PeLCV were compatible with the Rep-binding iterons of ToLCNDV-B. The results suggested that PeLCV and ToLCNDV DNA-B can interact synergistically and can be disastrous under field conditions. © 2021 Friends Science Publishers

scholarly journals A DNAβ Associated with Tomato Yellow Leaf Curl China Virus Is Required for Symptom Induction

2004 ◽  
Vol 78 (24) ◽  
pp. 13966-13974 ◽  
Author(s):  
Xiaofeng Cui ◽  
Xiaorong Tao ◽  
Yan Xie ◽  
Claude M. Fauquet ◽  
Xueping Zhou
Keyword(s):  
Transgenic Plants ◽  
Stop Codon ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Tomato Yellow Leaf ◽  
Cauliflower Mosaic Virus ◽  
Start Codon ◽  
35S Promoter ◽  
Yellow Leaf ◽  
Leaf Curl

ABSTRACT We report here that all 25 isolates of Tomato yellow leaf curl China virus (TYLCCNV) collected from tobacco, tomato, or Siegesbeckia orientalis plants in different regions of Yunnan Province, China, were associated with DNAβ molecules. To investigate the biological role of DNAβ, full-length infectious clones of viral DNA and DNAβ of TYLCCNV isolate Y10 (TYLCCNV-Y10) were agroinoculated into Nicotiana benthamiana, Nicotiana glutinosa, Nicotiana. tabacum Samsun (NN or nn), tomato, and petunia plants. We found that TYLCCNV-Y10 alone could systemically infect these plants, but no symptoms were induced. TYLCCNV-Y10 DNAβ was required, in addition to TYLCCNV-Y10, for induction of leaf curl disease in these hosts. Similar to TYLCCNV-Y10, DNAβ of TYLCCNV isolate Y64 was also found to be required for induction of typical leaf curl diseases in the hosts tested. When the βC1 gene of TYLCCNV-Y10 DNAβ was mutated, the mutants failed to induce leaf curl symptoms in N. benthamiana when coinoculated with TYLCCNV-Y10. However, Southern blot hybridization analyses showed that the mutated DNAβ molecules were replicated. When N. benthamiana and N. tabacum plants were transformed with a construct containing the βC1 gene under the control of the Cauliflower mosaic virus 35S promoter, many transgenic plants developed leaf curl symptoms similar to those caused by a virus, the severity of which paralleled the level of βC1 transcripts, while transgenic plants transformed with the βC1 gene containing a stop codon after the start codon remained symptomless. Thus, expression of a βC1 gene is adequate for induction of symptoms of viral infection in the absence of virus.

scholarly journals Plasmid-Borne Genes Code for an Angular Dioxygenase Involved in Dibenzofuran Degradation by Terrabacter sp. Strain YK3

2002 ◽  
Vol 68 (8) ◽  
pp. 3716-3723 ◽  
Author(s):  
Toshiya Iida ◽  
Yuki Mukouzaka ◽  
Kaoru Nakamura ◽  
Toshiaki Kudo
Keyword(s):  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Pcr Primers ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
The Novel ◽  
Southern Blot Hybridization Analysis ◽  
Angular Dioxygenation ◽  
Dioxygenase Gene ◽  
Angular Dioxygenase

ABSTRACT The genes responsible for angular dioxygenation of dibenzofuran in actinomycetes were cloned by using a degenerate set of PCR primers designed by using conserved sequences of the dioxygenase alpha subunit genes. One sequence of alpha subunit genes was commonly amplified from four dibenzofuran-utilizing actinomycetes: Terrabacter sp. strains YK1 and YK3, Rhodococcus sp. strain YK2, and Microbacterium sp. strain YK18. A 5.2-kb PstI fragment encoding the alpha and beta subunits of the terminal dioxygenase, ferredoxin, and ferredoxin reductase (designated dfdA1 to dfdA4, respectively) was cloned from the large circular plasmid pYK3 isolated from Terrabacter sp. strain YK3. We confirmed that transcription of the dfdA1 gene was induced by dibenzofuran in Terrabacter sp. strain YK3. Southern blot hybridization analysis revealed that this type of dioxygenase gene is distributed among diverse dibenzofuran-utilizing actinomycetes. However, genes homologous to dfdA1 were not detected in dibenzofuran utilization-deficient mutants of Terrabacter, Rhodococcus, and Microbacterium species. When the dfdA1 to dfdA4 genes were introduced into a non-dibenzofuran-degrading mutant of Rhodococcus sp. strain YK2, strain YK2-RD2, which had spontaneously lost the gene homologous to dfdA1, the ability to degrade dibenzofuran was restored. Analysis of the breakdown products indicated that DfdA has angular dioxygenase activity. This dfdA transformant degraded several aromatic compounds, indicating that the novel angular dioxygenase possesses broad substrate specificity.

scholarly journals Characterization of a New Monopartite Begomovirus with a Betasatellite Associated with Leaf Curl, Yellow Vein, and Vein Enation in Hibiscus rosa-sinensis

Plant Disease ◽  
2020 ◽  
Vol 104 (5) ◽  
pp. 1318-1327
Author(s):  
Chih-Hung Huang ◽  
Chia-Hsing Tai ◽  
Nabin Sharma ◽  
Chia-Hung Chao ◽  
Chung-Jan Chang ◽  
...  
Keyword(s):  
Open Reading Frames ◽  
Yellow Vein ◽  
Nucleotide Identity ◽  
Cotton Leaf ◽  
Species Demarcation ◽  
Conserved Sequence ◽  
Hibiscus Rosa Sinensis ◽  
A Genome ◽  
Species Demarcation Threshold ◽  
Leaf Curl

A new begomovirus, tentatively named hibiscus yellow vein leaf curl virus (HYVLCV), was identified in Hibiscus rosa-sinensis plants showing symptoms of leaf curl, yellow vein, and vein enation on the undersides of the leaf in Taiwan. Sequence analysis of the full-length HYVLCV genome from the rolling cycle amplicon revealed a genome of 2,740 nucleotides that contains six open reading frames and a conserved sequence (5′-TAATATTAC-3′) commonly found in geminiviral genomes. HYVLCV shares the highest nucleotide identity (88.8%) with cotton leaf curl Multan virus (CLCuMuV) genome, which is lower than the criteria (91%) set for species demarcation in the genus Begomovirus. No begomoviral DNA-B was detected; however, a begomovirus-associated DNA betasatellite (DNA-β) was detected. The DNA-β (1,355 nucleotides) shares the highest nucleotide identity (78.6%) with malvastrum yellow vein betasatellite (MaYVB). Because the identity is slightly higher than the criteria (78%) set for the species demarcation threshold for a distinct DNA-β species, the DNA-β of HYVLCV reported in this study is considered the same species of MaYVB and tentatively named MaYVB-Hib. An expected 1,498-bp fragment was amplified with two HYVLCV-specific primers from 10 of 11 field-collected samples. Four independent amplicons were sequenced, revealing 100% nucleotide identity with the HYVLCV genome. Agroinoculation of a dimer of the infectious monopartite genome alone to Nicotiana benthamiana resulted in mild symptoms at 28 days postinoculation (dpi); coagroinoculation with the DNA-β satellite resulted in severe symptoms at 12 dpi. HYVLCV could be transmitted to healthy H. rosa-sinensis by grafting, resulting in yellow vein symptoms at 30 dpi.

scholarly journals Pathogenicity of a naturally occurring recombinant DNA satellite associated with tomato yellow leaf curl China virus

2008 ◽  
Vol 89 (1) ◽  
pp. 306-311 ◽  
Author(s):  
Xiaorong Tao ◽  
Xueping Zhou
Keyword(s):  
Petunia Hybrida ◽  
Recombinant Dna ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Tomato Yellow Leaf ◽  
Yellow Leaf ◽  
Nicotiana Glutinosa ◽  
Naturally Occurring ◽  
Infected Tobacco ◽  
Leaf Curl

Recombinant DNA β molecules (RecDNA-Aβ) comprising parts of DNA A and DNA β associated with tomato yellow leaf curl China virus (TYLCCNV) have been identified in naturally infected tobacco plants. Several examples of the recombinant DNA have been cloned and characterized by sequence analysis. All are approximately half the size of TYLCCNV genomic DNA, and all contain the βC1 gene and the A-rich region from TYLCCNV DNA β as well as intergenic region sequences and the 5′ terminus of the AC1 gene from TYLCCNV DNA A. RecDNA-Aβ was detected by PCR in five of 25 TYLCCNV isolates. Co-inoculation of TYLCCNV DNA A and RecDNA-Aβ induced symptoms indistinguishable from those induced by TYLCCNV DNA A and DNA β in Nicotiana benthamiana, Nicotiana glutinosa, Solanum lycopersicum and Petunia hybrida plants, and Southern blot hybridization results showed that RecDNA-Aβ could replicate stably in N. benthamiana plants.

scholarly journals The transferable resistome of produce

2018 ◽  
Author(s):  
Khald Blau ◽  
Antje Bettermann ◽  
Sven Jechalke ◽  
Eva Fornefeld ◽  
Yann Vanrobaeys ◽  
...  
Keyword(s):  
Real Time ◽  
Resistance Genes ◽  
Real Time Pcr ◽  
Direct Detection ◽  
Blot Hybridization ◽  
Antibiotic Resistance Genes ◽  
Southern Blot Hybridization ◽  
Pcr Primers ◽  
Human Pathogens ◽  
E Coli

ABSTRACTProduce is increasingly recognized as a reservoir of human pathogens and transferable antibiotic resistance genes. This study aimed to explore methods to characterize the transferable resistome of bacteria associated with produce. Mixed salad, arugula, and cilantro purchased from supermarkets were analyzed by means of cultivation- and DNA-based methods. Before and after a nonselective enrichment step, tetracycline (tet) resistantEscherichia coliwere isolated and plasmids conferring tet resistance were captured by exogenous plasmid isolation. Tet resistantE. coliisolates, transconjugants and total community (TC)-DNA from the microbial fraction detached from leaves or after enrichment were analyzed for the presence of resistance genes, class 1 integrons and various plasmids by real-time PCR and PCR-Southern blot hybridization. Real-time PCR primers were developed for IncI and IncF plasmids. Tet resistantE. coliisolated from arugula and cilantro carried IncF, IncI1, IncN, IncH11, IncU and IncX1 plasmids. Three isolates from cilantro were positive for IncN plasmids andblaCTX-M-1. From mixed salad and cilantro, IncF, Inc11, and IncP-1β plasmids were captured exogenously. Importantly, whereas direct detection of IncI and IncF plasmids in TC-DNA failed, these plasmids became detectable in DNA extracted from enrichment cultures. This confirms that cultivation-independent DNA-based methods are not always sufficiently sensitive to detect the transferable resistome in the rare microbiome. In summary, this study showed that an impressive diversity of self-transmissible multiple resistance plasmids was detected in bacteria associated with produce that is consumed raw, and exogenous capturing intoE. colisuggests that they could transfer to gut bacteria as well.

scholarly journals Using Multiplexed CRISPR/Cas9 for Suppression of Cotton Leaf Curl Virus

2021 ◽  
Vol 22 (22) ◽  
pp. 12543
Author(s):  
Barkha Binyameen ◽  
Zulqurnain Khan ◽  
Sultan Habibullah Khan ◽  
Aftab Ahmad ◽  
Nayla Munawar ◽  
...  
Keyword(s):  
Genome Editing ◽  
Insect Pests ◽  
Restriction Analysis ◽  
Cotton Leaf ◽  
Cotton Production ◽  
Colony Pcr ◽  
Guide Rna ◽  
Cotton Leaf Curl Virus ◽  
Leaf Curl Virus ◽  
Leaf Curl

In recent decades, Pakistan has suffered a decline in cotton production due to several factors, including insect pests, cotton leaf curl disease (CLCuD), and multiple abiotic stresses. CLCuD is a highly damaging plant disease that seriously limits cotton production in Pakistan. Recently, genome editing through CRISPR/Cas9 has revolutionized plant biology, especially to develop immunity in plants against viral diseases. Here we demonstrate multiplex CRISPR/Cas-mediated genome editing against CLCuD using transient transformation in N. benthamiana plants and cotton seedlings. The genomic sequences of cotton leaf curl viruses (CLCuVs) were obtained from NCBI and the guide RNA (gRNA) were designed to target three regions in the viral genome using CRISPR MultiTargeter. The gRNAs were cloned in pHSE401/pKSE401 containing Cas9 and confirmed through colony PCR, restriction analysis, and sequencing. Confirmed constructs were moved into Agrobacterium and subsequently used for transformation. Agroinfilteration in N. benthamiana revealed delayed symptoms (3–5 days) with improved resistance against CLCuD. In addition, viral titer was also low (20–40%) in infected plants co-infiltrated with Cas9-gRNA, compared to control plants (infected with virus only). Similar results were obtained in cotton seedlings. The results of transient expression in N. benthamiana and cotton seedlings demonstrate the potential of multiplex CRISPR/Cas to develop resistance against CLCuD. Five transgenic plants developed from three experiments showed resistance (60−70%) to CLCuV, out of which two were selected best during evaluation and screening. The technology will help breeding CLCuD-resistant cotton varieties for sustainable cotton production.

scholarly journals Engineer complete resistance to Cotton Leaf Curl Multan virus by the CRISPR/Cas9 system in Nicotiana benthamiana

2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Kangquan Yin ◽  
Ting Han ◽  
Ke Xie ◽  
Jinping Zhao ◽  
Junqi Song ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Cotton Leaf ◽  
Complete Resistance ◽  
Leaf Curl

scholarly journals Leaf Curl Disease of Carica papaya from India May Be Caused by a Bipartite Geminivirus

Plant Disease ◽  
1998 ◽  
Vol 82 (1) ◽  
pp. 126-126 ◽  
Author(s):  
Sangeeta Saxena ◽  
Vipin Hallan ◽  
B. P. Singh ◽  
P. V. Sane
Keyword(s):  
Southern Blot ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Dna Probes ◽  
Economic Losses ◽  
Degenerate Primers ◽  
Tomato Leaf Curl ◽  
Leaf Curl Virus ◽  
Leaf Curl Disease ◽  
Leaf Curl

Papaya has considerable economic importance to agriculture in India. Papaya leaf curl disease was first reported in 1939 by Thomas and Krishnaswamy (3). This disease is of moderate incidence and widely distributed in India. Recent observations of papaya fields in India indicated that there has been a continued increase in the incidence of papaya leaf curl disease (as shown by symptoms), resulting in severe economic losses. The disease is characterized by downward curling and cupping of leaves followed by vein clearing and thickening. Enations develop in the form of frills on green veins. The affected leaves become leathery and brittle and the petioles become twisted in a zig-zag manner. Diseased plants may bear a few small fruits, which are distorted in shape and tend to fall prematurely. The disease could be transmitted by the whitefly Bemisia tabaci Genn. Therefore, possible involvement of a geminivirus was suspected. Three different cloned geminiviral DNAs, Indian tomato leaf curl virus (ITLCV) (2), tomato yellow leaf curl virus from Sardinia (TYLCV Sar), and tomato golden mosaic virus (TGMV), were used as probes (with radioactive labeling) to detect the presence of geminiviral DNA from infected papaya tissue in both slot-blot and Southern blot hybridization studies with high stringency washes. These DNA probes gave strong signals with DNA isolated from infected papaya tissue whereas they did not give any signals with DNA from healthy tissue. Further, successful polymerase chain reaction (PCR)-based amplification of fragments from both DNA-A and DNA-B components with geminivirus degenerate primers (1) was accomplished only from the DNA of infected papaya plants. The PCR-amplified DNA fragments gave positive signals in Southern blot hybridization with the three geminiviral DNA probes. These results suggest that the causal agent of papaya leaf curl disease is a bipartite geminivirus that may be provisionally called papaya leaf curl virus (PLCV). References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) K. M. Srivastava et al. J. Virol. Methods 51:297, 1995. (3) K. M. Thomas and C. S. Krishnaswamy. Curr. Sci. 8:316, 1939.

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