Biallelic editing of the LOB1 promoter via CRISPR/Cas9 creates canker-resistant ‘Duncan’ grapefruit

2021 ◽  
Author(s):  
Hongge Jia ◽  
Ahmad Omar ◽  
Vladimir Orbović ◽  
Nian Wang
Keyword(s):  
Susceptibility Gene ◽  
Citrus Canker ◽  
35S Promoter ◽  
Wild Type ◽  
Coding Region ◽  
Transcription Activator ◽  
Tal Effector ◽  
Conserved Sequence ◽  
Disease Resistant ◽  
Citrus Varieties

Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most devastating citrus diseases worldwide. Generating disease-resistant citrus varieties is considered one of the most efficient and environmentally friendly measures for controlling canker. Xcc causes canker symptoms by inducing the expression of canker susceptibility gene LOB1 via PthA4, a transcription activator-like (TAL) effector, by binding to the effector binding element (EBE) in the promoter region. In previous studies, canker-resistant plants were generated by mutating the coding region or the EBE of LOB1. However, homozygous or biallelic canker-resistant plants have not been generated for commercial citrus varieties, such as grapefruit (C. paradisi), which usually contain two alleles of LOB1 and thus have two types of LOB1 promoter sequences: TI LOBP and TII LOBP. Two different sgRNAs were used to target both EBE types. Both 35S promoter and Yao promoter were used to drive the expression of SpCas9p to modify EBEPthA4-LOBP in grapefruit. Using ‘Duncan’ grapefruit epicotyls as explants, 19 genome-edited grapefruit plants were generated with one biallelic mutant line (#DunYao7). Xcc caused canker symptoms on wild-type and non-biallelic mutant plants but not on #DunYao7. XccPthA4 mutant containing the designer TAL effector dLOB1.5, which recognizes a conserved sequence in both wild-type and #DunYao7, caused canker symptoms on both wild-type and #DunYao7. No off-target mutations were detected in #DunYao7. This study represents the first time that CRISPR-mediated genome editing has been successfully used to generate disease-resistant plants for ‘Duncan’ grapefruit, paving the way for utilizing disease-resistant varieties to control canker.

scholarly journals Postharvest Analysis of Lowland Transgenic Tomato Fruits Harboring hpRNAi-ACO1Construct

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Bita Behboodian ◽  
Zainon Mohd Ali ◽  
Ismanizan Ismail ◽  
Zamri Zainal
Keyword(s):  
Acc Oxidase ◽  
Pectin Methylesterase ◽  
Flower Senescence ◽  
35S Promoter ◽  
Specific Gene ◽  
Wild Type ◽  
Coding Region ◽  
Tomato Cultivar ◽  
Titratable Acid ◽  
Rnai Technology

The plant hormone, ethylene, is an important regulator which involved in regulating fruit ripening and flower senescence. In this study, RNA interference (RNAi) technology was employed to silence the genes involved in ethylene biosynthetic pathway. This was achieved by blocking the expression of specific gene encoding the ACC oxidase. Initially, cDNA corresponding toACO1of lowland tomato cultivar (MT1), which has high identity withACO1ofSolanum lycopersicumin GenBank, was cloned through RT-PCR. Using a partial coding region ofACO1, one hpRNAi transformation vector was constructed and expressed ectopically under the 35S promoter. Results showed that transgenic lines harboring the hpRNA-ACO1construct had lower ethylene production and a longer shelf life of 32 days as compared to 10 days for wild-type fruits. Changes in cell wall degrading enzyme activities were also investigated in cases where the transgenic fruits exhibited reduced rates of firmness loss, which can be associated with a decrease in pectin methylesterase (PME) and polygalacturonase (PG) activities. However, no significant change was detected in both transgenic and wild-type fruits in terms ofβ-galactosidase (β-Gal) activity and levels of total soluble solid, titratable acid and ascorbic acid.

scholarly journals PthA4 AT , a 7.5‐repeats transcription activator‐like (TAL) effector from Xanthomonas citri ssp. citri , triggers citrus canker resistance

2019 ◽  
Vol 20 (10) ◽  
pp. 1394-1407 ◽  
Author(s):  
Roxana Andrea Roeschlin ◽  
Facundo Uviedo ◽  
Lucila García ◽  
María Celeste Molina ◽  
María Alejandra Favaro ◽  
...  
Keyword(s):  
Citrus Canker ◽  
Transcription Activator ◽  
Xanthomonas Citri ◽  
Tal Effector

A Mutation in Paramecium tetraurelia Reveals Functional and Structural Features of Developmentally Excised DNA Elements

Genetics ◽  
1998 ◽  
Vol 148 (1) ◽  
pp. 139-149 ◽  
Author(s):  
Kimberly M Mayer ◽  
Kazuyuki Mikami ◽  
James D Forney
Keyword(s):  
Mutant Cell ◽  
Consensus Sequence ◽  
Surface Protein ◽  
Structural Features ◽  
Inverted Terminal Repeat ◽  
Paramecium Tetraurelia ◽  
Coding Region ◽  
Conserved Sequence ◽  
Variable Surface ◽  
Dna Elements

Abstract The excision of internal eliminated sequences (IESs) from the germline micronuclear DNA occurs during the differentiation of a new macronuclear genome in ciliated protozoa. In Paramecium, IESs are generally short (28–882 bp), AT rich DNA elements that show few conserved sequence features with the exception of an inverted-terminal-repeat consensus sequence that has similarity to the ends of mariner/Tc1 transposons (Klobutcher and Herrick 1995). We have isolated and analyzed a mutant cell line that cannot excise a 370-bp IESs (IES2591) from the coding region of the 51A variable surface protein gene. A single micronuclear C to T transition within the consensus sequence prevents excision. The inability to excise IES2591 has revealed a 28-bp IES inside the larger IES, suggesting that reiterative integration of these elements can occur. Together, the consensus sequence mutation and the evidence for reiterative integration support the theory that Paramecium IESs evolved from transposable elements. Unlike a previously studied Paramecium IES, the presence of this IES in the macronucleus does not completely inhibit excision of its wild-type micronuclear copy through multiple sexual generations.

scholarly journals Modified Monosaccharides Content of Xanthan Gum Impairs Citrus Canker Disease by Affecting the Epiphytic Lifestyle of Xanthomonas citri subsp. citri

2021 ◽  
Vol 9 (6) ◽  
pp. 1176
Author(s):  
Simone Cristina Picchi ◽  
Laís Moreira Granato ◽  
Maria Júlia Festa Franzini ◽  
Maxuel Oliveira Andrade ◽  
Marco Aurélio Takita ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Xanthan Gum ◽  
Wild Type Strain ◽  
Natural Infection ◽  
Citrus Canker ◽  
Wild Type ◽  
Xanthomonas Citri ◽  
Canker Disease ◽  
Key Enzyme ◽  
Citrus Canker Disease

Xanthomonas citri subsp. citri (X. citri) is a plant pathogenic bacterium causing citrus canker disease. The xanA gene encodes a phosphoglucomutase/phosphomannomutase protein that is a key enzyme required for the synthesis of lipopolysaccharides and exopolysaccharides in Xanthomonads. In this work, firstly we isolated a xanA transposon mutant (xanA::Tn5) and analyzed its phenotypes as biofilm formation, xanthan gum production, and pathogenesis on the sweet orange host. Moreover, to confirm the xanA role in the impaired phenotypes we further produced a non-polar deletion mutant (ΔxanA) and performed the complementation of both xanA mutants. In addition, we analyzed the percentages of the xanthan gum monosaccharides produced by X. citri wild-type and xanA mutant. The mutant strain had higher ratios of mannose, galactose, and xylose and lower ratios of rhamnose, glucuronic acid, and glucose than the wild-type strain. Such changes in the saccharide composition led to the reduction of xanthan yield in the xanA deficient strain, affecting also other important features in X. citri, such as biofilm formation and sliding motility. Moreover, we showed that xanA::Tn5 caused no symptoms on host leaves after spraying, a method that mimetics the natural infection condition. These results suggest that xanA plays an important role in the epiphytical stage on the leaves that is essential for the successful interaction with the host, including adaptive advantage for bacterial X. citri survival and host invasion, which culminates in pathogenicity.

scholarly journals Essential Function of the Pseudorabies Virus UL36 Gene Product Is Independent of Its Interaction with the UL37 Protein

2004 ◽  
Vol 78 (21) ◽  
pp. 11879-11889 ◽  
Author(s):  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Pseudorabies Virus ◽  
Rabbit Kidney ◽  
Tegument Protein ◽  
Wild Type ◽  
Coding Region ◽  
Ordered Structures ◽  
Ca 50 ◽  
Essential Function ◽  
Full Length Clone

ABSTRACT The large tegument protein encoded by the UL36 gene of pseudorabies virus (PrV) physically interacts with the product of the adjacent UL37 gene (B. G. Klupp, W. Fuchs, H. Granzow, R. Nixdorf, and T. C. Mettenleiter, J. Virol. 76:3065-3071, 2002). To analyze UL36 function, two PrV recombinants were generated by mutagenesis of an infectious PrV full-length clone in Escherichia coli: PrV-ΔUL36F exhibited a deletion of virtually the complete UL36 coding region, whereas PrV-UL36BSF contained two in-frame deletions of 238 codons spanning the predicted UL37 binding domain. Coimmunoprecipitation experiments confirmed that the mutated gene product of PrV-UL36BSF did not interact with the UL37 protein. Like the previously described PrV-ΔUL37 (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) but in contrast to PrV-ΔUL36F, PrV-UL36BSF was able to replicate in rabbit kidney (RK13) cells, although maximum virus titers were reduced ca. 50-fold and plaque diameters were reduced by ca. 45% compared to wild-type PrV. PrV-ΔUL36F was able to productively replicate after repair of the deleted gene or in a trans-complementing cell line. Electron microscopy of infected RK13 cells revealed that PrV-UL36BSF and phenotypically complemented PrV-ΔUL36F were capable of nucleocapsid formation and egress from the nucleus by primary envelopment and deenvelopment at the nuclear membrane. However, reenvelopment of nucleocapsids in the cytoplasm was blocked. Only virus-like particles without capsids were released efficiently from cells. Interestingly, cytoplasmic nucleocapsids of PrV-UL36BSF but not of PrV-ΔUL36F were found in large ordered structures similar to those which had previously been observed with PrV-ΔUL37. In summary, our results demonstrate that the interaction between the UL36 and UL37 proteins is important but not strictly essential for the formation of secondary enveloped, infectious PrV particles. Furthermore, UL36 possesses an essential function during virus replication which is independent of its ability to bind the UL37 protein.

scholarly journals Systemic Polyomavirus Genome Increase and Dissemination of Capsid-Defective Genomes in Mammary Gland Tumor-Bearing Mice

2000 ◽  
Vol 74 (15) ◽  
pp. 6975-6983 ◽  
Author(s):  
Julie J. Wirth ◽  
Li Chen ◽  
Michele M. Fluck
Keyword(s):  
Mammary Gland ◽  
Viral Genome ◽  
Wild Type Strain ◽  
Neonatal Infection ◽  
Wild Type ◽  
Coding Region ◽  
Live Virus ◽  
Viral Genomes ◽  
Mammary Gland Tumors ◽  
Low Levels

ABSTRACT BALB/c mice that developed tumors 7 to 8 months following neonatal infection by polyomavirus (PYV) wild-type strain A2 were characterized with respect to the abundance and integrity of the viral genome in the tumors and in 12 nontumorous organs. These patterns were compared to those found in tumor-free mice infected in parallel. Six mice were analyzed in detail including four sibling females with mammary gland tumors. In four of five mammary gland tumors, the viral genome had undergone a unique deletion and/or rearrangement. Three tumor-resident genomes with an apparently intact large T coding region were present in abundant levels in an unintegrated state. Two of these had undergone deletions and rearrangements involving the capsid genes and therefore lacked the capacity to produce live virus. In the comparative organ survey, the tumors harboring replication-competent genomes contained by far the highest levels of genomes of any tissue. However, the levels of PYV genomes in other organs were elevated by up to 1 to 2 orders of magnitude compared to those detected in the same organs of tumor-free mice. The genomes found in the nontumorous organs had the same rearrangements as the genomes residing in the tumors. The original wild-type genome was detected at low levels in a few organs, particularly in the kidneys. The data indicate that a systemic increase in the level of viral genomes occurred in conjunction with the induction of tumors by PYV. The results suggest two novel hypotheses: (i) that genomes may spread from the tumors to the usual PYV target tissues and (ii) that this dissemination may take place in the absence of capsids, providing an important path for a virus to escape from the immune response. This situation may offer a useful model for the spread of HPV accompanying HPV-induced oncogenesis.

scholarly journals Structural defects of a Pax8 mutant that give rise to congenital hypothyroidism

1999 ◽  
Vol 341 (1) ◽  
pp. 89-93 ◽  
Author(s):  
Gianluca TELL ◽  
Lucia PELLIZZARI ◽  
Gennaro ESPOSITO ◽  
Carlo PUCILLO ◽  
Paolo Emidio MACCHIA ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Congenital Hypothyroidism ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Structural Defects ◽  
Molecular Defect ◽  
Wild Type ◽  
Coding Region ◽  
Induced Fit ◽  
Helical Content

Pax proteins are transcriptional regulators that play important roles during embryogenesis. These proteins recognize specific DNA sequences via a conserved element: the paired domain (Prd domain). The low level of organized secondary structure, in the free state, is a general feature of Prd domains; however, these proteins undergo a dramatic gain in α-helical content upon interaction with DNA (‘induced fit’). Pax8 is expressed in the developing thyroid, kidney and several areas of the central nervous system. In humans, mutations of the Pax8 gene, which are mapped to the coding region of the Prd domain, give rise to congenital hypothyroidism. Here, we have investigated the molecular defects caused by a mutation in which leucine at position 62 is substituted for an arginine. Leu62 is conserved among Prd domains, and contributes towards the packing together of helices 1 and 3. The binding affinity of the Leu62Arg mutant for a specific DNA sequence (the C sequence of thyroglobulin promoter) is decreased 60-fold with respect to the wild-type Pax8 Prd domain. However, the affinities with which the wild-type and the mutant proteins bind to a non-specific DNA sequence are very similar. CD spectra demonstrate that, in the absence of DNA, both wild-type Pax8 and the Leu62Arg mutant possess a low α-helical content; however, in the Leu62Arg mutant, the gain in α-helical content upon interaction with DNA is greatly reduced with respect to the wild-type protein. Thus the molecular defect of the Leu62Arg mutant causes a reduced capability for induced fit upon DNA interaction.

scholarly journals TAL Effector Repertoires of Strains of Xanthomonas phaseoli pv. manihotis in Commercial Cassava Crops Reveal High Diversity at the Country Scale

2021 ◽  
Vol 9 (2) ◽  
pp. 315
Author(s):  
Carlos A. Zárate-Chaves ◽  
Daniela Osorio-Rodríguez ◽  
Rubén E. Mora ◽  
Álvaro L. Pérez-Quintero ◽  
Alexis Dereeper ◽  
...  
Keyword(s):  
Target Genes ◽  
Susceptibility Gene ◽  
Rflp Analysis ◽  
Transcription Activator ◽  
Cassava Bacterial Blight ◽  
Functional Convergence ◽  
Neutral Genetic Diversity ◽  
Functional Variant ◽  
Novel Variants ◽  
Host Metabolism

Transcription activator-like effectors (TALEs) play a significant role for pathogenesis in several xanthomonad pathosystems. Xanthomonas phaseoli pv. manihotis (Xpm), the causal agent of Cassava Bacterial Blight (CBB), uses TALEs to manipulate host metabolism. Information about Xpm TALEs and their target genes in cassava is scarce, but has been growing in the last few years. We aimed to characterize the TALE diversity in Colombian strains of Xpm and to screen for TALE-targeted gene candidates. We selected eighteen Xpm strains based on neutral genetic diversity at a country scale to depict the TALE diversity among isolates from cassava productive regions. RFLP analysis showed that Xpm strains carry TALomes with a bimodal size distribution, and affinity-based clustering of the sequenced TALEs condensed this variability mainly into five clusters. We report on the identification of 13 novel variants of TALEs in Xpm, as well as a functional variant with 22 repeats that activates the susceptibility gene MeSWEET10a, a previously reported target of TAL20Xam668. Transcriptomics and EBE prediction analyses resulted in the selection of several TALE-targeted candidate genes and two potential cases of functional convergence. This study provides new bases for assessing novel potential TALE targets in the Xpm–cassava interaction, which could be important factors that define the fate of the infection.

scholarly journals Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions

2011 ◽  
Vol 92 (12) ◽  
pp. 2838-2848 ◽  
Author(s):  
Shigeo Nagashima ◽  
Masaharu Takahashi ◽  
Suljid Jirintai ◽  
Toshinori Tanaka ◽  
Tsutomu Nishizawa ◽  
...  
Keyword(s):  
Susceptibility Gene ◽  
Multivesicular Body ◽  
Hepatitis E ◽  
Negative Control ◽  
Dominant Negative ◽  
Wild Type ◽  
Orf3 Protein ◽  
Virus Particles ◽  
Virion Release ◽  
Vacuolar Protein

We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wild-type ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4 % of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6 %, respectively, while the overexpression of wild-type Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles.

scholarly journals Comparative reactions of recombinant papaya ringspot viruses with chimeric coat protein (CP) genes and wild-type viruses on CP-transgenic papaya

2001 ◽  
Vol 82 (11) ◽  
pp. 2827-2836 ◽  
Author(s):  
Chu-Hui Chiang ◽  
Ju-Jung Wang ◽  
Fuh-Jyh Jan ◽  
Shyi-Dong Yeh ◽  
Dennis Gonsalves
Keyword(s):  
Coat Protein ◽  
Sequence Similarity ◽  
Transcriptional Gene Silencing ◽  
Papaya Ringspot Virus ◽  
Wild Type ◽  
Coding Region ◽  
Transgenic Papaya ◽  
Cp Gene ◽  
Post Transcriptional Gene Silencing

Transgenic papaya cultivars SunUp and Rainbow express the coat protein (CP) gene of the mild mutant of papaya ringspot virus (PRSV) HA. Both cultivars are resistant to PRSV HA and other Hawaii isolates through homology-dependent resistance via post-transcriptional gene silencing. However, Rainbow, which is hemizygous for the CP gene, is susceptible to PRSV isolates from outside Hawaii, while the CP-homozygous SunUp is resistant to most isolates but susceptible to the YK isolate from Taiwan. To investigate the role of CP sequence similarity in overcoming the resistance of Rainbow, PRSV HA recombinants with various CP segments of the YK isolate were constructed and evaluated on Rainbow, SunUp and non-transgenic papaya. Non-transgenic papaya were severely infected by all recombinants, but Rainbow plants developed a variety of symptoms. On Rainbow, a recombinant with the entire CP gene of YK caused severe symptoms, while recombinants with only partial YK CP sequences produced a range of milder symptoms. Interestingly, a recombinant with a YK segment from the 5′ region of the CP gene caused very mild, transient symptoms, whereas recombinants with YK segments from the middle and 3′ parts of the CP gene caused prominent and lasting symptoms. SunUp was resistant to all but two recombinants, which contained the entire CP gene or the central and 3′-end regions of the CP gene and the 3′ non-coding region of YK, and the resulting symptoms were mild. It is concluded that the position of the heterologous sequences in the recombinants influences their pathogenicity on Rainbow.

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