Analysis of the Involvement of an Inducible Arabidopsis RNA-Dependent RNA Polymerase in Antiviral Defense

RNA-dependent RNA polymerases (RdRPs) have been implicated in posttranscriptional gene silencing (PTGS) and antiviral defense. An Arabidopsis RdRP (SDE1/SGS2) has been previously shown to be required for transgene-induced PTGS but has no general role in antiviral defense. On the other hand, we have recently shown that transgenic tobacco deficient in an inducible RdRP (NtRdRP1) activity became more susceptible to both Tobacco mosaic virus and Potato virus X. Thus, different RdRPs may have distinct roles in closely related PTGS and antiviral defense. In the present study, we analyzed roles of a newly identified Arabidopsis RdRP gene (AtRdRP1) in plant antiviral defense. AtRdRP1 encodes an RdRP closely related structurally to NtRdRP1 and is also induced by salicylic acid treatment and virus infection. A T-DNA insertion mutant for AtRdRP1 has been isolated and analyzed for possible alterations in response to viral infection. When infected by a to-bamovirus and a tobravirus, the knockout mutant accumulated higher and more persistent levels of viral RNAs in both the lower, inoculated and in upper, systemically infected leaves than did wild-type plants. These results suggest that the inducible AtRdRP1 is the Arabidopsis ortholog of NtRdRP1 and plays a role in antiviral defense. Examination of short viral RNAs and silencing studies using a viral vector harboring an endogenous plant gene suggest that, while not required for virus-induced PTGS, AtRdRP1 can apparently promote turnover of viral RNAs in infected plants.

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OsASN1 Plays a Critical Role in Asparagine-Dependent Rice Development

International Journal of Molecular Sciences ◽

10.3390/ijms20010130 ◽

2018 ◽

Vol 20 (1) ◽

pp. 130 ◽

Cited By ~ 10

Author(s):

Le Luo ◽

Ruyi Qin ◽

Tao Liu ◽

Ming Yu ◽

Tingwen Yang ◽

...

Keyword(s):

Critical Role ◽

Tiller Number ◽

Insertion Mutant ◽

Wild Type ◽

Long Distance ◽

Long Distance Transport ◽

Similar Phenotype ◽

Dna Insertion ◽

Mutant Lines ◽

Distance Transport

Asparagine is one of the important amino acids for long-distance transport of nitrogen (N) in plants. However, little is known about the effect of asparagine on plant development, especially in crops. Here, a new T-DNA insertion mutant, asparagine synthetase 1 (asn1), was isolated and showed a different plant height, root length, and tiller number compared with wild type (WT). In asn1, the amount of asparagine decreased sharply while the total nitrogen (N) absorption was not influenced. In later stages, asn1 showed reduced tiller number, which resulted in suppressed tiller bud outgrowth. The relative expression of many genes involved in the asparagine metabolic pathways declined in accordance with the decreased amino acid concentration. The CRISPR/Cas9 mutant lines of OsASN1 showed similar phenotype with asn1. These results suggest that OsASN1 is involved in the regulation of rice development and is specific for tiller outgrowth.

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Gibberellin 20-Oxidase Gene OsGA20ox3 Regulates Plant Stature and Disease Development in Rice

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-12-0138-r ◽

2013 ◽

Vol 26 (2) ◽

pp. 227-239 ◽

Cited By ~ 61

Author(s):

Xue Qin ◽

Jun Hua Liu ◽

Wen Sheng Zhao ◽

Xu Jun Chen ◽

Ze Jian Guo ◽

...

Keyword(s):

Transgenic Plants ◽

Critical Role ◽

Insertion Site ◽

Insertion Mutant ◽

Wild Type Plant ◽

Wild Type ◽

Oxidase Gene ◽

Dna Insertion ◽

Flanking Sequences ◽

Plant Stature

Gibberellin (GA) 20-oxidase (GA20ox) catalyses consecutive steps of oxidation in the late part of the GA biosynthetic pathway. A T-DNA insertion mutant (17S-14) in rice, with an elongated phenotype, was isolated. Analysis of the flanking sequences of the T-DNA insertion site revealed that an incomplete T-DNA integration resulted in enhanced constitutively expression of downstream OsGA20ox3 in the mutant. The accumulation of bioactive GA1 and GA4 were increased in the mutant in comparison with the wild-type plant. Transgenic plants overexpressing OsGA20ox3 showed phenotypes similar to those of the 17S-14 mutant, and the RNA interference (RNAi) lines that had decreased OsGA20ox3 expression exhibited a semidwarf phenotype. Expression of OsGA20ox3 was detected in the leaves and roots of young seedlings, immature panicles, anthers, and pollens, based on β-glucuronidase (GUS) activity staining in transgenic plants expressing the OsGA20ox3 promoter fused to the GUS gene. The OsGA20ox3 RNAi lines showed enhanced resistance against rice pathogens Magnaporthe oryzae (causing rice blast) and Xanthomonas oryzae pv. oryzae (causing bacterial blight) and increased expression of defense-related genes. Conversely, OsGA20ox3-overexpressing plants were more susceptible to these pathogens comparing with the wild-type plants. The susceptibility of wild-type plants to X. oryzae pv. oryzae was increased by exogenous application of GA3 and decreased by S-3307 treatment. Together, the results provide direct evidence for a critical role of OsGA20ox3 in regulating not only plant stature but also disease resistance in rice.

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The ORF1 Products of Tombusviruses Play a Crucial Role in Lethal Necrosis of Virus-Infected Plants

Journal of Virology ◽

10.1128/jvi.74.23.10873-10881.2000 ◽

2000 ◽

Vol 74 (23) ◽

pp. 10873-10881 ◽

Cited By ~ 35

Author(s):

József Burgyán ◽

Csaba Hornyik ◽

György Szittya ◽

Dániel Silhavy ◽

György Bisztray

Keyword(s):

Transgenic Plants ◽

Viral Replication ◽

Potato Virus ◽

Nicotiana Benthamiana ◽

Crucial Role ◽

Viral Vector ◽

Potato Virus X ◽

Wild Type ◽

Systemic Necrosis

ABSTRACT Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided intrans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.

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Clarification of the dispensability of PDX1.2 for Arabidopsis viability using CRISPR/Cas9

BMC Plant Biology ◽

10.1186/s12870-019-2071-9 ◽

2019 ◽

Vol 19 (1) ◽

Author(s):

Elisa Dell’Aglio ◽

Ivan Dalvit ◽

Sylvain Loubéry ◽

Teresa B. Fitzpatrick

Keyword(s):

Heat Stress ◽

Stress Responses ◽

Gene Editing ◽

Growth Conditions ◽

Insertion Mutant ◽

Wild Type ◽

Biotic And Abiotic Stress ◽

Knockout Mutants ◽

Dna Insertion ◽

Mutant Lines

Abstract Background PDX1.2 has recently been shown to be a regulator of vitamin B6 biosynthesis in plants and is implicated in biotic and abiotic stress resistance. PDX1.2 expression is strongly and rapidly induced by heat stress. Interestingly, PDX1.2 is restricted to eudicota, wherein it behaves as a non-catalytic pseudoenzyme and is suggested to provide an adaptive advantage to this clade. A first report on an Arabidopsis insertion mutant claims that PDX1.2 is indispensable for viability, being essential for embryogenesis. However, a later study using an independent insertion allele suggests that knockout mutants of pdx1.2 are viable. Therefore, the essentiality of PDX1.2 for Arabidopsis viability is a matter of debate. Given the important implications of PDX1.2 in stress responses, it is imperative to clarify if it is essential for plant viability. Results We have studied the previously reported insertion alleles of PDX1.2, one of which is claimed to be essential for embryogenesis (pdx1.2–1), whereas the other is viable (pdx1.2–2). Our study shows that pdx1.2–1 carries multiple T-DNA insertions, but the T-DNA insertion in PDX1.2 is not responsible for the loss of embryogenesis. By contrast, the pdx1.2–2 allele is an overexpressor of PDX1.2 under standard growth conditions and not a null allele as previously reported. Nonetheless, upregulation of PDX1.2 expression under heat stress is impaired in this mutant line. In wild type Arabidopsis, studies of PDX1.2-YFP fusion proteins show that the protein is enhanced under heat stress conditions. To clarify if PDX1.2 is essential for Arabidopsis viability, we generated several independent mutant lines using the CRISPR-Cas9 gene editing technology. All of these lines are viable and behave similar to wild type under standard growth conditions. Reciprocal crosses of a subset of the CRISPR lines with pdx1.2–1 recovers viability of the latter line and demonstrates that knocking out the functionality of PDX1.2 does not impair embryogenesis. Conclusions Gene editing reveals that PDX1.2 is dispensable for Arabidopsis viability and resolves conflicting reports in the literature on its function.

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Molecular Mechanisms Controlling the Disease Cycle in the Vascular Pathogen Verticillium dahliae Characterized Through Forward Genetics and Transcriptomics

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-19-0228-r ◽

2020 ◽

Vol 33 (6) ◽

pp. 825-841

Author(s):

Jorge L. Sarmiento-Villamil ◽

Nicolás E. García-Pedrajas ◽

M. Carmen Cañizares ◽

María D. García-Pedrajas

Keyword(s):

G Protein ◽

Verticillium Dahliae ◽

Forward Genetics ◽

Phenotypic Characterization ◽

Insertion Mutant ◽

G Protein Signaling ◽

Protein Signaling ◽

Wild Type ◽

Disease Cycle ◽

Dna Insertion

The soil-borne pathogen Verticillium dahliae has a worldwide distribution and a plethora of hosts of agronomic value. Molecular analysis of virulence processes can identify targets for disease control. In this work, we compared the global gene transcription profile of random T-DNA insertion mutant strain D-10-8F, which exhibits reduced virulence and alterations in microsclerotium formation and polar growth, with that of the wild-type strain. Three genes identified as differentially expressed were selected for functional characterization. To produce deletion mutants, we developed an updated version of one-step construction of Agrobacterium-recombination-ready plasmids (OSCAR) that included the negative selection marker HSVtk (herpes simplex virus thymidine kinase gene) to prevent ectopic integration of the deletion constructs. Deletion of VdRGS1 (VDAG_00683), encoding a regulator of G protein signaling (RGS) protein and highly upregulated in the wild type versus D-10-8F, resulted in phenotypic alterations in development and virulence that were indistinguishable from those of the random T-DNA insertion mutant. In contrast, deletion of the other two genes selected, vrg1 (VDAG_07039) and vvs1 (VDAG_01858), showed that they do not play major roles in morphogenesis or virulence in V. dahliae. Taken together the results presented here on the transcriptomic analysis and phenotypic characterization of D-10-8F and ∆VdRGS1 strains provide evidence that variations in G protein signaling control the progression of the disease cycle in V. dahliae. We propose that G protein–mediated signals induce the expression of multiple virulence factors during biotrophic growth, whereas massive production of microsclerotia at late stages of infection requires repression of G protein signaling via upregulation of VdRGS1 activity.

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A Replicase of Potato virus X Acts as the Resistance-Breaking Determinant for JAX1-Mediated Resistance

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-04-13-0094-r ◽

2013 ◽

Vol 26 (9) ◽

pp. 1106-1112 ◽

Cited By ~ 11

Author(s):

Kyoko Sugawara ◽

Takuya Shiraishi ◽

Tetsuya Yoshida ◽

Naoko Fujita ◽

Osamu Netsu ◽

...

Keyword(s):

Amino Acid ◽

Potato Virus ◽

Potato Virus X ◽

Single Amino Acid ◽

Wild Type ◽

Methyl Transferase ◽

General Role ◽

Resistance Breaking ◽

Viral Replicase

Lectin-mediated resistance (LMR) has been suggested to comprise an uncharacterized branch of antiviral plant innate immunity. To unveil the feature of resistance conferred by jacalin-type lectin required for potexvirus resistance 1 (JAX1), a recently isolated LMR gene against potexviruses, we analyzed the resistance-breaking variants to find the viral component involved in resistance. We employed grafting-mediated inoculation, a high-pressure virus inoculation method, to obtain Potato virus X (PVX) variants that can overcome JAX1-mediated resistance. Whole-genome sequencing of the variants suggested that a single amino acid in the methyl transferase domain of the replicase encoded by PVX is responsible for this resistance-breaking property. Reintroduction of the amino-acid substitution to avirulent wild-type PVX was sufficient to overcome the JAX1-mediated resistance. These results suggest that viral replicase is involved in JAX1-mediated resistance. The residue that determines the resistance-breaking properties was highly conserved among potexviruses, suggesting a general role of the residue in potexvirus–JAX1 interactions.

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Characteristics of Artificial Virus-like Particles Assembled in vitro from Potato Virus X Coat Protein and Foreign Viral RNAs

Acta Naturae ◽

10.32607/20758251-2011-3-3-40-46 ◽

2011 ◽

Vol 3 (3) ◽

pp. 40-46 ◽

Cited By ~ 8

Author(s):

M V Arkhipenko ◽

E K Petrova ◽

N A Nikitin ◽

A D Protopopova ◽

E V Dubrovin ◽

...

Keyword(s):

Coat Protein ◽

Potato Virus ◽

Potato Virus X ◽

Viral Rnas ◽

Virus Like Particles ◽

Artificial Virus

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The contribution of PIP2-type aquaporins to photosynthetic response to increased vapour pressure deficit

Journal of Experimental Botany ◽

10.1093/jxb/erab187 ◽

2021 ◽

Author(s):

D Israel ◽

S Khan ◽

C R Warren ◽

J J Zwiazek ◽

T M Robson

Keyword(s):

Vapour Pressure Deficit ◽

Air Humidity ◽

Wild Type ◽

Knockout Mutant ◽

Evaporative Demand ◽

Co2 Transport ◽

Whole Plant ◽

Knockout Mutants ◽

Leaf Level ◽

Low Humidity

Abstract The roles of different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using knockout mutants. Since multiple Arabidopsis PIPs are implicated in CO2 transport across cell membranes, we focused on identifying the effects of the knockout mutations on photosynthesis, and whether they are mediated through the control of stomatal conductance of water vapour (gs), mesophyll conductance of CO2 (gm) or both. We grew Arabidopsis plants in low and high humidity environments and found that the contribution of PIPs to gs was larger under low air humidity when the evaporative demand was high, whereas any effect of lacking PIP function was minimal under higher humidity. The pip2;4 knockout mutant had 44% higher gs than the wild type plants under low humidity, which in turn resulted in an increased net photosynthetic rate (Anet). We also observed a 23% increase in whole-plant transpiration (E) for this knockout mutant. The lack of functional AtPIP2;5 did not affect gs or E, but resulted in homeostasis of gm despite changes of humidity, indicating a possible role in regulating CO2 membrane permeability. CO2 transport measurements in yeast expressing AtPIP2;5 confirmed that this aquaporin is indeed permeable to CO2.

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α-Galactosidase A Augmentation by Non-Viral Gene Therapy: Evaluation in Fabry Disease Mice

Pharmaceutics ◽

10.3390/pharmaceutics13060771 ◽

2021 ◽

Vol 13 (6) ◽

pp. 771

Author(s):

Julen Rodríguez-Castejón ◽

Ana Alarcia-Lacalle ◽

Itziar Gómez-Aguado ◽

Mónica Vicente-Pascual ◽

María Ángeles Solinís Aspiazu ◽

...

Keyword(s):

Gene Therapy ◽

Fabry Disease ◽

Viral Vector ◽

Viral Gene ◽

Lysosomal Storage ◽

Wild Type ◽

Dose Administration ◽

Viral Gene Therapy ◽

Liver Transaminases ◽

Low Levels

Fabry disease (FD) is a monogenic X-linked lysosomal storage disorder caused by a deficiency in the lysosomal enzyme α-Galactosidase A (α-Gal A). It is a good candidate to be treated with gene therapy, in which moderately low levels of enzyme activity should be sufficient for clinical efficacy. In the present work we have evaluated the efficacy of a non-viral vector based on solid lipid nanoparticles (SLN) to increase α-Gal A activity in an FD mouse model after intravenous administration. The SLN-based vector incremented α-Gal A activity to about 10%, 15%, 20% and 14% of the levels of the wild-type in liver, spleen, heart and kidney, respectively. In addition, the SLN-based vector significantly increased α-Gal A activity with respect to the naked pDNA used as a control in plasma, heart and kidney. The administration of a dose per week for three weeks was more effective than a single-dose administration. Administration of the SLN-based vector did not increase liver transaminases, indicative of a lack of toxicity. Additional studies are necessary to optimize the efficacy of the system; however, these results reinforce the potential of lipid-based nanocarriers to treat FD by gene therapy.

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