scholarly journals Crowdsourced Identification of Potential Target Genes for CTV Induced Gene Silencing for Controlling the Citrus Greening Vector Diaphorina citri

2021 ◽  
Vol 12 ◽  
Author(s):  
John E. Ramos ◽  
Ritesh G. Jain ◽  
Charles A. Powell ◽  
William O. Dawson ◽  
Siddarame Gowda ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Artificial Diet ◽  
Target Genes ◽  
Chain Complex ◽  
Heme Iron ◽  
Citrus Greening ◽  
Diaphorina Citri ◽  
Virus Induced Gene Silencing ◽  
Iron Sulfur Cluster ◽  
Citrus Production

Citrus Greening or Huanglongbing (HLB) is a disease of citrus, causing high reduction in citrus production and is transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama vectoring a phloem-limited bacterium Candidatus Liberibacter sp. We report research results using crowdsourcing challenge strategy identifying potential gene targets in D. citri to control the insect using RNA interference (RNAi). From 63 submitted sequences, 43 were selected and tested by feeding them to D. citri using artificial diet assays. After feeding on artificial diet, the three most effective dsRNAs causing 30% mortality above control silenced genes expressing iron-sulfur cluster subunit of the mitochondrial electron transport chain complex (Rieske), heme iron-binding terminal oxidase enzyme (Cytochrome P450) and tetrahydrobiopterin (BH4) pathway enzyme (Pterin 4α-Carbinolamine Dehydratase). These sequences were cloned into a citrus phloem-limited virus (Citrus tristeza virus, CTV T36) expressing dsRNA against these target genes in citrus. The use of a viral mediated “para-transgenic” citrus plant system caused higher mortality to adult D. citri than what was observed using artificial diet, reaching 100% when detached citrus leaves with the engineered CTV expressing dsRNA were fed to adult D. citri. Using this approach, a virus-induced gene silencing (VIGS) can be used to test future transgenic cultivars before genetically engineering citrus. RNA Seq analysis after feeding D. citri CTV-RIE on infected leaves identified transcriptionally modified genes located upstream and downstream of the targeted RIE gene. These genes were annotated showing that many are associated with the primary function of the Rieske gene that was targeted by VIGS.

scholarly journals Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Kunxin Wu ◽  
Yadan Wu ◽  
Chunwei Zhang ◽  
Yan Fu ◽  
Zhixin Liu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Double Mutant ◽  
Target Genes ◽  
Rna Binding ◽  
Protein Product ◽  
Antiviral Defense ◽  
Virus Induced Gene Silencing ◽  
Argonaute 2 ◽  
Plant Genes ◽  
Silencing Pathways

Abstract Background Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes. Results In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to validate the involvement of DICER-LIKE 4 (DCL4) in antiviral defense against TCV. We further revealed the involvement of ARGONAUTE 2 (AGO2) in PDS silencing and antiviral defense against TCV in dcl2drb4 double mutant plants. These results demonstrated that DOUBLE-STRANDED RNA-BINDING PROTEIN 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B derivative in dcl2drb4 double mutant plants. Conclusions The CPB1B-based vector developed in this work is a valuable tool with visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.

Simultaneous silencing of two target genes using virus-induced gene silencing technology in Nicotiana benthamiana

2019 ◽  
Vol 74 (5-6) ◽  
pp. 151-159
Author(s):  
Feng Zhu ◽  
Yanping Che ◽  
Fei Xu ◽  
Yangkai Zhou ◽  
Kun Qian ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Nicotiana Benthamiana ◽  
Target Genes ◽  
Function Analysis ◽  
Tobacco Rattle Virus ◽  
Pcr Analysis ◽  
Virus Induced Gene Silencing ◽  
Biotic Stresses ◽  
Overlap Extension

Abstract Virus-induced gene silencing (VIGS) is an effective strategy for rapid gene function analysis. It is well established that the NAC transcription factor and salicylic acid (SA) signal pathway play essential roles in response to biotic stresses. However, simultaneous silencing of two target genes using VIGS in plants has been rarely reported. Therefore, in this report, we performed VIGS to silence simultaneously the SA-binding protein 2 (NbSABP2) and NbNAC1 in Nicotiana benthamiana to investigate the gene silencing efficiency of simultaneous silencing of two genes. We first cloned the full-length NbNAC1 gene, and the characterization of NbNAC1 was also analysed. Overlap extension polymerase chain reaction (PCR) analysis showed that the combination of NbSABP2 and NbNAC1 was successfully amplified. Bacteria liquid PCR confirmed that the combination of NbSABP2 and NbNAC1 was successfully inserted into the tobacco rattle virus vector. The results showed that the leaves from the NbSABP2 and NbNAC1 gene-silenced plants collapsed slightly, with browning at the base of petiole or veina. Quantitative real-time PCR results showed that the expression of NbSABP2 and NbNAC1 were significantly reduced in 12 days post silenced plants after tobacco rattle virus infiltration compared with the control plants. Overall, our results suggest that VIGS can be used to silence simultaneously two target genes.

scholarly journals Simultaneous Silencing of Two Different Arabidopsis Genes With A Novel Virus-Induced Gene Silencing Vector

2020 ◽  
Author(s):  
Xiuchun Zhang ◽  
Yadan Wu ◽  
Chunwei Zhang ◽  
Kunxin Wu ◽  
Zhixin Liu
Keyword(s):  
Gene Silencing ◽  
Rna Silencing ◽  
Target Genes ◽  
Rna Binding ◽  
Protein Product ◽  
Turnip Crinkle Virus ◽  
Virus Induced Gene Silencing ◽  
Argonaute 2 ◽  
Plant Genes ◽  
Silencing Pathways

Abstract Background: Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes. Results: In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of Turnip crinkle virus (TCV) known as CPB that can be readily used in Arabidopsis thaliana to interrogate genes of this model plant. A CPB derivative, designated CPB1B, was produced by inserting a 46 nucleotide section of the Arabidopsis PHYTOENE DESATURASE (PDS) gene into CPB, in antisense orientation. CPB1B induced robust PDS silencing, causing easily visible photobleaching in systemically infected Arabidopsis leaves. More importantly, CPB1B can accommodate additional inserts, derived from other Arabidopsis genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the CPB1B vector to evaluate the relative importance of several known RNA silencing pathway genes in PDS VIGS. This approach allowed us to validate the involvement of DICER-LIKE 4 (DCL4) and ARGONAUTE 2 (AGO2) in PDS silencing. Notably, double-stranded RNA-binding protein 4 (DRB4), whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for PDS silencing induced by CPB1B. Conclusions: The CPB1B-based vector developed in this work is a valuable tool with tracable and visualizable indicator of the silencing penetrance for interrogating Arabidopsis genes, especially those involved in the RNA silencing pathways.

scholarly journals California TRV-based VIGS vectors mediate gene silencing at elevated temperatures but with greater growth stunting

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jamilur Rahman ◽  
Ian T. Baldwin ◽  
Klaus Gase
Keyword(s):  
Gene Silencing ◽  
Host Plant ◽  
Target Genes ◽  
Elevated Temperatures ◽  
Tobacco Rattle Virus ◽  
Vector System ◽  
Nicotiana Attenuata ◽  
Cdna Clones ◽  
Virus Induced Gene Silencing ◽  
In Planta

Abstract Background Tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS), a widely used functional genomics tool, requires growth temperatures typically lower than those of the plant’s native environment. Enabling VIGS under native conditions in the field according to applicable safety regulations could be a revolutionary advance for ecological research. Results Here, we report the development of an enhanced thermal tolerant VIGS vector system based on a TRV California isolate. cDNA clones representing the whole viral genome were sequenced and used to construct separate binary plant transformation vectors for functional elements of RNA1 (6765 nt) and RNA2 (3682 nt). VIGS of target genes was induced by transient transformation of the host plant with both vectors or by treating the host plant with sap from already VIGS induced plants. In Nicotiana attenuata the silencing efficiency of the PDS (phytoene desaturase) gene was 90% at 28 °C and 78% at 30 °C. Silencing at these temperatures was more prominent and durable than silencing induced by the widely used TRV PpK20-based pBINTRA6/pTV00 system, but was associated with a viral phenotype. Differences in the suppressor protein and RNA dependent RNA polymerase sequences between the TRV California isolate and PpK20 may be the reason for their different thermal tolerance. Conclusions The new TRV California-based VIGS vectors induce gene silencing in Nicotiana attenuata at higher temperatures than the existing pBINTRA6/pTV00 vector system, but cause greater growth defects. The new vector system opens up an avenue to study genes functions in planta under field conditions.

scholarly journals Development of resistant crop plants to parasitic weeds based on trans-specific gene silencing

2013 ◽  
Author(s):  
Radi Aly ◽  
John I. Yoder
Keyword(s):  
Gene Silencing ◽  
Host Plants ◽  
Target Genes ◽  
Tobacco Rattle Virus ◽  
Genetic Resistance ◽  
Transgenic Tomato ◽  
Specific Gene ◽  
Virus Induced Gene Silencing ◽  
Parasitic Weeds ◽  
Experimental Systems

Broomrapes (Orobanche/Phelipanchespp.) are holo parasitic plants that subsist on the roots of a variety of agricultural crops and cause severe losses to the yield quality and quantity. Effective methods for controlling parasitic weeds are scarce, with only a few known cases of genetic resistance. In the current study, we proposed an improved strategy for the control of parasitic weeds based on trans-specific gene-silencing of three parasite genes at once. We used two strategies to express dsRNA containing selected sequences of three Phelipancheaegyptiacagenes PaACS, PaM6PR and PaPrx1 (pma): transient expression using Tobacco rattle virus (TRV:pma) as a virus-induced gene-silencing (VIGS) vector and stable expression in transgenic tomato Solanumlycopersicum(Mill.) plants harboring a hairpin construct (pBINPLUS35:pma). siRNA-mediated transgene-silencing (20–24 nt) was detected in the host plants. Our results demonstrate that the quantities of PaACSand PaM6PR transcripts from P. aegyptiacatubercles grown on transgenic tomato or on Tobacco rattle virus-infected Nicotianabenthamianaplants were significantly reduced. However, only partial reductions in the quantity of PaPrx1 transcripts were observed in the parasite tubercles grown on tomato and on N. benthamianaplants. Concomitant with the suppression of the target genes, there were significant decreases in the number and weight of the parasite tubercles that grew on the host plants, in both the transient and the stable experimental systems. The results of the work carried out using both strategies point to the movement of mobile exogenous siRNA from the host to the parasite, leading to the impaired expression of essential parasite target genes. In light of the importance of parasitic weeds to world agriculture and the difficulty of obtaining resistance by conventional methods, we assume that genetic resistance based on the silencing of key metabolic genes in the parasite is now feasible. BARD Report - Project4622 Page 2 of 60 

scholarly journals Annotation of yellow genes in Diaphorina citri, the vector for Huanglongbing disease

Gigabyte ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Crissy Massimino ◽  
Chad Vosburg ◽  
Teresa Shippy ◽  
Prashant S. Hosmani ◽  
Mirella Flores-Gonzalez ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Gene Families ◽  
Citrus Greening ◽  
Manual Annotation ◽  
Diaphorina Citri ◽  
In Planta ◽  
Molecular Control ◽  
Citrus Greening Disease ◽  
Depth Analysis ◽  
Citrus Production

Huanglongbing (HLB), also known as citrus greening disease, is caused by the bacterium Candidatus Liberibacter asiaticus (CLas). It is a serious threat to global citrus production. This bacterium is transmitted by the Asian citrus psyllid, Diaphorina citri (Hemiptera). There are no effective in planta treatments for CLas. Therefore, one strategy is to manage the psyllid population. Manual annotation of the D. citri genome can identify and characterize gene families that could be novel targets for psyllid control. The yellow gene family is an excellent target because yellow genes, which have roles in melanization, are linked to development and immunity. Combined analysis of the genome with RNA-seq datasets, sequence homology, and phylogenetic trees were used to identify and annotate nine yellow genes in the D. citri genome. Manual curation of genes in D. citri provided in-depth analysis of the yellow family among hemipteran insects and provides new targets for molecular control of this psyllid pest. Manual annotation was done as part of a collaborative Citrus Greening community annotation project.

scholarly journals First Evidence of Feeding-Induced RNAi in Banana Weevil via Exogenous Application of dsRNA

Insects ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 40
Author(s):  
Henry Shaykins Mwaka ◽  
Olivier Christiaens ◽  
Priver Namanya Bwesigye ◽  
Jerome Kubiriba ◽  
Wilberforce Kateera Tushemereirwe ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Artificial Diet ◽  
Target Genes ◽  
Pupal Stage ◽  
Current Control ◽  
Control Measures ◽  
Banana Weevil ◽  
Water Control ◽  
Environmentally Sound ◽  
Species Specific

Banana weevil (Cosmopolites sordidus) is the most devastating pest of banana and plantain worldwide, yet current control measures are neither effective, sustainable, nor environmentally sound, and no resistant farmer-preferred cultivars are known to date. In this paper, we examined the ability to induce RNA interference (RNAi) in the banana weevil via feeding. We first developed an agar- and banana corm (rhizome) flour-based artificial diet in a multi-well plate setup that allowed the banana weevils to complete their life cycle from egg through the larval instars to the pupal stage in an average period of 53 days. Adults emerged about 20 days later. The artificial diet allowed the tunneling and burrowing habits of the larvae and successful metamorphosis up to adult eclosion. Adding dsRNA for laccase2 to the artificial diet resulted in albino phenotypes, confirming gene-silencing. Finally, C. sordidus was fed with dsRNA against a selection of essential target genes: snf7, rps13, mad1, vha-a, vha-d, and lgl for a period of 45 days. 100% mortality within 9–16 days was realized with dssnf7, dsrps13, and dsmad1 at 200 ng/mL artificial diet, and this corresponded to a strong reduction in gene expression. Feeding the dsRNA targeting the two vha genes resulted in 100% mortality after about 3–4 weeks, while treatment with dslgl resulted in no mortality above the dsgfp-control and the water-control. Our results have implications for the development of RNAi approaches for managing important crop pests, in that banana weevils can be controlled based on the silencing of essential target genes as snf7, rps13, and mad1. They also highlight the need for research into the development of RNAi for banana protection, eventually the engineering of host-induced gene-silencing (HIGS) cultivars, given the high RNAi efficacy and its species-specific mode of action, adding the RNAi approach to the armory of integrated pest management (IPM).

scholarly journals Candidatus Liberibacter Asiaticus Manipulates the Expression of Vitellogenin, Cytoskeleton, and Endocytotic Pathway-Related Genes to Become Circulative in Its Vector, Diaphorina citri (Hemiptera: Psyllidae)

2020 ◽  
Author(s):  
Damini Jaiswal ◽  
V. Kavi Sidharthan ◽  
Susheel Kumar Sharma ◽  
Richa Rai ◽  
Nandlal Choudhary ◽  
...  
Keyword(s):  
Differential Regulation ◽  
Citrus Greening ◽  
Insect Vector ◽  
Diaphorina Citri ◽  
Citrus Greening Disease ◽  
Physiological Processes ◽  
Citrus Production ◽  
Liberibacter Asiaticus ◽  
Greening Disease ◽  
Cellular Components

Citrus greening disease or huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) limits the citrus production worldwide. CLas is transmitted by the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera: Psyllidae) in a persistent-propagative manner. Application of insecticides to manage the psyllid vectors and disease is the most common practice. Understanding the molecular interaction between CLas and ACP and interrupting the interrelationship can provide an alternative to insecticides for managing citrus greening disease. Transcriptome analysis of ACP in response to CLas showed differential expression of 3911 genes (2196 up-regulated, and 1715 down-regulated) including the key genes of ACP involved in cytoskeleton synthesis and nutrition-related proteins. Majority of the differentially expressed genes were categorized under molecular function followed by cellular components and biological processes. KEGG pathway analysis showed differential regulation of carbohydrate, nucleotide and energy metabolic pathways, the endocytotic pathway and the defense-related pathways. Differential regulation of genes associated with the key pathways might favors CLas to become systemic and propagate in its insect vector. The study provides an understanding of genes involved in circulation of CLas in ACP. The candidate genes involved in key physiological processes and CLas transmission by ACP would be potential targets for sustainable management of ACP and CLas.

2020–2021 Florida Citrus Production Guide: Huanglongbing (Citrus Greening)

EDIS ◽  
2020 ◽  
Author(s):  
Megan M. Dewdney ◽  
Tripti Vashisth ◽  
Lauren M. Diepenbrock
Keyword(s):  
Citrus Greening ◽  
Citrus Production ◽  
Florida Citrus

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