Genomic Resources and Tools for Gene Function Analysis in Potato

Potato, a highly heterozygous tetraploid, is undergoing an exciting phase of genomics resource development. The potato research community has established extensive genomic resources, such as large expressed sequence tag (EST) data collections, microarrays and other expression profiling platforms, and large-insert genomic libraries. Moreover, potato will now benefit from a global potato physical mapping effort, which is serving as the underlying resource for a full potato genome sequencing project, now well underway. These tools and resources are having a major impact on potato breeding and genetics. The genome sequence will provide an invaluable comparative genomics resource for cross-referencing to the other Solanaceae, notably tomato, whose sequence is also being determined. Most importantly perhaps, a potato genome sequence will pave the way for the functional analysis of the large numbers of potato genes that await discovery. Potato, being easily transformable, is highly amenable to the investigation of gene function by biotechnological approaches. Recent advances in the development of Virus Induced Gene Silencing (VIGS) and related methods will facilitate rapid progress in the analysis of gene function in this important crop.

Download Full-text

A cassava common mosaic virus vector for virus-induced gene silencing in cassava

Plant Methods ◽

10.1186/s13007-021-00775-w ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Decai Tuo ◽

Peng Zhou ◽

Pu Yan ◽

Hongguang Cui ◽

Yang Liu ◽

...

Keyword(s):

Gene Silencing ◽

Mosaic Virus ◽

Gene Function ◽

Viral Vector ◽

Function Analysis ◽

Systemic Infection ◽

Cdna Clones ◽

Virus Induced Gene Silencing ◽

Efficient Gene ◽

Gene Function Analysis

Abstract Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

Download Full-text

An Efficient Virus-Induced Gene Silencing System for Functional Genomics Research in Walnut (Juglans regia L.) Fruits

Frontiers in Plant Science ◽

10.3389/fpls.2021.661633 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yifan Wang ◽

Ning Huang ◽

Niu Ye ◽

Lingyu Qiu ◽

Yadong Li ◽

...

Keyword(s):

Gene Silencing ◽

Gene Function ◽

Function Analysis ◽

Juglans Regia ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Tobacco Rattle Virus ◽

Post Inoculation ◽

Virus Induced Gene Silencing ◽

Gene Function Analysis

The Persian walnut (Juglans regia L.) is a leading source of woody oil in warm temperate regions and has high nutritional and medicinal values. It also provides both tree nuts and woody products. Nevertheless, incomplete characterization of the walnut genetic system limits the walnut gene function analysis. This study used the tobacco rattle virus (TRV) vector to construct an infectious pTRV-JrPDS recombinant clone. A co-culture inoculation method utilizing Agrobacterium was screened out from four inoculation methods and optimized to set up an efficient virus-induced gene silencing (VIGS) system for J. regia fruit. The optimized VIGS-TRV system induced complete photobleaching phenotype on the walnut fruits of four cultivars, and the JrPDS transcript levels decreased by up to 88% at 8 days post-inoculation (dpi). While those of browning-related J. regia polyphenol oxidase (PPO) genes JrPPO1 and JrPPO2 decreased by 67 and 80% at 8 dpi, respectively, accompanied by a significant reduction in fruit browning phenotype. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis screening and Western Blot showed that the PPO protein levels were significantly reduced. Moreover, a model of TRV-mediated VIGS system for inoculating J. regia fruit with efficient silence efficiency via co-culture was developed. These results indicate that the VIGS-TRV system is an efficient tool for rapid gene function analysis in J. regia fruits.

Download Full-text

A Cassava Common Mosaic Virus Vector for Virus-induced Gene Silencing in Cassava

10.21203/rs.3.rs-154080/v1 ◽

2021 ◽

Author(s):

Decai Tuo ◽

Peng Zhou ◽

Pu Yan ◽

Yang Liu ◽

Hongguang Cui ◽

...

Keyword(s):

Gene Silencing ◽

Mosaic Virus ◽

Gene Function ◽

Viral Vector ◽

Function Analysis ◽

Systemic Infection ◽

Cdna Clones ◽

Virus Induced Gene Silencing ◽

Efficient Gene ◽

Gene Function Analysis

Abstract Background: Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing.Results:In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus strain CM (CsCMV-CM) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions: This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

Download Full-text

Optimisation of tobacco rattle virus-induced gene silencing in Arabidopsis

Functional Plant Biology ◽

10.1071/fp05096 ◽

2006 ◽

Vol 33 (4) ◽

pp. 347 ◽

Cited By ~ 46

Author(s):

Changchun Wang ◽

Xinzhong Cai ◽

Xuemin Wang ◽

Zhong Zheng

Keyword(s):

Gene Silencing ◽

High Throughput ◽

Function Analysis ◽

Tobacco Rattle Virus ◽

Phytoene Desaturase ◽

Growth Stages ◽

Virus Induced Gene Silencing ◽

Gene Functions ◽

Genes Encoding ◽

Gene Function Analysis

Arabidopsis thaliana (L.) Heynh. is a model plant species in which to study plant gene functions. Recently developed virus-induced gene silencing (VIGS) offers a rapid and high-throughput technique platform for gene function analysis. In this paper we report optimisation of tobacco rattle virus (TRV)-induced gene silencing in Arabidopsis. The parameters potentially affecting the efficiency of VIGS in Arabidopsis were investigated. These included the concentration and pre-incubation of Agrobacterium inocula (agro-inocula), the concentration of acetosyringone included in agro-inocula, the Agrobacterium inoculation (agro-inoculation) method, the ecotypes and the growth stages of Arabidopsis plants for agro-inoculation, and the growth temperature of agro-inoculated plants. The optimised VIGS procedure involves preparing the agro-inocula with OD600 of 2.0, pre-incubating for 2 h in infiltration buffer containing 200 μm acetosyringone, agro-inoculating by vacuum infiltration, and growth of agro-inoculated plants at 22 −24°C. Following this procedure consistent and highly efficient VIGS was achieved for the genes encoding phytoene desaturase (PDS) and actin in Arabidopsis. The silencing phenotype lasts for at least 6 weeks, and is applicable in at least seven ecotypes, including Col-0, Cvi-0, Sd, Nd-1, Ws-0, Bay-0 and Ler. TRV-induced VIGS was expressed not only in leaves, but also in stems, inflorescences and siliques. However, VIGS was not transmissible through seed to the subsequent generation. The optimised procedure of the TRV-induced gene silencing should facilitate high-throughput functional analysis of genes in Arabidopsis.

Download Full-text

Draft Genome Sequence of Xylella fastidiosa subsp. fastidiosa Strain Stag’s Leap

Genome Announcements ◽

10.1128/genomea.00240-16 ◽

2016 ◽

Vol 4 (2) ◽

Cited By ~ 8

Author(s):

J. Chen ◽

F. Wu ◽

Z. Zheng ◽

X. Deng ◽

L. P. Burbank ◽

...

Keyword(s):

Disease Resistance ◽

Genome Sequence ◽

Gene Function ◽

Xylella Fastidiosa ◽

Draft Genome ◽

Draft Genome Sequence ◽

Pierce's Disease ◽

Pierce’S Disease ◽

Knockout Mutants ◽

Fastidiosa Strain

Xylella fastidiosa subsp. fastidiosa causes Pierce’s disease of grapevine. Presented here is the draft genome sequence of the Stag’s Leap strain, previously used in pathogenicity/virulence assays to evaluate grapevine germplasm bearing Pierce’s disease resistance and a phenotypic assessment of knockout mutants to determine gene function.

Download Full-text

A Draft Genome Sequence of Nicotiana benthamiana to Enhance Molecular Plant-Microbe Biology Research

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-12-0148-ta ◽

2012 ◽

Vol 25 (12) ◽

pp. 1523-1530 ◽

Cited By ~ 247

Author(s):

Aureliano Bombarely ◽

Hernan G. Rosli ◽

Julia Vrebalov ◽

Peter Moffett ◽

Lukas A. Mueller ◽

...

Keyword(s):

Genome Sequence ◽

Nicotiana Benthamiana ◽

Draft Genome ◽

Draft Genome Sequence ◽

Virus Induced Gene Silencing ◽

Transient Protein Expression ◽

Mitogen Activated Protein ◽

Microbe Interactions ◽

Solanaceae Family ◽

Biology Research

Nicotiana benthamiana is a widely used model plant species for the study of fundamental questions in molecular plant-microbe interactions and other areas of plant biology. This popularity derives from its well-characterized susceptibility to diverse pathogens and, especially, its amenability to virus-induced gene silencing and transient protein expression methods. Here, we report the generation of a 63-fold coverage draft genome sequence of N. benthamiana and its availability on the Sol Genomics Network for both BLAST searches and for downloading to local servers. The estimated genome size of N. benthamiana is 3 Gb (gigabases). The current assembly consists of approximately 141,000 scaffolds, spanning 2.6 Gb with 50% of the genome sequence contained within scaffolds >89 kilobases. Of the approximately 16,000 N. benthamiana unigenes available in GenBank, >90% are represented in the assembly. The usefulness of the sequence was demonstrated by the retrieval of N. benthamiana orthologs for 24 immunity-associated genes from other species including Ago2, Ago7, Bak1, Bik1, Crt1, Fls2, Pto, Prf, Rar1, and mitogen-activated protein kinases. The sequence will also be useful for comparative genomics in the Solanaceae family as shown here by the discovery of microsynteny between N. benthamiana and tomato in the region encompassing the Pto and Prf genes.

Download Full-text

Cloning and function analysis of BAG family genes in wheat

Functional Plant Biology ◽

10.1071/fp15317 ◽

2016 ◽

Vol 43 (5) ◽

pp. 393

Author(s):

Shiming Ge ◽

Zhen Kang ◽

Ying Li ◽

Fuzhen Zhang ◽

Yinzhu Shen ◽

...

Keyword(s):

Heat Stress ◽

Salinity Stress ◽

Heat Tolerance ◽

Expressed Sequence Tag ◽

Function Analysis ◽

Pcr Analysis ◽

Subcellular Localisation ◽

Hybrid Technique ◽

Rt Pcr ◽

Conserved Domain

By analysing the cDNA microarray of the salt tolerant mutant of wheat RH8706–49 under salinity stress, our results showed an expressed sequence tag fragment and acquired an unknown gene (designated as TaBAG) with a BAG conserved domain through electronic cloning and RT–PCR technology. The gene was registered into GenBank (No. FJ599765). After homologous alignment analysis, electronic cloning, and amplifying with RT–PCR, the other gene with a BAG conserved domain, TaBAG2, was obtained and registered into GenBank (No. GU471210). Quantitative PCR analysis demonstrated that TaBAG2 expression was induced by saline and heat stress. TaBAG gene expression under salinity stress increased remarkably but showed an insignificant response to heat stress. The adversity stress detection results showed that Arabidopsis overexpressing TaBAG and TaBAG2 exhibited an obvious salt tolerance increase. Under heat stress, Arabidopsis overexpressing TaBAG2 showed increased heat tolerance; however, the heat tolerance of Arabidopsis overexpressing TaBAG did not vary significantly under heat stress. Subcellular localisation results demonstrated that TaBAGs were mainly located in the cytoplasm and the cell nucleus. We applied fluorescence complementation and yeast two-hybrid technique to prove that TaBAG2 can obviously bond with TaHsp70 and TaCaMs. After the respective mutation of aspartic acid (D) and arginine (R) at high conservation in BAG domain of TaBAG2, the bonding interaction between TaBAG2 and TaHsp70 disappeared, indicating that the two amino acids were the key loci for the interaction between TaBAG2 and TaHsp70. Heat tolerance detection results demonstrated that the heat tolerance of Arabidopsis overexpressing and cotransfected with TaBAG2 and TaHsp70 was much higher than that of Arabidopsis overexpressing TaBAG2 and Arabidopsis overexpressing TaHSP70. This finding implies that the synergistic use of TaBAG2 and TaHSP70 can improve heat tolerance of plants.

Download Full-text