scholarly journals IDA (INFLORESCENCE DEFICIENT IN ABSCISSION)-like peptides and HAE (HAESA)-like receptors regulate corolla abscission in Nicotiana benthamiana flowers

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Daniel Ventimilla ◽  
Karelia Velázquez ◽  
Susana Ruiz-Ruiz ◽  
Javier Terol ◽  
Miguel A. Pérez-Amador ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Viral Vector ◽  
Corolla Tube ◽  
Proteolytic Processing ◽  
Virus Induced Gene Silencing ◽  
Small Peptides ◽  
Gene Suppression ◽  
Obvious Effect ◽  
Active Peptides ◽  
Cell To Cell Adhesion

Abstract Background Abscission is an active, organized, and highly coordinated cell separation process enabling the detachment of aerial organs through the modification of cell-to-cell adhesion and breakdown of cell walls at specific sites on the plant body known as abscission zones. In Arabidopsis thaliana, abscission of floral organs and cauline leaves is regulated by the interaction of the hormonal peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), a pair of redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE2 (HSL2), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors. However, the functionality of this abscission signaling module has not yet been demonstrated in other plant species. Results The expression of the pair of NbenIDA1 homeologs and the receptor NbenHAE.1 was supressed at the base of the corolla tube by the inoculation of two virus-induced gene silencing (VIGS) constructs in Nicotiana benthamiana. These gene suppression events arrested corolla abscission but did not produce any obvious effect on plant growth. VIGS plants retained a higher number of corollas attached to the flowers than control plants, an observation related to a greater corolla breakstrength. The arrest of corolla abscission was associated with the preservation of the parenchyma tissue at the base of the corolla tube that, in contrast, was virtually collapsed in normal corollas. In contrast, the inoculation of a viral vector construct that increased the expression of NbenIDA1A at the base of the corolla tube negatively affected the growth of the inoculated plants accelerating the timing of both corolla senescence and abscission. However, the heterologous ectopic overexpression of citrus CitIDA3 and Arabidopsis AtIDA in N. benthamiana did not alter the standard plant phenotype suggesting that the proteolytic processing machinery was unable to yield active peptides. Conclusion Here, we demonstrate that the pair of NbenIDA1 homeologs encoding small peptides of the IDA-like family and the receptor NbenHAE.1 control cellular breakdown at the base of the corolla tube awhere an adventitious AZ should be formed and, therefore, corolla abscission in N. benthamiana flowers. Altogether, our results provide the first evidence supporting the notion that the IDA-HAE/HSL2 signaling module is conserved in angiosperms.

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

Plant Methods ◽  
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.

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

2012 ◽  
Vol 25 (12) ◽  
pp. 1523-1530 ◽  
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.

scholarly journals Genome-Wide microRNA Profiling Using Oligonucleotide Microarray Reveals Regulatory Networks of microRNAs in Nicotiana benthamiana During Beet Necrotic Yellow Vein Virus Infection

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 310 ◽  
Author(s):  
Junying Liu ◽  
Huiyan Fan ◽  
Ying Wang ◽  
Chenggui Han ◽  
Xianbing Wang ◽  
...  
Keyword(s):  
Nicotiana Benthamiana ◽  
Regulatory Networks ◽  
Target Genes ◽  
Tobacco Rattle Virus ◽  
Yellow Vein ◽  
Differentially Expressed ◽  
Virus Induced Gene Silencing ◽  
Developmental Defects ◽  
Differentially Expressed Mirnas ◽  
Species Specific

Beet necrotic yellow vein virus (BNYVV) infections induce stunting and leaf curling, as well as root and floral developmental defects and leaf senescence in Nicotiana benthamiana. A microarray analysis with probes capable of detecting 1596 candidate microRNAs (miRNAs) was conducted to investigate differentially expressed miRNAs and their targets upon BNYVV infection of N. benthamiana plants. Eight species-specific miRNAs of N. benthamiana were identified. Comprehensive characterization of the N. benthamiana microRNA profile in response to the BNYVV infection revealed that 129 miRNAs were altered, including four species-specific miRNAs. The targets of the differentially expressed miRNAs were predicted accordingly. The expressions of miR164, 160, and 393 were up-regulated by BNYVV infection, and those of their target genes, NAC21/22, ARF17/18, and TIR, were down-regulated. GRF1, which is a target of miR396, was also down-regulated. Further genetic analysis of GRF1, by Tobacco rattle virus-induced gene silencing, assay confirmed the involvement of GRF1 in the symptom development during BNYVV infection. BNYVV infection also induced the up-regulation of miR168 and miR398. The miR398 was predicted to target umecyanin, and silencing of umecyanin could enhance plant resistance against viruses, suggesting the activation of primary defense response to BNYVV infection in N. benthamiana. These results provide a global profile of miRNA changes induced by BNYVV infection and enhance our understanding of the mechanisms underlying BNYVV pathogenesis.

scholarly journals Plant sterol biosynthesis: identification of two distinct families of sterol 4alpha-methyl oxidases

2004 ◽  
Vol 378 (3) ◽  
pp. 889-898 ◽  
Author(s):  
Sylvain DARNET ◽  
Alain RAHIER
Keyword(s):  
Viral Vector ◽  
Gene Families ◽  
Amino Acid Sequences ◽  
Virus Induced Gene Silencing ◽  
Qualitative And Quantitative ◽  
Sequence Identity ◽  
Photosynthetic Eukaryotes ◽  
Membrane Bound ◽  
Haem Iron ◽  
Cdna Fragments

In plants, the conversion of cycloartenol into functional phytosterols requires the removal of the two methyl groups at C-4 by an enzymic complex including a sterol 4α-methyl oxidase (SMO). We report the cloning of candidate genes for SMOs in Arabidopsis thaliana, belonging to two distinct families termed SMO1 and SMO2 and containing three and two isoforms respectively. SMO1 and SMO2 shared low sequence identity with each other and were orthologous to the ERG25 gene from Saccharomyces cerevisiae which encodes the SMO. The plant SMO amino acid sequences possess all the three histidine-rich motifs (HX3H, HX2HH and HX2HH), characteristic of the small family of membrane-bound non-haem iron oxygenases that are involved in lipid oxidation. To elucidate the precise functions of SMO1 and SMO2 gene families, we have reduced their expression by using a VIGS (virus-induced gene silencing) approach in Nicotiana benthamiana. SMO1 and SMO2 cDNA fragments were inserted into a viral vector and N. benthamiana inoculated with the viral transcripts. After silencing with SMO1, a substantial accumulation of 4,4-dimethyl-9β,19-cyclopropylsterols (i.e. 24-methylenecycloartanol) was obtained, whereas qualitative and quantitative levels of 4α-methylsterols were not affected. In the case of silencing with SMO2, a large accumulation of 4α-methyl-Δ7-sterols (i.e. 24-ethylidenelophenol and 24-ethyllophenol) was found, with no change in the levels of 4,4-dimethylsterols. These clear and distinct biochemical phenotypes demonstrate that, in contrast with animals and fungi, in photosynthetic eukaryotes, these two novel families of cDNAs are coding two distinct types of C-4-methylsterol oxidases controlling the level of 4,4-dimethylsterol and 4α-methylsterol precursors respectively.

scholarly journals Cell Death of Nicotiana benthamiana Is Induced by Secreted Protein NIS1 of Colletotrichum orbiculare and Is Suppressed by a Homologue of CgDN3

2012 ◽  
Vol 25 (5) ◽  
pp. 625-636 ◽  
Author(s):  
Kae Yoshino ◽  
Hiroki Irieda ◽  
Fumie Sugimoto ◽  
Hirofumi Yoshioka ◽  
Tetsuro Okuno ◽  
...  
Keyword(s):  
Cell Death ◽  
Nicotiana Benthamiana ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Expression System ◽  
Functional Screening ◽  
Secreted Protein ◽  
Virus Induced Gene Silencing ◽  
Colletotrichum Orbiculare ◽  
Green Fluorescent

Colletotrichum orbiculare, the causal agent of cucumber anthracnose, infects Nicotiana benthamiana. Functional screening of C. orbiculare cDNAs in a virus vector-based plant expression system identified a novel secreted protein gene, NIS1, whose product induces cell death in N. benthamiana. Putative homologues of NIS1 are present in selected members of fungi belonging to class Sordariomycetes, Dothideomycetes, or Orbiliomycetes. Green fluorescent protein–based expression studies suggested that NIS1 is preferentially expressed in biotrophic invasive hyphae. NIS1 lacking signal peptide did not induce NIS1-triggered cell death (NCD), suggesting apoplastic recognition of NIS1. NCD was prevented by virus-induced gene silencing of SGT1 and HSP90, indicating the dependency of NCD on SGT1 and HSP90. Deletion of NIS1 had little effect on the virulence of C. orbiculare against N. benthamiana, suggesting possible suppression of NCD by C. orbiculare at the postinvasive stage. The CgDN3 gene of C. gloeosporioides was previously identified as a secreted protein gene involved in suppression of hypersensitive-like response in Stylosanthes guianensis. Notably, we found that NCD was suppressed by the expression of a CgDN3 homologue of C. orbiculare. Our findings indicate that C. orbiculare expresses NIS1 at the postinvasive stage and suggest that NCD could be repressed via other effectors, including the CgDN3 homologue.

scholarly journals The involvement of two epoxide hydrolase genes, NbEH1.1 and NbEH1.2, of Nicotiana benthamiana in the interaction with Colletotrichum destructivum, Colletotrichum orbiculare or Pseudomonas syringae pv. tabaci

2008 ◽  
Vol 35 (11) ◽  
pp. 1112 ◽  
Author(s):  
C. P. Wijekoon ◽  
P. H. Goodwin ◽  
T. Hsiang
Keyword(s):  
Pseudomonas Syringae ◽  
Nicotiana Benthamiana ◽  
Epoxide Hydrolase ◽  
Virus Induced Gene Silencing ◽  
Colletotrichum Orbiculare ◽  
Hypersensitive Resistance ◽  
Resistance Response ◽  
Colletotrichum Destructivum ◽  
Vicinal Diols ◽  
Epoxy Alcohols

Epoxide hydrolase hydrates epoxides to vicinal diols in the phyto-oxylipin peroxygenase pathway resulting in the production of epoxy alcohols, dihydrodiols, triols and epoxides, including many lipid epoxides associated with resistance. Two epoxide hydrolase genes from Nicotiana benthamiana L., NbEH1.1 and NbEH1.2, were amplified from coding DNA of leaves during a susceptible response to the hemibiotrophic pathogens, Colletotrichum destructivum O’Gara, Colletotrichum orbiculare Berk. and Mont. von Arx. or Pseudomonas syringae pv. tabaci Wolf and Foster, or the hypersensitive resistance response to P. syringae pv. tabaci expressing avrPto. Increases in expression of NbEH1.1 generally occurred during the late biotrophic and necrotrophic stages in the susceptible responses and before the hypersensitive response. NbEH1.2 expression was not significantly induced by C. orbiculare but was induced by C. destructivum, P. syringae pv. tabaci and P. syringae pv. tabaci expressing avrPto, although to a lesser degree than NbEH1.1. Virus-induced gene silencing of NbEH1.1 delayed the appearance of lesions for C. destructivum, reduced populations of P. syringae pv. tabaci and increased populations of P. syringae pv. tabaci expressing avrPto. The importance of epoxide hydrolase during pathogen attack may be related to its roles in detoxification, signalling, or metabolism of antimicrobial compounds.

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