Impairment of Tomato WAT1 Enhances Resistance to Vascular Wilt Fungi Despite Severe Growth Defects

Verticillium dahliae is a particularly notorious vascular wilt pathogen of tomato and poses a reoccurring challenge to crop protection as limited qualitative resistance is available. Therefore, alternative approaches for crop protection are pursued. One such strategy is the impairment of disease susceptibility (S) genes, which are plant genes targeted by pathogens to promote disease development. In Arabidopsis and cotton, the Walls Are Thin 1 (WAT1) gene has shown to be a S gene for V. dahliae. In this study, we identified the tomato WAT1 homolog Solyc04g080940 (SlWAT1). Transient and stable silencing of SlWAT1, based on virus-induced gene silencing (VIGS) and RNAi, respectively, did not consistently lead to reduced V. dahliae susceptibility in tomato. However, CRISPR-Cas9 tomato mutant lines carrying targeted deletions in SlWAT1 showed significantly enhanced resistance to V. dahliae, and furthermore also to Verticillium albo-atrum and Fusarium oxysporum f. sp. lycopersici (Fol). Thus, disabling the tomato WAT1 gene resulted in broad-spectrum resistance to various vascular pathogens in tomato. Unfortunately these tomato CRISPR mutant lines suffered from severe growth defects. In order to overcome the pleiotropic effect caused by the impairment of the tomato WAT1 gene, future efforts should be devoted to identifying tomato SlWAT1 mutant alleles that do not negatively impact tomato growth and development.

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

Plant Methods ◽

10.1186/s13007-020-00701-6 ◽

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.

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Arabidopsis mtHSC70-1 plays important roles in the establishment of COX-dependent respiration and redox homeostasis

Journal of Experimental Botany ◽

10.1093/jxb/erz357 ◽

2019 ◽

Vol 70 (20) ◽

pp. 5575-5590 ◽

Cited By ~ 2

Author(s):

Shan-Shan Wei ◽

Wei-Tao Niu ◽

Xiao-Ting Zhai ◽

Wei-Qian Liang ◽

Meng Xu ◽

...

Keyword(s):

Alternative Oxidase ◽

Redox Homeostasis ◽

Superoxide Dismutase 1 ◽

Respiratory Pathway ◽

Cellular Processes ◽

Growth Defects ◽

Alternative Respiratory Pathway ◽

Abnormal Mitochondria ◽

Shock Proteins ◽

Severe Growth

Abstract The 70 kDa heat shock proteins function as molecular chaperones and are involved in diverse cellular processes. However, the functions of the plant mitochondrial HSP70s (mtHSC70s) remain unclear. Severe growth defects were observed in the Arabidopsis thaliana mtHSC70-1 knockout lines, mthsc70-1a and mthsc70-1b. Conversely, the introduction of the mtHSC70-1 gene into the mthsc70-1a background fully reversed the phenotypes, indicating that mtHSC70-1 is essential for plant growth. The loss of mtHSC70-1 functions resulted in abnormal mitochondria and alterations to respiration because of an inhibition of the cytochrome c oxidase (COX) pathway and the activation of the alternative respiratory pathway. Defects in COX assembly were observed in the mtHSC70-1 knockout lines, leading to decreased COX activity. The mtHSC70-1 knockout plants have increased levels of reactive oxygen species (ROS). The introduction of the Mn-superoxide dismutase 1 (MSD1) or the catalase 1 (CAT1) gene into the mthsc70-1a plants decreased ROS levels, reduced the expression of alternative oxidase, and partially rescued growth. Taken together, our data suggest that mtHSC70-1 plays important roles in the establishment of COX-dependent respiration.

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Redundant Roles of Tead1 and Tead2 in Notochord Development and the Regulation of Cell Proliferation and Survival

Molecular and Cellular Biology ◽

10.1128/mcb.01759-07 ◽

2008 ◽

Vol 28 (10) ◽

pp. 3177-3189 ◽

Cited By ~ 107

Author(s):

Atsushi Sawada ◽

Hiroshi Kiyonari ◽

Kanako Ukita ◽

Noriyuki Nishioka ◽

Yu Imuta ◽

...

Keyword(s):

Cell Proliferation ◽

Paraxial Mesoderm ◽

Mouse Development ◽

Lateral Plate ◽

Growth Defects ◽

Mouse Mutants ◽

Severe Growth

ABSTRACT Four members of the TEAD/TEF family of transcription factors are expressed widely in mouse embryos and adult tissues. Although in vitro studies have suggested various roles for TEAD proteins, their in vivo functions remain poorly understood. Here we examined the role of Tead genes by generating mouse mutants for Tead1 and Tead2. Tead2 −/− mice appeared normal, but Tead1 −/−; Tead2 −/− embryos died at embryonic day 9.5 (E9.5) with severe growth defects and morphological abnormalities. At E8.5, Tead1 −/−; Tead2 −/− embryos were already small and lacked characteristic structures such as a closed neural tube, a notochord, and somites. Despite these overt abnormalities, differentiation and patterning of the neural plate and endoderm were relatively normal. In contrast, the paraxial mesoderm and lateral plate mesoderm were displaced laterally, and a differentiated notochord was not maintained. These abnormalities and defects in yolk sac vasculature organization resemble those of mutants for Yap, which encodes a coactivator of TEAD proteins. Moreover, we demonstrated genetic interactions between Tead1 and Tead2 and Yap. Finally, Tead1 −/−; Tead2 −/− embryos showed reduced cell proliferation and increased apoptosis. These results suggest that Tead1 and Tead2 are functionally redundant, use YAP as a major coactivator, and support notochord maintenance as well as cell proliferation and survival in mouse development.

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Expression of α-DIOXYGENASE 1 in Tomato and Arabidopsis Contributes to Plant Defenses Against Aphids

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-13-0031-r ◽

2013 ◽

Vol 26 (8) ◽

pp. 977-986 ◽

Cited By ~ 21

Author(s):

Carlos Augusto Avila ◽

Lirio Milenka Arevalo-Soliz ◽

Argelia Lorence ◽

Fiona L. Goggin

Keyword(s):

Green Peach Aphid ◽

Macrosiphum Euphorbiae ◽

Insertion Mutant ◽

Plant Signaling ◽

Virus Induced Gene Silencing ◽

Basal Resistance ◽

Potato Aphid ◽

Dna Insertion ◽

Mutant Lines ◽

Increased Susceptibility

Plant α-dioxygenases (α-DOX) are fatty acid–hydroperoxidases that contribute to the synthesis of oxylipins, a diverse group of compounds primarily generated through oxidation of linoleic (LA) and linolenic acid (LNA). Oxylipins are implicated in plant signaling against biotic and abiotic stresses. We report here that the potato aphid (Macrosiphum euphorbiae) induces Slα-DOX1 but not Slα-DOX2 expression in tomato (Solanum lycopersicum). Slα-DOX1 upregulation by aphids does not require either jasmonic acid (JA) or salicylic acid (SA) accumulation, since tomato mutants deficient in JA (spr2, acx1) or SA accumulation (NahG) still show Slα-DOX1 induction. Virus-induced gene silencing of Slα-DOX1 enhanced aphid population growth in wild-type (WT) plants, revealing that Slα-DOX1 contributes to basal resistance to aphids. Moreover, an even higher percent increase in aphid numbers occurred when Slα-DOX1 was silenced in spr2, a mutant line characterized by elevated LA levels, decreased LNA, and enhanced aphid resistance as compared with WT. These results suggest that aphid reproduction is influenced by oxylipins synthesized from LA by Slα-DOX1. In agreement with our experiments in tomato, two independent α-dox1 T-DNA insertion mutant lines in Arabidopsis thaliana also showed increased susceptibility to the green peach aphid (Myzus persicae), indicating that the role α-DOX is conserved in other plant-aphid interactions.

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Stepwise Exposure of Staphylococcus aureus to Pleuromutilins Is Associated with Stepwise Acquisition of Mutations in rplC and Minimally Affects Susceptibility to Retapamulin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01066-06 ◽

2007 ◽

Vol 51 (6) ◽

pp. 2048-2052 ◽

Cited By ~ 51

Author(s):

Daniel R. Gentry ◽

Stephen F. Rittenhouse ◽

Lynn McCloskey ◽

David J. Holmes

Keyword(s):

Staphylococcus Aureus ◽

Second Step ◽

Wild Type ◽

Fast Growing ◽

Growth Defects ◽

Mutational Step ◽

Drug Concentrations ◽

Negative Effect ◽

Ribosomal Protein L3 ◽

Severe Growth

ABSTRACT To assess their effects on susceptibility to retapamulin in Staphylococcus aureus, first-, second-, and third-step mutants with elevated MICs to tiamulin and other investigational pleuromutilin compounds were isolated and characterized through exposure to high drug concentrations. All first- and second-step mutations were in rplC, encoding ribosomal protein L3. Most third-step mutants acquired a third mutation in rplC. While first- and second-step mutations did cause an elevation in tiamulin and retapamulin MICs, a significant decrease in activity was not seen until a third mutation was acquired. All third-step mutants exhibited severe growth defects, and faster-growing variants arose at a high frequency from most isolates. These faster-growing variants were found to be more susceptible to pleuromutilins. In the case of a mutant with three alterations in rplC, the fast-growing variants acquired an additional mutation in rplC. In the case of fast-growing variants of isolates with two mutations in rplC and at least one mutation at an unmapped locus, one of the two rplC mutations reverted to wild type. These data indicate that mutations in rplC that lead to pleuromutilin resistance have a direct, negative effect on fitness. While reduction in activity of retapamulin against S. aureus can be seen through mutations in rplC, it is likely that target-specific resistance to retapamulin will be slow to emerge due to the need for three mutations for a significant effect on activity and the fitness cost of each mutational step.

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Mutations at pipX Suppress Lethality of PII-Deficient Mutants of Synechococcus elongatus PCC 7942

Journal of Bacteriology ◽

10.1128/jb.00557-09 ◽

2009 ◽

Vol 191 (15) ◽

pp. 4863-4869 ◽

Cited By ~ 17

Author(s):

Javier Espinosa ◽

Miguel Angel Castells ◽

Karim Boumediene Laichoubi ◽

Asunción Contreras

Keyword(s):

Molecular Basis ◽

Synechococcus Elongatus ◽

Wild Type ◽

Growth Defects ◽

Genetic Inactivation ◽

Domains Of Life ◽

Synechococcus Elongatus Pcc 7942 ◽

Pcc 7942 ◽

Severe Growth ◽

Null Mutants

ABSTRACT The PII proteins are found in all three domains of life as key integrators of signals reflecting the balance of nitrogen and carbon. Genetic inactivation of PII proteins is typically associated with severe growth defects or death. However, the molecular basis of these defects depends on the specific functions of the proteins with which PII proteins interact to regulate nitrogen metabolism in different organisms. In Synechococcus elongatus PCC 7942, where PII forms complexes with the NtcA coactivator PipX, attempts to engineer PII-deficient strains failed in a wild-type background but were successful in pipX null mutants. Consistent with the idea that PII is essential to counteract the activity of PipX, four different spontaneous mutations in the pipX gene were found in cultures in which glnB had been genetically inactivated.

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Ric1p and the Ypt6p GTPase Function in a Common Pathway Required for Localization of Trans-Golgi Network Membrane Proteins

Molecular Biology of the Cell ◽

10.1091/mbc.12.1.13 ◽

2001 ◽

Vol 12 (1) ◽

pp. 13-26 ◽

Cited By ~ 58

Author(s):

Eric S. Bensen ◽

Bonny G. Yeung ◽

Gregory S. Payne

Keyword(s):

Membrane Proteins ◽

Protein Localization ◽

Rab Gtpase ◽

Temperature Sensitive ◽

Common Pathway ◽

Multicopy Suppressor ◽

Trans Golgi Network ◽

Growth Defects ◽

Golgi Network ◽

Severe Growth

In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Δ) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Δ cells also harboringend3Δ to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes.SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Δ. Further comparison of ric1Δ and ypt6Δ cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Δ andypt6Δ cells. SLY1–20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.

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CRISPR-TSKO facilitates efficient cell type-, tissue-, or organ-specific mutagenesis in Arabidopsis

10.1101/474981 ◽

2018 ◽

Cited By ~ 5

Author(s):

Ward Decaestecker ◽

Rafael Andrade Buono ◽

Marie L. Pfeiffer ◽

Nick Vangheluwe ◽

Joris Jourquin ◽

...

Keyword(s):

Lateral Roots ◽

Cell Types ◽

Loss Of Function ◽

Cellular Functions ◽

Functional Studies ◽

Organ Specific ◽

Plant Genes ◽

Mutant Lines ◽

Functional Analyses ◽

Efficient Selection

AbstractDetailed functional analyses of many fundamentally-important plant genes via conventional loss-of-function approaches are impeded by severe pleiotropic phenotypes. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a CRISPR-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis, CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The underlying modular cloning system allows for efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens new avenues to discover and analyze gene functions in spatial and temporal contexts of plant life while avoiding pleiotropic effects of system-wide loss of gene function.

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