scholarly journals RNA-spray-mediated silencing of Fusarium graminearum AGO and DCL genes improve barley disease resistance

2019 ◽  
Author(s):  
B Werner ◽  
FY Gaffar ◽  
J Schuemann ◽  
D Biedenkopf ◽  
A Koch
Keyword(s):  
Gene Silencing ◽  
Fusarium Graminearum ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
Plant Protection ◽  
Future Application ◽  
In Planta ◽  
Plant Host ◽  
Barley Leaves ◽  
Potential Alternative

AbstractOver the last decade, several studies have revealed the enormous potential of RNA-silencing strategies as a potential alternative to conventional pesticides for plant protection. We have previously shown that targeted gene silencing mediated by an in planta expression of non-coding inhibitory double-stranded RNAs (dsRNAs) can protect host plants against various diseases with unprecedented efficiency. In addition to the generation of RNA-silencing (RNAi) signals in planta, plants can be protected from pathogens and pests by spray-applied RNA-based biopesticides. Despite the striking efficiency of RNA-silencing-based technologies holds for agriculture, the molecular mechanisms underlying spray-induced gene silencing (SIGS) strategies are virtually unresolved, a requirement for successful future application in the field. Based on our previous work, we predict that the molecular mechanism of SIGS is controlled by the fungal-silencing machinery. In this study, we used SIGS to compare the silencing efficiencies of computationally-designed versus manually-designed dsRNA constructs targeting ARGONAUTE and DICER genes of Fusarium graminearum (Fg). We found that targeting key components of the fungal RNAi machinery via SIGS could protect barley leaves from Fg infection and that the manual design of dsRNAs resulted in higher gene-silencing efficiencies than the tool-based design. Moreover, our results indicate the possibility of cross-kingdom RNA silencing in the Fg-barley interaction, a phenomenon in which sRNAs operate as effector molecules to induce gene silencing between species from different kingdoms, such as a plant host and their interacting pathogens.

scholarly journals Study on the efficacy of dsRNAs with increasing length in RNAi-based silencing of the Fusarium CYP51 genes

2019 ◽  
Author(s):  
L Höfle ◽  
A Shrestha ◽  
B Werner ◽  
L Jelonek ◽  
A Koch
Keyword(s):  
Disease Resistance ◽  
Gene Silencing ◽  
Fungal Infection ◽  
Disease Control ◽  
Fusarium Graminearum ◽  
Rna Silencing ◽  
Transgenic Arabidopsis ◽  
Plant Protection ◽  
Field Application ◽  
Rnai Technology

AbstractPreviously, we have demonstrated that transgenic Arabidopsis and barley plants, expressing a 791 nucleotide (nt) dsRNA (CYP3RNA) that targets all three CYP51 genes (FgCYP51A, FgCYP51B, FgCYP51C) in Fusarium graminearum (Fg), inhibited fungal infection via a process designated as host-induced gene silencing (HIGS). More recently, we have shown that spray applications of CYP3RNA also protect barley from fungal infection via a process termed spray-induced gene silencing (SIGS). Thus, RNAi technology may have the potential to revolutionize plant protection in agriculture. Therefore, successful field application will require optimization of RNAi design necessary to maximize the efficacy of the RNA silencing construct for making RNAi-based strategies a realistic and sustainable approach.Previous studies indicate that silencing is correlated with the number of siRNAs generated from a dsRNA precursor. To prove the hypothesis that silencing efficiency is correlated with the number of siRNAs processed out of the dsRNA precursor, we tested in a HIGS and SIGS approach dsRNA precursors of increasing length ranging from 400 nt to 1500 nt to assess gene silencing efficiency of individual FgCYP51 genes. Concerning HIGS-mediated disease control, we found that there is no significant correlation between the length of the dsRNA precursor and the reduction of Fg infection on CYP51-dsRNA expressing Arabidopsis plants. Importantly and in clear contrast to HIGS, we measured a decrease in SIGS-mediated Fg disease resistance that significantly correlates with the length of the dsRNA construct that was sprayed, indicating that the size of the dsRNA interferes with a sufficient uptake of dsRNAs by the fungus.

scholarly journals Perturbations of a Kinase-Pseudokinase Module Activate Plant Immunity

2019 ◽  
Author(s):  
D. Patrick Bastedo ◽  
Derek Seto ◽  
Alexandre Martel ◽  
Madiha Khan ◽  
Inga Kireeva ◽  
...  
Keyword(s):  
Immune Response ◽  
Arabidopsis Thaliana ◽  
Immune Responses ◽  
Pseudomonas Syringae ◽  
Molecular Mechanisms ◽  
Kinase Domain ◽  
Host Cells ◽  
In Planta ◽  
Plant Host ◽  
Model Plant

ABSTRACTThe Pseudomonas syringae acetyltransferase HopZ1a is delivered directly into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the ZED1 pseudokinase. Here we demonstrate that HopZ1a can acetylate members of a family of ‘receptor-like cytoplasmic kinases’ (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1/ZED1/PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger immunity in planta, all independently of HopZ1a. Our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family and activating ZAR1-mediated ETI.AUTHOR SUMMARYAll plants must ward off potentially infectious microbes, and those grown in large-scale crop operations are especially vulnerable to the rapid spread of disease by successful pathogens. Although many bacteria and fungi can supress plant immune responses by producing specialized virulence proteins called ‘effectors’, these effectors can also trigger immune responses that render plants resistant to infection. We studied the molecular mechanisms underlying one such effector-triggered immune response elicited by the bacterial effector HopZ1a in the model plant host Arabidopsis thaliana. We have shown that HopZ1a promotes binding between a ZED1, a ‘pseudokinase’ required for HopZ1a-triggered immunity, and several ‘true kinases’ (known as PBLs) that are likely targets of HopZ1a activity in planta. HopZ1a-induced ZED1-PBL interactions also recruit ZAR1, an Arabidopsis ‘resistance protein’ previously implicated in HopZ1a-triggered immunity. Importantly, ZED1 mutants that restore degenerate kinase motifs can bridge interactions between PBLs and ZAR1 (independently of HopZ1a) and trigger immunity in planta. Our results suggest that equilibria between active and inactive kinase domain conformations regulate ZED1-PBL interactions and formation of ternary complexes with ZAR1. Improved models describing molecular interactions between immunity determinants, effectors and effector targets will inform efforts to exploit natural diversity for development of crops with enhanced disease resistance.

scholarly journals Perspectives of RNAi studies in plant pathogenic fungi

2016 ◽  
Vol 14 (1) ◽  
pp. 157-168
Author(s):  
Nguyễn Bảo Quốc ◽  
Nguyễn Ngọc Bảo Châu
Keyword(s):  
Rna Silencing ◽  
Molecular Mechanisms ◽  
Plant Protection ◽  
Pathogenic Fungi ◽  
Transcriptional Gene Silencing ◽  
Agricultural Industry ◽  
Global Food Security ◽  
Post Transcriptional Gene Silencing ◽  
Intrinsic Roles ◽  
Potential Applications

RNA silencing, the phenomenon known as RNA interference (RNAi), co-suppression or post-transcriptional gene silencing (PTGS) and quelling, has become more popular in studies of its intrinsic roles and applications in many organisms or of gene functions in a whole genomic scale. Since the discovery of RNA silencing more two decades ago, this powerful technology has demonstrated its applicability in developing RNAi-based drugs for various diseases in human. RNA silencing is also of interest in basic and applied studies in agriculture, especially in plant protection to create crop varieties that are resistant to biotic and abiotic stresses. This review provides an overview of RNA silencing studies in filamentous fungi, the molecular mechanisms of RNA silencing in fungi, and also describes potential applications in plant protection potentially important for the agricultural industry and for global food security.

scholarly journals Extracellular vesicles isolated from dsRNA sprayed barley plants exhibit no growth inhibition or gene silencing in Fusarium graminearum

2021 ◽  
Author(s):  
Timo Schlemmer ◽  
Richard Lischka ◽  
Dagmar Biedenkopf ◽  
Aline Koch
Keyword(s):  
Gene Silencing ◽  
Fusarium Graminearum ◽  
Extracellular Vesicles ◽  
Fungal Growth ◽  
Host Cells ◽  
Double Stranded Rna ◽  
Microtiter Plates ◽  
Barley Leaves ◽  
Or Gene

Abstract Incorporating a double-stranded RNA (dsRNA)-expressing transgene into plants or applying dsRNA by spraying it onto plant leaves successfully protects plants against invading pathogens with RNA interference (RNAi). How dsRNAs or siRNAs are transferred between donor host cells and recipient fungal cells is largely unknown It is speculated that plant extracellular vesicles (EVs) function as RNA shuttles between plants and their interacting pathogens. Recently, we found that EVs isolated from HIGS or SIGS plants contained dsRNA-derived siRNAs. In this study, we evaluated whether isolated EVs from RNA-sprayed barley ( Hordeum vulgare ) plants affected the growth of the phytopathogenic ascomycete Fusarium graminearum ( Fg ). Encouraged by our previous finding that dropping barley-derived EVs on Fg cultures caused fungal stress phenotypes, we conducted an in vitro growth experiment in microtiter plates where we co-cultivated Fg with plant EVs isolated from dsRNA-sprayed barley leaves. We observed that co-cultivation of Fg macroconidia with barley EVs did not affect fungal growth. Furthermore, plant EVs containing SIGS-derived siRNA appeared not to affect Fg growth and showed no gene silencing activity on FgCYP51 genes. We conclude that either the amount of spray-derived sRNA was insufficient to induce target gene silencing (SIGS) in Fg or Fg uptake of plant EVs from liquid cultures was inefficient or impossible.

scholarly journals Suppressors of RNA Silencing Encoded by the Components of the Cotton Leaf Curl Begomovirus-BetaSatellite Complex

2011 ◽  
Vol 24 (8) ◽  
pp. 973-983 ◽  
Author(s):  
Imran Amin ◽  
Khadim Hussain ◽  
Rashid Akbergenov ◽  
Jitender S. Yadav ◽  
Javaria Qazi ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Rna Silencing ◽  
Cotton Leaf ◽  
Small Interfering Rnas ◽  
In Planta ◽  
Suppressor Activity ◽  
Natural Defense ◽  
Or Gene ◽  
First Time ◽  
Leaf Curl

Begomoviruses (family Geminiviridae) are single-stranded DNA viruses transmitted by the whitefly Bemisia tabaci. Many economically important diseases in crops are caused by begomoviruses, particularly in tropical and subtropical environments. These include the betasatellite-associated begomoviruses causing cotton leaf curl disease (CLCuD) that causes significant losses to a mainstay of the economy of Pakistan, cotton. RNA interference (RNAi) or gene silencing is a natural defense response of plants against invading viruses. In counter-defense, viruses encode suppressors of gene silencing that allow them to effectively invade plants. Here, we have analyzed the ability of the begomovirus Cotton leaf curl Multan virus (CLCuMV) and its associated betasatellite, Cotton leaf curl Multan β-satellite (CLCuMB) which, together, cause CLCuD, and the nonessential alphasatellite (Cotton leaf curl Multan alphasatellite [CLCuMA]) for their ability to suppress gene silencing in Nicotiana benthamiana. The results showed that CLCuMV by itself was unable to efficiently block silencing. However, in the presence of the betasatellite, gene silencing was entirely suppressed. Silencing was not affected in any way when infections included CLCuMA, although the alphasatellite was, for the first time, shown to be a target of RNA silencing, inducing the production in planta of specific small interfering RNAs, the effectors of silencing. Subsequently, using a quantitative real-time polymerase chain reaction assay and Northern blot analysis, the ability of all proteins encoded by CLCuMV and CLCuMB were assessed for their ability to suppress RNAi and the relative strengths of their suppression activity were compared. The analysis showed that the V2, C2, C4, and βC1 proteins exhibited suppressor activity, with the V2 showing the strongest activity. In addition, V2, C4, and βC1 were examined for their ability to bind RNA and shown to have distinct specificities. Although each of these proteins has, for other begomoviruses or betasatellites, been previously shown to have suppressor activity, this is the first time all proteins encoded by a geminiviruses (or begomovirus-betasatellite complex) have been examined and also the first for which four separate suppressors have been identified.

scholarly journals Copy number-dependent DNA methylation of the Pyricularia oryzae MAGGY retrotransposon is triggered by DNA damage

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ba Van Vu ◽  
Quyet Nguyen ◽  
Yuki Kondo-Takeoka ◽  
Toshiki Murata ◽  
Naoki Kadotani ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Copy Number ◽  
Rna Silencing ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Pyricularia Oryzae ◽  
Transcriptional Gene Silencing ◽  
Physical Interaction ◽  
Uncharacterized Protein ◽  
Form Complex

AbstractTransposable elements are common targets for transcriptional and post-transcriptional gene silencing in eukaryotic genomes. However, the molecular mechanisms responsible for sensing such repeated sequences in the genome remain largely unknown. Here, we show that machinery of homologous recombination (HR) and RNA silencing play cooperative roles in copy number-dependent de novo DNA methylation of the retrotransposon MAGGY in the fungusPyricularia oryzae. Genetic and physical interaction studies revealed thatRecAdomain-containing proteins, includingP. oryzaehomologs ofRad51, Rad55, andRad57, together with an uncharacterized protein, Ddnm1, form complex(es) and mediate either the overall level or the copy number-dependence of de novo MAGGY DNA methylation, likely in conjunction with DNA repair. Interestingly,P. oryzaemutants of specific RNA silencing components (MoDCL1andMoAGO2)were impaired in copy number-dependence of MAGGY methylation. Co-immunoprecipitation of MoAGO2 and HR components suggested a physical interaction between the HR and RNA silencing machinery in the process.

Elicitor Recognition and Signal Transduction in Plant Defense Gene Activation

1990 ◽  
Vol 45 (6) ◽  
pp. 569-575 ◽  
Author(s):  
Dierk Scheel ◽  
Jane E. Parker
Keyword(s):  
Signal Transduction ◽  
Plant Defense ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Plant Protection ◽  
Plant Cells ◽  
Defense Responses ◽  
Plant Defense Responses ◽  
Defense Genes ◽  
Plant Defense Genes

Abstract Plants defend themselves against pathogen attack by activating a whole set of defense responses, most of them relying on transcriptional activation of plant defense genes. The same responses are induced by treatment of plant cells with elicitors released from the pathogen or from the plant surface. Several plant/elicitor combinations have been used successfully as experimental systems to investigate the molecular basis of plant defense responses. Receptor-like structures on the plasma membrane of plant cells appear to bind the elicitors. Thereby, intracellular signal transduction chains are initiated which finally result in the activation of plant defense genes. A better understanding of the molecular mechanisms of early processes in plant defense responses, as provided by these studies, may in the long term help to develop environmentally safe plant protection methods for agriculture.

scholarly journals Interactions with Hosts at Cool Temperatures, Not Cold Tolerance, Explain the Unique Epidemiology of Ralstonia solanacearum Race 3 Biovar 2

2009 ◽  
Vol 99 (10) ◽  
pp. 1127-1134 ◽  
Author(s):  
Annett Milling ◽  
Fanhong Meng ◽  
Timothy P. Denny ◽  
Caitilyn Allen
Keyword(s):  
North American ◽  
Ralstonia Solanacearum ◽  
Potato Tubers ◽  
In Planta ◽  
Plant Host ◽  
Tomato Plants ◽  
North American Strain ◽  
Tropical Highlands ◽  
American Strain ◽  
Warm Temperate

Most strains of the bacterial wilt pathogen Ralstonia solanacearum are tropical, but race 3 biovar 2 (R3bv2) strains can attack plants in temperate zones and tropical highlands. The basis of this distinctive ecological trait is not understood. We compared the survival of tropical, R3bv2, and warm-temperate North American strains of R. solanacearum under different conditions. In water at 4°C, North American strains remained culturable the longest (up to 90 days), whereas tropical strains remained culturable for the shortest time (≈40 days). However, live/dead staining indicated that cells of representative strains remained viable for >160 days. In contrast, inside potato tubers, R3bv2 strain UW551 survived >4 months at 4°C, whereas North American strain K60 and tropical strain GMI1000 were undetectable after <70 days in tubers. GMI1000 and UW551 grew similarly in minimal medium at 20 and 28°C and, although both strains wilted tomato plants rapidly at 28°C, UW551 was much more virulent at 20°C, killing all inoculated plants under conditions where GMI100 killed just over half. Thus, differences among the strains in the absence of a plant host were not predictive of their behavior in planta at cooler temperatures. These data indicate that interaction with plants is required for expression of the temperate epidemiological trait of R3bv2.

scholarly journals Cytorhabdovirus P protein suppresses RISC-mediated cleavage and RNA silencing amplification in planta

Virology ◽  
2016 ◽  
Vol 490 ◽  
pp. 27-40 ◽  
Author(s):  
Krin S. Mann ◽  
Karyn N. Johnson ◽  
Bernard J. Carroll ◽  
Ralf G. Dietzgen
Keyword(s):  
Rna Silencing ◽  
In Planta ◽  
P Protein
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