scholarly journals Phytobacterial Type III Effectors HopX1, HopAB1 and HopF2 Enhance Sense-Post-Transcriptional Gene Silencing Independently of Plant R Gene-Effector Recognition

2011 ◽  
Vol 24 (8) ◽  
pp. 907-917 ◽  
Author(s):  
Panagiotis F. Sarris ◽  
Shang Gao ◽  
Konstantinos Karademiris ◽  
Hailing Jin ◽  
Kriton Kalantidis ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Pathogenic Bacteria ◽  
Fluorescent Protein ◽  
Effector Proteins ◽  
Transcriptional Gene Silencing ◽  
Type Iii ◽  
Post Transcriptional Gene Silencing ◽  
Rna Pathways ◽  
Green Fluorescent ◽  
Effector Recognition

Plant- and animal-pathogenic bacteria deploy a variable arsenal of type III effector proteins (T3EP) to manipulate host defense. Specific biochemical functions and molecular or subcellular targets have been demonstrated or proposed for a growing number of T3EP but remain unknown for the majority of them. Here, we show that transient expression of genes coding certain bacterial T3EP (HopAB1, HopX1, and HopF2), which did not elicit hypersensitive response (HR) in transgenic green fluorescent protein (GFP) Nicotiana benthamiana 16C line, enhanced the sense post-transcriptional gene silencing (S-PTGS) triggered by agrodelivery of a GFP-expressing cassette and the silencing enhancement could be blocked by two well-known viral silencing suppressors. Further analysis using genetic truncations and site-directed mutations showed that the receptor recognition domains of HopAB1 and HopX1 are not involved in enhancing silencing. Our studies provide new evidence that phytobacterial pathogen T3EP manipulate the plant small interfering RNA pathways by enhancing silencing efficiency in the absence of effector-triggered immunity signaling and suggest that phytopathogenic bacterial effectors affect host RNA silencing in yet other ways than previously described.

scholarly journals Pseudomonas syringae HrpP Is a Type III Secretion Substrate Specificity Switch Domain Protein That Is Translocated into Plant Cells but Functions Atypically for a Substrate-Switching Protein

2009 ◽  
Vol 191 (9) ◽  
pp. 3120-3131 ◽  
Author(s):  
Joanne E. Morello ◽  
Alan Collmer
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Fluorescent Protein ◽  
Plant Cells ◽  
Effector Proteins ◽  
Type Iii ◽  
Native Promoter ◽  
C Terminus ◽  
Green Fluorescent

ABSTRACT Pseudomonas syringae delivers virulence effector proteins into plant cells via an Hrp1 type III secretion system (T3SS). P. syringae pv. tomato DC3000 HrpP has a C-terminal, putative T3SS substrate specificity switch domain, like Yersinia YscP. A ΔhrpP DC3000 mutant could not cause disease in tomato or elicit a hypersensitive response (HR) in tobacco, but the HR could be restored by expression of HrpP in trans. Though HrpP is a relatively divergent protein in the T3SS of different P. syringae pathovars, hrpP from P. syringae pv. syringae 61 and P. syringae pv. phaseolicola 1448A restored HR elicitation and pathogenicity to DC3000 ΔhrpP. HrpP was translocated into Nicotiana benthamiana cells via the DC3000 T3SS when expressed from its native promoter, but it was not secreted in culture. N- and C-terminal truncations of HrpP were tested for their ability to be translocated and to restore HR elicitation activity to the ΔhrpP mutant. No N-terminal truncation completely abolished translocation, implying that HrpP has an atypical T3SS translocation signal. Deleting more than 20 amino acids from the C terminus abolished the ability to restore HR elicitation. HrpP fused to green fluorescent protein was no longer translocated but could restore HR elicitation activity to the ΔhrpP mutant, suggesting that translocation is not essential for the function of HrpP. No T3SS substrates were detectably secreted by DC3000 ΔhrpP except the pilin subunit HrpA, which unexpectedly was secreted poorly. HrpP may function somewhat differently than YscP because the P. syringae T3SS pilus likely varies in length due to differing plant cell walls.

scholarly journals A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing

2000 ◽  
Vol 81 (8) ◽  
pp. 2103-2109 ◽  
Author(s):  
Fuh-Jyh Jan ◽  
Carmen Fagoaga ◽  
Sheng-Zhi Pang ◽  
Dennis Gonsalves
Keyword(s):  
Gene Silencing ◽  
Virus Resistance ◽  
Fluorescent Protein ◽  
Gene Segment ◽  
N Gene ◽  
Transcriptional Gene Silencing ◽  
Multiple Resistance ◽  
Cp Gene ◽  
Transgenic Lines ◽  
Green Fluorescent

We showed previously that 218 and 110 bp N gene segments of tomato spotted wilt virus (TSWV) that were fused to the non-target green fluorescent protein (GFP) gene were able to confer resistance to TSWV via post-transcriptional gene silencing (PTGS). N gene segments expressed alone did not confer resistance. Apparently, the GFP DNA induced PTGS that targetted N gene segments and the incoming homologous TSWV for degradation, resulting in a resistant phenotype. These observations suggested that multiple resistance could be obtained by replacing the GFP DNA with a viral DNA that induces PTGS. The full-length coat protein (CP) gene of turnip mosaic virus (TuMV) was linked to 218 or 110 bp N gene segments and transformed into Nicotiana benthamiana. A high proportion (4 of 18) of transgenic lines with the 218 bp N gene segment linked to the TuMV CP gene were resistant to both viruses, and resistance was transferred to R2 plants. Nuclear run-on and Northern experiments confirmed that resistance was via PTGS. In contrast, only one of 14 transgenic lines with the TuMV CP linked to a 110 bp N gene segment yielded progeny with multiple resistance. Only a few R1 plants were resistant and resistance was not observed in R2 plants. These results clearly show the applicability of multiple virus resistance through the fusion of viral segments to DNAs that induce PTGS.

scholarly journals Exploring resveratrol dimers as virulence blocking agents – Attenuation of type III secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Charlotta Sundin ◽  
Caroline E. Zetterström ◽  
Duc Duy Vo ◽  
Robert Brkljača ◽  
Sylvia Urban ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Bacterial Infections ◽  
Pathogenic Bacteria ◽  
Yersinia Pseudotuberculosis ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Resistant Bacteria ◽  
Type Iii ◽  
Green Fluorescent

AbstractBacterial infections continue to threaten humankind and the rapid spread of antibiotic resistant bacteria is alarming. Current antibiotics target essential bacterial processes and thereby apply a strong selective pressure on pathogenic and non-pathogenic bacteria alike. One alternative strategy is to block bacterial virulence systems that are essential for the ability to cause disease but not for general bacterial viability. We have previously show that the plant natural product (-)-hopeaphenol blocks the type III secretion system (T3SS) in the Gram-negative pathogens Yersinia pseudotuberculosis and Pseudomonas aeruginosa. (-)-Hopeaphenol is a resveratrol tetramer and in the present study we explore various resveratrol dimers, including partial structures of (-)-hopeaphenol, as T3SS inhibitors. To allow rapid and efficient assessment of T3SS inhibition in P. aeruginosa, we developed a new screening method by using a green fluorescent protein reporter under the control of the ExoS promoter. Using a panel of assays we showed that compounds with a benzofuran core structure i.e. viniferifuran, dehydroampelopsin B, anigopreissin A, dehydro-δ-viniferin and resveratrol-piceatannol hybrid displayed significant to moderate activities towards the T3SS in Y. pseudotuberculosis and P. aeruginosa.

scholarly journals Mastrevirus Rep and RepA Proteins Suppress de novo Transcriptional Gene Silencing

2021 ◽  
Vol 22 (21) ◽  
pp. 11462
Author(s):  
Kikyo Watanabe ◽  
Masashi Ugaki
Keyword(s):  
Gene Silencing ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
De Novo ◽  
Transcriptional Gene Silencing ◽  
35S Promoter ◽  
Gene Encoding ◽  
Suppressor Activity ◽  
Yellow Dwarf Virus ◽  
Green Fluorescent

Transcriptional gene silencing (TGS) in plants is a defense mechanism against DNA virus infection. The genomes of viruses in the Geminiviridae family encode several TGS suppressors. In this study, we induced de novo TGS against the transgenic GFP gene encoding green fluorescent protein by expressing a hairpin-shaped self-complementary RNA corresponding to the enhancer region of the 35S promoter (hpE35S). In addition, we examined the TGS suppression activity of proteins encoded in the genome of Tobacco yellow dwarf virus (TYDV, genus Mastrevirus). The results show that the replication-associated protein (Rep) and RepA encoded by TYDV have TGS suppressor activity and lead to decreased accumulation of 24-nt siRNAs. These results suggest that Rep and RepA can block the steps before the loading of siRNAs into Argonaute (AGO) proteins. This is the first report of TGS suppressors in the genus Mastrevirus.

scholarly journals Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts

2021 ◽  
Author(s):  
Johannes W. Debler ◽  
Bernadette M. Henares ◽  
Robert C. Lee
Keyword(s):  
Broad Spectrum ◽  
Faba Bean ◽  
Fluorescent Protein ◽  
Transient Gene Expression ◽  
Low Frequency ◽  
Effector Proteins ◽  
Field Pea ◽  
Grain Legume ◽  
Pathogen Effectors ◽  
Green Fluorescent

Abstract Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.

scholarly journals Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1187
Author(s):  
Michael Wassenegger ◽  
Athanasios Dalakouras
Keyword(s):  
Dna Methylation ◽  
Rna Interference ◽  
Gene Silencing ◽  
Plant Cells ◽  
Transcriptional Gene Silencing ◽  
Rnai Machinery ◽  
Double Stranded Rna ◽  
Post Transcriptional Gene Silencing ◽  
Cumulative Amount

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

scholarly journals Epigenetic Changes in Host Ribosomal DNA Promoter Induced by an Asymptomatic Plant Virus Infection

Biology ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 91 ◽  
Author(s):  
Miryam Pérez-Cañamás ◽  
Elizabeth Hevia ◽  
Carmen Hernández
Keyword(s):  
Gene Silencing ◽  
Ribosomal Dna ◽  
Plant Virus ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Methylation Pattern ◽  
Transcriptional Gene Silencing ◽  
Post Transcriptional Gene Silencing ◽  
The Impact

DNA cytosine methylation is one of the main epigenetic mechanisms in higher eukaryotes and is considered to play a key role in transcriptional gene silencing. In plants, cytosine methylation can occur in all sequence contexts (CG, CHG, and CHH), and its levels are controlled by multiple pathways, including de novo methylation, maintenance methylation, and demethylation. Modulation of DNA methylation represents a potentially robust mechanism to adjust gene expression following exposure to different stresses. However, the potential involvement of epigenetics in plant-virus interactions has been scarcely explored, especially with regard to RNA viruses. Here, we studied the impact of a symptomless viral infection on the epigenetic status of the host genome. We focused our attention on the interaction between Nicotiana benthamiana and Pelargonium line pattern virus (PLPV, family Tombusviridae), and analyzed cytosine methylation in the repetitive genomic element corresponding to ribosomal DNA (rDNA). Through a combination of bisulfite sequencing and RT-qPCR, we obtained data showing that PLPV infection gives rise to a reduction in methylation at CG sites of the rDNA promoter. Such a reduction correlated with an increase and decrease, respectively, in the expression levels of some key demethylases and of MET1, the DNA methyltransferase responsible for the maintenance of CG methylation. Hypomethylation of rDNA promoter was associated with a five-fold augmentation of rRNA precursor levels. The PLPV protein p37, reported as a suppressor of post-transcriptional gene silencing, did not lead to the same effects when expressed alone and, thus, it is unlikely to act as suppressor of transcriptional gene silencing. Collectively, the results suggest that PLPV infection as a whole is able to modulate host transcriptional activity through changes in the cytosine methylation pattern arising from misregulation of methyltransferases/demethylases balance.

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