| 1. |
- Lukhovitskaya, Nina, et al.
(författare)
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A viral transcription factor exhibits antiviral RNA silencing suppression activity independent of its nuclear localization
- 2014
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Ingår i: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 95, s. 2831-2837
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Tidskriftsartikel (refereegranskat)abstract
- Viral suppressors of RNA silencing (VSRs) are critical for the success of virus infection and efficient accumulation of virus progeny. The chrysanthemum virus B p12 protein acts as a transcription factor to regulate cell size and proliferation favourable for virus infection. Here, we showed that the p12 protein suppressed RNA silencing and was able to complement a VSR-deficient unrelated virus. Moreover, p12 counter-silencing activity could be uncoupled from its function as a transcription factor in the nucleus. The altered p12 protein, which lacked a nuclear localization signal and was not imported into the nucleus, was able to suppress RNA silencing as efficiently as the native protein. The data revealed new aspects of p12 functioning and identified a novel role for this viral zinc-finger transcription factor. The results provided a general insight into one of the activities of the p12 protein, which appeared to possess more than one function.
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| 2. |
- Ramesh, Vetukuri, et al.
(författare)
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Can silencing of transposons contribute to variation in effector gene expression in Phytophthora infestans?
- 2012
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Ingår i: Landes Bioscience. - : Informa UK Limited. ; 2, s. 110-114
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Tidskriftsartikel (refereegranskat)abstract
- Transposable elements are ubiquitous residents in eukaryotic genomes. Often considered to be genomic parasites, they can lead to dramatic changes in genome organization, gene expression, and gene evolution. The oomycete plant pathogen Phytophthora infestans has evolved a genome organization where core biology genes are predominantly located in genome regions that have relatively few resident transposons. In contrast, disease effector-encoding genes are most frequently located in rapidly evolving genomic regions that are rich in transposons. P. infestans, as a eukaryote, likely uses RNA silencing to minimize the activity of transposons. We have shown that fusion of a short interspersed element (SINE) to an effector gene in P. infestans leads to the silencing of both the introduced fusion and endogenous homologous sequences. This is also likely to occur naturally in the genome of P. infestans, as transcriptional inactivation of effectors is known to occur, and over half of the translocated “RXLR class” of effectors are located within 2 kb of transposon sequences in the P. infestans genome. In this commentary, we review the diverse transposon inventory of P. infestans, its control by RNA silencing, and consequences for expression modulation of nearby effector genes in this economically important plant pathogen.
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| 3. |
- Ramesh, Vetukuri, et al.
(författare)
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Evidence for involvement of Dicer-like, Argonaute and histone deacetylase proteins in gene silencing in Phytophthora infestans
- 2011
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Ingår i: Molecular Plant Pathology. - 1464-6722 .- 1364-3703. ; 12, s. 772-785
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Tidskriftsartikel (refereegranskat)abstract
- Gene silencing may have a direct or indirect impact on many biological processes in eukaryotic cells, and is a useful tool for the determination of the roles of specific genes. In this article, we report silencing in Phytophthora infestans, an oomycete pathogen of potato and tomato. Gene silencing is known to occur in P. infestans, but its genetic basis has yet to be determined. Genes encoding the major components of the RNA interference (RNAi) pathway, Dicer-like (Pidcl1), Argonaute (Piago1-5) and RNA-directed RNA polymerase (Pirdr1), were identified in the P. infestans genome by comparative genomics, together with families of other genes potentially involved in gene silencing, such as histone deacetylases, histone methyltransferases, DEAD heli-cases, chromodomain proteins and a class 1 RNaseIII. Real-time reverse transcription-polymerase chain reaction demonstrated transcript accumulation for all candidate genes throughout the asexual lifecycle and plant infection, but at different levels of mRNA abundance. A functional assay was developed in which silencing of the sporulation-associated Picdc14 gene was released by the treatment of protoplasts with in vitro-synthesized double-stranded RNAs homologous to Pidcl1, Piago1/2 and histone deacetylase Pihda1. These results suggest that the components of gene silencing, namely Dicer-like, Argonaute and histone deacetylase, are functional in P. infestans. Our data demonstrate that this oomycete possesses canonical gene silencing pathways similar to those of other eukaryotes.
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| 4. |
- Ramesh, Vetukuri, et al.
(författare)
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Evidence for Small RNAs Homologous to Effector-Encoding Genes and Transposable Elements in the Oomycete Phytophthora infestans
- 2012
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:12, s. e51399-
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Tidskriftsartikel (refereegranskat)abstract
- Phytophthora infestans is the oomycete pathogen responsible for the devastating late blight disease on potato and tomato. There is presently an intense research focus on the role(s) of effectors in promoting late blight disease development. However, little is known about how they are regulated, or how diversity in their expression may be generated among different isolates. Here we present data from investigation of RNA silencing processes, characterized by non-coding small RNA molecules (sRNA) of 19-40 nt. From deep sequencing of sRNAs we have identified sRNAs matching numerous RxLR and Crinkler (CRN) effector protein genes in two isolates differing in pathogenicity. Effector gene-derived sRNAs were present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Small RNAs in P. infestans grouped into three clear size classes of 21, 25/26 and 32 nt. Small RNAs from all size classes mapped to RxLR effector genes, but notably 21 nt sRNAs were the predominant size class mapping to CRN effector genes. Some effector genes, such as PiAvr3a, to which sRNAs were found, also exhibited differences in transcript accumulation between the two isolates. The P. infestans genome is rich in transposable elements, and the majority of sRNAs of all size classes mapped to these sequences, predominantly to long terminal repeat (LTR) retrotransposons. RNA silencing of Dicer and Argonaute genes provided evidence that generation of 21 nt sRNAs is Dicer-dependent, while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, we identified six microRNA (miRNA) candidates from our sequencing data, their precursor sequences from the genome sequence, and target mRNAs. These miRNA candidates have features characteristic of both plant and metazoan miRNAs.
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| 5. |
- Ramesh, Vetukuri
(författare)
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Gene silencing mechanisms in Phytophthora infestans
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The hemibiotrophic oomycete, Phytophthora infestans, causes late blight on potato and tomato. This destructive pathogen is well known for the rapidity with which it overcomes host resistance. This thesis focuses on the molecular basis of gene (RNA) silencing in P. infestans and the role it may have in its genome biology and pathogenesis. A comparative genomic approach identified 51 genes from nine gene families, encoding Dicer-like (PiDcl1), Argonaute (PiAgo), RNA-dependent RNA polymerase (PiRdr1), Histone eacetylases, histone methyltransferases, DEAD helicases, chromodomain proteins,and a class 1 RNaseIII with a possible role in gene silencing. It was shown by knockdown of PiDcl1, PiAgo1, and PiHda1 (Histone deacetylase) expression, that these components were involved in maintaining transcriptional (heterochromatic) silencing in P. infestans. The large genome of P. infestans (~ 240 Mb) is composed of approximately 75% transposons and repeats. The genome has a bimodal architecture, consisting of a highly conserved and tightly packed core genome that is interrupted by transposons and repeats. The genes encoding the RxLR and Crinkler (CRN) effector classes that are critical to plant infection are predominantly located in the repeat and transposon rich regions. Small RNAs (sRNA) of 40 nt, homologous to a non-autonomous short interspersed retrotransposable element infSINEm were found. A sense orientation transcriptional fusion of infSINEm to the PiAvr3a (RxLR) effector gene led to silencing of both the introduced fusion and endogenous homologous sequences. Hence, it was concluded that P. infestans likely uses RNA silencing to minimize the activity of transposons and might influence expression of effector encoding genes in close proximity. Deep sequencing of sRNA from different life cycle stages of two P. infestans isolates, that differ in their disease causing abilities, revealed 21, 25/26, and 32 nt size classes of sRNAs that were predominantly derived from transposons. Effector gene-derived sRNAs were also present in both isolates, but exhibited marked differences in abundance, especially for CRN effectors. Knockdown of Dicer and Argonaute gene expression provided evidence that biogenesis of 21 nt sRNAs is Dicerdependent,while accumulation of longer sRNAs was impacted by silencing of Argonaute genes. Additionally, six miRNA candidates, and sRNAs that mapped to mitochondrial, tRNA and genomic hotspots were identified. sRNAs and RNA silencing in P. infestans have features characteristic of both plants and animals.
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| 6. |
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| 7. |
- Ramesh, Vetukuri, et al.
(författare)
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Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1
- 2010
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 107, s. 9909-9914
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Tidskriftsartikel (refereegranskat)abstract
- Fungal and oomycete plant pathogens translocate effector proteins into host cells to establish infection. However, virulence targets and modes of action of their effectors are unknown. Effector AVR3a from potato blight pathogen Phytophthora infestans is translocated into host cells and occurs in two forms: AVR3a(KI), which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3a(EM), which evades recognition by R3a, weakly suppresses host ICD. Here we show that AVR3a interacts with and stabilizes host U-box E3 ligase CMPG1, which is required for ICD. In contrast, AVR3a(KI/Y147del), a mutant with a deleted C-terminal tyrosine residue that fails to suppress ICD, cannot interact with or stabilize CMPG1. CMPG1 is stabilized by the inhibitors MG132 and epoxomicin, indicating that it is degraded by the 26S proteasome. CMPG1 is degraded during ICD. However, it is stabilized by mutations in the U-box that prevent its E3 ligase activity. In stabilizing CMPG1, AVR3a thus modifies its normal activity. Remarkably, given the potential for hundreds of effector genes in the P. infestans genome, silencing Avr3a compromises P. infestans pathogenicity, suggesting that AVR3a is essential for virulence. Interestingly, Avr3a silencing can be complemented by in planta expression of Avr3a(KI) or Avr3a(EM) but not the Avr3a(KI/Y147del) mutant. Our data provide genetic evidence that AVR3a is an essential virulence factor that targets and stabilizes the plant E3 ligase CMPG1, potentially to prevent host cell death during the biotrophic phase of infection.
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| 8. |
- Ramesh, Vetukuri, et al.
(författare)
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Silencing of the PiAtvr3a effector-encoding gene from Phytophthora infestans by transcriptional fusion to a short interspersed element
- 2011
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Ingår i: Fungal Biology. - : Elsevier BV. - 1878-6146. ; 115, s. 1225-1233
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Tidskriftsartikel (refereegranskat)abstract
- Phytophthora infestans is the notorious oomycete causing late blight of potato and tomato. A large proportion of the P. infestans genome is composed of transposable elements, the activity of which may be controlled by RNA silencing. Accumulation of small RNAs is one of the hallmarks of RNA silencing. Here we demonstrate the presence of small RNAs corresponding to the sequence of a short interspersed retrotransposable element (SINE) suggesting that small RNAs might be involved in silencing of SINEs in P. infestans. This notion was exploited to develop novel tools for gene silencing in P. infestans by engineering transcriptional fusions of the PiAvr3a gene, encoding an RXLR avirulence effector, to the infSINEm retroelement. Transgenic P. infestans lines expressing either 5'-infSINEm::PiAvr3a-3' or 5'-PiAvr3a::SINEm-3' chimeric transcripts initially exhibited partial silencing of PiAvr3a. Over time, PiAvr3a either recovered wild type transcript levels in some lines, or became fully silenced in others. Introduction of an inverted repeat construct was also successful in yielding P. infestans transgenic lines silenced for PiAvr3a. In contrast, constructs expressing antisense or aberrant RNA transcripts failed to initiate silencing of PiAvr3a. Lines exhibiting the most effective silencing of PiAvr3a were either weakly or non-pathogenic on susceptible potato cv. Bintje. This study expands the repertoire of reverse genetics tools available for P. infestans research, and provides insights into a possible mode of variation in effector expression through spread of silencing from adjacent retroelements. Crown Copyright (C) 2011 Published by Elsevier Ltd on behalf of The British Mycological Society. All rights reserved.
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| 9. |
- Vetukuri, Ramesh R., et al.
(författare)
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Evidence for involvement of Dicer-like, Argonaute, and Histone Deacetylase proteins in gene silencing in Phytophthora infestans
- 2011
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Ingår i: Molecular plant pathology. - : Wiley. - 1464-6722 .- 1364-3703. ; 12:8, s. 772-785
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Tidskriftsartikel (refereegranskat)abstract
- Gene silencing may have a direct or indirect impact on many biological processes in eukaryotic cells, and is a useful tool for the determination of the roles of specific genes. In this article, we report silencing in Phytophthora infestans, an oomycete pathogen of potato and tomato. Gene silencing is known to occur in P. infestans, but its genetic basis has yet to be determined. Genes encoding the major components of the RNA interference (RNAi) pathway, Dicer-like (Pidcl1), Argonaute (Piago1-5) and RNA-directed RNA polymerase (Pirdr1), were identified in the P. infestans genome by comparative genomics, together with families of other genes potentially involved in gene silencing, such as histone deacetylases, histone methyltransferases, DEAD heli-cases, chromodomain proteins and a class 1 RNaseIII. Real-time reverse transcription-polymerase chain reaction demonstrated transcript accumulation for all candidate genes throughout the asexual lifecycle and plant infection, but at different levels of mRNA abundance. A functional assay was developed in which silencing of the sporulation-associated Picdc14 gene was released by the treatment of protoplasts with in vitro-synthesized double-stranded RNAs homologous to Pidcl1, Piago1/2 and histone deacetylase Pihda1. These results suggest that the components of gene silencing, namely Dicer-like, Argonaute and histone deacetylase, are functional in P. infestans. Our data demonstrate that this oomycete possesses canonical gene silencing pathways similar to those of other eukaryotes.
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| 10. |
- Åsman, Anna, et al.
(författare)
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Fragmentation of tRNA in Phytophthora infestans asexual life cycle stages and during host plant infection
- 2014
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 14, s. 1-12
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Tidskriftsartikel (refereegranskat)abstract
- The oomycete Phytophthora infestans possesses active RNA silencing pathways, which presumably enable this plant pathogen to control the large numbers of transposable elements present in its 240 Mb genome. Small RNAs (sRNAs), central molecules in RNA silencing, are known to also play key roles in this organism, notably in regulation of critical effector genes needed for infection of its potato host.Results: To identify additional classes of sRNAs in oomycetes, we mapped deep sequencing reads to transfer RNAs (tRNAs) thereby revealing the presence of 19-40 nt tRNA-derived RNA fragments (tRFs). Northern blot analysis identified abundant tRFs corresponding to half tRNA molecules. Some tRFs accumulated differentially during infection, as seen by examining sRNAs sequenced from P. infestans-potato interaction libraries. The putative connection between tRF biogenesis and the canonical RNA silencing pathways was investigated by employing hairpin RNA-mediated RNAi to silence the genes encoding P. infestans Argonaute (PiAgo) and Dicer (PiDcl) endoribonucleases. By sRNA sequencing we show that tRF accumulation is PiDcl1-independent, while Northern hybridizations detected reduced levels of specific tRNA-derived species in the PiAgo1 knockdown line.Conclusions: Our findings extend the sRNA diversity in oomycetes to include fragments derived from non-protein-coding RNA transcripts and identify tRFs with elevated levels during infection of potato by P. infestans.
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