RNAi silencing: A tool for functional genomics research on fungi

Armas-Tizapantzi, Anahí; Montiel-González, Alba Mónica

doi:10.1016/j.fbr.2016.05.003

Cited by 7 publications

(3 citation statements)

References 96 publications

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“…This phenomenon was named RNAi. However, RNAi in animals is equivalent to post-transcriptional gene silencing in plants or gene quelling in fungi since they all work using a series of basic components, such as Dicer, Argonaute and RNA-dependent RNA polymerase ( Fagard et al, 2000 ; Ipsaro and Joshua-Tor, 2015 ; Armas-Tizapantzi and Montiel-González, 2016 ). In the RNAi pathway, Dicer cleaves long dsRNA into short RNA duplexes of approximately 21–25 nucleotide (nt) small non-coding RNAs (sRNAs) ( Zamore et al, 2000 ; Bernstein et al, 2001 ; Chapman and Carrington, 2007 ).…”

Section: Introductionmentioning

confidence: 99%

Dicer-Like Genes Are Required for H2O2 and KCl Stress Responses, Pathogenicity and Small RNA Generation in Valsa mali

Liu

et al. 2017

Front. Microbiol.

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Valsa mali (V. mali) is the causative agent of apple tree Valsa canker, which heavily damages the production of apples in China. However, the biological roles of the RNA interfering (RNAi) pathway in the pathogenicity of V. mali remain unknown. Dicer-like proteins (DCLs) are important components that control the initiation of the RNAi pathway. In this study, VmDCL1 and VmDCL2 were isolated and functionally characterized in V. mali. VmDCL1 and VmDCL2 are orthologous in evolution to the DCLs in Cryphonectria parasitica. The deletion of VmDCL1 and VmDCL2 did not affect vegetative growth when the mutants (ΔVmDCL1, ΔVmDCL2 and ΔVmDCL1DCL2) and wild type strain 03–8 were grown on a PDA medium at 25°C in the dark. However, the colony of ΔVmDCL1 increased by 37.1% compared to the 03–8 colony in a medium containing 0.05% H2O2 3 days after inoculation, and the growth of ΔVmDCL1 was significantly inhibited in a medium containing 0.5 M KCl at a ratio of 25.7%. Meanwhile, in the presence of 0.05% H2O2, the growth of ΔVmDCL2 decreased by 34.5% compared with the growth of 03–8, but ΔVmDCL2 grew normally in the presence of 0.5 M KCl. More importantly, the expression of VmDCL2 was up-regulated 125-fold during the pathogen infection. In the infection assays using apple twigs, the pathogenicity of ΔVmDCL2 and ΔVmDCL1DCL2 was significantly reduced compared with that of 03–8 at a ratio of 24.7 and 41.3%, respectively. All defective phenotypes could be nearly rescued by re-introducing the wild type VmDCL1 and VmDCL2 alleles. Furthermore, the number and length distribution of unique small RNAs (unisRNAs) in the mutants and 03–8 were analyzed using deep sequencing. The number of unisRNAs was obviously lower in ΔVmDCL1, ΔVmDCL2 and ΔVmDCL1DCL2 than that in 03–8, and the length distribution of the sRNAs also markedly changed after the VmDCLs were deleted. These results indicated that VmDCLs function in the H2O2 and KCl stress response, pathogenicity and generation of sRNAs.

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Section: Introductionmentioning

confidence: 99%

Dicer-Like Genes Are Required for H2O2 and KCl Stress Responses, Pathogenicity and Small RNA Generation in Valsa mali

Liu

et al. 2017

Front. Microbiol.

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“…These strategies include engineering mosquitoes with (i) impaired competency to transmit pathogens, based on RNA interference (RNAi) (ii) lethal genes, involving the release of insects carrying dominant lethal genes (RIDL) (iii) editing genes using the CRISPR/Cas9 tool to implement a gene-drive to replace the wild population. These strategies can steadily overcome the challenges imparted by mosquitoes to humans and other animals [13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Multiple dengue virus serotypes resistant transgenicAedes aegyptifitness evaluated under laboratory conditions

et al. 2020

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Dengue viruses (DENV) are the wildest transmitted arbovirus members of the family Flaviviridae, genus Flavivirus. Dengue viruses are composed of four serotypes, DENV1, 2, 3, and 4, and these viruses can cause dengue fever and dengue haemorrhagic fever or dengue shock syndrome, when infecting humans. RNA interference (RNAi) is a self-defence mechanism, which can be used to prevent invasions of RNA viruses to the host. Genetically engineering a host with an RNAi molecule that targets a single virus serotype may develop escape mutants, and can cause unusual dominance over other serotypes. Therefore, the simultaneous targeting of multiple serotypes is necessary to block DENV transmission. Here, we report the development of transgenic Aedes aegypti based on a bioinformatically designed multiple miRshRNA (microRNA-based shRNA) DNA sequence under the control of a blood-meal induced promoter, Carboxypeptidase A, to induce RNAi for DENV in Aedes aegypti, and demonstrate the expression of a synthetic multiple shRNA polycistronic cluster having RNA interference sequences to target DENV genomes. The transgenic mosquitoes have lower rates of infection, dissemination, and transmission for DENV2 and DENV4 compared to wild mosquitoes, with a significant reduction of dengue copy number and antigen levels in the midgut. These levels of DENV were low enough to make transgenic mosquitoes stop the DENV transmission from infected host to a susceptible host and refractory to DENV2 and DENV4 infection. Such multiple resistance in Ae. aegypti has not been documented previously. Laboratory fitness measurement of transgenic Ae. aegypti showed results comparable to other reported transgenic mosquitoes.

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“…In this context, Post-Transcriptional Gene Silencing (PTGS) emerges as a powerful alternative. Since its discovery, it has been used for the targeted downregulation of genes in numerous fungal species ( Armas-Tizapantzi and Montiel-González, 2016 ; Bashyal and Aggarwal, 2016 ), including B. cinerea . Sense-antisense ( Schumacher et al, 2008 ; Patel et al, 2010 ; Giesbert et al, 2012 ; Espino et al, 2014 ) and stem-loop ( Rolland and Bruel, 2008 ; Schumacher et al, 2008 ; Giesbert et al, 2012 ; Espino et al, 2014 ) strategies have been developed to generate specific double-stranded RNA (dsRNA) in B. cinerea cells.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous Silencing of Xylanase Genes in Botrytis cinerea

García

González

et al. 2017

Front. Plant Sci.

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The endo-β-1,4-xylanase BcXyn11A is one of several plant cell-wall degrading enzymes that the phytopathogenic fungus Botrytis cinerea secretes during interaction with its hosts. In addition to its enzymatic activity, this protein also acts as an elicitor of the defense response in plants and has been identified as a virulence factor. In the present work, other four endoxylanase coding genes (Bcxyn11B, Bcxyn11C, Bcxyn10A, and Bcxyn10B) were identified in the B. cinerea genome and the expression of all five genes was analyzed by Q-RT- PCR in vitro and in planta. A cross-regulation between xylanase genes was identified analyzing their expression pattern in the ΔBcxyn11A mutant strain and a putative BcXyn11A-dependt induction of Bcxyn10B gene was found. In addition, multiple knockdown strains were obtained for the five endoxylanase genes by transformation of B. cinerea with a chimeric DNA construct composed of 50-nt sequences from the target genes. The silencing of each xylanase gene was analyzed in axenic cultures and during infection and the results showed that the efficiency of the multiple silencing depends on the growth conditions and on the cross-regulation between them. Although the simultaneous silencing of the five genes was observed by Q-RT-PCR when the silenced strains were grown on medium supplemented with tomato extract, the endoxylanase activity measured in the supernatants was reduced only by 40%. Unexpectedly, the silenced strains overexpressed the Bcxyn11A and Bcxyn11C genes during the infection of tomato leaves, making difficult the analysis of the role of the endo-β-1,4-xylanases in the virulence of the fungus.

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RNAi silencing: A tool for functional genomics research on fungi

Cited by 7 publications

References 96 publications

Dicer-Like Genes Are Required for H2O2 and KCl Stress Responses, Pathogenicity and Small RNA Generation in Valsa mali

Dicer-Like Genes Are Required for H2O2 and KCl Stress Responses, Pathogenicity and Small RNA Generation in Valsa mali

Multiple dengue virus serotypes resistant transgenicAedes aegyptifitness evaluated under laboratory conditions

Simultaneous Silencing of Xylanase Genes in Botrytis cinerea

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