Small RNA-based antiviral defense in the phytopathogenic fungus Colletotrichum higginsianum

Even though the fungal kingdom contains more than 3 million species, little is known about the biological roles of RNA silencing in fungi. The Colletotrichum genus comprises fungal species that are pathogenic for a wide range of crop species worldwide. To investigate the role of RNA silencing in the ascomycete fungus Colletotrichum higginsianum, knock-out mutants affecting genes for three RNA-dependent RNA polymerase (RDR), two Dicer-like (DCL), and two Argonaute (AGO) proteins were generated by targeted gene replacement. No effects were observed on vegetative growth for any mutant strain when grown on complex or minimal media. However, Δdcl1, Δdcl1Δdcl2 double mutant, and Δago1 strains showed severe defects in conidiation and conidia morphology. Total RNA transcripts and small RNA populations were analyzed in parental and mutant strains. The greatest effects on both RNA populations was observed in the Δdcl1, Δdcl1Δdcl2, and Δago1 strains, in which a previously uncharacterized dsRNA mycovirus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)] was derepressed. Phylogenetic analyses clearly showed a close relationship between ChNRV1 and members of the segmented Partitiviridae family, despite the non-segmented nature of the genome. Immunoprecipitation of small RNAs associated with AGO1 showed abundant loading of 5'U-containing viral siRNA. C. higginsianum parental and Δdcl1 mutant strains cured of ChNRV1 revealed that the conidiation and spore morphology defects were primarily caused by ChNRV1. Based on these results, RNA silencing involving ChDCL1 and ChAGO1 in C. higginsianum is proposed to function as an antiviral mechanism

Preparation of multiplexed small RNA libraries from plants

[EN] High-throughput sequencing is a powerful tool for exploring small RNA populations in plants. The ever-increasing output from an Illumina Sequencing System allows for multiplexing multiple samples while still obtaining sufficient data for small RNA discovery and characterization. Here we describe a protocol for generating multiplexed small RNA libraries for sequencing up to 12 samples in one lane of an Illumina HiSeq System single-end, 50 base pair run. RNA ligases are used to add the 3¿ and 5¿ adaptors to purified small RNAs; ligation products that lack a small RNA molecule (adaptor-adaptor products) are intentionally depleted. After cDNA synthesis, a linear PCR step amplifies the DNA fragments. The 3¿ PCR primers used here include unique 6- nucleotide sequences to allow for multiplexing up to 12 samples.The original version of this protocol was described in Carbonell et al. (2012). The updated version of the protocol was described in Carbonell et al. (2014). This work was supported by grants from the National Science Foundation (MCB-0956526, MCB-1231726) and National Institutes of Health (AI043288)Gilbert, KB.; Fahlgren, N.; Kasschau, KD.; Chapman, EJ.; Carrington, JC.; Carbonell, A. (2014). Preparation of multiplexed small RNA libraries from plants. Bio-protocol. 4(21):1-17. https://doi.org/10.21769/BioProtoc.1275S11742

Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants

[EN] In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.We thank Goretti Nguyen for excellent technical assistance. A. C. was supported by a postdoctoral fellowship from the Ministerio de Ciencia e Innovacion (BMC-2008-0188). H.G.-R. was the recipient of a Helen Hay Whitney Postdoctoral fellowship (F-972). 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Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection

[EN] In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro.National Institutes of Health (www.nih.gov) grant AI43288 to JCC. National Science Foundation (www.nsf.gov) grant MCB-0956526 to JCC. Helen Hay Whitney (www.hhwf.org) Post-Doctoral fellowship (F-972) to HGR. USDA AFRI NIFA (www.csrees.usda.gov) Postdoctoral Fellowship (MOW-2012-01361) to NF. NSF (www.nsf.gov) Graduate Research Fellowship (DGE-1143954) to JSH Japan Society for the Promotion of Science (www.jsps.go.jp) Postdoctoral Fellowship to AT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Garcia-Ruiz, H.; Carbonell, A.; Hoyer, JS.; Fahlgren, N.; Gilbert, KB.; Takeda, A.; Giampetruzzi, A.... (2015). Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection. 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Roles and Programming of Arabidopsis ARGONAUTE Proteins During Turnip Mosaic Virus Infection

In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro

Functional Analysis of Three Arabidopsis

In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses

Functional Analysis of Three Arabidopsis ARGONAUTES Using Slicer-Defective Mutants

In RNA-directed silencing pathways, ternary complexes result from small RNA-guided ARGONAUTE (AGO) associating with target transcripts. Target transcripts are often silenced through direct cleavage (slicing), destabilization through slicer-independent turnover mechanisms, and translational repression. Here, wild-type and active-site defective forms of several Arabidopsis thaliana AGO proteins involved in posttranscriptional silencing were used to examine several AGO functions, including small RNA binding, interaction with target RNA, slicing or destabilization of target RNA, secondary small interfering RNA formation, and antiviral activity. Complementation analyses in ago mutant plants revealed that the catalytic residues of AGO1, AGO2, and AGO7 are required to restore the defects of Arabidopsis ago1-25, ago2-1, and zip-1 (AGO7-defective) mutants, respectively. AGO2 had slicer activity in transient assays but could not trigger secondary small interfering RNA biogenesis, and catalytically active AGO2 was necessary for local and systemic antiviral activity against Turnip mosaic virus. Slicer-defective AGOs associated with miRNAs and stabilized AGO-miRNA-target RNA ternary complexes in individual target coimmunoprecipitation assays. In genome-wide AGO-miRNA-target RNA coimmunoprecipitation experiments, slicer-defective AGO1-miRNA associated with target RNA more effectively than did wild-type AGO1-miRNA. These data not only reveal functional roles for AGO1, AGO2, and AGO7 slicer activity, but also indicate an approach to capture ternary complexes more efficiently for genome-wide analyses.Keywords: Antiviral defense, Micrornas, Thaliana, Binding sites, Trans acting sirnas, Directed DNA methylation, Translational repression, Interfering RNA biogenesis, Messenger RNA, GeneKeywords: Antiviral defense, Micrornas, Thaliana, Binding sites, Trans acting sirnas, Directed DNA methylation, Translational repression, Interfering RNA biogenesis, Messenger RNA, Gen

PlantCV v2: Image analysis software for high-throughput plant phenotyping

Systems for collecting image data in conjunction with computer vision techniques are a powerful tool for increasing the temporal resolution at which plant phenotypes can be measured non-destructively. Computational tools that are flexible and extendable are needed to address the diversity of plant phenotyping problems. We previously described the Plant Computer Vision (PlantCV) software package, which is an image processing toolkit for plant phenotyping analysis. The goal of the PlantCV project is to develop a set of modular, reusable, and repurposable tools for plant image analysis that are open-source and community-developed. Here we present the details and rationale for major developments in the second major release of PlantCV. In addition to overall improvements in the organization of the PlantCV project, new functionality includes a set of new image processing and normalization tools, support for analyzing images that include multiple plants, leaf segmentation, landmark identification tools for morphometrics, and modules for machine learning

Mutations in DNA polymerase δ subunit 1 co-segregate with CMD2-type resistance to Cassava Mosaic Geminiviruses

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses

Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection

In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro