Final Report Summary - SMALLRNATRANSFER (Intergenomic Relationships during plant-pathogen interactions)
In nature, plants interact with a wide range of microorganisms, such as fungi, viruses and bacteria. When the outcome of this interaction is detrimental, they produce plant disease. A perpetual arms race between host and pathogens has driven the evolution of host defensive and pathogen counter-defensive strategies. A first layer of plant defense involves pathogen recognition by plant receptors. In turn, pathogens counter-defense producing effector proteins that interfere and manipulate host defense pathways, causing disease. In response to this, plants activate and produce a specific and potent defensive strategy based on the production of resistance (R) proteins. These strategies have long been considered protein-based. However, recent studies indicate that small RNAs are crucial for this battle. Although antiviral defense mediated by virus-derived small RNAs has been well studied in both plants and animal systems, the role of plant endogenous small RNAs during interaction with bacterial and fungal pathogens has recently come to light.
Small RNAs are short pieces of RNA that are anywhere from 20-25 base pairs in length. They are key regulators of gene expression by guiding transcriptional and post-transcriptional gene silencing activities. Among the components of the silencing process, Argonaute (AGO) proteins are the core constituents of the silencing effector complex. Small RNAs have been found mobile in organisms. In this project we hypothesized that small RNA, or their precursors, can move and function across the plant-pathogen interface as effector proteins do.
Our main goal was to decipher if reciprocal transfer of small RNAs between species occurs in the fungal-plant interaction, and to understand the biological significance of such transfer. To accomplish these objectives, we took advantage of the high-throughput sequencing technology, and methods to identify small RNAs associated with AGO proteins. As the interaction of the fungus Colletotrichum higginsianum with the Arabidopsis thaliana plant is well-studied, and both pathogen and host have complete genome sequences, we used this model to reach our goals. However, although small RNAs have been well-characterized in both plants and animals, very little was known in fungi.
Achievements: First, we obtained small RNAs libraries from Arabidopsis plants infected with C. higginsianum. By informatics analysis we noticed that fungal small RNAs were present in a very low proportion. Furthermore, deeper analysis revealed that these fungal reads did not follow the criteria to be bona fide small RNAs. To enrich our libraries with fungal reads, we obtained small RNA libraries from fungal mycelia grown in the absence of the plant. Again, our attempts to detect bona fide small RNAs were unsuccessful. Despite our negative results, the components of the silencing machinery, namely two AGOs, two DICER and three RNA-depedent RNA polymerases, were identified in the C. higginsianum genome. We confirmed their expression in mycelia and plant-infected tissues, suggesting fungal silencing activity.
To better characterize the C. higginsianum RNA silencing pathway, we generated a collection of gene silencing-defective as well as epitope-tagged AGO1 and AGO2 fungal strains. Identification of small RNAs directly bound to AGO proteins has proven a tool to confidently identify small RNAs. Phenotypic analysis revealed that Δago1 and Δdcl1 showed severe defects on conidiation (production of asexual spores) and conidia morphology. Genome-wide analysis of small RNAs and RNA transcripts was done in mutant and parental strains. The greatest effect on both populations was observed in Δago1 and Δdcl1 fungal mutants, which showed abundance of reads with no homology to any genome. Through transcriptomic analysis, we demonstrated that this effect was due to a de-repression of a previously uncharacterized resident virus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)]. Phylogenetic analysis showed a clear relationship to members of the Partitiviridae family despite the non-segmented nature of its genome. Immunoprecipitation of small RNAs bound to fungal AGO1 showed abundant loading of viral small RNAs, indicating antiviral activity against ChNRV1. C. higginsianum Δdcl1 strains cured of the virus were normal, indicating that the phenotypical defects previously observed in parental Δdcl1 strains were mainly caused by viral de-repression, rather than silencing endogenous activities.
Viral presence in C. higginisianum parental strains showed no negative effects in growth, conidiation and germination when grown in the absence of the plant. Conversely, viral-containing fungal strains were slightly less pathogenic in Arabidopsis than those cured of the virus. Both strains activated the expression of plant defense genes at the same level, suggesting that viral-induced decrease in pathogenesis was no related to suppression of plant host immunity.
In this project we have generated novel and valuable information in the study of RNA silencing in fungi, genome evolution and regulatory mechanisms involved in the plant-fungal interactions.
Our findings will now inspire more research efforts in the plant/fungus/virus interaction area. Future research activities will explore the impact of viral small RNA in plant host targets. It remains to be discovered if viral small RNAs act as signaling molecules during pathogenesis. Tools generated in this project will be useful for basic and applied research and will open up new avenues for the understanding of resistance mechanisms in plants. Future applications from these research activities should finally help to define new strategies to improve disease resistance in plants that will also benefit breeding programs for crop protection.
Small RNAs are short pieces of RNA that are anywhere from 20-25 base pairs in length. They are key regulators of gene expression by guiding transcriptional and post-transcriptional gene silencing activities. Among the components of the silencing process, Argonaute (AGO) proteins are the core constituents of the silencing effector complex. Small RNAs have been found mobile in organisms. In this project we hypothesized that small RNA, or their precursors, can move and function across the plant-pathogen interface as effector proteins do.
Our main goal was to decipher if reciprocal transfer of small RNAs between species occurs in the fungal-plant interaction, and to understand the biological significance of such transfer. To accomplish these objectives, we took advantage of the high-throughput sequencing technology, and methods to identify small RNAs associated with AGO proteins. As the interaction of the fungus Colletotrichum higginsianum with the Arabidopsis thaliana plant is well-studied, and both pathogen and host have complete genome sequences, we used this model to reach our goals. However, although small RNAs have been well-characterized in both plants and animals, very little was known in fungi.
Achievements: First, we obtained small RNAs libraries from Arabidopsis plants infected with C. higginsianum. By informatics analysis we noticed that fungal small RNAs were present in a very low proportion. Furthermore, deeper analysis revealed that these fungal reads did not follow the criteria to be bona fide small RNAs. To enrich our libraries with fungal reads, we obtained small RNA libraries from fungal mycelia grown in the absence of the plant. Again, our attempts to detect bona fide small RNAs were unsuccessful. Despite our negative results, the components of the silencing machinery, namely two AGOs, two DICER and three RNA-depedent RNA polymerases, were identified in the C. higginsianum genome. We confirmed their expression in mycelia and plant-infected tissues, suggesting fungal silencing activity.
To better characterize the C. higginsianum RNA silencing pathway, we generated a collection of gene silencing-defective as well as epitope-tagged AGO1 and AGO2 fungal strains. Identification of small RNAs directly bound to AGO proteins has proven a tool to confidently identify small RNAs. Phenotypic analysis revealed that Δago1 and Δdcl1 showed severe defects on conidiation (production of asexual spores) and conidia morphology. Genome-wide analysis of small RNAs and RNA transcripts was done in mutant and parental strains. The greatest effect on both populations was observed in Δago1 and Δdcl1 fungal mutants, which showed abundance of reads with no homology to any genome. Through transcriptomic analysis, we demonstrated that this effect was due to a de-repression of a previously uncharacterized resident virus [termed Colletotrichum higginsianum non-segmented dsRNA virus 1 (ChNRV1)]. Phylogenetic analysis showed a clear relationship to members of the Partitiviridae family despite the non-segmented nature of its genome. Immunoprecipitation of small RNAs bound to fungal AGO1 showed abundant loading of viral small RNAs, indicating antiviral activity against ChNRV1. C. higginsianum Δdcl1 strains cured of the virus were normal, indicating that the phenotypical defects previously observed in parental Δdcl1 strains were mainly caused by viral de-repression, rather than silencing endogenous activities.
Viral presence in C. higginisianum parental strains showed no negative effects in growth, conidiation and germination when grown in the absence of the plant. Conversely, viral-containing fungal strains were slightly less pathogenic in Arabidopsis than those cured of the virus. Both strains activated the expression of plant defense genes at the same level, suggesting that viral-induced decrease in pathogenesis was no related to suppression of plant host immunity.
In this project we have generated novel and valuable information in the study of RNA silencing in fungi, genome evolution and regulatory mechanisms involved in the plant-fungal interactions.
Our findings will now inspire more research efforts in the plant/fungus/virus interaction area. Future research activities will explore the impact of viral small RNA in plant host targets. It remains to be discovered if viral small RNAs act as signaling molecules during pathogenesis. Tools generated in this project will be useful for basic and applied research and will open up new avenues for the understanding of resistance mechanisms in plants. Future applications from these research activities should finally help to define new strategies to improve disease resistance in plants that will also benefit breeding programs for crop protection.