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Time course of trans-species miRNAs from Cuscuta campestris grown on Solanum lycopersicum

ABSTRACT: Cuscuta campestris is an obligate stem parasite which uses an organ called the haustoria to divert water and photosynthates from the host. Previously, we have identified that at the haustorial interface between Cuscuta campestris and Arabidopsis thaliana, miRNAs generated by the parasite are able to move into the host and regulate host gene expression. This study identifies how long after attachment does trans-species miRNA transcription begin in Arabidopsis thaliana and Solanum lycopersicum, as well as identifying which stage of haustoria development they become detectable. A time course was performed by harvesting interfaces every 24 hours post attachment, and samples were subjected to RNA extraction and sRNA sequencing. We have identified that in Arabidopsis thaliana, trans-species miRNAs become detectable two days post attachment. In S. lycopersicum, some trans-species miRNAs are detectable one day post attachment, but all become detectable by day 2. Secondary siRNA accumulation was detected four days post attachment in both hosts. In order to determine which stage of haustoria development trans-species miRNAs become detectable, vibratome sectioning was performed on the haustorial interfaces of both hosts. By looking at the morphology of the developing haustoria, it was determined that trans-species miRNAs become detectable during the adhesive phase. This suggests that trans-species miRNA production is one of the first steps of haustoria development, as the parasite tissue has not started to invade host tissue before they become detectable.

ORGANISM(S): Solanum lycopersicum

PROVIDER: GSE184642 | GEO | 2022/07/06

REPOSITORIES: GEO

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Time course of trans-species miRNAs from Cuscuta campestris grown on Arabidopsis thaliana

Project description:Cuscuta campestris is an obligate stem parasite which uses an organ called the haustoria to divert water and photosynthates from the host. Previously, we have identified that at the haustorial interface between Cuscuta campestris and Arabidopsis thaliana, miRNAs generated by the parasite are able to move into the host and regulate host gene expression. This study identifies how long after attachment does trans-species miRNA transcription begin in Arabidopsis thaliana and Solanum lycopersicum, as well as identifying which stage of haustoria development they become detectable. A time course was performed by harvesting interfaces every 24 hours post attachment, and samples were subjected to RNA extraction and sRNA sequencing. We have identified that in Arabidopsis thaliana, trans-species miRNAs become detectable two days post attachment. In S. lycopersicum, some trans-species miRNAs are detectable one day post attachment, but all become detectable by day 2. Secondary siRNA accumulation was detected four days post attachment in both hosts. In order to determine which stage of haustoria development trans-species miRNAs become detectable, vibratome sectioning was performed on the haustorial interfaces of both hosts. By looking at the morphology of the developing haustoria, it was determined that trans-species miRNAs become detectable during the adhesive phase. This suggests that trans-species miRNA production is one of the first steps of haustoria development, as the parasite tissue has not started to invade host tissue before they become detectable.

2022-07-06 | GSE184641 | GEO

microRNA accumulation from Cuscuta campestris in vitro haustoria

Project description:The parasitic plant Cuscuta campestris produces specialized microRNAs that are specifically expressed at the haustorial interface. Some of these "Interface-Induced MicroRNAs" function to target host mRNAs. C. campestris haustoria can be induced in the absence of any host tissues using a combination of light and physical pressure. This experiment tested with such in vitro, host-free haustoria produced interface-induced microRNAs. Small RNA-seq was performed from three different treatments: Shoot tips of C. campestris without any haustoria formation, in vitro haustoria formed in the presence of host tissue (detached leaves of Arabidopsis thaliana) and in vitro haustoria formed in the absence of any host tissue.

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Small RNAs from Cuscuta campestris parasitizing Arabidopsis thaliana

Project description:Dodders (Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bi-directional movement of viruses, proteins, and mRNAs between host and parasite, but the functional effects of these movements are not clear. Here we show that C. campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana hosts. Many of these miRNAs are 22 nts long, a usually rare size of plant miRNA associated with amplification of target silencing through secondary small interfering RNA (siRNA) production. Several A. thaliana mRNAs are targeted by C. campestris 22 nt miRNAs during parasitism, resulting in high levels of secondary siRNA production. The targeted mRNAs function in hormone perception, pathogen-defense signaling, phloem function, and stem-cell identity. Homologs of these target mRNAs from diverse plants also have high-confidence complementary sites to C. campestris miRNAs, suggesting that homologous mRNAs are targeted by C. campestris across its very broad host range. These data show that C. campestris miRNAs act as trans-species regulators of host gene expression, and suggest that they may act as virulence factors during parasitism.

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An artificial host system enables the obligate parasitic plant Cuscuta campestris to grow and complete its life cycle in vitro

Project description:Cuscuta campestris is an obligate parasitic plant that requires a host to complete its lifecycle. Parasite-host connections occur via an haustorium, a unique organ that acts as a bridge for the uptake of water, nutrients and macromolecules. Research on Cuscuta is often complicated by host influences, but comparable systems for growing the parasite in the absence of a host do not exist. We developed an axenic method to grow C. campestris on an Artificial Host System (AHS). We evaluated the effects of nutrients and phytohormones on parasite haustoria development and growth. Haustorium morphology and gene expression were characterized. The AHS consists of an inert, fibrous stick that mimics a host stem, wicking water and nutrients to the parasite. It enables C. campestris to exhibit a parasitic habit and develop through all stages of its lifecycle, including production of new shoots and viable seeds. Phytohormones NAA and BA affect haustoria morphology, and increase parasite fresh weight and biomass. Gene expression in AHS haustoria reflect process similar to those in haustoria on actual host plants. The AHS is a methodological improvement for studying Cuscuta biology by avoiding specific host effects on parasite and giving researchers full control of the parasite environment.

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Parasitic plant sRNome analyses unveils parasite-specific signatures of miRNA retention, loss and gain

Project description:Parasitism is a successful life strategy that has evolved independently in several families of Higher Plants. In parasitic plants, nutrients and water are obtained from their hosts through specifically adapted haustoria. The genera Cuscuta and Orobanche represent examples for the two profoundly different groups of parasites, one parasitizing host shoots and the other infecting host roots. In this study, we sequenced and described the overall repertoire of small RNAs (sRNome) from Cuscuta campestris and compared it to an exploratory sRNome from Orobanche aegyptiaca. Because they regulate the genomic information by mechanisms like RNA interference, these sRNomes add important bricks to our understanding of the success of the parasitic lifestyle. We could show that Cuscuta campestris contains a number of novel microRNAs (miRNAs) in addition to a conspicuous retention of miRNAs that are typically lacking in other Solanales, while several typically conserved miRNAs seem to have become obsolete in the parasite. One new miRNA appears to be derived from a horizontal gene transfer event, a process never reported in plants for the acquisition of new miRNA sequences. The exploratory miRNome (exploratory due to the absence of a full genomic sequence for reference) from the root parasitic Orobanche aegyptiaca also revealed a loss of a number of miRNAs compared to established miRNomes of photosynthetic species from the same order. In summary, our study shows for both parasites partly similar evolutionary signatures in the RNA silencing machinery. Our data bear proof for the dynamism of this regulatory mechanism in parasitic plants.

2022-07-22 | GSE181576 | GEO

Time course of trans-species miRNAs from Cuscuta campestris grown on Arabidopsis thaliana

Project description:Time course of trans-species miRNAs from Cuscuta campestris grown on Arabidopsis thaliana

| PRJNA765592 | ENA

Time course of trans-species miRNAs from Cuscuta campestris grown on Solanum lycopersicum

Project description:Time course of trans-species miRNAs from Cuscuta campestris grown on Solanum lycopersicum

| PRJNA765594 | ENA

microRNA accumulation from Cuscuta campestris in vitro haustoria

Project description:microRNA accumulation from Cuscuta campestris in vitro haustoria

| PRJNA844285 | ENA

LC-MSMS of Hordeum vulgare epidermis containing haustoria from Blumeria graminis f.sp. hordei

Project description:Blumeria graminis f.sp. hordei is an obligate biotrohic fungal pathogen causing powdery mildew in barley. As for other biotrophic fungi, haustorial structures are at the centre of the biotrophic interaction and molecular exchanges, delivering fungal effectors or virulence factors, and taking nutrient from the host. Haustoria are originiated by the fungus, following successful penetration of the initial penetration peg through the plant cell call. Haustorial structures mainly of fungal origin, but they are surrounding by a plant component, the extrauhaustorial membrane and matrix (EHM and EHMx) forming the extrahuastorial complex (EHMc). The plant protein make-up of the plant extrahaustorial components remained unexplored, and this is a first study trying to describe plant proteome associated with haustoria using samples enriched for these structures. Therefore, proteomes of haustoria enriched samples from the epidermis of barley leaves infected with Blumeria graminins f.sp. hordei, the causing agent of barley powdery mildew, were compared to infected epidermis and un-infected epidermis to identify haustoria associated plant proteins. Haustoria were enriched from infected epidermis by digesting epidermal cell walls with cell wall degrading enzymes prior to enrichment for haustorial structures. Proteins identified in these samples were compared to infected and uninfected epidermis samples using a non-targeted label free semi-quantitation method.

2019-11-01 | PXD012684 | Pride

Full-length RNA-seq of short RNAs from Cuscuta campestris

Project description:Cuscuta campestris is an obligate parasitic plant that attaches to the stems of host plants to obtain water and nutrients. C. campestris produces a specialized set of microRNAs specifically at the host-parasite interface. Many of these interface-induced microRNAs target mRNAs from the host. This experiment was designed to capture full-length primary transcripts that give rise to C. campestris interface-induced microRNAs. C. campestris shoot tips were stimulated to produce haustoria, then harvested to extract total RNA. RNA was then treated with various enzymes to modify 5' ends. Libraries were then made and sequenced. Importantly the library method only captures RNAs with a 5'-monophosphate. Therefore the specific enzymatic treatments can reveal the native 5' ends of the sequenced RNAs. Data were used to identify the primary transcripts for many of the known C. campestris primary microRNA transcripts. The data were also interrogated to tally specific snRNAs (U1, U2, U4, and U5) that are known to have a 2,2,7mGppp cap, and the tally pre-tRNAs, which are known to have a ppp 5' end. The analyzed results indicated that C. campestris interface-induced microRNA primary transcripts likely have a 5' ppp, not a cap, consistent with transcription by RNA polymerase III. The analysis also demonstrated that C. campestris interface-induced microRNA primary transcripts mostly terminate within stretches of polyT and lack polyA tails, also features consistent with Pol III transcription. Finally, the analysis indicated that the transcriptional start sites were located ~ 55 bp downstream of a common ten-base pair promoter element (the upstream sequence element). The 55bp spacing is also consistent with Pol III transcription.

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