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.
Project description:Cuscuta campestris parasitized on various hosts including bamboo stick, wt Nicotiana tabacum, amiR targeting CcJKD-expressing N. tabacum
Project description:An intriguing new paradigm in plant biology is that systemically-mobile mRNAs play a role in coordinating development. In this process, specific mRNAs are loaded into the phloem transport stream for translocation to distant tissues, where they may impact developmental processes. However, despite its potential significance for plant growth regulation, mRNA trafficking remains poorly understood and challenging to study. Here we show that phloem-mobile mRNAs can also traffic between widely divergent species from a host to the plant parasite, lespedeza dodder (Cuscuta pentagona Engelm.). Reverse transcriptase PCR (RT-PCR) and microarray analysis were used to detect specific tomato transcripts in dodder grown on tomato (Lycopersicon esculentum Mill.) that were not present in control dodder grown on other host species. The foreign transcripts included LeGAI, which has been previously shown to be translocated in the phloem, as well as nine other transcripts not reported to be mobile. Dodders are parasitic plants that obtain resources by drawing from the phloem of a host plant, and have joint plasmodesmata with host cortical cells. Although viruses are known to move between dodder and its hosts, translocation of endogenous plant mRNA has not been reported. These results point to a potentially new level of interspecies communication, and raise questions about the ability of parasites to recognize, use, and respond to transcripts acquired from their hosts. Experiment Overall Design: In order to identify potential tomato transcripts in dodder, microarray analysis was performed on RNA from dodder and hosts. Total RNA was extracted from the tomato host and from dodder grown on tomato, Arabidopsis, tobacco, or pumpkin. The host tomato RNA was included to verify that any transcripts detected in the parasite were in fact expressed in the host. The dodder samples grown on tobacco, Arabidopsis, and pumpkin served as controls for dodder genes that may cross-hybridize with tomato array probes, with three different host species used to minimize any host-specific effects on dodder gene expression. Samples were analyzed using the Affymetrix GeneChip Tomato Array and transcripts scored for presence or absence in each sample. Considering that host transcripts present in dodder would be at low levels and diluted with dodder transcripts, a P-value of 0.06 in at least two of three biological replicates was used as the threshold for scoring a transcript as being present.
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.
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.