
Editor's Introduction
Genomic-scale exchange of mRNA between a parasitic plant and its hosts
Communication is at the center of our existence. Every day we communicate with a number of people around us to live productive and meaningful lives. Meanwhile, within our own human bodies, millions of cells act in unison, never ceasing to listen or talk to each other, maintaining a perfectly synchronized biological system. Communication between the individual units of a single organism is an essential part of its existence and survival. In plants, cellular and tissue communication triggers growth, adaptation to environmental stress, transfer of nutrients, and many other vital processes. Parasitic plants are known to bind to their hosts and trigger trafficking of different nutrients and molecules. This paper investigates the scope of genomic mobility between host and parasitic plants. Do different species communicate with each other by exchanging genetic material? Remarkably, more than half of all messenger RNAs (intermediary molecules with critical functions in transmitting genetic information from DNA) get trafficked between the parasite and its hosts. Characterizing the properties, localization, and abundance of mobile mRNAs, this study highlights potential mechanisms of cross-talk between distinct species.
Paper Details
Abstract
Movement of RNAs between cells of a single plant is well documented, but cross-species RNA transfer is largely unexplored. Cuscuta pentagona (dodder) is a parasitic plant that forms symplastic connections with its hosts and takes up host messenger RNAs (mRNAs). We sequenced transcriptomes of Cuscuta growing on Arabidopsis and tomato hosts to characterize mRNA transfer between species and found that mRNAs move in high numbers and in a bidirectional manner. The mobile transcripts represented thousands of different genes, and nearly half the expressed transcriptome of Arabidopsis was identified in Cuscuta. These findings demonstrate that parasitic plants can exchange large proportions of their transcriptomes with hosts, providing potential mechanisms for RNA-based interactions between species and horizontal gene transfer.
Report
Cuscuta species (dodders) are parasitic plants that obtain water and nutrients from their plant hosts by using specialized organs termed haustoria. The haustoria of Cuscuta develop from the stem of the parasite, where it coils around the host, penetrating host tissues and ultimately forming vascular connections (1, 2). These connections allow transfer of not only water and nutrients into the parasite but macromolecules, including mRNAs (3–5) and proteins (6), and even pathogens, such as viruses (7, 8), viroids (9), and phytoplasmas (10). Here, we characterize the scope and directionality of mRNA movement.
mRNA trafficking between cells regulates plant development (11, 12), with potential for controlling processes such as leaf shape (13, 14), time of flowering (15), tuber formation (16, 17), and root growth (18). Small RNAs can also act systemically to influence plant development (19), and a construct encoding a silencing RNA and expressed in a host plant can silence a Cuscuta gene (20). Although this last example is from an artificial construct, it supports the idea that RNA movement between separate plant individuals can function as a type of organismal communication (21). We used transcriptomics to investigate the RNA transfer between Cuscuta and its hosts.
Cuscuta species parasitize a wide range of broad-leaved plants (often simultaneously) and are destructive to crops, such as tomato (Solanum lycopersicum) (22). We grew Cuscuta on Arabidopsis thaliana and tomato hosts because the sequenced genomes of these species facilitates confident identification of host and parasite transcripts from mixed RNA populations. We harvested three distinct regions for each parasite-host association for analysis (Fig. 1A). The Cuscuta haustorium grows toward the center of the host stem and does not spread systemically inside the host (2), so the tissues harvested did not risk inclusion of endophytic haustorial tissue that could lead to cross-contamination of samples. Furthermore, the growth habit of Cuscuta allows unattached stem regions to be easily collected separate from the interface regions where haustoria bind tightly to host tissues (Fig. 1A and fig. S1).
Fig. 1. Transcriptome compositions of host and parasite tissues at and near the region of haustorial attachment. (A) Tissues analyzed were the host stem above the region of attachment (HS), interface region where parasite is connected to the host (I), and the parasite stem near the region of attachment (PS). Scale bars represent 1 mm. (B) Pie charts show the proportions of reads mapped to host and parasite transcriptomes in each tissue. Arabidopsis with Cuscuta data are means (±SE) of three separate sequencing runs; tomato with Cuscuta data are from one run.
Objective
Determine if foreign transcripts are detected in either host or parasite plants, suggesting possible transcript trafficking between the two.
Experimental Approach
Identify what proportion of host or parasite transcripts are present at the point of host/parasite contact and regions adjacent to it.
A. Collect plant tissue samples from the region where the host plant and parasite interact (I), or the ones leading to this junction point (HS, PS)
B. Analyze the identity of detected transcripts in the three regions of interest. Match all transcripts to known DNA sequences in tomato, Cuscuta, or Arabidopsis.
Results
A very small proportion of host transcripts (<1%) were detected in the parasite in tissue regions not adjacent to the point of contact between the parasite and the host plant. This result was consistent for both tomato and Arabidopsis, and was also observed in the opposite scenario—parasite transcripts present into host tissues outside of the region of host/parasite contact.
As expected, in tissues where the parasite and host plants make a physical interaction, transcripts from either plant were detected, with higher host percentage (85%) in the case of tomato/parasite junction compared with Arabidopsis/parasite (~50-50%).
Conclusions
Transcripts from the host plants tomato and Arabidopsis are detected in the Cuscuta parasite, and vice versa.
To identify host and parasite mobile transcriptomes, we sequenced cDNA libraries derived from each of the three tissues. The rate of RNA mobility was expected to be low, so the first libraries were sequenced with a full lane of Illumina GAIIx (Illumina, Incorporated, San Diego, CA) for one run each of Cuscuta growing on Arabidopsisand tomato hosts. Second and third biological replicates of Cuscutawith Arabidopsis were sequenced with a full lane and one-sixth lane of the higher output HiSeq 2000 (Illumina, Incorporated) platform, respectively. This yielded over 1.6 billion high-quality reads, which were subjected to various controls by which we filtered out contaminating reads and poor quality reads and trimmed away adapters and primers. Reads were then identified as host, parasite, or too highly conserved to assign (fig. S2). The parasite reads were used to reconstruct a Cuscuta transcriptome assembly.
Reads from each library were stringently mapped to host and parasite transcriptomes to estimate RNA movement between the species. Arabidopsis read proportions in parasite tissue averaged 1.1% of total mapped reads across the three sequencing runs, whereas host stems contained 0.6% Cuscuta reads (Fig. 1B). Read mapping in the tomato-Cuscuta association suggested somewhat lower rates of transfer, but the pattern was similar to that ofArabidopsis with the exception of interface tissue, where the greater mass of the tomato stem likely resulted in a higher proportion of reads. Bidirectional mobility in transcript movement is consistent with the long-established ability of Cuscuta to transmit viruses between plants bridged by the parasite (7) and suggests that Cuscuta is capable of transmitting mRNAs between different plants.
Independent confirmation of mobility was shown by reverse transcription polymerase chain reaction (RT-PCR) amplification and subsequent sequencing of selected transcripts. Mobility of 24 tomato transcripts into Cuscuta has been documented this way (3,23), so we analyzed Arabidopsis transcripts moving into Cuscutaand Cuscuta transcripts moving into Arabidopsis and tomato hosts (fig. S3). Such confirmation is not practical for all mobile transcripts, but the output of read mapping itself produced a compelling picture of RNA transfer (Fig. 2). The read sequences and coverage from parasite stem tissue closely matched those of the interface tissue, with the exception that mobile mRNAs in the parasite occurred in fully spliced mature form; introns were only found in libraries derived from host stem or interface tissues.
Fig. 2. Example of read assemblies of an Arabidopsis gene,TRANSLATIONALLY CONTROLLED TUMOR PROTEIN(AtTCTP), in host stem (HS), interface (I), and parasite stem (PS) tissues. Intron sequences were not found in sequences derived from parasite tissue. The gene model at top indicates coding sequence as blue bars and introns as line bridges. Dark and light purple lines indicate forward and reverse paired reads, respectively, as mapped to the gene model. UTR, untranslated region; CDS, coding sequence.
Purpose of this experiment
Validate the observation of transcript mobility between parasite and host.
Experimental Approach
Fig.1 shows that foreign mobile transcripts were detected in both host and parasite indicating bidirectional mobility. The sequence of such mobile transcripts was resolved and matched to known host and parasite gene sequences.
Here, one of these matched and sequenced mobile transcripts (AtTCTP) was used as an example to illustrate how its sequence aligns (matches) with transcripts detected in the regions from parasite only (PS), host only (HS), or the region shared between the host and parasite (I).
Results
The sequence of this representative mobile transcript (that of the AtCTP gene), is closely conserved between transcripts detected in all three tested regions (HS, I, and PS).
As expected, in tissues where the parasite and host plants make a physical interaction, transcripts from either plant were detected, with higher host percentage (85%) in the case of tomato/parasite junction compared with Arabidopsis/parasite (~50-50%).
Interesting Observation
Parasite AtCTP transcripts lack introns which are otherwise present in AtCTP transcripts detected in HS or I.
The diversity of transcripts represented by the mobile reads was determined by high-stringency mapping of reads from the three species (Arabidopsis, tomato, and Cuscuta) to their combined reference sequences. The criterion for transcript mobility was set by using fragment counts where one fragment represents either a matched pair of reads or a single unpaired read. The threshold for mobile transcripts was set at a mean of four fragments per transcript because this level was found to produce positive RT-PCR confirmation (fig. S3), whereas eight fragments per transcript was considered strong evidence of mobility. The greatest number of mobile transcripts originated from Arabidopsis hosts, with 45% (9518) of the genes in the expressed Arabidopsis transcriptome found in Cuscuta, and most of these (5983) showed strong evidence of mobility (Table 1). In contrast, tomato hosts produced substantially fewer mobile transcripts than Arabidopsis, with 347 (1.6% of total expressed) detected in the parasite. Part of the difference between tomato and Arabidopsis transcript mobility into Cuscuta may be attributed to the single sample of tomato-Cuscuta sequenced and the lack of deep sequencing from a full lane of HiSeq 2000 data, but even allowing for these differences there appear to be differences in RNA transfer to the parasite from different host species.
Table 1. Numbers of genes and unigenes with mobile transcripts from hosts intoCuscuta or Cuscuta into hosts. The numbers represent transcript reference (the Arabidopsis Information Resource reference annotation TAIR 10/International Tomato Annotation Group annotation ITAG 2.4/Cuscuta unigenes) sequences as categorized by number of fragments detected in self and nonself tissues. A threshold of four fragments per gene was used to determine transcript detection, with four fragments detected in nonself tissues considered evidence for mobility and eight fragments providing strong evidence for mobility.
What is summarized in Table 1?
The sum total of all mobile and nonmobile transcripts detected in the hosts (Arabidopsis or Tomato) or the parasite (Cuscuta).
Mobile transcripts, eight versus four fragments: What does this mean?
All transcripts were mapped to host or parasite libraries of known gene sequences. Matched pair or single unpaired reads corresponds to one fragment. Thus, the more you have, the higher the probability of correct transcript detection.
Four - minimum threshold for detection of mobile transcripts
Eight - very strong evidence for detection of mobile transcripts
Main observations
The number of mobile transcripts trafficked between Arabidopsis and Cuscuta is much higher than between Tomato and Cuscuta (~ 8,500 to 9,500 versus ~300).
At the same time, the number of nonmobile transcripts in Arabidopsis is ~2x lower than in either Cuscuta or tomato.
Caveat to the difference in transcript reads between Arabidopsis and tomato?
Only one sample of tomato-Cuscuta pairs was analyzed, and the deep sequencing data was incomplete. Still, the difference of transcript mobility for the two hosts appears to be significant.
With respect to movement from parasite to host, 8655 Cuscuta unigenes were classified as mobile into Arabidopsis stem, and 5973 unigenes showed strong evidence of mobility (Table 1). This is 24% of the 35,614 unigenes expressed in Cuscuta. Tomato host uptake ofCuscuta transcripts was again lower than that of Arabidopsis, with 288 unigenes showing evidence of mobility. The rates of transcript movement between Cuscuta and the two hosts were consistent in both directions, with a much freer exchange occurring between Cuscuta and Arabidopsis than between Cuscuta and tomato, suggesting that mechanisms regulating haustorial selectivity may be host-specific.
We asked whether mobile and nonmobile RNAs have distinctive properties that provide insight into mechanisms of mobility. One characteristic common to mobile transcripts was high abundance, as measured by fragments per kilobase per million mapped reads (FPKM) in the interface region, and this was especially pronounced for the Arabidopsis interaction with Cuscuta (Fig. 3A). FPKM (24) was used because it normalizes fragment counts to transcript length and depth of transcriptome sequencing to better estimate transcript levels. The patterns were similar for mobile and nonmobile transcripts in the tomato-Cuscuta interaction, although tomato nonmobile transcripts spanned the spectrum from low to high abundance (Fig. 3B). This indicates that one aspect of transcript mobility is related to their high abundance in the cells near the host-parasite boundary, but it is not the only factor influencing mobility, as evidenced by the many transcripts with similar expression levels yet differing mobility.
Fig. 3. Properties of mobile and nonmobile RNAs. (A) Distribution of transcript expression levels in interface tissue as related to mobility in Arabidopsis-Cuscuta associations. (B) Same as (A), but for mobility in tomato-Cuscuta associations. (C) Venn diagrams showing common sets of transcripts that were either mobile or nonmobile out of Arabidopsis, tomato, orCuscuta. Numbers are orthologous clusters as determined by OrthoMCL. (D) Pie charts showing Gene Ontology (GO) slim terms as proportions of sets of 11 mobile and 23 nonmobile GO terms that were enriched for multiple species. The full lists of GO slim terms for these data sets and all terms significantly overrepresented and underrepresented in each of the three species are given in table S2.
Expression levels of host/parasite transcripts
Panels A and B represent plots of expression levels (how much) and frequency (how often) of detected mobile and non-mobile host/parasite transcripts.
Legend:
Cp - parasite (Cuscuta pentagona)
At - host (Arabidopsis thaliana)
Sl - host tomato (Solanum lycopersicum)
FPKM - transcript fragment per kilobase per million mapped read
Note: FPKM normalizes transcript numbers to the transcript length and depth of sequencing so that these parameters do not affect the plotted results)
Results, Panels A and B
Mobile and nonmobile transcripts detected in host or parasite have distinct properties. Mobile transcript are more highly expressed but detected much less frequently than nonmobile ones. This is consistent for both hosts (tomato and Arabidopsis).
Panel C
Number of overlapping or unique transcripts (mobile or nonmobile) observed in the two hosts and the parasite.
Panel D
A plot of the likely biological functions/roles of mobile and nonmobile transcripts, as determined by analysis of Gege Ontology annotations (term explained earlier in the manuscript text).
To consider whether transcript mobility is associated with gene function, we used OrthoMCL software to generate orthologous clusters of mobile and nonmobile gene classes that were common to all three species (25) (Fig. 3C). Assigning genes from these clusters to gene ontology terms led to the identification of terms enriched among mobile and nonmobile classes (table S2). Restricting the list to those terms that were only enriched for multiple species (e.g., transcripts from both Arabidopsis and Cuscuta) yielded smaller sets of terms that may reflect core mobile and nonmobile categories (Fig. 3D). These results demonstrate that mobility can be correlated with gene function, but the mechanistic basis for such correlations remains obscure. For example, a large proportion of mobile transcripts are assigned to the response-to-stimulus term; it is possible that these transcripts are specifically targeted for intercellular mobility, but it is also possible that characteristics of transcript accumulation or localization in the cytoplasm makes them especially prone to host-parasite exchange.
Further evidence for selective mobility of RNAs comes from plots of transcript abundance in the interface region versus abundance of the same transcripts in the parasite stem (Fig. 4). The plot of Arabidopsis to Cuscuta mobile transcripts showed that levels of most transcripts in the parasite were about one-hundredth of those in the interface tissues, indicating that most transcripts follow the same dynamics of movement (Fig. 4A). However, some host RNAs appear to move more readily into the parasite and occurred at FPKM levels in the parasite nearly equal to those in the interface (seen as outliers above the main group in Fig. 4). The tomato-Cuscuta data showed a more-dispersed pattern of mobilities that supports the idea that dynamics of movement differ between tomato and Arabidopsis hosts (Fig. 4B).
Fig. 4. Scatter plots of transcript abundance (FPKM) in parasite stem versus host-parasite interface. (A) A total of 9518 Arabidopsis transcripts identified as mobile into Cuscuta (Table 1). (B) A total of 347 tomato transcripts identified as mobile into Cuscuta. Lines are linear regressions of the data.
Mobile transcript abundance in stem vs. interface
Most transcripts follow the same trend and stick around at the interface. A small proportion (about 1%) however, are found in the parasite stem.
What about the tomato/parasite pair?
The mobility of transcripts in tomato is more dispersed indicating that the movement dynamics of transcripts for the two hosts follows different patterns.
Did you notice the outliers in each plot?
There are a few transcripts that are as highly abundant in the parasite as at the interface between host and parasite (top 5 dots towards the upper right corner, panel A) (top cluster of dots in the upper right corner, panel B).
An unresolved question regarding Cuscuta haustoria is the precise route used to acquire material from the host. Substantial physiological evidence points to symplastic connections consistent with direct transfer between phloem tissues of host and parasite [e.g., (8)], but no open phloem connections have been observed (26). Rather, Cuscuta haustorial cells share plasmodesmata with hosts across chimeric cell walls (2, 8), and these have been implicated in host-parasite mobility of RNA (4). The long-distance movement of RNAs in the parasite suggest phloem involvement (4,5), but our data indicate that the situation is complex. We compared transcripts moving from Arabidopsis into Cuscuta to published phloem transcriptome data from Arabidopsis and four other species (27–31), finding significant associations between the data sets (table S3). Further analysis using the subset of Arabidopsis transcripts with especially high mobility into Cuscuta (i.e., those significantly above the mass of data points in Fig. 4A) indicated correlations with the more-robust data sets (Arabidopsis and ash) but did not demonstrate a linkage between phloem-associated transcripts and high mobility into Cuscuta (table S4). Our data also indicate that Cuscuta acquires transcripts such as the ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) small subunit, which is not considered part of an authentic phloem transcriptome (32). Taken together, these data suggest that host-parasite RNA exchange includes RNAs known to occur in phloem but also many RNAs from other cells.
We can only speculate about the importance of large-scale mRNA movement between individuals of different species. For example, some specific mRNAs transmit information long distances in plants (13, 14, 18, 33), and these same information molecules could help the parasite track host physiological status or, in the other direction, use its own mRNA to manipulate the host to facilitate parasitism. However, it is not known whether mobile mRNAs act through translation into protein or through another mechanism, so it is unclear whether mRNAs could even function across widely different species. Host mRNAs disappear within several hours inside Cuscuta(5), but this could be due to processes such as translation into protein or degradation for nucleotide recycling. In this regard, the question of whether Cuscuta can distinguish its own transcripts from those of its hosts is interesting.
This widespread exchange of mRNA raises the possibility of horizontal gene transfer (HGT). Given what appears to be a constant exchange of mRNA between Cuscuta and its hosts, the relative prevalence of cases of HGT involving Cuscuta is not surprising (34–38). Although most documented cases of HGT in parasitic plants suggest a mechanism involving direct transfer of DNA, at least one case of HGT into a parasitic plant (Striga hermonthica) exhibits evidence of an RNA intermediate in the mechanism (39). The ability of one Cuscuta plant to bridge many different host individuals raises the possibility that this parasite could mediate RNA exchange across different individuals and even across hosts of different species.
Supplementary Materials
www.sciencemag.org/content/345/6198/808/suppl/DC1
Materials and Methods
Figs. S1 to S4
Tables S1 to S5
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