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• W4313253676 abstract "•Ten thousand mRNAs transfer between Haloxylon ammodendron and root parasitic Cistanche deserticola•RNA mobility and functional analysis were performed in sunflower-Orobanche cumana system•CdNLR1 and CdNLR2 would cause root-specific HR and affect parasitic equilibrium Exchanges of mRNA were shown between host and stem parasites but not root parasites. Cistanche deserticola (Orobanchaceae) is a holoparasitic herb which parasitizes on the roots of woody plant Haloxylon ammodendron (Chenopodiaceae). We used transcriptome sequencing and bioinformatic analyses to identify nearly ten thousand mobile mRNAs. Transcript abundance appears to be a driving force for transfer event and mRNA exchanges occur through haustorial junction. Mobility of selected mRNAs was confirmed in situ and in sunflower-Orobanche cumana heterologous parasitic system. Four C. deserticola →H. ammodendron mobile mRNAs appear to facilitate haustorium development. Of interest, two mobile mRNAs of putative resistance genes CdNLR1 and CdNLR2 cause root-specific hypersensitive response and retard parasite development, which might contribute to parasitic equilibrium. The present study provides evidence for the large-scale mRNA transfer event between a woody host and a root parasite, and demonstrates the functional relevance of six C. deserticola genes in host-parasite interactions. Exchanges of mRNA were shown between host and stem parasites but not root parasites. Cistanche deserticola (Orobanchaceae) is a holoparasitic herb which parasitizes on the roots of woody plant Haloxylon ammodendron (Chenopodiaceae). We used transcriptome sequencing and bioinformatic analyses to identify nearly ten thousand mobile mRNAs. Transcript abundance appears to be a driving force for transfer event and mRNA exchanges occur through haustorial junction. Mobility of selected mRNAs was confirmed in situ and in sunflower-Orobanche cumana heterologous parasitic system. Four C. deserticola →H. ammodendron mobile mRNAs appear to facilitate haustorium development. Of interest, two mobile mRNAs of putative resistance genes CdNLR1 and CdNLR2 cause root-specific hypersensitive response and retard parasite development, which might contribute to parasitic equilibrium. The present study provides evidence for the large-scale mRNA transfer event between a woody host and a root parasite, and demonstrates the functional relevance of six C. deserticola genes in host-parasite interactions. Increasing evidence suggests the importance of macromolecules such as proteins and mRNAs in both short- and long-distance communication in plants.1Turnbull C.G.N. Lopez-Cobollo R.M. Heavy traffic in the fast lane: long-distance signalling by macromolecules.New Phytol. 2013; 198: 33-51https://doi.org/10.1111/nph.12167Crossref PubMed Scopus (72) Google Scholar Plasmodesmata are considered as short-distance transport channels, whereas vascular system carries out long-distance molecular transport between distal tissues.2Kehr J. Kragler F. Long distance RNA movement.New Phytol. 2018; 218: 29-40https://doi.org/10.1111/nph.15025Crossref PubMed Scopus (111) Google Scholar,3Kondhare K.R. Patil N.S. Banerjee A.K. A historical overview of long-distance signalling in plants.J. Exp. Bot. 2021; 72: 4218-4236https://doi.org/10.1093/jxb/erab048Crossref PubMed Scopus (15) Google Scholar mRNA was reported to transfer in phloem.4Ruiz-Medrano R. Xoconostle-Cázares B. Lucas W.J. Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants.Development. 1999; 126: 4405-4419Crossref PubMed Google Scholar Recent studies have shown that the stem-loop structure of tRNA origin endowed mRNA with long-distance transport capacity.5Zhang W. Thieme C.J. Kollwig G. Apelt F. Yang L. Winter N. Andresen N. Walther D. Kragler F. tRNA-related sequences trigger systemic mRNA transport in plants.Plant Cell. 2016; 28: 1237-1249https://doi.org/10.1105/tpc.15.01056Crossref PubMed Scopus (121) Google Scholar AtRRP44A, a subunit of the RNA exosome, was reported to interact with plasmodesmata and to mediate the cell-to-cell trafficking of KNOTTED1 (KN1) mRNA.6Kitagawa M. Wu P. Balkunde R. Cunniff P. Jackson D. An RNA exosome subunit mediates cell-to-cell trafficking of a homeobox mRNA via plasmodesmata.Science. 2022; 375: 177-182https://doi.org/10.1126/science.abm0840Crossref PubMed Scopus (20) Google Scholar Parasitic plants account for about 1% of flowering plants. The most important feature of parasitic plants is a specialized structure called haustorium, which establishes physical and physiological connection channels between the host and parasitic plants and so dominates most of their interactions.7Yoshida S. Cui S. Ichihashi Y. Shirasu K. The haustorium, a specialized invasive organ in parasitic plants.Annu. Rev. Plant Biol. 2016; 67: 643-667https://doi.org/10.1146/annurev-arplant-043015-111702Crossref PubMed Scopus (167) Google Scholar Parasitic plants rely on the host to sustain their growth, absorbing water and the nutrients such as photosynthates, amino acids and other intermediate metabolites from the host through the haustorium.8Hibberd J.M. Jeschke W.D. Solute flux into parasitic plants.J. Exp. Bot. 2001; 52: 2043-2049https://doi.org/10.1093/jexbot/52.363.2043Crossref PubMed Scopus (103) Google Scholar Haustoria also function as the connection bridge between the hosts to transmit signaling molecule such as herbivore signal.9Hettenhausen C. Li J. Zhuang H. Sun H. Xu Y. Qi J. Zhang J. Lei Y. Qin Y. Sun G. et al.Stem parasitic plant Cuscuta australis (dodder) transfers herbivory-induced signals among plants.Proc. Natl. Acad. Sci. USA. 2017; 114: E6703-E6709https://doi.org/10.1073/pnas.1704536114Crossref PubMed Scopus (52) Google Scholar It was also shown that the biomolecules including proteins and RNAs exchange bidirectionally through host-parasite connections. The earliest example of RNA transfer between the host and parasitic plants is the transmission of RNA virus from the infected host to the uninfected plant through dodder.10Hosford R.M. Transmission of plant viruses by dodder.Bot. Rev. 1967; 33: 387-406https://doi.org/10.1007/Bf02858742Crossref Scopus (0) Google Scholar Small RNAs (sRNAs) such as small interference RNA (siRNA) and microRNA (miRNA) were shown to move between host-parasite to regulate the target gene expression in recipient organisms.11Alakonya A. Kumar R. Koenig D. Kimura S. Townsley B. Runo S. Garces H.M. Kang J. Yanez A. David-Schwartz R. et al.Interspecific RNA interference of SHOOT MERISTEMLESS-Like disrupts Cuscuta pentagona plant parasitism.Plant Cell. 2012; 24: 3153-3166https://doi.org/10.1105/tpc.112.099994Crossref PubMed Scopus (112) Google Scholar,12Shahid S. Kim G. Johnson N.R. Wafula E. Wang F. Coruh C. Bernal-Galeano V. Phifer T. dePamphilis C.W. Westwood J.H. Axtell M.J. MicroRNAs from the parasitic plant Cuscuta campestris target host messenger RNAs.Nature. 2018; 553: 82-85https://doi.org/10.1038/nature25027Crossref PubMed Scopus (218) Google Scholar,13Hou Y. Ma W. Natural host-induced gene silencing offers new opportunities to engineer disease resistance.Trends Microbiol. 2020; 28: 109-117https://doi.org/10.1016/j.tim.2019.08.009Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar As for mRNA transfer, mobile mRNAs from the hosts tomato, pumpkin and alfalfa to the parasite Cuscuta chinensis have been demonstrated for several genes in the initial test.14Roney J.K. Khatibi P.A. Westwood J.H. Cross-species translocation of mRNA from host plants into the parasitic plant dodder.Plant Physiol. 2007; 143: 1037-1043https://doi.org/10.1104/pp.106.088369Crossref PubMed Scopus (127) Google Scholar Through microarray analysis, Roney et al. found that 474 mRNAs were transferred from tomato to dodder.14Roney J.K. Khatibi P.A. Westwood J.H. Cross-species translocation of mRNA from host plants into the parasitic plant dodder.Plant Physiol. 2007; 143: 1037-1043https://doi.org/10.1104/pp.106.088369Crossref PubMed Scopus (127) Google Scholar However, large-scale identification of transfer mRNAs was achieved only on the uses of next-generation sequencing technology. Kim et al. used transcriptome sequencing technology to identify large-scale and bidirectional mRNA transfer between the parasite Cuscuta pentagona and the hosts such as Arabidopsis thaliana and tomato.15Kim G. LeBlanc M.L. Wafula E.K. Depamphilis C.W. Westwood J.H. Genomic-scale exchange of mRNA between a parasitic plant and its hosts.Science. 2014; 345: 808-811https://doi.org/10.1126/science.1253122Crossref PubMed Scopus (197) Google Scholar But the functional relevance of transfer mRNAs in host-parasite interactions was not clear. Orobanchaceae is the largest parasitic angiosperm family in which many are facultative or obligate root parasites.16Bennett J.R. Mathews S. Phylogeny of the parasitic plant family Orobanchaceae inferred from phytochrome A.Am. J. Bot. 2006; 93: 1039-1051https://doi.org/10.3732/ajb.93.7.1039Crossref PubMed Scopus (164) Google Scholar Cistanche is a worldwide genus of holoparasitic desert plants in Orobanchaceae. C. deserticola is a holoparasite which parasitizes on the roots of a woody psammophyte, Haloxylon ammodendron (Chenopodiaceae).17Wang T. Zhang X. Xie W. Cistanche deserticola Y. C. Ma, Desert Ginseng: a Review.Am. J. Chin. Med. 2012; 40: 1123-1141https://doi.org/10.1142/S0192415x12500838Crossref PubMed Scopus (0) Google Scholar It was shown that chloroplast rpoC2 gene was transferred from H. ammodendron to C. deserticola by horizontal gene transfer.18Li X. Zhang T.C. Qiao Q. Ren Z. Zhao J. Yonezawa T. Hasegawa M. Crabbe M.J.C. Li J. Zhong Y. Complete chloroplast genome sequence of holoparasite Cistanche deserticola (Orobanchaceae) reveals gene loss and horizontal gene transfer from its host Haloxylon ammodendron (Chenopodiaceae).PLoS One. 2013; 8: e58747https://doi.org/10.1371/journal.pone.0058747Crossref PubMed Scopus (94) Google Scholar Here, we identified nearly ten thousand mobile mRNAs between C. deserticola and H. ammodendron through next-generation transcriptome sequencing and bioinformatic analysis. The mobile mRNAs were ascertained by multiple sequence alignment, PCR validation, and especially utilization of a sunflower-Orobanche cumana parasitic system. The function of mobile mRNAs was also demonstrated for several genes by this heterologous parasitic system. To date, the reports on functional mRNAs exchange between host and parasite were very limited. Therefore, our study provides new insights into the parasitic mechanism from the view point of mobile mRNAs. Tight physical connection of host H. ammodendron root and parasite C. deserticola makes it impossible to separate them at the haustorium. Therefore, we combined high-throughput RNA-sequencing (RNA-seq) and stepwise bioinformatic classification to identify the mobile transcripts between host and parasite. Four different set of samples were collected for RNA-seq analyses on Illumina HiSeq2500 platform. These include the root tissue of parasite-free host H. ammodendron (HA), succulent stem of C. deserticola (CD), the root tissue of host H. ammodendron parasitized with C. deserticola (HC), and the haustorial interface (HI). For HC and CD samples, the respective host root and parasite stem were collected 1 cm away from the parasitic junction (Figure 1A ). Reliable identification of source tissue for a given transcript from interacting organisms is a challenging issue. Therefore Ikeue et al. have developed a useful bioinformatic method to distinguish host and parasite transcripts.19Ikeue D. Schudoma C. Zhang W. Ogata Y. Sakamoto T. Kurata T. Furuhashi T. Kragler F. Aoki K. A bioinformatics approach to distinguish plant parasite and host transcriptomes in interface tissue by classifying RNA-Seq reads.Plant Methods. 2015; 11: 34https://doi.org/10.1186/s13007-015-0066-6Crossref PubMed Scopus (15) Google Scholar Here we used a modified bioinformatic approach which is based on their method to identify the mobile mRNAs between C. deserticola and H. ammodendron (Figure 1B). A grand total of 723,382,940 clean reads were generated from above libraries (Table S2), and these reads were assembled via different strategies (Figure 1B). As a result, all ten samples were hybrid assembled into 222,899 unigenes (termed “Combined” hereafter, Table S3). C. deserticola and H. ammodendron samples were also assembled into 107,752 and 194,720 unigenes respectively (termed “Cis” and “HAC” hereafter, Table S3). To visualize the variation as well as the similarity for all samples, we performed a principal component analysis (PCA) on the normalized FPKM values of all the detected genes in “Combined unigenes”. The PCA plot showed that the data for three biological replicates were clustered closely and were separated in different samples, especially between H. ammodendron and C. deserticola (Figure S1). Meanwhile, ORF prediction showed that 149,825 (67.23%) of the “Combined” unigenes were assumed to encode putative proteins (Figure 1C). The individually assembled unigenes with the expression threshold value of FPKM≥0.3 from four different samples were cross-checked with the ORF prediction results (Tables S4–S6). This analysis identified that 69.01–75.90% of the unigenes from the four samples CD, HC, HA and HI had putative ORF (Figure 1C). Full-length transcriptome sequencing was carried out and the data, CD_FL for C. deserticola and HA_FL for H. ammodendron, were used for assembly error correction for both host H. ammodendron and parasite C. deserticola as both species lack genomic data (Tables S8–S11). When the assigned unigenes from above four samples were filtered with dual BLASTN (E value = 1e−10) against Cis, HAC, CD_FL and HA_FL, 94.78–99.00% of them had reliable consensus sequences (Figure 1C and Table S7). Based on the above analysis, 17,379 (HA-exclusive 14,810 and HA-containing 2,569) common unigenes were finally retrieved from HC and CD on Venn diagram analysis of the unigenes from CD, HC and HA (Figure 1D). Among them, the former 14,810 unigenes are most probably of parasite CD origin because of their absence in HA, but a proportion of them might also represent HC→CD mobile mRNAs which are present only in HC but not in HA owing to their up-regulation on parasite attachment. The latter 2,569 unigenes most probably represent unidirectional HC→CD transfer mRNAs as they are also present in HA. The other unigenes were excluded from candidate mobile mRNAs as they were not shared between HC and CD. One should also note that 14,810 common unigenes in [HC, CD] outnumbered 166 common unigenes in [HA, CD] by 89 times, highly suggesting the reliability of our analyses as the latter was logically impossible and was caused by the inevitable errors from unigene assembly or bioinformatic analyses (Figure 1D). The origin of putative mobile mRNAs was further confirmed via dual BLAST analyses to assign their sequence origin (Figure 1B and Table S12). Because of the lack of genomic data for both host and parasite, we utilized available sequence data for Chenopodiaceae and Orobanchaceae family organisms to which H. ammodendron and C. deserticola belong respectively. Our hypothesis is that mobile mRNAs of H. ammodendron origin most probably have higher homologies with their orthologs from other Chenopodiaceae species than those from Orobanchaceae species, while it is vice versa for mobile mRNA of C. deserticola origin, and the conserved orthologs probably have high similarity with both Orobanchaceae and Chenopodiaceae. This analysis could help us to ascertain the origins of the mobile mRNAs as they could be assigned to family level via homology searching. To prove our hypothesis, phylogenetic tree of thirty-seven species, including six Orobanchaceae and four Chenopodiaceae species, was constructed using OrthoFinder. The far evolutionary distance between Orobanchaceae and Chenopodiaceae species ascertained the reliability of our hypothesis (Figure 2A and Table S13). Gene loss analysis showed that the transcripts for many orthogroups and photosynthesis related genes were absent in C. deserticola (Figures 2B and 2C, Table S14, Data S2 and S3). Moreover, the Venn diagram analysis between C. deserticola and three other sequenced parasitic plants also showed that 53.84% (926/1,720) of the missing orthogroups in C. deserticola were also absent in other three parasitic plants, namely 50.81% (755/1486), 48.09% (377/784) and 50.00% (260/520) in C. australis, Striga asiatica and Phtheirospermum japonicum respectively (Figure S2). These results not only indicated the parasitic property of this species but also the reliability of unigene assembly from our analyses, and also supported our hypothesis to use relative plant species for confirmation of sequence origin. Therefore, the candidate transfer unigenes from above analyses were separated with dual BLASTN (E value = 1e−10) against a collection of public available sequence datasets from Orobanchaceae (transcriptome of Triphysaria versicolor, Striga hermonthica and Phelipanche aegyptiaca; EST and mRNA sequences from NCBI) and Chenopodiaceae (next-generation and full-length transcriptome of H. ammodendron; EST and mRNA sequences from NCBI) (Figure 1B and Table S12). As a result, 7,496 unigenes were confirmed to originate from the parasite C. deserticola, accounting for 9.66% (7,496/77,615) and 13.70% (7,496/54,721) of the unigenes in destination HC samples and source CD, respectively; and 2,370 unigenes were assigned to host H. ammodendron, accounting for 3.38% (2,370/70,119) and 4.15% (2,370/57,091) of the unigenes in source HC and destination CD samples, respectively (Figures 2D and 2E, Tables S7, S12, and S15). The common 2,931 unigenes were too homologous to be assigned to the exact source organism. These results indicated that nearly ten thousand (7,496 + 2,370 = 9,866) unigenes could transfer between root parasitic plant C. deserticola and woody plant host H. ammodendron. Much more (7,496/2,370 = 3.16-folds) mobile RNAs were of parasite C. deserticola origin than of host H. ammodendron origin, showing the mRNA transfer bias in a parasite→host direction. Although mRNA transfer has been documented between stem parasitic plant in Cuscuta species and their hosts,14Roney J.K. Khatibi P.A. Westwood J.H. Cross-species translocation of mRNA from host plants into the parasitic plant dodder.Plant Physiol. 2007; 143: 1037-1043https://doi.org/10.1104/pp.106.088369Crossref PubMed Scopus (127) Google Scholar,15Kim G. LeBlanc M.L. Wafula E.K. Depamphilis C.W. Westwood J.H. Genomic-scale exchange of mRNA between a parasitic plant and its hosts.Science. 2014; 345: 808-811https://doi.org/10.1126/science.1253122Crossref PubMed Scopus (197) Google Scholar,20LeBlanc M. Kim G. Patel B. Stromberg V. Westwood J. Quantification of tomato and Arabidopsis mobile RNAs trafficking into the parasitic plant Cuscuta pentagona.New Phytol. 2013; 200: 1225-1233https://doi.org/10.1111/nph.12439Crossref PubMed Scopus (40) Google Scholar the transfer mechanism remains poorly understood. It was hypothesized that mRNA transfer might be driven by specific selective determinants, such as abundance, size, featured motif, or specific modification of the mobile mRNAs.5Zhang W. Thieme C.J. Kollwig G. Apelt F. Yang L. Winter N. Andresen N. Walther D. Kragler F. tRNA-related sequences trigger systemic mRNA transport in plants.Plant Cell. 2016; 28: 1237-1249https://doi.org/10.1105/tpc.15.01056Crossref PubMed Scopus (121) Google Scholar,15Kim G. LeBlanc M.L. Wafula E.K. Depamphilis C.W. Westwood J.H. Genomic-scale exchange of mRNA between a parasitic plant and its hosts.Science. 2014; 345: 808-811https://doi.org/10.1126/science.1253122Crossref PubMed Scopus (197) Google Scholar,21Ham B.K. Brandom J.L. Xoconostle-Cázares B. Ringgold V. Lough T.J. Lucas W.J. A polypyrimidine tract binding protein, Pumpkin RBP50, forms the basis of a phloem-mobile ribonucleoprotein complex.Plant Cell. 2009; 21: 197-215https://doi.org/10.1105/tpc.108.061317Crossref PubMed Scopus (165) Google Scholar,22Yang L. Perrera V. Saplaoura E. Apelt F. Bahin M. Kramdi A. Olas J. Mueller-Roeber B. Sokolowska E. Zhang W. et al.m(5)C methylation guides systemic transport of messenger RNA over graft junctions in plants.Curr. Biol. 2019; 29: 2465-2476.e5https://doi.org/10.1016/j.cub.2019.06.042Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar Therefore, we asked whether these characteristics of mobile mRNAs could influence the transfer events between root parasitic plant C. deserticola and host H. ammodendron or not. FPKM analyses showed that transcript abundance of the majority of mobile unigenes of C. deserticola origin was higher than that of non-mobile unigenes from same species. However, the transcript abundance of mobile mRNAs of H. ammodendron origin showed divergent patterns of comparable to higher FPKM values than those of non-mobile mRNAs from same species (Figure 3A ). Moreover, mobile mRNAs of CD origin showed higher abundance in source than in destination tissue, where we speculate that the mobile mRNAs might be diluted or degraded after transfer event (Figure 3B). Compared with this, mobile mRNAs of HC origin showed no uniform correlation of transcript abundances between source and destination tissues (Figure 3B). These results demonstrated that mRNA transfer could be correlated with unigene abundance, but the mechanistic basis for such correlations remains obscure. We assume that mobile mRNAs might be present in haustorium as they should translocate through parasitic interface. Most of the mobile unigenes could be found in haustoria unigene dataset, accounting for 99.21% (7,437/7,496) and 80.89% (1,917/2,370) of the transfer unigenes originated from C. deserticola and H. ammodendron, respectively (Figures 3C and 3D). This result indicated that root parasitic plant and its host exchanged their transcripts through haustorial junction, which serves as mechanical bridge between host and parasite and mediates the transfer event. Further evidence for haustorium-mediated selective mobility of unigenes came from plots of unigene abundance in haustorium versus abundance of the same unigenes in source C. deserticola and H. ammodendron, respectively. The plot of CD→HC mobile mRNAs showed that expression levels of most unigenes in the source CD sample were positively correlated with those in haustorium (Figure 3E). This result was similar to previous report about mobile mRNA between Arabidopsis and Cuscuta (Kim et al., 2014), indicating that most unigenes followed the same dynamics of movement. The plot of HC→CD mobile mRNAs showed a more-dispersed pattern of transcript abundances without obvious correlation between source HC and haustoria (Figure 3F). These results demonstrated that root parasitic plant C. deserticola and its host H. ammodendron could exchange their transcripts in a haustorium-mediated selective manner, but the dynamics of movement differed between two directions. KEGG analyses showed the overall functions of the mobile mRNAs (Figures 4A and 4B ). In the KEGG analysis, the terms related to RNA transport and metabolism, such as “RNA transport”, “spliceosome” and “mRNA surveillance pathway” were enriched in CD→HC mobile RNAs, whereas none of them were detected in HC→CD mobile RNAs. The enrichment of above pathways is consistent with the more frequent transfer of mRNA in CD→HC direction than that in the opposite direction (Figure 4A). “RNA transport” related mobile mRNAs in parasite C. deserticola might facilitate the large-scale transfer of mRNAs in the CD→HC direction. Furthermore, the presence of “spliceosome” and “mRNA surveillance pathway” related mobile RNAs from the parasite into host indicates that these mobile mRNAs might be involved in the maturation of parasite-derived mRNAs, or they may modify the mRNAs of host origin to influence the recipient plant’s physiology.23Jamar N.H. Kritsiligkou P. Grant C.M. Loss of mRNA surveillance pathways results in widespread protein aggregation.Sci. Rep. 2018; 8: 3894https://doi.org/10.1038/s41598-018-22183-2Crossref PubMed Scopus (19) Google Scholar In addition to “RNA transport”, “protein export” pathway was also enriched in CD→HC mobile RNAs (Figure 4A). More frequent transfer of proteins than RNAs was observed in the association of stem parasite dodder and its host Arabidopsis and soybean.24Liu N. Shen G. Xu Y. Liu H. Zhang J. Li S. Li J. Zhang C. Qi J. Wang L. Wu J. Extensive inter-plant protein transfer between Cuscuta parasites and their host plants.Mol. Plant. 2020; 13: 573-585https://doi.org/10.1016/j.molp.2019.12.002Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar The term “autophagy”, a process involved in programmed cell death (PCD) and immune response, was enriched in CD→HC mobile RNAs (Figure 4A). In HC→CD direction, metabolic pathway genes for protein biosynthesis are significantly enriched compared with CD→HC direction. The term “ribosome”, “protein processing in endoplasmic reticulum”, “ribosome biogenesis in eukaryotes” and “aminoacyl-tRNA biosynthesis” are all related to protein biosynthesis and related genes were highly represented in HC→CD mobile RNAs (Figure 4B). We tried to test the independent transfer event but it was impossible to experimentally validate all of these mobile mRNAs. Therefore, we randomly selected some of the unigenes to verify the transfer event (Tables 1 and S3–S14). Polymerase chain reaction (PCR) assay was carried out to validate the mobile mRNAs and genomic DNAs (gDNAs) of both host and parasite were used as control templates. We reasoned that the PCR product could only be amplified from gDNA of source organism other than gDNA of destination organism for a specific mobile mRNA, as the mobile mRNA can only be transcribed from source gDNA but not from destination gDNA. To avoid the amplicon size differences between gDNA and cDNA, we used the gene fragments with no putative intron for PCR assay. We confirmed ten CD→HC and three HC→CD mobile mRNAs using this assay (Table 1 and Figures S3–S14). We could amplify HC→CD mobile sequences from HA gDNA, HA cDNA, HC cDNA and less abundantly from HI cDNA and CD cDNA while not from CD gDNA. Similarly, we could amplify CD→HC transfer sequence from CD gDNA, CD cDNA, and less abundantly from HI cDNA and HC cDNA while not from HA cDNA and HA gDNA (Figure 5). The presence of corresponding nucleic acids for mobile mRNAs in source gDNA but not in destination gDNA confirmed the transfer direction. Less abundant transcript level in destination than in source sample indicates that mobile mRNA is less represented in the recipient sample, which is consistent with the sequencing results (Figure 3B). As a negative control for this PCR experiment, we tested two HA non-mobile mRNAs (HabHLH110, HabZIP60) and two CD non-mobile mRNAs (CdbHLH47, CdMYB3), and we could not amplify them from CD cDNA or HC cDNA respectively (Figure 5). Above data indicated the reliability of the PCR results.Table 1Information of thirteen mobile and four non-mobile mRNAs for experimental validationGene AnnotationTranscriptome IDGene SymbolPredicted ORF length (bp)Transfer directionPeptidase_M1 domain-containing proteinc121402_g2CdAPM510CD→HCDNA ligasec114708_g2CdLIG2, 247CD→HC26S rRNAc85065_g1CdRRN26NACD→HCATP synthase subunit Ac126123_g1CdATPA774CD→HCGlucose-6-phosphate isomerasec119887_g2CdPGI747CD→HC50S ribosomal protein L22c79535_g1CdRPL22507CD→HCRetrovirus-related pol polyprotein from transposonc90862_g1CdRE1591CD→HCMaturase Kc55576_g1CdMatK1, 548CD→HCNB-ARC domain-containing proteinc137753_g2CdNLR12, 247CD→HCNB-ARC domain-containing proteinc137753_g3CdNLR21, 407CD→HCProgrammed cell death proteinc139367_g1HaPCDP1, 959HC→CDGlyceraldehyde-3-phosphate dehydrogenasec127689_g1HaGAPDH450HC→CDPectinesterasec127482_g1HaPE1, 068HC→CDTranscription factor bHLH110c107962_g1HabHLH1101, 425Non-transferred in HCTranscription factor bZIP60c108491_g1HabZIP60984Non-transferred in HCTranscription factor MYB3-likec104027_g1CdMYB3897Non-transferred in CDTranscription factor bHLH47c112633_g1CdbHLH47669Non-transferred in CDThe ORF was predicted from unigenes by performing BLAST (E value = 1e−5) in the databases such as NR (NCBI non-redundant protein sequences) and SwissProt. ESTScan software was also used to confirm the annotation. Cd, Cistanche deserticola; Ha, Haloxylon ammodendron;NA, not applicable. Open table in a new tab The ORF was predicted from unigenes by performing BLAST (E value = 1e−5) in the databases such as NR (NCBI non-redundant protein sequences) and SwissProt. ESTScan software was also used to confirm the annotation. Cd, Cistanche deserticola; Ha, Haloxylon ammodendron;NA, not applicable. Based on the above results, we aimed to further validate the transfer event by in planta overexpression and to characterize the function of the mobile mRNAs. We mainly studied CD→HC mobile mRNAs for validation as they were more broadly and confidently identified than HC→CD mobile RNAs. H. ammodendron-C. deserticola parasitization system is not applicable for this purpose as the transgenic approaches are not available for both the host and parasite. Therefore, we utilized sunflower-O. cumana parasitic system in which the latter belongs to Orobanchae species as the same with C. deserticola. First, we evaluated the phylogenetic relationship between root parasitic plants C. deserticola and O. cumana, as well as corresponding hosts H. ammodendron and Helianthus annuus via phylogenetic analyses using rbcL (RuBisCO large subunit) sequence. The evolutionary tree showed that C. deserticola had a close phylogenetic relationship with O. cumana, as was also the same for the two hosts. However, the stem parasitic plant Cuscuta australis had a far phylogenetic relationship with both parasitic plants and their hosts, which" @default.
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• W4313253676 title "Large-scale mRNA transfer between Haloxylon ammodendron (Chenopodiaceae) and herbaceous root holoparasite Cistanche deserticola (Orobanchaceae)" @default.
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