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Over-expression of Oryza sativa Xrn4 confers plant resistance to virus infection
Accepted Manuscript Over-expression of Oryza sativa Xrn4 confers plant resistance to virus infection
Shanshan Jiang, Liangliang Jiang, Jian Yang, Jiejun Peng, Yuwen Lu, Hongying Zheng, Lin Lin, Jianping Chen, Fei Yan PII: DOI: Reference:
S0378-1119(17)30817-X doi:10.1016/j.gene.2017.10.004 GENE 42223
To appear in:
Gene
Received date: Revised date: Accepted date:
30 June 2017 30 September 2017 3 October 2017
Please cite this article as: Shanshan Jiang, Liangliang Jiang, Jian Yang, Jiejun Peng, Yuwen Lu, Hongying Zheng, Lin Lin, Jianping Chen, Fei Yan , Over-expression of Oryza sativa Xrn4 confers plant resistance to virus infection. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.10.004
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ACCEPTED MANUSCRIPT Over-expression of Oryza sativa Xrn4 confers plant resistance to virus infection
Shanshan Jianga, b, #, Liangliang Jianga, #, Jian Yangc, Jiejun Pengc, Yuwen
College of Plant Protection, Nanjing Agricultural University, Nanjing 210095,
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a
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Luc, Hongying Zhengc, Lin Linc, Jianping Chenc, *, Fei Yanc, *
China;
Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan
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b
c
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250100, China;
Stake Key laboratory Breeding Base for Sustainable Control of Pest and Disease,
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MOA and Zhejiang Provincial Key Laboratory of Plant Virology, Institute of Virology
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and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021,
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China;
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# These authors contributed equally to this work * Corresponding author. Tel: +86-571-86404313; Fax: +86-571-86404258. E-mail address: [email protected] (Jianping Chen) or [email protected] (Fei Yan)
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ACCEPTED MANUSCRIPT Abstract Plant Xrn4 is a cytoplasmic 5’ to 3’ exoribonuclease that is reported to play an antiviral role during viral infection as demonstrated by experiments using the Xrn4s of Nicotiana benthamiana and Arabidopsis thaliana. Meanwhile, little is known about
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the anti-viral activity of Xrn4 from other plants. Here, we cloned the cytoplasmic
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Xrn4 gene of Oryza sativa (OsXrn4), and demonstrated that its over-expression
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elevated the 5’-3’ exoribonuclease activity in rice plants and conferred resistance to rice stripe virus, a negative-sense RNA virus causing serious losses in East Asia. The
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accumulation of viral RNAs was also decreased. Moreover, the ectopic expression of
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OsXrn4 in N. benthamiana also conferred plant resistance to tobacco mosaic virus infection. These results show that the monocotyledonous plant cytoplasmic Xrn4 also
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resistant to viral infection.
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has an antiviral role and thus provides a strategy for producing transgenic plants
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Keywords: Xrn4; rice stripe virus; resistance; transgenic plant
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ACCEPTED MANUSCRIPT 1. Introduction The 5’-3’ exoribonucleases (XRNs) play key roles in degrading several classes of RNA including mRNA, rRNA, and small RNAs. In Arabidopsis thaliana (At), three Xrns genes have been identified, named Xrn2, Xrn3 and Xrn4; no Xrn1, homologous
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to yeast Xrn1p, has been detected (Kastenmayer and Green, 2000). AtXrn2 and
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AtXrn3 are localized in the nucleus where they degrade miRNA-derived loops that
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are excised during miRNA maturation (Kastenmayer and Green, 2000; Gy et al., 2007). In contrast, AtXrn4 is targeted to the cytoplasm where it degrades cytoplasmic,
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uncapped mRNA and miRNA target cleavage products (Kastenmayer and Green,
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2000; Souret et al., 2004). Previous work showed that silencing of Nicotiana benthamiana Xrn4 (NbXrn4) resulted in the accumulation of cucumber necrosis virus
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(CNV) RNAs and facilitated the infection of tobacco mosaic virus (TMV), while
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over-expression of AtXrn4 in N. benthamiana accelerated the degradation of the viral RNAs (Cheng et al., 2007; Jaag and Nagy, 2009; Peng et al., 2011). These results
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suggest that plant cytoplasmic Xrn4 probably affects the replication of the viral RNAs
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and hence plays an antiviral role during viral infection. However, little is known about the anti-viral activity of Xrn4 from other plants. Here, we cloned the cytoplasmic Xrn4 gene of Oryza sativa (OsXrn4), and found that its over-expression in rice improved the resistance of plants against rice stripe virus (RSV), a negative-strand RNA virus causing serious losses in East Asia. Moreover, the ectopic expression of OsXrn4 in N. benthamiana also conferred resistance against TMV infection. These results indicate that a monocotyledonous plant cytoplasmic Xrn4 also plays an 3
ACCEPTED MANUSCRIPT antiviral role, and thus provides a strategy for producing plants resistant to viral infection.
2. Materials and methods
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2.1 Plasmid construction and sequence analysis
from
complementary
DNA
(cDNA)
with
the
primers
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amplified
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The coding region of the rice (Oryza sativa Japonica Group) OsXrn4 gene was
5’-CCGAGACTCCGAGAGAGATTAG-3’
and
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5’-GCTTACACATTTACAATGGCCG-3’. The sequence of OsXrn4 was divided into
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three fragments and successively ligated into pCV-1300 (Lu et al., 2011), producing vector pCV-OsXrn4. The primers are listed in Table 1.
and
motifs
were
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domains
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The coding sequence of the OsXrn4 gene was analyzed with ClustalW. The analyzed
by
the
conserved
domains
tool
(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Xrn members were searched
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in the Rice Genome annotation project database (http://rice.plantbiology.msu.edu/)
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using the XRN_N domain (pfam03159). 2.2 Microscopy
The plant binary vectors used in subcellular localization assay experiments have been described previously (Lu et al., 2011). A Leica TCS SP5 (Leica Microsystems) confocal laser scanning microscope system was used to examine the fluorescence of green fluorescent protein (GFP) between 2 and 4 days post infiltration (dpi). All images were processed using Adobe Photoshop version 7.0 software (Adobe Systems 4
ACCEPTED MANUSCRIPT Inc.). 2.3 Plant transformation Plasmid pCV-OsXrn4 was constructed and transferred to Agrobacterium tumefaciens strain EHA105 for use in transformation of rice calli. Rice (O. sativa) subsp. japonica,
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cv. Nipponbare, was used as the wild type to generate the transgenic plants
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over-expressing OsXrn4. The transformants were selected on 50 mg/L hygromycin
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medium. Rice seeds were immersed in water for 2 days to induce germination, and then the plants were grown in a greenhouse under 14 h light/10 h dark and 30/28°C
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(day/night) conditions.
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Leaf disks of N. benthamiana were inoculated with Agrobacterium strain EHA105 containing the vector pCV-OsXrn4. Transformed cells were selected and regenerated
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in MS medium containing 50 mg/L hygromycin, and roots were induced after transfer
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of plantlets to hormone-free medium. Rooted transformants were transferred to soil and grown under greenhouse conditions. All N. benthamiana plants were grown in a
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conditions.
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glasshouse at 23-25°C, with a 12 h day/night light cycle under well-watered
For agroinfiltration, N. benthamiana plants were maintained in growth chambers at 20-25°C with a 12 h day/night light cycle throughout the assays. Agrobacterium infiltration and GFP fluorescence detection were performed as previously described (He et al., 2012). In co-infiltration experiments, equal volumes of each suspension were mixed before infiltration.
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ACCEPTED MANUSCRIPT 2.4 Southern blotting Genomic DNA was extracted from frozen rice samples using standard methods. After quantification by UV spectrometry, approximately 40 µg of DNA were digested with EcoRI at 37°C overnight, electrophoresed and transferred to Hybond N+ nylon
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membrane (Amersham) using 20 × SSC. The membrane was then baked at 80°C for 2
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h and hybridized with DIG-labeled probes specific to OsXrn4, which was amplified
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with primers 5’-CTGCTAGTGGTGGAATGAAC-3’ and
5’-CATACTGATTGGGATGTGGCTG-3’. Membranes were pre-hybridized for at
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least 1 h and hybridized overnight at 42°C using a DIG High Prime Labeling and
exposure to X-ray film (Kodak).
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2.5 Virus infection assay
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Detection Starter Kit II (Roche). The hybridization signals were visualized by
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RSV-infected rice plants were obtained as described previously (Yan et al., 2010). Briefly, viruliferous adult small brown planthoppers (Laodelphax striatellus Fallen)
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were transferred onto rice seedlings at the two-leaf stage for virus inoculation. The
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ratio of plants to insects was 1:3. After 48-72 h, the planthoppers were removed. All rice plants were grown in a glasshouse as described above. The numbers of rice plants with symptoms on their newly developing leaves was recorded at 7 day intervals until maturity. The plasmid pCV-OsXrn4 was introduced into A. tumefaciens strain EHA105 and the cells were used to infiltrate the leaves of 3-week-old N. benthamiana plants. The injection areas were 1 cm2 in half leaves. The infiltrated leaves were mechanically 6
ACCEPTED MANUSCRIPT inoculated with 20 μl of crude extracts from TMV-infected leaves. Sap inoculation of TMV-GFP was prepared from N. benthamiana leaves that became infected after infiltration with A. tumefaciens strain EHA105 harbouring an infectious cDNA clone p35S-30B:GFP (Jia et al., 2003). Agrobacterium cultures containing the plasmid
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p35S-30B: GFP were also infiltrated into the upper leaves of 3-week-old T2
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transgenic N. benthamiana plants expressing OsXrn4. All treatments were repeated at
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least three times. 2.6 RNA analysis
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Total RNA was isolated from rice plants using TRI-ZOL (Invitrogen) following the
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manufacturer’s instructions. The RNA that was used for the quantitative real-time PCR was treated with RNase-free DNaseI (TaKaRa). First-strand cDNA was prepared
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from total RNA using KOD-Plus (ToYoBo) with random primers according to the
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manufacturer’s instructions. OsXrn4 expression was detected by real-time PCR using the primers (in rice: 5’-GGCTATCCAGAACAATGAGGAG-3’ and
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5’-GGATAAAACCACTGCCATGAGC-3’, in N. benthamiana:
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5’-AAAGAAGAGGCAGAACTAAAACCCA-3’ and 5’-CAAATCCCCATAGTACTTTGGCAAC-3’). O. sativa actin gene (Accession number: AK068380.1) and N. benthamiana Ubiquitin C gene (UBC) gene (Accession number: AB026056.1) were used as the internal reference genes for analysis (Actin primers: 5’-CTTGTGAAAGATGAAGATCTTGT-3’ and 5’-GTACTCAGCCTTGGCAATCCACA-3’; UBC primers: 5’-GAGGAAGAGACTGGTGAGGGAT-3’ and 7
ACCEPTED MANUSCRIPT 5’-CACAGAGCAAAGACTGGATTGA-3’). A Roche LightCycler® 480 Real-Time PCR System was used for the reaction and the results were analyzed by the ΔΔCT method. For northern blot analyses, DNA probes were synthesized with primers (RSV CP:
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5’-CTCCTGGTCATCACATGCAAG-3’ and
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5’-GCCAGCGCATCGAAGATTGTG-3’; GFP:
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GTTGAATTAGATGGTGATGTTA and 5’-CCTTAAGCTCGATCCTGTTG-3’) and labeled with DIG according to the manufacturer’s protocol (DIG Oligonucleotide
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3’-end labeling Kit, Roche). Pre-hybridization, hybridization and signal detection
Detection Starter Kit II (Roche).
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were done according to the protocol of the DIG High Prime DNA Labeling and
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2.7 5’-3’ exoribonuclease activity assay
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Transcript half-life measurements were used to analyze the 5’-3’ exoribonuclease activity of OsXrn4 on total RNAs in plants (Seeley et al., 1992). O. sativa were grown
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on liquid nutrient medium for 3 weeks. The seedlings were then transferred to a flask
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with incubation buffer including 0.6 mM cordycepin (Sigma-Aldrich) and incubated for up to 100 min. Tissue samples were harvested at various time points and quickly frozen in liquid nitrogen. Total RNAs were isolated followed by formaldehyde denaturing gel electrophoresis. The effect of OsXrn4 on degradation of viral RNAs was also determined. The total active proteins of plant samples were extracted with lysis buffer (10% glycerol, 25mM Tris·HCl, 1mM EDTA,150mM NaCl, 10mM DTT, 1mM PMSF, 0.1% 8
ACCEPTED MANUSCRIPT Nonidet P-40). The full-length TMV-GFP RNA was synthesized in vitro by the T7 RivoMAX express large scale RNA production system (Promega), and was added into the prepared protein solutions, and the mixtures were incubated at 37°C for 45 min~150 min followed by northern blot analyses. Ribonuclease Inhibitor (ToYoBo)
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was added into the mixtures to prevent environmental degradation of RNAs (Tomecki
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et al., 2015).
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2.8 Western blotting
Proteins were separated in a 12% SDS-PAGE gel, transferred onto nitrocellulose
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(Amersham) by wet electroblotting, and were detected with anti-Actin (Abbkine)
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primary antibody and anti-mouse (Sigma) secondary antibody. The antigen-antibody complexes were visualized using NBT/BCIP buffer (Sigma) under standard
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conditions.
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3. Results
3.1 Cloning of OsXrn4 and its subcellular localization
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To identify and clone OsXrn4, AtXrn4 (NM_104327) containing 2844 nucleotides in
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its open reading frame (ORF), was used as query for BLAST analysis against the Oryza sativa Japonica Group nucleotide database. A sequence (Accession number: AK070933) with 2967 nucleotides in its ORF was identified to have high identity to AtXrn4,
and
recognized
as
OsXrn4
(5’-CCGAGACTCCGAGAGAGATTAG-3’
(Fig.
1A
and
B).
Primers and
5’-GCTTACACATTTACAATGGCCG-3’) were then designed to amplify OsXrn4 from Oryza sativa cDNA by PCR. Sequencing confirmed that the cloned OsXrn4 9
ACCEPTED MANUSCRIPT ORF sequence was similar to AK070933 (data not shown). It was predicted to encode a protein of 988 amino acids with the conserved XRN_N domain (Fig. 1A). The amino acid sequences of Xrns contain two highly conserved regions (CR1 and CR2) in their N-terminal segment (Page et al., 1998). Sequence alignment showed that
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OsXrn4 had a higher similarity to cytoplasmic AtXrn4 than to nuclear AtXrn2
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(NM_123619) or AtXrn3 (NM_106217), and the conserved N-terminus of OsXrn4
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contains a XRN_N domain comprised of 256 amino acids and a zinc finger domain, implying that the cloned sequence was the cytoplasmic Xrn of rice (Fig. 1A and B).
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The highly conserved Xrn family is typically represented by cytoplasmic enzymes
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(Xrn1 or Xrn4) and nuclear enzymes (Xrn2 and Xrn3). To detect other Xrn-like sequences of O. sativa, we conducted a search of the Rice Genome annotation project
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database for sequences containing the XRN_N domain. Two further sequences
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(Os01g65220 and Os02g28074) were identified. Os01g65220 was most similar to AtXrn3, Zea mays Xrn3 (ZmXrn3, Gene symbol: LOC103649964) and Setaria italic
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Xrn3-like (SiXrn3-L, Gene symbol: LOC101758007), and it was therefore designated
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OsXrn3. Os02g28074 was most similar to ZmXrn4 (Gene symbol: LOC103641862) and SiXrn4-like (SiXrn4-L, Gene symbol: LOC101777938), but not to AtXrn4 and OsXrn4 (Supp. Table 1). We named it as OsXrn4-like (OsXrn4-L), and suppose that this gene may be specific to monocot plants. To confirm the cytoplasmic location of OsXrn4, GFP-fused OsXrn4 was expressed in N. benthamiana epidermal cells and the subsequent fluorescence was examined under confocal microscopy. In cells expressing only GFP (control), green 10
ACCEPTED MANUSCRIPT fluorescence was present in both the cytoplasm and nucleus, while in cells expressing GFP-fused OsXrn4, fluorescence was only in the cytoplasm, demonstrating that the cloned OsXrn4 was indeed cytoplasmic (Fig. 1C). 3.2 Over-expression of OsXrn4 in rice inhibits the infection of rice stripe virus
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To determine whether over-expression of the monocot OsXrn4 could confer plant
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resistance against viral infection, we used Agrobacterium-mediated transformation to
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produce transgenic rice plants over-expressing OsXrn4 driven by the CaMV 35S promoter (Fig. 2A). All lines over-expressing OsXrn4 had a normal phenotype (data
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not shown). The integration of foreign OsXrn4 into the rice genome in five
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independent T3 transgenic lines (5-5, 11-2, 21-1, 50-2 and 60-1) was verified by southern blot using the OsXrn4 gene as the probe (Fig. 2B). Real-time PCR analysis
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of the T3 transgenic rice plants showed that OsXrn4 was highly expressed in all
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transgenic lines, and that line 11-2 had the highest levels of expression (Fig. 2C). To determine the effect of OsXrn4 over-expression on viral infection, T3 plants
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from five transgenic lines (5-5, 11-2, 21-1, 50-2 and 60-1) were inoculated with RSV
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using viruliferous L. striatellus (Yan et al., 2010; Guo et al., 2012). The non-transgenic plants served as controls. Twenty days post inoculation (dpi), typical symptoms of RSV appeared on wild type plants and some transgenic plants. Symptoms were monitored during the whole experiment and at 90 dpi the symptoms on plants were assigned to one of four categories: no symptoms (-), chlorotic stripes without stunting (+), chlorotic stripes with mild stunting (++), and chlorotic stripes with severe stunting (+++). The average numbers of plants with each class of 11
ACCEPTED MANUSCRIPT symptom from three independent replicates (50 plants per replicate) are shown in Fig. 2D. Survival rates were significantly greater (p<0.01) in lines 21-1 and 50-2 than in the wild type and there were more plants with "-" and "+" symptoms. Lines 11-2 and 60-1 also survived better than the wild type (p<0.05), but line 5-5 did not differ
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significantly from the controls. Accumulation of RSV RNAs in the transgenic plants
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from lines 21-1 and 50-2 with "++" symptoms was less than that in wild type plants
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(Fig. 2E).
To further determine whether the 5’-3’ exoribonuclease activity in transgenic plants
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was enhanced, the accumulation of rRNAs was detected by the half-life measurement
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(Seeley et al., 1992). There were no differences between the plants treated with 0.6 mM cordycepin for 20 min, but after 80 min rRNAs were degraded more in
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transgenic line 50-2 than in wild type plants. After 100 min, rRNAs in transgenic
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plants were further degraded but those in wild type plants were reduced only a little compared with those at 20 min (Fig. 3). Line 11-2 had no more 5’-3’ exoribonuclease
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in lines 50-2.
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activity than line 50-2 even though the RNA levels of OsXrn4 were higher than those
Taken together, the results demonstrate that the over-expression of OsXrn4 enhanced 5’-3’ exoribonuclease activity and conferred rice resistance to RSV infection by decreasing the accumulation of viral RNAs. 3.3 Transient expression of OsXrn4 in Nicotiana benthamiana leaves inhibits the infection of TMV It has been reported that the ectopic expression of AtXrn4 in N. benthamiana leaves 12
ACCEPTED MANUSCRIPT led to degradation of CNV RNAs (Cheng et al., 2007). To determine whether the monocot OsXrn4 had the same ability to degrade viral RNAs in a dicotyledonous plant, we over-expressed OsXrn4 in N. benthamiana leaves transiently by agroinfiltration and then (12 h later) mechanically inoculated the leaves with TMV. To
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ensure a high level of expression of OsXrn4, it was co-expressed with the suppressor
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of RNA silencing, p19 of tomato bushy stunt virus (TBSV). TMV infection was
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visualized by inoculation with the modified TMV vector expressing GFP. Five days post infiltration (dpi) of TMV-GFP, fewer fluorescent loci were present in zones
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expressing OsXrn4 than in zones expressing the unrelated GUS protein that was used
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as a control (Fig. 4A and B). Moreover, northern blot (using Gfp probe) revealed that TMV RNAs accumulated less in the zones expressing OsXrn4 than in the control (Fig.
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4C). Real-time PCR confirmed the expression of OsXrn4 in the OsXrn4-infiltrated
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zones (Fig. 4D). These results indicate that transient expression of OsXrn4 in the dicotyledon N. benthamiana leaves can inhibit infection by TMV.
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3.4 Transgenic expression of OsXrn4 in Nicotiana benthamiana inhibits the
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infection of TMV
To investigate the effects of constitutive expression of OsXrn4 on infection by TMV in N. benthamiana, we produced transgenic N. benthamiana plants expressing CaMV 35S-driven OsXrn4. PCR detection and RT-PCR analysis of the T3 transgenic N. benthamiana plants confirmed that OsXrn4 was transferred into N. benthamiana plants and could express in all transgenic lines (Fig. 5A and B). For viral challenge, Agrobacterium containing the plasmid p35S-30B:GFP (the infectious clone of 13
ACCEPTED MANUSCRIPT TMV-GFP) was infiltrated into the upper leaves of three lines of 3-week-old T4 transgenic plants (10 plants per treatment). Wild type N. benthamiana plants served as controls. From 5 dpi, GFP fluorescence was monitored every 24 h under UV to detect the TMV infection status. Fluorescence first appeared on the new top leaves of control
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plants at 6 dpi but not until at least 2 days later in those expressing OsXrn4 (Fig. 5C).
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TMV RNAs accumulated at lower levels in plants expressing OsXrn4 than in wild
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type plants at either 6 or 8 dpi (Fig. 5D). These results further demonstrate that OsXrn4 inhibits infection of TMV in N. benthamiana.
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To examine the 5'-3' exoribonuclease activity of OsXrn4 on TMV RNAs, TMV
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genomic RNAs were synthesized in vitro and mixed with the total proteins extracted from transgenic or wild type plants for different incubation times. The levels of TMV
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RNAs from the different plants were similar at 45 min, but at 150 min TMV RNA
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levels were significantly lower in the transgenic plant extracts than in those from wild type plants (Fig. 5E). This demonstrates that OsXrn4 in transgenic plants has specific
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5'-3' exoribonuclease activity against TMV RNAs.
4. Discussion
Plant Xrn4 is a key protein in RNA metabolic pathways and can also degrade exogenous nucleic acid (transgene or virus RNAs) in the cytoplasm (Souret et al., 2004; Cheng et al., 2007). Meanwhile, there have been few studies of the Xrn4 of plants other than A. thaliana and N. benthamiana. We here cloned Xrn4 from O. sativa and investigated its function in inhibiting viral infection, and found that the 14
ACCEPTED MANUSCRIPT monocot Xrn4 also inhibits viral infection and that this function is retained when it is ectopically expressed in a dicotyledon (Fig. 3 and Fig. 4). While this supports the view that plant Xrn4 plays an antiviral role it has also been reported that AtXrn4 can act as an endogenous suppressor of post-transcriptional gene silencing (PTGS) by
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degrading the substrate of the RNA-induced silencing complex (RISC) (Gazzani et al.,
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2004). This means that Xrn4 could enhance viral infection by suppressing the antiviral
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RNA silencing mechanism in plants. Our results do not exclude this possibility since the transgenic rice line 11-2 had the highest OsXrn4 expression levels, but did not
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have the highest resistance against RSV infection. We suppose that the final effects of
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OsXrn4 over-expression in plants results from a balance between its functions of inhibiting viral infection by degrading viral RNAs and assisting viral infection by
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suppressing antiviral RNA silencing.
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The use of virus-derived genes including coat protein, movement protein and replicase to produce transgenic plants resistant to viral infection has been explored
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since the first transgenic resistant tobacco expressing TMV coat protein (CP) gene
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was created in 1986 (Abel et al., 1986; Sudarshana et al., 2007). About ten years ago, resistance strategies based on the RNA silencing mechanism, such as double-stranded RNAs (dsRNAs), hairpin RNAs and artificial miRNAs began to be developed (Kalantidis et al., 2002; Tenllado et al., 2004; Yin et al., 2005; Niu et al., 2006; Qu et al., 2007; Sudarshana et al., 2007). Only a few studies have attempted to examine transgenic strategies to produce rice with RSV resistance (Sasaya et al., 2014; Li et al., 2016) but Shimizu et al. found that expression of dsRNAs targeting RSV pc3 and pc4 15
ACCEPTED MANUSCRIPT genes conferred near immunity to RSV infection, strongly suggesting that a dsRNA strategy could provide high resistance against RSV (Shimizu et al., 2011). Recently, several host genes have been identified that might be used to enhance plant resistance against viruses. Transgenic rice with elevated expression of two host transcription
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factors, RF2a and RF2b, had weak or no symptoms after inoculation with rice tungro
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bacilliform virus (Dai et al., 2008). Transgenic soybean plants over-expressing
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GmAKT2, the ortholog of Arabidopsis K+ weak channel encoding gene AKT2, showed significantly enhanced resistance to soybean mosaic virus (SMV) (Zhou et al., 2014)
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and transgenic Brassica rapa plants over-expressing eIF(iso)4E variants had
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broad-spectrum turnip mosaic virus resistance (Kim et al., 2014). Genes from plants pose less biological safety risks than genes or sequences from the virus genome, and
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our results show that OsXrn4 has the potential to provide such a host gene-based
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resistance strategy against RSV.
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ACCEPTED MANUSCRIPT Conflict of interests The authors declare no conflict of interest.
Acknowledgements
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This work was financially supported by the State Basic Research Program of China
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(2014CB138400), the International Science & Technology Cooperation Program of
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China (2015DFA30700), the Major Project of New Varieties of Genetically Modified Organism of China (2013ZX08001-002, 2014ZX0800104B), the National Natural
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Science Foundation of China (31272016). This work is also supported by the Program
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for Leading Team of Agricultural Research and Innovation of Ministry of Agriculture, China. We thank Professor M. J. Adams, Stevenage, UK for help in correcting the
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English of the manuscript.
Author contribution statement
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Fei Yan, Shanshan Jiang, Liangliang Jiang and Jianpin Chen designed the experiments.
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Jian Yang, Jiejun Peng and Yuwen Lu cloned the sequence of OsXrn4 and analyzed the subcellular localization of OsXrn4. Shanshan Jiang, Liangliang Jiang and Lin Lin prepared the transgenic rice and tobacco plants over-expressing of OsXrn4. Shanshan Jiang, Liangliang Jiang and Hongying Zheng carried out the antiviral analysis of transgenic plants. Fei Yan, Shanshan Jiang and Jianping Chen prepared the manuscript.
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ACCEPTED MANUSCRIPT 13985-90. Kim, J., Kang, W.H., Hwang, J., Yang, H.B., Dosun, K., Oh, C.S. and Kang, B.C., 2014. Transgenic Brassica rapa plants over-expressing eIF(iso)4E variants show broad-spectrum Turnip mosaic virus (TuMV) resistance. Mol Plant
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Lu, Y., Yan, F., Guo, W., Zheng, H., Lin, L., Peng, J., Adams, M.J. and Chen, J., 2011.
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ACCEPTED MANUSCRIPT confers virus resistance. Nat Biotechnol 24, 1420-8. Page, A.M., Davis, K., Molineux, C., Kolodner, R.D. and Johnson, A.W., 1998. Mutational analysis of exoribonuclease I from Saccharomyces cerevisiae. Nucleic Acids Res 26, 3707-16.
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Parker, R. and Song, H., 2004. The enzymes and control of eukaryotic mRNA
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Peng, J., Yang, J., Yan, F., Lu, Y., Jiang, S., Lin, L., Zheng, H., Chen, H. and Chen, J., 2011. Silencing of NbXrn4 facilitates the systemic infection of Tobacco
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Qu, J., Ye, J. and Fang, R., 2007. Artificial microRNA-mediated virus resistance in
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Sasaya, T., Nakazono-Nagaoka, E., Saika, H., Aoki, H., Hiraguri, A., Netsu, O.,
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Uehara-Ichiki, T., Onuki, M., Toki, S., Saito, K. and Yatou, O., 2014. Transgenic strategies to confer resistance against viruses in rice plants. Front
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Seeley, K.A., Byrne, D.H. and Colbert, J.T., 1992. Red Light-Independent Instability of Oat Phytochrome mRNA in Vivo. Plant Cell 4, 29-38. Shimizu, T., Nakazono-Nagaoka, E., Uehara-Ichiki, T., Sasaya, T. and Omura, T., 2011. Targeting specific genes for RNA interference is crucial to the development of strong resistance to rice stripe virus. Plant Biotechnol J 9, 503-12. Souret, F.F., Kastenmayer, J.P. and Green, P.J., 2004. AtXRN4 degrades mRNA in 21
ACCEPTED MANUSCRIPT Arabidopsis and its substrates include selected miRNA targets. Mol Cell 15, 173-83. Sudarshana, M.R., Roy, G. and Falk, B.W., 2007. Methods for engineering resistance to plant viruses. Methods Mol Biol 354, 183-95.
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Tenllado, F., Llave, C. and Diaz-Ruiz, J.R., 2004. RNA interference as a new
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Yan, F., Zhang, H., Adams, M.J., Yang, J., Peng, J., Antoniw, J.F., Zhou, Y. and Chen,
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J., 2010. Characterization of siRNAs derived from rice stripe virus in infected rice plants by deep sequencing. Arch Virol 155, 935-40.
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Yin, Y., Chory, J. and Baulcombe, D., 2005. RNAi in transgenic plants. Curr Protoc
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Mol Biol Chapter 26, Unit 26 6. Zhou, L., He, H., Liu, R., Han, Q., Shou, H. and Liu, B., 2014. Overexpression of GmAKT2 potassium channel enhances resistance to soybean mosaic virus. BMC Plant Biol 14, 154.
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ACCEPTED MANUSCRIPT Tables Table 1 Primers used for construction of pCV-OsXrn4.
Figure legends
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Figure 1 Alignment of OsXrn4 with AtXrn2, AtXrn3 and AtXrn4, and subcellular
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localization of GFP-fused OsXrn4. A, amino acid alignment of OsXrn4 with
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AtXrn2, AtXrn3 and AtXrn4. The putative conserved XRN domain and zinc finger motif is underlined. Two highly conserved regions (CR1and CR2) are
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labeled. B, amino acid (numbers shadowed with color light brown) and
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nucleotide identities (numbers shadowed with light blue) of OsXrn4 to AtXrn2, AtXrn3 and AtXrn4, showing the high identity of OsXrn4 with AtXrn4. C,
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subcellular localization of GFP-fused OsXrn4 in N. benthamiana epidermal cells.
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GFP-fused OsXrn4 was localized in the cytoplasm, while the GFP control was localized in both the cytoplasm and nucleus. Fluorescence photographs were
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taken at 4 dpi. Scale bar: 25 μm.
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Figure 2 Resistance to RSV infection of T3 transgenic rice over-expressing OsXrn4. A, map of the binary vector used for transformation with OsXrn4. B, southern blot analysis of T3 transgenic plants demonstrating the integration of foreign OsXrn4 in the genomic DNA. The upper band represents the internal OsXrn4 in the rice genome, and the lower one shows the integrated foreign OsXrn4. DNA sizes are indicated by black arrowheads. Wt: wild type plant. C, quantitative RT-PCR indicated the expression level of OsXrn4 transcripts in 23
ACCEPTED MANUSCRIPT transgenic and wild type rice plants, showing that OsXrn4 was over-expressed in all transgenic lines. Bars represent the standard errors of the means. A two-sample unequal variance directional t-test was used to test the significance of the difference (*P < 0.05, **P <0.01). D, analysis of RSV incidence in wild
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type plants and in those over-expressing OsXrn4 at 90 dpi (50 plants per
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treatment). Symptoms were divided into four classes according to severity. -: no
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symptom, +: chlorotic stripes without stunting, ++: chlorotic stripes with mild stunting, +++: chlorotic stripes with severe stunting, and D represents dead
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plants. The numbers of each class and the numbers of surviving plants (NSP) are
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arrowed. E, northern blot (using RSV CP probe) showing that RSV RNAs accumulated less in OsXrn4 transgenic plants than in wild type plants at 50 dpi.
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Wt: wild type plant; 21-1 and 50-2: OsXrn4 transgenic plants. RNA sizes are
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indicated by black arrowheads.
Figure 3 5’-3’ exoribonuclease activity assay of transgenic plants overexpressing
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OsXrn4. Leaf samples from transgenic (lines 11-2 and 50-2) and wild type (wt)
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plants were collected and treated with cordycepin for 20, 80 and 100 minutes (min). rRNAs were extracted from the treated leaves and electrophoresed in 1.5% agarose/5.7% formaldehyde gel with ethidium bromide. The rRNAs (28S and 18S) in transgenic plants were difficult to detect at 100 min, but those in wild type plants were only slightly less than in those sampled at 20 min. RNA sizes are indicated by black arrowheads. Figure 4 Resistance to TMV infection of N. benthamiana leaves transiently 24
ACCEPTED MANUSCRIPT expressing OsXrn4. A, images taken under long-wavelength UV-light at 5 dpi of TMV-GFP, showing fewer fluorescent loci present in zones expressing OsXrn4 than in zones expressing the unrelated GUS protein (control). The infiltrated zones were indicated by circles with broken white line. B, compared with the
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control (GUS), the number of fluorescent loci in zones expressing OsXrn4 was
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reduced. C, northern blot showed that TMV RNAs accumulated less in zones
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expressing OsXrn4 than in the control. The GFP probe used in the experiment could detect all genomic and subgenomic RNAs of TMV. RNA sizes are
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indicated by black arrowheads. D, Quantitative real-time PCR confirmed the
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expression of OsXrn4 in the OsXrn4-infiltrated zones. Bars represent the standard errors of the means. A two-sample unequal variance directional t-test
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was used to test the significance of the difference (*P < 0.05, **P <0.01).
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Figure 5 Resistance of T4 OsXrn4 transgenic N. benthamiana plants to TMV infection. A, PCR detection confirmed that OsXrn4 has been transferred into N.
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benthamiana. Wild type (wt) plants were used as the negative control and the
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plasmid of pCV-OsXrn4 was used as positive control for PCR. No bands could be obtained from the wild type plants, demonstrating the specificity of the primers. B, RT-PCR confirmed the expression of OsXrn4 in transgenic N. benthamiana
plants.
C
shows
the
development
of
fluorescence
in
TMV-GFP-infected wild type plants and in OsXrn4 transgenic plants at 6 and 8 dpi (10 plants per treatment). Boxes with a broken white line indicate the leaves with TMV-GFP systemic infection. D, TMV RNAs accumulated at a lower level 25
ACCEPTED MANUSCRIPT in plants expressing OsXrn4 than in wild type plants at either 6 dpi or 8 dpi of TMV-GFP. Total RNAs were isolated from systemic leaves at the same position. The GFP probe used in the experiment could detect all genomic and subgenomic RNAs of TMV. RNA sizes are indicated by arrows. E, equal amounts of TMV
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genomic RNAs were incubated with total protein extracted from transgenic and
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wild type plants for 45 min and 150 min. The levels of TMV RNAs among
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different treatments were similar at 45 min, but at 150 min the TMV RNAs mixed with total proteins from transgenic plants were reduced significantly
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compared to those treated with total proteins from wild type, showing that
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OsXrn4 in transgenic plants has 5'-3' exoribonuclease activity specifically
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against TMV RNAs. Actin detected by its antibody was used as loading control.
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Electronic Supplementary Material Supplementary Table S1 Sequence alignments of OsXrns with AtXrns, ZmXrns
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and SiXrns.
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ACCEPTED MANUSCRIPT Table 1 Primers used for construction of pCV-OsXrn4.
Primers
Sequences 5’-TCTAGAATGGGAGTCCCGGCGTTCTACC-3’ (XbaI)
OsXrn4-1r:
5’-TCTAGACCTCTGCTGATTCAT-3’ (XbaI)
OsXrn4-3f:
5’-GGATCCTAGGATGGCCATGGATA-3’ (BamHI)
OsXrn4-3r:
5’-GAGCTCTCACTCACTCAGGCCATTGG-3’ (SacI)
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OsXrn4-1f:
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ACCEPTED MANUSCRIPT Abbreviation list Os, Oryza sativa At, Arabidopsis thaliana Si, Setaria italic
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Zm, Zea mays
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TMV, tobacco mosaic virus
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CNV, cucumber necrosis virus RSV, rice stripe virus
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TBSV, tomato bushy stunt virus
GFP, green fluorescent protein
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UBC, Ubiquitin C gene
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dpi, days post infiltration
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cDNA, complementary DNA
ORF, open reading frame
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CR, conserved region
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PTGS, post-transcriptional gene silencing RISC, RNA-induced silencing complex CP, coat protein
dsRNAs, double-stranded RNAs
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ACCEPTED MANUSCRIPT Highlights ●
The cytoplasmic Xrn4 gene of Oryza sativa (OsXrn4) was cloned
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Over-expression of OsXrn4 enhances the resistance of rice to rice stripe virus
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Ectopic expression of OsXrn4 enhances the resistance of N. benthamiana to TMV
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● OsXrn4 could be used to produce plants resistant to viral infection
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Shanshan Jiang, Liangliang Jiang, Jian Yang, Jiejun Peng, Yuwen Lu, Hongying Zheng, Lin Lin, Jianping Chen, Fei Yan PII: DOI: Reference:
S0378-1119(17)30817-X doi:10.1016/j.gene.2017.10.004 GENE 42223
To appear in:
Gene
Received date: Revised date: Accepted date:
30 June 2017 30 September 2017 3 October 2017
Please cite this article as: Shanshan Jiang, Liangliang Jiang, Jian Yang, Jiejun Peng, Yuwen Lu, Hongying Zheng, Lin Lin, Jianping Chen, Fei Yan , Over-expression of Oryza sativa Xrn4 confers plant resistance to virus infection. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Gene(2017), doi:10.1016/j.gene.2017.10.004
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ACCEPTED MANUSCRIPT Over-expression of Oryza sativa Xrn4 confers plant resistance to virus infection
Shanshan Jianga, b, #, Liangliang Jianga, #, Jian Yangc, Jiejun Pengc, Yuwen
College of Plant Protection, Nanjing Agricultural University, Nanjing 210095,
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a
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Luc, Hongying Zhengc, Lin Linc, Jianping Chenc, *, Fei Yanc, *
China;
Institute of Plant Protection, Shandong Academy of Agricultural Sciences, Jinan
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b
c
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250100, China;
Stake Key laboratory Breeding Base for Sustainable Control of Pest and Disease,
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MOA and Zhejiang Provincial Key Laboratory of Plant Virology, Institute of Virology
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and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021,
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China;
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# These authors contributed equally to this work * Corresponding author. Tel: +86-571-86404313; Fax: +86-571-86404258. E-mail address: [email protected] (Jianping Chen) or [email protected] (Fei Yan)
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ACCEPTED MANUSCRIPT Abstract Plant Xrn4 is a cytoplasmic 5’ to 3’ exoribonuclease that is reported to play an antiviral role during viral infection as demonstrated by experiments using the Xrn4s of Nicotiana benthamiana and Arabidopsis thaliana. Meanwhile, little is known about
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the anti-viral activity of Xrn4 from other plants. Here, we cloned the cytoplasmic
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Xrn4 gene of Oryza sativa (OsXrn4), and demonstrated that its over-expression
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elevated the 5’-3’ exoribonuclease activity in rice plants and conferred resistance to rice stripe virus, a negative-sense RNA virus causing serious losses in East Asia. The
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accumulation of viral RNAs was also decreased. Moreover, the ectopic expression of
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OsXrn4 in N. benthamiana also conferred plant resistance to tobacco mosaic virus infection. These results show that the monocotyledonous plant cytoplasmic Xrn4 also
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resistant to viral infection.
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has an antiviral role and thus provides a strategy for producing transgenic plants
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Keywords: Xrn4; rice stripe virus; resistance; transgenic plant
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ACCEPTED MANUSCRIPT 1. Introduction The 5’-3’ exoribonucleases (XRNs) play key roles in degrading several classes of RNA including mRNA, rRNA, and small RNAs. In Arabidopsis thaliana (At), three Xrns genes have been identified, named Xrn2, Xrn3 and Xrn4; no Xrn1, homologous
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to yeast Xrn1p, has been detected (Kastenmayer and Green, 2000). AtXrn2 and
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AtXrn3 are localized in the nucleus where they degrade miRNA-derived loops that
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are excised during miRNA maturation (Kastenmayer and Green, 2000; Gy et al., 2007). In contrast, AtXrn4 is targeted to the cytoplasm where it degrades cytoplasmic,
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uncapped mRNA and miRNA target cleavage products (Kastenmayer and Green,
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2000; Souret et al., 2004). Previous work showed that silencing of Nicotiana benthamiana Xrn4 (NbXrn4) resulted in the accumulation of cucumber necrosis virus
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(CNV) RNAs and facilitated the infection of tobacco mosaic virus (TMV), while
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over-expression of AtXrn4 in N. benthamiana accelerated the degradation of the viral RNAs (Cheng et al., 2007; Jaag and Nagy, 2009; Peng et al., 2011). These results
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suggest that plant cytoplasmic Xrn4 probably affects the replication of the viral RNAs
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and hence plays an antiviral role during viral infection. However, little is known about the anti-viral activity of Xrn4 from other plants. Here, we cloned the cytoplasmic Xrn4 gene of Oryza sativa (OsXrn4), and found that its over-expression in rice improved the resistance of plants against rice stripe virus (RSV), a negative-strand RNA virus causing serious losses in East Asia. Moreover, the ectopic expression of OsXrn4 in N. benthamiana also conferred resistance against TMV infection. These results indicate that a monocotyledonous plant cytoplasmic Xrn4 also plays an 3
ACCEPTED MANUSCRIPT antiviral role, and thus provides a strategy for producing plants resistant to viral infection.
2. Materials and methods
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2.1 Plasmid construction and sequence analysis
from
complementary
DNA
(cDNA)
with
the
primers
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amplified
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The coding region of the rice (Oryza sativa Japonica Group) OsXrn4 gene was
5’-CCGAGACTCCGAGAGAGATTAG-3’
and
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5’-GCTTACACATTTACAATGGCCG-3’. The sequence of OsXrn4 was divided into
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three fragments and successively ligated into pCV-1300 (Lu et al., 2011), producing vector pCV-OsXrn4. The primers are listed in Table 1.
and
motifs
were
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domains
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The coding sequence of the OsXrn4 gene was analyzed with ClustalW. The analyzed
by
the
conserved
domains
tool
(http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). Xrn members were searched
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in the Rice Genome annotation project database (http://rice.plantbiology.msu.edu/)
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using the XRN_N domain (pfam03159). 2.2 Microscopy
The plant binary vectors used in subcellular localization assay experiments have been described previously (Lu et al., 2011). A Leica TCS SP5 (Leica Microsystems) confocal laser scanning microscope system was used to examine the fluorescence of green fluorescent protein (GFP) between 2 and 4 days post infiltration (dpi). All images were processed using Adobe Photoshop version 7.0 software (Adobe Systems 4
ACCEPTED MANUSCRIPT Inc.). 2.3 Plant transformation Plasmid pCV-OsXrn4 was constructed and transferred to Agrobacterium tumefaciens strain EHA105 for use in transformation of rice calli. Rice (O. sativa) subsp. japonica,
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cv. Nipponbare, was used as the wild type to generate the transgenic plants
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over-expressing OsXrn4. The transformants were selected on 50 mg/L hygromycin
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medium. Rice seeds were immersed in water for 2 days to induce germination, and then the plants were grown in a greenhouse under 14 h light/10 h dark and 30/28°C
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(day/night) conditions.
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Leaf disks of N. benthamiana were inoculated with Agrobacterium strain EHA105 containing the vector pCV-OsXrn4. Transformed cells were selected and regenerated
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in MS medium containing 50 mg/L hygromycin, and roots were induced after transfer
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of plantlets to hormone-free medium. Rooted transformants were transferred to soil and grown under greenhouse conditions. All N. benthamiana plants were grown in a
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conditions.
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glasshouse at 23-25°C, with a 12 h day/night light cycle under well-watered
For agroinfiltration, N. benthamiana plants were maintained in growth chambers at 20-25°C with a 12 h day/night light cycle throughout the assays. Agrobacterium infiltration and GFP fluorescence detection were performed as previously described (He et al., 2012). In co-infiltration experiments, equal volumes of each suspension were mixed before infiltration.
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ACCEPTED MANUSCRIPT 2.4 Southern blotting Genomic DNA was extracted from frozen rice samples using standard methods. After quantification by UV spectrometry, approximately 40 µg of DNA were digested with EcoRI at 37°C overnight, electrophoresed and transferred to Hybond N+ nylon
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membrane (Amersham) using 20 × SSC. The membrane was then baked at 80°C for 2
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h and hybridized with DIG-labeled probes specific to OsXrn4, which was amplified
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with primers 5’-CTGCTAGTGGTGGAATGAAC-3’ and
5’-CATACTGATTGGGATGTGGCTG-3’. Membranes were pre-hybridized for at
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least 1 h and hybridized overnight at 42°C using a DIG High Prime Labeling and
exposure to X-ray film (Kodak).
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2.5 Virus infection assay
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Detection Starter Kit II (Roche). The hybridization signals were visualized by
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RSV-infected rice plants were obtained as described previously (Yan et al., 2010). Briefly, viruliferous adult small brown planthoppers (Laodelphax striatellus Fallen)
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were transferred onto rice seedlings at the two-leaf stage for virus inoculation. The
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ratio of plants to insects was 1:3. After 48-72 h, the planthoppers were removed. All rice plants were grown in a glasshouse as described above. The numbers of rice plants with symptoms on their newly developing leaves was recorded at 7 day intervals until maturity. The plasmid pCV-OsXrn4 was introduced into A. tumefaciens strain EHA105 and the cells were used to infiltrate the leaves of 3-week-old N. benthamiana plants. The injection areas were 1 cm2 in half leaves. The infiltrated leaves were mechanically 6
ACCEPTED MANUSCRIPT inoculated with 20 μl of crude extracts from TMV-infected leaves. Sap inoculation of TMV-GFP was prepared from N. benthamiana leaves that became infected after infiltration with A. tumefaciens strain EHA105 harbouring an infectious cDNA clone p35S-30B:GFP (Jia et al., 2003). Agrobacterium cultures containing the plasmid
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p35S-30B: GFP were also infiltrated into the upper leaves of 3-week-old T2
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transgenic N. benthamiana plants expressing OsXrn4. All treatments were repeated at
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least three times. 2.6 RNA analysis
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Total RNA was isolated from rice plants using TRI-ZOL (Invitrogen) following the
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manufacturer’s instructions. The RNA that was used for the quantitative real-time PCR was treated with RNase-free DNaseI (TaKaRa). First-strand cDNA was prepared
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from total RNA using KOD-Plus (ToYoBo) with random primers according to the
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manufacturer’s instructions. OsXrn4 expression was detected by real-time PCR using the primers (in rice: 5’-GGCTATCCAGAACAATGAGGAG-3’ and
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5’-GGATAAAACCACTGCCATGAGC-3’, in N. benthamiana:
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5’-AAAGAAGAGGCAGAACTAAAACCCA-3’ and 5’-CAAATCCCCATAGTACTTTGGCAAC-3’). O. sativa actin gene (Accession number: AK068380.1) and N. benthamiana Ubiquitin C gene (UBC) gene (Accession number: AB026056.1) were used as the internal reference genes for analysis (Actin primers: 5’-CTTGTGAAAGATGAAGATCTTGT-3’ and 5’-GTACTCAGCCTTGGCAATCCACA-3’; UBC primers: 5’-GAGGAAGAGACTGGTGAGGGAT-3’ and 7
ACCEPTED MANUSCRIPT 5’-CACAGAGCAAAGACTGGATTGA-3’). A Roche LightCycler® 480 Real-Time PCR System was used for the reaction and the results were analyzed by the ΔΔCT method. For northern blot analyses, DNA probes were synthesized with primers (RSV CP:
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5’-CTCCTGGTCATCACATGCAAG-3’ and
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5’-GCCAGCGCATCGAAGATTGTG-3’; GFP:
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GTTGAATTAGATGGTGATGTTA and 5’-CCTTAAGCTCGATCCTGTTG-3’) and labeled with DIG according to the manufacturer’s protocol (DIG Oligonucleotide
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3’-end labeling Kit, Roche). Pre-hybridization, hybridization and signal detection
Detection Starter Kit II (Roche).
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were done according to the protocol of the DIG High Prime DNA Labeling and
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2.7 5’-3’ exoribonuclease activity assay
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Transcript half-life measurements were used to analyze the 5’-3’ exoribonuclease activity of OsXrn4 on total RNAs in plants (Seeley et al., 1992). O. sativa were grown
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on liquid nutrient medium for 3 weeks. The seedlings were then transferred to a flask
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with incubation buffer including 0.6 mM cordycepin (Sigma-Aldrich) and incubated for up to 100 min. Tissue samples were harvested at various time points and quickly frozen in liquid nitrogen. Total RNAs were isolated followed by formaldehyde denaturing gel electrophoresis. The effect of OsXrn4 on degradation of viral RNAs was also determined. The total active proteins of plant samples were extracted with lysis buffer (10% glycerol, 25mM Tris·HCl, 1mM EDTA,150mM NaCl, 10mM DTT, 1mM PMSF, 0.1% 8
ACCEPTED MANUSCRIPT Nonidet P-40). The full-length TMV-GFP RNA was synthesized in vitro by the T7 RivoMAX express large scale RNA production system (Promega), and was added into the prepared protein solutions, and the mixtures were incubated at 37°C for 45 min~150 min followed by northern blot analyses. Ribonuclease Inhibitor (ToYoBo)
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was added into the mixtures to prevent environmental degradation of RNAs (Tomecki
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et al., 2015).
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2.8 Western blotting
Proteins were separated in a 12% SDS-PAGE gel, transferred onto nitrocellulose
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(Amersham) by wet electroblotting, and were detected with anti-Actin (Abbkine)
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primary antibody and anti-mouse (Sigma) secondary antibody. The antigen-antibody complexes were visualized using NBT/BCIP buffer (Sigma) under standard
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3. Results
3.1 Cloning of OsXrn4 and its subcellular localization
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To identify and clone OsXrn4, AtXrn4 (NM_104327) containing 2844 nucleotides in
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its open reading frame (ORF), was used as query for BLAST analysis against the Oryza sativa Japonica Group nucleotide database. A sequence (Accession number: AK070933) with 2967 nucleotides in its ORF was identified to have high identity to AtXrn4,
and
recognized
as
OsXrn4
(5’-CCGAGACTCCGAGAGAGATTAG-3’
(Fig.
1A
and
B).
Primers and
5’-GCTTACACATTTACAATGGCCG-3’) were then designed to amplify OsXrn4 from Oryza sativa cDNA by PCR. Sequencing confirmed that the cloned OsXrn4 9
ACCEPTED MANUSCRIPT ORF sequence was similar to AK070933 (data not shown). It was predicted to encode a protein of 988 amino acids with the conserved XRN_N domain (Fig. 1A). The amino acid sequences of Xrns contain two highly conserved regions (CR1 and CR2) in their N-terminal segment (Page et al., 1998). Sequence alignment showed that
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OsXrn4 had a higher similarity to cytoplasmic AtXrn4 than to nuclear AtXrn2
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(NM_123619) or AtXrn3 (NM_106217), and the conserved N-terminus of OsXrn4
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contains a XRN_N domain comprised of 256 amino acids and a zinc finger domain, implying that the cloned sequence was the cytoplasmic Xrn of rice (Fig. 1A and B).
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The highly conserved Xrn family is typically represented by cytoplasmic enzymes
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(Xrn1 or Xrn4) and nuclear enzymes (Xrn2 and Xrn3). To detect other Xrn-like sequences of O. sativa, we conducted a search of the Rice Genome annotation project
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database for sequences containing the XRN_N domain. Two further sequences
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(Os01g65220 and Os02g28074) were identified. Os01g65220 was most similar to AtXrn3, Zea mays Xrn3 (ZmXrn3, Gene symbol: LOC103649964) and Setaria italic
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Xrn3-like (SiXrn3-L, Gene symbol: LOC101758007), and it was therefore designated
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OsXrn3. Os02g28074 was most similar to ZmXrn4 (Gene symbol: LOC103641862) and SiXrn4-like (SiXrn4-L, Gene symbol: LOC101777938), but not to AtXrn4 and OsXrn4 (Supp. Table 1). We named it as OsXrn4-like (OsXrn4-L), and suppose that this gene may be specific to monocot plants. To confirm the cytoplasmic location of OsXrn4, GFP-fused OsXrn4 was expressed in N. benthamiana epidermal cells and the subsequent fluorescence was examined under confocal microscopy. In cells expressing only GFP (control), green 10
ACCEPTED MANUSCRIPT fluorescence was present in both the cytoplasm and nucleus, while in cells expressing GFP-fused OsXrn4, fluorescence was only in the cytoplasm, demonstrating that the cloned OsXrn4 was indeed cytoplasmic (Fig. 1C). 3.2 Over-expression of OsXrn4 in rice inhibits the infection of rice stripe virus
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To determine whether over-expression of the monocot OsXrn4 could confer plant
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resistance against viral infection, we used Agrobacterium-mediated transformation to
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produce transgenic rice plants over-expressing OsXrn4 driven by the CaMV 35S promoter (Fig. 2A). All lines over-expressing OsXrn4 had a normal phenotype (data
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not shown). The integration of foreign OsXrn4 into the rice genome in five
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independent T3 transgenic lines (5-5, 11-2, 21-1, 50-2 and 60-1) was verified by southern blot using the OsXrn4 gene as the probe (Fig. 2B). Real-time PCR analysis
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of the T3 transgenic rice plants showed that OsXrn4 was highly expressed in all
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transgenic lines, and that line 11-2 had the highest levels of expression (Fig. 2C). To determine the effect of OsXrn4 over-expression on viral infection, T3 plants
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from five transgenic lines (5-5, 11-2, 21-1, 50-2 and 60-1) were inoculated with RSV
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using viruliferous L. striatellus (Yan et al., 2010; Guo et al., 2012). The non-transgenic plants served as controls. Twenty days post inoculation (dpi), typical symptoms of RSV appeared on wild type plants and some transgenic plants. Symptoms were monitored during the whole experiment and at 90 dpi the symptoms on plants were assigned to one of four categories: no symptoms (-), chlorotic stripes without stunting (+), chlorotic stripes with mild stunting (++), and chlorotic stripes with severe stunting (+++). The average numbers of plants with each class of 11
ACCEPTED MANUSCRIPT symptom from three independent replicates (50 plants per replicate) are shown in Fig. 2D. Survival rates were significantly greater (p<0.01) in lines 21-1 and 50-2 than in the wild type and there were more plants with "-" and "+" symptoms. Lines 11-2 and 60-1 also survived better than the wild type (p<0.05), but line 5-5 did not differ
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significantly from the controls. Accumulation of RSV RNAs in the transgenic plants
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from lines 21-1 and 50-2 with "++" symptoms was less than that in wild type plants
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(Fig. 2E).
To further determine whether the 5’-3’ exoribonuclease activity in transgenic plants
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was enhanced, the accumulation of rRNAs was detected by the half-life measurement
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(Seeley et al., 1992). There were no differences between the plants treated with 0.6 mM cordycepin for 20 min, but after 80 min rRNAs were degraded more in
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transgenic line 50-2 than in wild type plants. After 100 min, rRNAs in transgenic
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plants were further degraded but those in wild type plants were reduced only a little compared with those at 20 min (Fig. 3). Line 11-2 had no more 5’-3’ exoribonuclease
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in lines 50-2.
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activity than line 50-2 even though the RNA levels of OsXrn4 were higher than those
Taken together, the results demonstrate that the over-expression of OsXrn4 enhanced 5’-3’ exoribonuclease activity and conferred rice resistance to RSV infection by decreasing the accumulation of viral RNAs. 3.3 Transient expression of OsXrn4 in Nicotiana benthamiana leaves inhibits the infection of TMV It has been reported that the ectopic expression of AtXrn4 in N. benthamiana leaves 12
ACCEPTED MANUSCRIPT led to degradation of CNV RNAs (Cheng et al., 2007). To determine whether the monocot OsXrn4 had the same ability to degrade viral RNAs in a dicotyledonous plant, we over-expressed OsXrn4 in N. benthamiana leaves transiently by agroinfiltration and then (12 h later) mechanically inoculated the leaves with TMV. To
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ensure a high level of expression of OsXrn4, it was co-expressed with the suppressor
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of RNA silencing, p19 of tomato bushy stunt virus (TBSV). TMV infection was
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visualized by inoculation with the modified TMV vector expressing GFP. Five days post infiltration (dpi) of TMV-GFP, fewer fluorescent loci were present in zones
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expressing OsXrn4 than in zones expressing the unrelated GUS protein that was used
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as a control (Fig. 4A and B). Moreover, northern blot (using Gfp probe) revealed that TMV RNAs accumulated less in the zones expressing OsXrn4 than in the control (Fig.
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4C). Real-time PCR confirmed the expression of OsXrn4 in the OsXrn4-infiltrated
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zones (Fig. 4D). These results indicate that transient expression of OsXrn4 in the dicotyledon N. benthamiana leaves can inhibit infection by TMV.
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3.4 Transgenic expression of OsXrn4 in Nicotiana benthamiana inhibits the
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infection of TMV
To investigate the effects of constitutive expression of OsXrn4 on infection by TMV in N. benthamiana, we produced transgenic N. benthamiana plants expressing CaMV 35S-driven OsXrn4. PCR detection and RT-PCR analysis of the T3 transgenic N. benthamiana plants confirmed that OsXrn4 was transferred into N. benthamiana plants and could express in all transgenic lines (Fig. 5A and B). For viral challenge, Agrobacterium containing the plasmid p35S-30B:GFP (the infectious clone of 13
ACCEPTED MANUSCRIPT TMV-GFP) was infiltrated into the upper leaves of three lines of 3-week-old T4 transgenic plants (10 plants per treatment). Wild type N. benthamiana plants served as controls. From 5 dpi, GFP fluorescence was monitored every 24 h under UV to detect the TMV infection status. Fluorescence first appeared on the new top leaves of control
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plants at 6 dpi but not until at least 2 days later in those expressing OsXrn4 (Fig. 5C).
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TMV RNAs accumulated at lower levels in plants expressing OsXrn4 than in wild
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type plants at either 6 or 8 dpi (Fig. 5D). These results further demonstrate that OsXrn4 inhibits infection of TMV in N. benthamiana.
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To examine the 5'-3' exoribonuclease activity of OsXrn4 on TMV RNAs, TMV
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genomic RNAs were synthesized in vitro and mixed with the total proteins extracted from transgenic or wild type plants for different incubation times. The levels of TMV
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RNAs from the different plants were similar at 45 min, but at 150 min TMV RNA
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levels were significantly lower in the transgenic plant extracts than in those from wild type plants (Fig. 5E). This demonstrates that OsXrn4 in transgenic plants has specific
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5'-3' exoribonuclease activity against TMV RNAs.
4. Discussion
Plant Xrn4 is a key protein in RNA metabolic pathways and can also degrade exogenous nucleic acid (transgene or virus RNAs) in the cytoplasm (Souret et al., 2004; Cheng et al., 2007). Meanwhile, there have been few studies of the Xrn4 of plants other than A. thaliana and N. benthamiana. We here cloned Xrn4 from O. sativa and investigated its function in inhibiting viral infection, and found that the 14
ACCEPTED MANUSCRIPT monocot Xrn4 also inhibits viral infection and that this function is retained when it is ectopically expressed in a dicotyledon (Fig. 3 and Fig. 4). While this supports the view that plant Xrn4 plays an antiviral role it has also been reported that AtXrn4 can act as an endogenous suppressor of post-transcriptional gene silencing (PTGS) by
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degrading the substrate of the RNA-induced silencing complex (RISC) (Gazzani et al.,
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2004). This means that Xrn4 could enhance viral infection by suppressing the antiviral
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RNA silencing mechanism in plants. Our results do not exclude this possibility since the transgenic rice line 11-2 had the highest OsXrn4 expression levels, but did not
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have the highest resistance against RSV infection. We suppose that the final effects of
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OsXrn4 over-expression in plants results from a balance between its functions of inhibiting viral infection by degrading viral RNAs and assisting viral infection by
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suppressing antiviral RNA silencing.
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The use of virus-derived genes including coat protein, movement protein and replicase to produce transgenic plants resistant to viral infection has been explored
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since the first transgenic resistant tobacco expressing TMV coat protein (CP) gene
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was created in 1986 (Abel et al., 1986; Sudarshana et al., 2007). About ten years ago, resistance strategies based on the RNA silencing mechanism, such as double-stranded RNAs (dsRNAs), hairpin RNAs and artificial miRNAs began to be developed (Kalantidis et al., 2002; Tenllado et al., 2004; Yin et al., 2005; Niu et al., 2006; Qu et al., 2007; Sudarshana et al., 2007). Only a few studies have attempted to examine transgenic strategies to produce rice with RSV resistance (Sasaya et al., 2014; Li et al., 2016) but Shimizu et al. found that expression of dsRNAs targeting RSV pc3 and pc4 15
ACCEPTED MANUSCRIPT genes conferred near immunity to RSV infection, strongly suggesting that a dsRNA strategy could provide high resistance against RSV (Shimizu et al., 2011). Recently, several host genes have been identified that might be used to enhance plant resistance against viruses. Transgenic rice with elevated expression of two host transcription
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factors, RF2a and RF2b, had weak or no symptoms after inoculation with rice tungro
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bacilliform virus (Dai et al., 2008). Transgenic soybean plants over-expressing
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GmAKT2, the ortholog of Arabidopsis K+ weak channel encoding gene AKT2, showed significantly enhanced resistance to soybean mosaic virus (SMV) (Zhou et al., 2014)
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and transgenic Brassica rapa plants over-expressing eIF(iso)4E variants had
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broad-spectrum turnip mosaic virus resistance (Kim et al., 2014). Genes from plants pose less biological safety risks than genes or sequences from the virus genome, and
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our results show that OsXrn4 has the potential to provide such a host gene-based
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resistance strategy against RSV.
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ACCEPTED MANUSCRIPT Conflict of interests The authors declare no conflict of interest.
Acknowledgements
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This work was financially supported by the State Basic Research Program of China
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(2014CB138400), the International Science & Technology Cooperation Program of
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China (2015DFA30700), the Major Project of New Varieties of Genetically Modified Organism of China (2013ZX08001-002, 2014ZX0800104B), the National Natural
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Science Foundation of China (31272016). This work is also supported by the Program
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for Leading Team of Agricultural Research and Innovation of Ministry of Agriculture, China. We thank Professor M. J. Adams, Stevenage, UK for help in correcting the
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English of the manuscript.
Author contribution statement
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Fei Yan, Shanshan Jiang, Liangliang Jiang and Jianpin Chen designed the experiments.
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Jian Yang, Jiejun Peng and Yuwen Lu cloned the sequence of OsXrn4 and analyzed the subcellular localization of OsXrn4. Shanshan Jiang, Liangliang Jiang and Lin Lin prepared the transgenic rice and tobacco plants over-expressing of OsXrn4. Shanshan Jiang, Liangliang Jiang and Hongying Zheng carried out the antiviral analysis of transgenic plants. Fei Yan, Shanshan Jiang and Jianping Chen prepared the manuscript.
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ACCEPTED MANUSCRIPT Tables Table 1 Primers used for construction of pCV-OsXrn4.
Figure legends
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Figure 1 Alignment of OsXrn4 with AtXrn2, AtXrn3 and AtXrn4, and subcellular
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localization of GFP-fused OsXrn4. A, amino acid alignment of OsXrn4 with
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AtXrn2, AtXrn3 and AtXrn4. The putative conserved XRN domain and zinc finger motif is underlined. Two highly conserved regions (CR1and CR2) are
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labeled. B, amino acid (numbers shadowed with color light brown) and
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nucleotide identities (numbers shadowed with light blue) of OsXrn4 to AtXrn2, AtXrn3 and AtXrn4, showing the high identity of OsXrn4 with AtXrn4. C,
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subcellular localization of GFP-fused OsXrn4 in N. benthamiana epidermal cells.
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GFP-fused OsXrn4 was localized in the cytoplasm, while the GFP control was localized in both the cytoplasm and nucleus. Fluorescence photographs were
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taken at 4 dpi. Scale bar: 25 μm.
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Figure 2 Resistance to RSV infection of T3 transgenic rice over-expressing OsXrn4. A, map of the binary vector used for transformation with OsXrn4. B, southern blot analysis of T3 transgenic plants demonstrating the integration of foreign OsXrn4 in the genomic DNA. The upper band represents the internal OsXrn4 in the rice genome, and the lower one shows the integrated foreign OsXrn4. DNA sizes are indicated by black arrowheads. Wt: wild type plant. C, quantitative RT-PCR indicated the expression level of OsXrn4 transcripts in 23
ACCEPTED MANUSCRIPT transgenic and wild type rice plants, showing that OsXrn4 was over-expressed in all transgenic lines. Bars represent the standard errors of the means. A two-sample unequal variance directional t-test was used to test the significance of the difference (*P < 0.05, **P <0.01). D, analysis of RSV incidence in wild
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type plants and in those over-expressing OsXrn4 at 90 dpi (50 plants per
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treatment). Symptoms were divided into four classes according to severity. -: no
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symptom, +: chlorotic stripes without stunting, ++: chlorotic stripes with mild stunting, +++: chlorotic stripes with severe stunting, and D represents dead
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plants. The numbers of each class and the numbers of surviving plants (NSP) are
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arrowed. E, northern blot (using RSV CP probe) showing that RSV RNAs accumulated less in OsXrn4 transgenic plants than in wild type plants at 50 dpi.
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Wt: wild type plant; 21-1 and 50-2: OsXrn4 transgenic plants. RNA sizes are
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indicated by black arrowheads.
Figure 3 5’-3’ exoribonuclease activity assay of transgenic plants overexpressing
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OsXrn4. Leaf samples from transgenic (lines 11-2 and 50-2) and wild type (wt)
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plants were collected and treated with cordycepin for 20, 80 and 100 minutes (min). rRNAs were extracted from the treated leaves and electrophoresed in 1.5% agarose/5.7% formaldehyde gel with ethidium bromide. The rRNAs (28S and 18S) in transgenic plants were difficult to detect at 100 min, but those in wild type plants were only slightly less than in those sampled at 20 min. RNA sizes are indicated by black arrowheads. Figure 4 Resistance to TMV infection of N. benthamiana leaves transiently 24
ACCEPTED MANUSCRIPT expressing OsXrn4. A, images taken under long-wavelength UV-light at 5 dpi of TMV-GFP, showing fewer fluorescent loci present in zones expressing OsXrn4 than in zones expressing the unrelated GUS protein (control). The infiltrated zones were indicated by circles with broken white line. B, compared with the
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control (GUS), the number of fluorescent loci in zones expressing OsXrn4 was
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reduced. C, northern blot showed that TMV RNAs accumulated less in zones
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expressing OsXrn4 than in the control. The GFP probe used in the experiment could detect all genomic and subgenomic RNAs of TMV. RNA sizes are
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indicated by black arrowheads. D, Quantitative real-time PCR confirmed the
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expression of OsXrn4 in the OsXrn4-infiltrated zones. Bars represent the standard errors of the means. A two-sample unequal variance directional t-test
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Figure 5 Resistance of T4 OsXrn4 transgenic N. benthamiana plants to TMV infection. A, PCR detection confirmed that OsXrn4 has been transferred into N.
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benthamiana. Wild type (wt) plants were used as the negative control and the
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plasmid of pCV-OsXrn4 was used as positive control for PCR. No bands could be obtained from the wild type plants, demonstrating the specificity of the primers. B, RT-PCR confirmed the expression of OsXrn4 in transgenic N. benthamiana
plants.
C
shows
the
development
of
fluorescence
in
TMV-GFP-infected wild type plants and in OsXrn4 transgenic plants at 6 and 8 dpi (10 plants per treatment). Boxes with a broken white line indicate the leaves with TMV-GFP systemic infection. D, TMV RNAs accumulated at a lower level 25
ACCEPTED MANUSCRIPT in plants expressing OsXrn4 than in wild type plants at either 6 dpi or 8 dpi of TMV-GFP. Total RNAs were isolated from systemic leaves at the same position. The GFP probe used in the experiment could detect all genomic and subgenomic RNAs of TMV. RNA sizes are indicated by arrows. E, equal amounts of TMV
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genomic RNAs were incubated with total protein extracted from transgenic and
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wild type plants for 45 min and 150 min. The levels of TMV RNAs among
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different treatments were similar at 45 min, but at 150 min the TMV RNAs mixed with total proteins from transgenic plants were reduced significantly
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compared to those treated with total proteins from wild type, showing that
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OsXrn4 in transgenic plants has 5'-3' exoribonuclease activity specifically
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against TMV RNAs. Actin detected by its antibody was used as loading control.
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Electronic Supplementary Material Supplementary Table S1 Sequence alignments of OsXrns with AtXrns, ZmXrns
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and SiXrns.
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ACCEPTED MANUSCRIPT Table 1 Primers used for construction of pCV-OsXrn4.
Primers
Sequences 5’-TCTAGAATGGGAGTCCCGGCGTTCTACC-3’ (XbaI)
OsXrn4-1r:
5’-TCTAGACCTCTGCTGATTCAT-3’ (XbaI)
OsXrn4-3f:
5’-GGATCCTAGGATGGCCATGGATA-3’ (BamHI)
OsXrn4-3r:
5’-GAGCTCTCACTCACTCAGGCCATTGG-3’ (SacI)
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OsXrn4-1f:
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ACCEPTED MANUSCRIPT Abbreviation list Os, Oryza sativa At, Arabidopsis thaliana Si, Setaria italic
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Zm, Zea mays
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TMV, tobacco mosaic virus
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CNV, cucumber necrosis virus RSV, rice stripe virus
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TBSV, tomato bushy stunt virus
GFP, green fluorescent protein
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UBC, Ubiquitin C gene
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dpi, days post infiltration
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cDNA, complementary DNA
ORF, open reading frame
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CR, conserved region
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PTGS, post-transcriptional gene silencing RISC, RNA-induced silencing complex CP, coat protein
dsRNAs, double-stranded RNAs
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ACCEPTED MANUSCRIPT Highlights ●
The cytoplasmic Xrn4 gene of Oryza sativa (OsXrn4) was cloned
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Over-expression of OsXrn4 enhances the resistance of rice to rice stripe virus
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Ectopic expression of OsXrn4 enhances the resistance of N. benthamiana to TMV
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● OsXrn4 could be used to produce plants resistant to viral infection
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