Sugarcane mosaic virus infection of model plants Brachypodium distachyon and Nicotiana benthamiana

XU Jing-sheng , DENG Yu-qing , CHENG Guang-yuan , ZHAl Yu-shan , PENG Lei , DONG Meng , XU Qian , YANG Yong-qing,

1 Key Laboratory of Sugarcane Biology and Genetic Breeding (Fujian) of Ministry of Agriculture and Rural Affairs, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R.China

2 Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, College of Crop Science, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R.China

3 Root Biology Center, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R.China

Abstract Sugarcane mosaic virus (SCMV; genus Potyvirus, family Potyviridae) is a causal pathogen of sugarcane mosaic disease,and it is widespread in regions where sugarcane (Saccharum spp. hybrids) is grown. It is difficult to investigate the molecular mechanism of pathogen infection in sugarcane because of limited genomic information. Here, we demonstrated that SCMV strain FZ1 can systemically infect Brachypodium distachyon inbred line Bd21 and Nicotiana benthamiana through inoculation,double antibody sandwich enzyme-linked immunosorbent, transmission electron microscopy, and reverse transcription PCR assays. The leaves of Bd21 developed mosaic symptoms, while the leaves of N. benthamiana showed no obvious symptoms under the challenge of SCMV-FZ1. We concluded that B. distachyon inbred line Bd21 is a promising experimental model plant compared with N. benthamiana for study on the infectivity of SCMV. This is the first report on the SCMV infection of model plants B. distachyon inbred line Bd21 and N. benthamiana, which will shed light on the mechanism of SCMV infection of sugarcane and benefit sugarcane breeding against sugarcane mosaic disease.

Keywords: SCMV, infection, sugarcane, Brachypodium distachyon, Nicotiana benthamiana

1. lntroduction

Sugarcane (Saccharum spp. hybrids), a C4plant, is the principal sugar and energy crop around the world (Lam et al. 2009; Rae et al. 2009). Sugarcane mosaic disease is widely distributed in sugarcane-growing countries (Wu et al. 2012) and causes heavy yield losses, which can be as high as 50% (Viswanathan and Balamurlikrishnana 2005;Putra et al. 2014). This disease almost collapsed the sugar industry in Argentina, Brazil, and Louisiana in the 1920s(Koike and Gillaspie 1989) and continues to threaten the sugarcane industry (Wu et al. 2012). The main pathogens of sugarcane mosaic disease are Sugarcane mosaic virus(SCMV), Sorghum mosaic virus (SrMV), and Sugarcane streak mosaic virus (SCSMV). All of these viruses are in the Potyviridae family, with SCMV and SrMV in the genus Potyvirus (Ward and Shukla 1991) and SCSMV in the genus Poacevirus (Xu et al. 2010; Putra et al. 2014).

It is important to elucidate the molecular mechanism of mosaic infections in sugarcane to identify and utilize resistant genes, thus enabling the ability to breed against mosaic disease in sugarcane. However, the molecular mechanism of mosaic infections in sugarcane is poorly understood (Zhai et al. 2015; Cheng et al. 2017). There are two main reasons for this. One reason is that Saccharum species are polyploids with huge genomes (D’Hont et al.1998). The genome size of S. officinarum ranges from 7.50 to 8.55 Gb, and that of Saccharum spontaneum ranges from 3.36 to 12.64 Gb (Zhang et al. 2012). Modern sugarcane cultivars widely planted around the world are derived from interspecific hybridization between S. officinarum(2n=8x=80) and S. spontaneum (2n=5x-16x=40-128), with 80-90% of the genome from S. officinarum and 10-20% of the genome from S. spontaneum (Grivet et al. 1996; Hoarau et al. 2002). However, the whole genome of sugarcane has not yet been sequenced. The other reason is that sugarcane plants are large and have long generation times, which makes it more difficult to genetically transform sugarcane than to transform model plants (Yao et al. 2004; Wu and Birch 2007; Srikanth et al. 2011).

It is therefore necessary to identify suitable hosts for sugarcane mosaic pathogens that can be used to investigate the molecular mechanism of pathogen infectivity. This will allow researchers to isolate and identify genes involved in infection by homologous cloning and transformation.Brachypodium distachyon inbred line Bd21 is an emerging temperate monocot model plant for cereal-pathogen interactions (Fitzgerald et al. 2015). The B. distachyon inbred line Bd21 has a short stature (15-30 cm), a short life cycle (80-90 days) and a small diploid genome (～272 Mb)(Vogel et al. 2010). It is self-fertile and readily transformed by Agrobacterium tumefaciens-mediated transformation(Brkljacic et al. 2011). Many cereal pathogens, including bacteria, fungi, oomycetes, and viruses, infect B. distachyon(Fitzgerald et al. 2015). To date, seven different viruses have been shown to infect B. distachyon: Panicum mosaic virus (PMV, genus Panicovirus, family Tombusviridae) and its satellite virus (SPMV) (Mandadi and Scholthof 2012),Barley stripe mosaic virus (BSMV, genus Hordeivirus, family Virgaviridae) (Cui et al. 2012), Wheat streak mosaic virus(WSMV, genus Tritimovirus, family Potyviridae), Brome mosaic virus (BMV, genus Bromovirus, family Bromoviridae),Sorghum yellow banding virus (SYBV, unassigned),Foxtail mosaic virus (FoMV, genus Potexvirus, family Alphaflexiviridae) (Mandadi et al. 2014), and Barley yellow dwarf virus (BYDV, genus Luteovirus, family Luteoviridae)(Tao et al. 2016). To date, no potyvirus has been shown to infect B. distachyon. Nicotiana benthamiana has been used as a model plant for the study of host-virus interactions for potyviruses (Goodin et al. 2008), such as Potato virus Y(PVY), Turnip mosaic virus (TuMV), Clover yellow vein virus(C1YVV) (Fukuzawa et al. 2010), and Soybean mosaic virus (SMV) (Gao et al. 2015). Moreover, transgene in N. benthamiana is highly efficient and easy to operate.

Sugarcane mosaic pathogens have a narrow host range,and they are almost completely confined to members of Poaceae, such as sugarcane, sorghum (Sorghum bicolor),and maize (Zea mays). Some weed grasses (Brachiaria moniliformis, Panicum repens, Paspalum conjugatum, and Rottboelia exaltata) can be infected by SCSMV through rubinoculation (Putra et al. 2015). Nevertheless, there have been no detailed reports of SCMV infecting B. distachyon or Nicotiana spp. naturally or artificially. In this study, we successfully inoculated the B. distachyon inbred line Bd21 and N. benthamiana with SCMV. The present work will benefit for study on the infectivity of sugarcane mosaic pathogens and breeding resistant sugarcane cultivars.

2. Materials and methods

2.1. Plant materials and SCMV inoculum

The virus-free tissue cultured sugarcane cv. Badila seedlings(highly susceptible to sugarcane mosaic pathogens), the B. distachyon inbred line Bd21, the N. benthamiana and the SCMV strain FZ1 (GenBank accession number: KR108212)were provided by the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, China. N. benthamiana seeds were germinated directly in moistened nutritive soil mixed with vermiculite at 24-25°C under 16-h light/8-h dark photoperiod in a growth chamber. N. benthamiana seedlings were transplanted into plastic cups with one plant per cup after germination.Bd21 seeds were sown in nutritive soil, stratified at 4°C for 7-14 days, and then grown under the following conditions:14-h light/10-h dark photoperiod (21°C/18°C day/night)under an illumination of approximately 250-280 μmol m-2s-1with 60% relative humidity. The SCMV-FZ1 was propagated on the Badila.

2.2. Virus inoculation

SCMV inoculum was prepared from the sugarcane cultivar Badila inoculated with SCMV-FZ1 by homogenizing approximately 1 g of symptomatic leaf tissue in 3 mL of 0.01 mol L-1phosphate buffer (pH 7.0). This inoculum was used to inoculate Badila, N. benthamiana and Bd21.N. benthamiana plants were inoculated with virus inoculum at the 5-6 leaf stage. A sterilized hairbrush dipped with SCMV-FZ1 inoculum was used to gently brush on the young leaves of N. benthamiana. The Bd21 and Badila plants were inoculated with virus inoculum at the 3-5 or 5-6 leaf stage, respectively. We dipped the sterilized gauze into SCMV-FZ1 inoculum, sequentially the sterilized 30-50 mesh quartz sand, and then rubbed the young leaves of the Bd21 or Badila plants individually from base to tip. Inoculated Badila plants were used as the positive control, while mock inoculated Badila, Bd21 or N. benthamiana plants with 0.01 mol L-1phosphate buffer (pH 7.0) were used as the negative controls. The inoculated plants were covered to maintain humidity for 24 h in the dark, and then water spray treatments were conducted before being transferred to the growth conditions described above. Phenotypes were observed once per week until the mosaic symptoms became visible on the leaves.

2.3. RT-PCR analysis

Reverse transcription PCR (RT-PCR) was deployed to detect the virus genome. Total RNA was isolated using a kit (Tiangen, Beijing, China). The RNA quality was checked by electrophoresis on a 1.5% agarose gel. Then,the cDNA was synthesized using a PrimeScript®RT Master Mix Kit (TaKaRa, Otsu, Japan) according to the manufacturer’s instructions. Forward primer SCMV-CP-F(5´-TACAGAGAGACACACAGCTG-3´) and reverse primer SCMV-CP-R (5´-ACGCTACACCAGAAGACACT-3´) were designed based on the sequence of coating protein (CP)gene from SCMV-FZ1. The size of the expected amplicon was 226 bp. PCR was conducted on a thermal cycler(Eppendorf, Germany) to detect the presence of SCMVFZ1 in the samples with the cDNA as a template. The PCR mixture (25 μL total volume) contained 1 μL of template cDNA(＜1 μg), 1 μL of each primer (10 μmol L-1), 0.125 μL Ex-Taq DNA polymerase, 2.5 μL of 10× PCR buffer, 2 μL of dNTPs,and 17.375 μL sterilized ddH2O. The PCR amplification program was as follows: one cycle of 94°C for 4 min; 34 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 1 min; final extension at 72°C for 7 min; and hold at 4°C. The amplified products were separated by electrophoresis on a 1% agarose gel, stained with ethidium bromide and photographed.

2.4. DAS-ELlSA

Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was applied to detect the presence of SCMV CP using commercial kits (Agdia, Elkhart, IN, USA)according to the manufacturer’s instructions. For each inoculation assay, three biological replicates were set. All samples for DAS-ELISA test were replicated three times, and the average values of optical density at 405 nm (OD405nm)were compared to the negative controls (mock-inoculated plants). The tested sample was considered to be positive for SCMV when its OD405nmvalue was at least twice the value of the negative controls; otherwise, the test sample was considered to be a failed infection.

2.5. Transmission electron microscopy

Leaves were sampled, fixed, dehydrated, and embedded,and then ultrathin sections were prepared for transmission electron microscopy (TEM) detection, as described previously (Wei et al. 2006). Finally, the sections were imaged by TEM (H-7650, HITACHI, Japan).

2.6. Back-inoculation

Back-inoculation was performed with the inoculum prepared from systemically infected leaves of N. benthamiana plants or Bd21 plants that tested positive in the RT-PCR and DASELISA analyses by homogenizing approximately 1 g of leaf tissue in 3 mL of 0.01 mol L-1phosphate buffer (pH 7.0).The inoculum were back-inoculated onto virus-free Badila sugarcane plantlets at the 5-6 leaf stage. Sugarcane plants mock-inoculated with 0.01 mol L-1phosphate buffer (pH 7.0)were used as the negative controls.

3. Results

3.1. Symptoms of B. distachyon and N. benthamiana infected by SCMV-FZ1

We examined the infectivity of SCMV using two model plants, the monocot B. distachyon inbred line Bd21 and the dicot N. benthamiana. The virus used was SCMV isolate FZ1. The susceptible sugarcane cultivar Badila, known to be systemically infected by the SCMV-FZ1, was used as the positive control (CK). Approximately 6-8 weeks after SCMVFZ1 infection, the CK line of Badila showed yellow stripes and spots on newly unfolded leaves (Fig. 1-A) compared with the mock-inoculated plants (Fig. 1-B). Approximately 4-8 weeks after infection, yellowing and necrotic symptoms developed on the leaves of Bd21 (Fig. 1-C) compared with the mock-inoculated plants (Fig. 1-D). However, no obvious symptoms were observed on the leaves of N. benthamiana(Fig. 1-E and F). These observations indicated that SCMVFZ1 can systemically infect Bd21, but infection rates for N. benthamiana are uncertain.

3.2. lmmunological and biological evidence for SCMV infectivity of model plants

Fig. 1 Symptoms on plants after infection with Sugarcane mosaic virus (SCMV)-FZ1. Leaves of cv. Badila inoculated with virus inoculum (A) and phosphate buffer (B). Brachypodium distachyon inbred line Bd21 inoculated with virus inoculum (C)and phosphate buffer (D). Nicotiana benthamiana inoculated with virus inoculum (E) and phosphate buffer (F).

RT-PCR and DAS-ELISA were applied to detect the presence of SCMV-FZ1 in the inoculated N. benthamiana plants and Bd21 plants. The inoculated and systemically infected leaves of N. benthamiana plants and Bd21 plants were sampled after 6-8 weeks once mosaic symptoms were observed. Total RNAs were isolated and RT-PCR assays were conducted to amplify the partial CP gene of SCMVFZ1 with the primer pairs SCMV-CP-F and SCMV-CP-R.The RT-PCR assays showed that the anticipated bands of the CP gene were observed (Fig. 2, L1-4). Sequencing of the PCR products confirmed that the sequences showed 100% identity with SCMV-FZ1 (data not shown). In the DAS-ELISA assays, a commercial kit (Agdia, Elkhart, IN,USA) was used to detect the infections of SCMV on the N. benthamiana plants and Bd21 plants according to the manufacturer’s instructions. The results showed that the OD405nmvalues of extracts from leaves developping disease symptoms were twice that of the negative controls (Table 1),which also confirmed the presence of SCMV in the two model plants (Fig. 3 and Table 1), although N. benthamiana plants showed no obvious symptoms.

To confirm the infection of SCMV on N. benthamiana and Bd21, we conducted the TEM assay. SCMV particles and pinwheel inclusion bodies were found in the systematic leaves of N. benthamiana (Fig. 3-A and B) and Bd21(Fig. 3-C, D and E). Disintegrated chloroplasts were also found in the leaves of Bd21 (Fig. 3-E).

To further explore the infectivity of SCMV in the two model plants, we conducted back-inoculation experiments. The virus inoculum were prepared from the systematic leaves of N. benthamiana or Bd21 and then inoculated onto the leaves of Badila. Approximately 6 weeks after inoculation,Badila plants showed typical yellow stripes and spots on newly unfolded leaves (Fig. 4-A and B) compared with mockinoculated Badila plants (Fig. 4-C). These symptoms were very similar to those on Badila plants directly inoculated with SCMV-FZ1 (Fig. 1-A). The symptomatic leaves of backinoculated Badila were sampled and detected by RT-PCR and DAS-ELISA. These results confirmed the presence of SCMV-FZ1 (Fig. 2, L5-6). These results also indicated that SCMV-FZ1 collected from both N. benthamiana and Bd21 retained its ability to infect Badila and could be recovered from infected Badila plants. Combining the evidence above, we concluded that SCMV could establish systematic infection of N. benthamiana or Bd21.

Fig. 2 RT-PCR detection of Sugarcane mosaic virus (SCMV). M, marker; + and -, positive and negative control, respectively;Lanes 1-6, samples from Nicotiana benthamiana inoculated leaves, N. benthamiana newly unfolded leaves, Bd21 inoculated leaves, Bd21 newly unfolded leaves, newly unfolded leaves of Badila back-inoculated from N. benthamiana, and newly unfolded leaves of Badila back-inoculated from Bd21, respectively.

Table 1 Serological determinations of Sugarcane mosaic virus (SCMV)-inoculated plants1)

Fig. 3 Transmission electron microscopy observation of Sugarcane mosaic virus (SCMV)-infected leaves of Nicotiana benthamiana and cv. Bd21. N. benthamiana leaves infected by SCMV with the virus particles (A, arrow) and the pinwheel inclusion bodies and integrated chloroplast (B). Bd21 leaves infected by SCMV with the virus particles (C, arrow),the pinwheel inclusion bodies (D) and the disintegrated chloroplast (E). CW, cell wall; PW, pinwheel inclusion body;M, mitochondria; CH, chloroplast. Bar=500 nm.

Fig. 4 Symptoms on Badila leaves back-inoculated with inoculum prepared from Sugarcane mosaic virus (SCMV)-FZ1 infected Nicotiana benthamiana and cv. Bd21, respectively.Photos were taken 6 weeks after back-inoculation. A, leaves of Badila inoculated with inoculum prepared from N. benthamiana.B, leaves of Badila inoculated with inoculum prepared from Brachypodium distachyon inbred line Bd21. C, healthy leaves of Badila.

4. Discussion

Among the fully sequenced monocot C4plants, sorghum(S. bicolor) is the closest relative of sugarcane and is also a host of SCMV. The sorghum genome is small and shows good colinearity with that of sugarcane (Wang et al. 2010).However, like sugarcane, sorghum plants are large with long generation times, and they are difficult to genetically transform (Grootboom et al. 2014). Maize is another host of SCMV, but like sorghum, it is yet not an ideal model plant for sugarcane. The genomes of two small C4plants in Paniceae, green bristle grass (Setaria viridis) and foxtail millet (Setaria italica), have been sequenced recently(Bennetzen et al. 2012; Jia et al. 2013). These two plants have small genomes and short life cycles and are presumed to be model C4plants (Brutnell et al. 2010; Mandadi et al.2014). However, their use as model plants is restricted by the low transformation efficiencies (Muthamilarasan and Prasad 2015). Until now, no suitable C4model plant has been available for research on SCMV infectivity of sugarcane. Among the C3plants whose entire genomes have been sequenced, rice (Oryza sativa) has been used extensively as a model monocot. However, its large plant size, demanding growth requirements, and long generation time have restricted its use as a model plant (Jung et al.2008). Additionally, the colinearity between the rice genome with those of other Poaceae plants is poor (Bennetzen et al.1998, 2003; Devos and Beales 1999; Guyot and Keller 2004), especially for genes controlling important agricultural traits such as resistance (Gallego et al. 1998; Leister et al.1999). The B. distachyon inbred line Bd21 has been used as a model grass plant (Vain 2011). Although the genome of Bd21 is more closely related to those of Triticeae than to those of sorghum and maize (Paterson et al. 2009), Bd21 is considered to be a suitable model plant for sugarcane at the present time.

N. benthamiana is a model plant for virus-plant interactions because of its susceptibility to many viruses from different genera or families (Goodin et al. 2008).N. benthamiana has been extensively used in experiments such as transient expression, subcellular location, intra- or intercellular transport, and protein interaction to explore the viral infection mechanism (Zhai et al. 2015; Cheng et al.2017). The successful establishment of SCMV infection of N. benthamiana will make it easier to identify genes involved in SCMV infection in sugarcane. However, the asymptomatic phenotype of N. benthamiana infected by SCMV limits its application in evaluating the mechanism of SCMV infection in sugarcane. Although virus particles and pinwheels inclusion bodies were found in the SCMVinoculated N. benthamiana plants, almost no degraded chloroplasts were observed by TEM assays, which partially accounts for the asymptomatic phenotype of SCMVinoculated N. benthamiana plants compared with that of Bd21 plants.

5. Conclusion

We demonstrated that SCMV can establish systemic infection on N. benthamiana and B. distachyon in bred line Bd21. The Bd21 developed obvious mosaic symptoms compared with N. benthamiana under the infection of SCMV-FZ1, indicating that B. distachyon inbred line Bd21 is a promising experimental model plant for study on the infectivity of SCMV. Considering that SCMV, SrMV, and SCSMV are the main causal pathogens of sugarcane mosaic disease and share the same genomic structure,we speculated that SrMV and SCSMV could also infect N. benthamiana or Bd21. The present work may shed light on the molecular mechanism of sugarcane mosaic pathogens infectivity, thereby benefiting sugarcane breeding against sugarcane mosaic disease.

Acknowledgements

We thank Dr. Mao Qianzhuo, Fujian Agriculture and Forestry University, China, for the transmission electron microscope observation of plant samples. Financial support was provided by the National Natural Science Foundation of China (31371688).