Further characterization of Maize chlorotic mottle virus and its synergistic interaction with Sugarcane mosaic virus in maize

Maize chlorotic mottle virus (MCMV) was first reported in maize in China in 2009. In this study we further analyzed the epidemiology of MCMV and corn lethal necrosis disease (CLND) in China. We determined that CLND observed in China was caused by co-infection of MCMV and sugarcane mosaic virus (SCMV). Phylogenetic analysis using four full-length MCMV cDNA sequences obtained in this study and the available MCMV sequences retrieved from GenBank indicated that Chinese MCMV isolates were derived from the same source. To screen for maize germplasm resistance against MCMV infection, we constructed an infectious clone of MCMV isolate YN2 (pMCMV) and developed an Agrobacterium-mediated injection procedure to allow high throughput inoculations of maize with the MCMV infectious clone. Electron microscopy showed that chloroplast photosynthesis in leaves was significantly impeded by the co-infection of MCMV and SCMV. Mitochondria in the MCMV and SCMV co-infected cells were more severely damaged than in MCMV-infected cells. The results of this study provide further insight into the epidemiology of MCMV in China and shed new light on physiological and cytopathological changes related to CLND in maize.


Maize chlorotic mottle virus (MCMV) is the only member of the genus Machlomovirus in the family
Tombusviridae. The 4436-nucleotide, sense, single-stranded RNA of the MCMV genome is encapsidated in isometric particles about 30 nm in diameter. Six open reading frames (ORFs) have been reported for the MCMV genome 1,2 . ORF1 encodes a 32-kDa hypothetic protein of unknown function. ORF2 encodes a 50-kDa protein (P50) and an N-terminus-overlapped 111 kDa protein (P111) produced by translational read-through of the UAG stop codon of ORF2. ORF4 encodes a 7-kDa protein, which functions as the movement protein of MCMV. A 31-kDa protein is expressed from ORF5 when the UGA stop codon of ORF4 is suppressed; it also has a role in cell-to-cell movement of MCMV 3 . The coat protein (CP) of MCMV is expressed from the 3′ proximal ORF (Fig. 1). When maize plants are co-infected with MCMV and one of several potyviruses including maize dwarf mosaic virus (MDMV), wheat streak mosaic virus (WSMV) or sugarcane mosaic virus (SCMV), leaves and stems of infected plants develop a severe systemic necrosis known as corn lethal necrosis disease (CLND). CLND is an important disease in maize industry in many countries 4 .
MCMV was first reported in Peru and later in the United States [5][6][7] . MCMV is transmitted by mechanical inoculation and seed, and it was also reported to be transmitted by chrysomelid beetles and thrips 8 . In 2009, MCMV was first observed in China in maize with necrotic and chlorotic leaves and stems 9 . At that time, an unknown virus with flexuous rod-shaped particles was also observed in the cytoplasm of infected leaf cells using electron microscopy. In the present study, we surveyed the distribution of MCMV and CLND in ten provinces of China by ELISA and RT-PCR. The results show that MCMV currently occurs only in the Yunnan and Sichuan Provinces of China, and the CLND disease observed in China is caused by co-infection with MCMV and SCMV. An infectious clone of MCMV was constructed by inserting the full length cDNA of representative MCMV isolate YN2 (MCMV-YN2) downstream a duplicated cauliflower mosaic virus (CaMV) 35S promoter, and an Phylogenetic relationship among MCMV isolates. Four full-length genomic cDNAs representing four respective MCMV isolates were cloned and sequenced. These full-length sequences have been deposited in the GenBank database (GU138674 for isolate YN1; JQ982468 for isolate YN2; JQ982469 for isolate YN3 and JQ982470 for isolate Sch1). The phylogenetic relationship among these four isolates, a sugarcane MCMV isolate found in Yunnan 10 (KF010583), a Taiwan MCMV isolate 11 (KJ782300) and two American MCMV isolates 1,12 (X14736 and EU358605) were analyzed. The results indicated that the isolates from Chinese mainland and Taiwan clustered in one group and the two isolates from America in the second group (Fig. 2), even though the sequence identity between the two groups is high (96.9-97.3%). Because the five isolates in group I shared 99.1%-99.7% sequence identity with each other, it is likely that all these isolates came from the same ancestor virus strain.
Agrobacterium-mediated injection of MCMV in maize. The MCMV infectious clone pMCMV was constructed (Fig. 1). To establish an efficient inoculation procedure for maize, we transformed Agrobacterium tumefaciens GV3101 with the pMCMV plasmid and the transformed Agrobacterium cells were injected into stems of maize cv. B73 seedlings. Fifteen among the 29 inoculated maize plants developed light chlorotic mottling on newly developed leaves at 10 days post inoculation (dpi) (Fig. 3A,D), the symptoms were similar to that observed on MCMV-YN1-inoculated plants (data not shown) at 6 dpi, but were very distinct from that of MCMV and SCMV co-infected plants (Fig. 3B,E). All of the leaf samples showing mottling symptoms were infected with MCMV when analyzed by RT-PCR using primers MCMV/CP-F and MCMV/CP-R. No mottling symptoms were observed on the leaves of plants inoculated with Agrobacterium harboring the pCB301 empty vector (Fig. 3C,F). Inoculation experiments were repeated three times, and infectivity of pMCMV on B73 is given in Table 2.
To determine the levels of MCMV in the infected maize plants, crude leaf extracts were isolated from the pMCMV-inoculated or MCMV-YN1-inoculated plants and analyzed by ELISA using MCMV antibody. Results showed that at 10 dpi the virus levels in the pMCMV-inoculated and MCMV-YN1-inoculated (wt) plants were similar (Fig. 4A). Western blot analysis of leaf extracts harvested at 10 dpi gave similar results for the pMCMV-inoculated or MCMV-YN1-inoculated plants (Fig. 4B). The Northern blots indicated that the relative level of viral RNA level in plants inoculated with pMCMV was comparable to that in plants inoculated with MCMV-YN1 (Fig. 4C). Numerous isometric particles of about 30 nm in diameter were also observed in negatively stained leaf extracts when checked with transmission electron microscope (Fig. 4D).
To investigate the infectivity of MCMV in different maize cultivars, we agro-inoculated maize cvs. B73, Zhengdan958, Suyu No.1, Huatian-Waxy Corn 072, Urban Beauty, Nongda108, Zhe-Waxy Corn no.5 with pMCMV. Virus infection in these cultivars were 72.2%, 50.0%, 58.3%, 83.3%, 77.8%, 77.8% and 97.2%, respectively, indicating that different maize cultivars have different tolerance reaction.  Ultrastructural changes in infected maize leaf cells. Because MCMV and SCMV co-infection is common in maize fields in Yunnan and Sichuan Provinces, we investigated the ultrastructural damage caused by MCMV infection and by MCMV and SCMV co-infection. Chloroplasts in bundle sheath cells of maize leaves are known to contain large starch grains and unstacked thylakoid membranes 13 . In the present study, bundle sheath cells infected with MCMV alone had starch grains in chloroplasts similar to those observed in the mock-inoculated plants ( Fig. 5A and B), but cells co-infected with MCMV and SCMV, however, had much smaller starch grains in the chloroplasts (Fig. 5C). Results of qRT-PCR agreed with these ultrastructural observations and showed that the mRNA level of pyruvate orthophosphate dikinase (PPDK) gene, a rate-limiting factor for CO 2 fixation in the C 4 -photosynthesis pathway 14 , was 7-fold lower in the co-infected leaf tissues than in the mock-inoculated or MCMV-infected plants (Fig. 5D). Mitochondria in the MCMV-infected ( Fig. 5F) or MCMV and SCMV co-infected cells ( Fig. 5G to I) had disorganized cristae (white arrows) and fine, fibrous materials (white solid arrowheads) comparable to the case in mock-inoculated plant cells (Fig. 5E). In MCMV and SCMV co-infected cells, some mitochondria were heavily disrupted, leading to leakages of internal content (    5O and P, black solid arrows). Some spherical viral particles were observed within or between MVBs (Fig. 5N, black open arrowheads), suggesting they are potential sites for viral replication.

Discussion
MCMV was first reported in Peru in 1974 and later in America and Thailand 1,3,5,6,15-17 . In the first report of MCMV in China in 2011 9 , an unknown flexious rod-shaped virus was observed in tissue samples by electron microscopy. Because MCMV was recently reported in several East Africa countries and Asia, and CLND has caused significant yield losses to maize production 18-21 , we decided to further investigate the occurrence of MCMV and CLND in China and to identify the unknown flexuous, rod-shaped virus that was observed in MCMV-infected maize samples 9 . The results of our field survey, started in 2009, of MCMV and CLND in maize-growing provinces in China, showed that MCMV currently occurs only in Yunnan and Sichuan Provinces in China. Phylogenic analysis indicated that the Chinese MCMV isolates were distinct from the reported American MCMV isolates. Studies on the interactions between a virus and its host plant require an easy and effective inoculation procedure. Scheets and co-workers 22 reported previously that in vitro transcribed MCMV RNA could be used to infect maize protoplasts and plants. Although this inoculation procedure is effective, it is costly and labor intensive. To develop an easy, low cost inoculation protocol for MCMV, we constructed a DNA-based MCMV vector and adopted an Agrobacterium-mediated injection procedure for maize. Because a large quantity of Agrobacterium can be cultured overnight at a very low cost, this protocol should benefit researchers doing large-scale screenings for maize genotypes resistant to MCMV infection.
Scheets et al. 22 reported previously that MCMV RNA transcripts transcribed by an in vitro T7 promoter had an extra 5′ m 7 GpppG and were less infectious in maize protoplasts and did not infect maize plants. The transcription site of T7 polymerase might start at the + 2 position on the MCMV cDNA. Consequently, the 5′ end of the first adenylate residue was eliminated during the in vitro transcription 22 . The pMCMV vector was constructed by inserting the full-length MCMV cDNA between a duplicated CaMV 35S promoter and an HDV-ribozyme sequence. Transcription of MCMV genomic RNA through this 35S promoter ensures transcription from the 5′ end of MCMV, while the HDV-ribozyme cuts the 3′ end sequence of MCMV RNA to give an authentic 3′ end ( Fig. 1) 23,24 . The MCMV RNA transcripts produced in vivo by the 35S promoter can infect maize plants as efficiently as the MCMV virus does.
A previous study showed that CLND-infected cells contain vacuolated MCMV viroplasms and pinwheel structures of SCMV 25 . In this study, chloroplasts in cells co-infected with MCMV and SCMV contained much smaller starch grains than that in the MCMV-infected cells, suggesting that photosynthesis in these cells was significantly impeded. We analyzed PPDK expression in MCMV or MCMV and SCMV co-infected cells through quantitative RT-PCR and the expression of this gene in leaves co-infected with MCMV and SCMV was indeed decreased. Mitochondria generate usable energy through the Krebs or tricarboxylic acid (TCA) cycle during plant growth and development. We observed that mitochondria in the co-infected leaf cells were severely damaged much earlier in the infection than in MCMV-infected cells. We consider that earlier disruption of chloroplast photosynthesis and mitochondrial respiration in the co-infected plants might be the main cause of the systemic necrosis in the CLND plants.
Interestingly, Multi-vesicular bodies (MVBs) were observed in both singly infected and co-infected plants. The MVBs found in MCMV-infected plants were morphologically similar to the peroxisomal multi-vesicular bodies (pMVBs) induced in N. benthamiana cells by tomato bushy stunt virus (TBSV, type species of the genus Tombusvirus in the family Tombusviridae) 26 . The pMVBs are the site of TBSV replication, we thus speculated that the MVBs are the scaffold for MCMV replication.
The concentration of MCMV increases over 5 fold in plants that are co-infected with SCMV, but the concentration of SCMV in co-infected plants is no difference than in singly infected plants 15 . Similar synergistic interactions were also observed in plants infected with potato virus X (PVX) and potato virus Y (PVY) or tobacco etch virus (TEV) 27,28 . Double infection of tobacco plants with PVX and PVY, PVX and TEV, or PVX with another potyvirus all induced much more severe symptoms and increased the level of PVX RNA. Numerous studies have also shown that potyviral HC-Pro, an RNA silencing suppressor, plays a critical role in the synergism during co-infection with PVX and a potyvirus 29,30 . Further study using transgenic tobacco expressing the TEV P1/HC-Pro showed a synergistic response upon infection of the plant with PVX. In contrast, tobacco plants expressing a mutant HC-Pro that could not suppress RNA silencing also failed to enhance PVX infection in the co-infected plants 29 . HC-Pro of SCMV is also a suppressor of RNA silencing 31 . This finding may explain why MCMV alone causes only mild disease symptoms in the infected plant, and the systemic necrosis and the increased MCMV RNA accumulation that result from the synergism between MCMV and SCMV in co-infected plants may be due to the presence of SCMV HC-Pro protein.

Materials and Methods
Virus sources. A Yunnan isolate of MCMV was previously identified in maize in Yunnan Province, China 9 and maintained in maize cv. B73 in an insect-proof greenhouse. The maize plant was co-infected with MCMV and one potyvirus and developed lethal necrosis. Because MCMV can tolerate temperatures up to 85 °C for 10 min but potyviruses can tolerate only about 50-60 °C, leaf samples with CLND symptoms were treated for 10 min at 70 °C before inoculation of maize seedlings. Single infection with MCMV was thus obtained, and the virus isolate was named MCMV-YN1 and used as a control for MCMV inoculation.
To determine the distribution of viruses in maize in China, we collected 171 maize leaf samples with virus-like symptoms between 2009 and 2013 from 10 maize-growing provinces (Yunnan, Sichuan, Guizhou, Guangxi, Shandong, Liaoning, Heilongjiang, Henan, Hebei and Zhejiang).

ELISA, RT-PCR and cloning.
Monoclonal antibodies against MCMV 32 and SCMV 33 , previously produced in our laboratory, were used to analyze leaf samples for the presence of MCMV and SCMV according to the procedure described previously 32 . MDMV and WSMV were identified using ELISA kits specific for MDMV or WSMV from Agdia (Elkhart, IN, USA) as instructed by the manufacturer.
Three MCMV samples (YN1, YN2 and YN3) from Yunnan province and one (Sch1) from Sichuan province showing lethal necrosis symptom were selected randomly for full-length genomic cDNA cloning and sequencing. RNA for RT-PCR was extracted from approximately 100 mg tissue from each leaf sample using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Two microliters of total RNA solution was then used in each 20-μ L reverse transcription reaction using AMV Reverse Transcriptase XL (TAKARA, Kyoto, Japan) and the specific primer M2R. MCMV fragments were amplified by PCR using the KOD FX DNA polymerase (TOYOBO, Osaka, Japan). Primers M1F and M2R were designed based on the MCMV genome sequences available in the GenBank database (accessions GU138674, X14736 and EU358605) and used to amplify the full length MCMV cDNAs. The resulting cDNAs were inserted into separate pGEM-T Easy Vector (Promega, Madison, WI, USA). Primers MCMV/ CP-F and MCMV/CP-R were used to amplify the 887-bp fragment covering the CP region of MCMV. Primer sequences were listed in Table 3.
For potyvirus identification, a set of universal primers for potyviruses was used for RT-PCR as previously reported 34

Construction of infectious MCMV clone.
Isolate MCMV-YN2 was randomly selected to construct an infectious clone. MCMV-YN2 cDNA was inserted behind a duplicated 35S promoter in the pCB301 vector. The binary vector pCB301 36 harboring a duplicated 35S promoter (2 × 35S) and a hepatitis delta virus ribozyme (HDV_Rz) and a NOS terminator was kindly provided by Professor Xiaorong Tao (Nanjing Agriculture University, Nanjing, China). A full-length PCR product representing MCMV-YN2 was further amplified and digested with XhoI restriction enzyme. The 5′ fragment of the digested PCR product (MCMV nucleotides 1 to Scientific RepoRts | 7:39960 | DOI: 10.1038/srep39960 3579) was ligated into the pCB301 vector pre-digested with StuI and SalI enzymes to generate pCB301-SX. The full-length PCR product was digested again with NheI enzyme, and the 3′ fragment product (MCMV nucleotides 748 to 4436) was ligated into the pCB301-SX vector at the NheI and SmaI site to generate pMCMV. This final clone contained a full-length MCMV-YN2 cDNA between the StuI and SmaI sites (Fig. 1). Primer sequences were listed in Table 3.
Inoculation. Isolate MCMV-YN1 was used as a control (wild-type virus, wt). MCMV-YN1-infected maize leaves were ground in 0.1 M phosphate buffer (pH 7.0), and the sap was rubbed onto leaves of maize plants with 5-6 leaves 37 .
pMCMV described above were used to transform Agrobacterium tumefaciens GV3101 via electroporation. The transformants were then cultured overnight at 28 °C in YEP broth supplemented with 25 mg/L rifampicin, 50 mg/L kanamycin and 25 mg/L gentamicin. The Agrobacterium cells were pelleted by 10 min centrifugation at 6000× g and then re-suspended in an infiltration buffer (10 mM MES, pH 5.6, 10 mM MgCl 2 , and 200 μ M acetosyringone) until the OD 600 reached 1.0 38 . Maize seedlings of cultivars B73, Zhengdan958, Suyu No. 1, Huatian-Waxy Corn 072, Urban Beauty, Nongda108 and Zhe-Waxy Corn No. 5 were inoculated with Agrobacterium cells transformed with pMCMV. A 1-mL syringe was then used to inject maize seedlings 3 mm above the coleoptilar node with 0.5 ml of Agrobacterium cells harboring pMCMV. This injection site was previously reported to contain meristematic tissue and is susceptible to Agrobacterium invasion 39 .
Northern blot assay and quantitative RT-PCR. Total RNA (10 μ g) extracted from maize leaf tissue was electrophoresed in 0.8% (w/v) agarose formaldehyde gels and transferred to the Hybond N+ membranes (GE Amersham, PA, USA) by capillary transfer. RNA agarose gels stained with ethidium bromide were imaged under a UV light to estimate the loadings for each sample. The membranes were hybridized with a 32 P-dCTP labeled MCMV-specific probe prepared with the random priming method (Prime-a-Gene Labeling System, Promega) as instructed by the manufacturer.
Quantitative RT-PCR (qRT-PCR) for the Pyruvate orthophosphate dikinase gene (PPDK) was performed using primers PPDK/F and PPDK/R. The Elongation factor 1-alpha (EF1α ) gene was used as the internal controls. All qRT-PCR experiments were done in triplicate using three independent samples as described previously 40 . Western blot. Approximately 100 mg tissue from each maize leaf sample was homogenized in 0.2 ml protein extraction buffer (50 mM Tris-HCl, pH 6.8, 9 M urea, 4.5% sodium dodecyl sulfate [SDS] and 7.5% β -mercaptoethanol). The crude extracts were centrifuged at 12,000× g for 15 min at room temperature, and the resulting supernatant (15 μ l per sample plus 15 μ l 2× SDS loading buffer) was electrophoresed in 12.5% SDS-PAGE gels followed by transferring proteins to nitrocellulose membranes performed as previously reported 41 . The membranes were then probed with the MCMV-specific monoclonal antibody 31 followed by a goat anti-mouse secondary antibody conjugated to horseradish peroxidase (Bio-Rad, Los Angeles, CA, USA).
Electron microscopy. Symptomatic leaves were collected from the MCMV-infected plants and ground in water. The leaf extracts were loaded onto formvar-coated copper grids. After negative staining with 2% phosphotungstic acid, pH 6.7, the grids were examined for MCMV particles with a transmission electron microscope (TEM; H-7650, Hitachi, Japan) at 80 kV accelerating voltage.
For cytopathological studies, leaves were harvested from maize plants infected with MCMV or coinfected with MCMV and SCMV, then cut into small pieces (about 1 × 3 mm). The tissues were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide in 100 mM phosphate buffer, pH 7.0, as described previously 42 . The fixed tissues were dehydrated through an ethanol series, then embedded in Epon 812 resin as instructed by the manufacturer (SPI-EM, Division of Structure Probe, West Chester, PA, USA). Ultrathin sections were cut and placed onto formvar-coated grids and stained with 2% uranyl acetate for 10 min followed by 2.5% lead citrate solution for 10 min. The stained sections were examined using the TEM as described.  Table 3. Primers and sequences used for PCR.