Sequence analysis of RNA3 of Maize stripe virus associated with stripe disease of sorghum (Sorghum bicolor) in India

Abstract

Maize stripe virus (MSpV), one of the distinct species of the genus Tenuivirus has been associated with stripe disease of sorghum in India. In this study, the authors have reported the complete sequence analysis of ambisense RNA3 of four MSpV isolates associated with this disease, to confirm its correct identity. The RNA3 of four MSpV-Sorg isolates is 2357 nucleotides in length with two ORFs, one in virion sense (594 nucleotides, non-structural protein 3, NS3) and the other in complementary sense (951 nucleotides, coat protein, CP). The intergenic region between these two ORFs is 653 nucleotides in length, which is rich in U and A residues. The deduced molecular weights of NS3 and CP are [asymptotically =]22 and [asymptotically =]34 kDa, respectively. RNA3 has [asymptotically =]82% sequence identity at nucleotide level with RNA3 of MSpV infecting maize in Florida, US and Reunion. NS3 and CP ORFs shared [asymptotically =]94% and [asymptotically =]95% identities at amino acid levels, respectively with MSpV isolates of maize from Florida and Reunion.

Headnote

Summary. Maize stripe virus (MSpV), one of the distinct species of the genus Tenuivirus, has been associated with stripe disease of sorghum in India. In this study, we report the complete sequence analysis of ambisense RNA3 of four MSpV isolates associated with this disease, to confirm its correct identity. The RNA3 of four MSpV-Sorg isolates is 2357 nucleotides in length with two ORFs, one in virion sense (594 nucleotides, non-structural protein 3, NS3) and the other in complementary sense (951 nucleotides, coat protein, CP). The intergenic region between these two ORFs is 653 nucleotides in length, which is rich in U and A residues. The deduced molecular weights of NS3 and CP are [asymptotically =]22 and [asymptotically =]34 kDa, respectively. RNA3 has [asymptotically =]82% sequence identity at nucleotide level with RNA3 of MSpV infecting maize in Florida, USA and Reunion. NS3 and CP ORFs shared [asymptotically =]94% and [asymptotically =]95% identities at amino acid levels, respectively with MSpV isolates of maize from Florida and Reunion. The internal non-coding region between two ORFs has 67-68% identity at nucleotide level with the reported MSpV isolates from Florida and Reunion. The sequence identity was more than [asymptotically =]98% among the four isolates of MSpV-Sorg. Compared to maize-infecting MSpV isolates in USA and Reunion, the sorghum-infecting MSpV isolates in India had more amino acid substitutions in both NS3 and CP. This is the first report of complete sequence analysis of MSpV RNA3 from Asia.

Key words: Sorghum stripe disease, MSpV-Sorg, India, RNA3 and Genetic relationships.

Introduction

Maize stripe virus (MSpV) belongs to the unusu- al plant virus genus Tenuivirus. These viruses have characteristic thin (3-6 nm in width) highly flexuous thread-like particles of undefined lengths. MSpV in- fecting maize has been reported from tropical regions of Asia, Africa, Australia and Americas (Ramirez et al, 1994; Falk and Tsai, 1998; Ramirez, 2008). The virus is transmitted by the plant hopper Peregrinus maidis in a persistent manner. Most of the MSpV isolates from maize were found to be serologically related (Huiet et al., 1992; Falk and Tsai, 1998; Mahmoud et al., 2007). The MSpV genome consists of five segments of either negative or ambisense ssRNA with a total estimated size of [asymptotically =] 18 kb (RNA1- 8.8 kb, RNA2- 3.4 kb, RNA3- 2.3 kb, RNA4- 2.2 kb and RNA5- 1.3 kb) (Ramirez, 2008). The MSpV RNAs have 5' and 3' terminal base comple- mentarity, a characteristic feature of all the tenuivirus- es (Ramirez, 2008). The RNA3 of all the tenuiviruses including MSpV have ambisense gene organization. MSpV RNA3 contains coat protein (CP) and non- structural protein 3 (NS3) ORFs in an ambisense gene organization, both ORFs separated by large intergenic regions (IR). The reported Mr of structural protein of virus particles ranges from 32-35 kDa. So far, the com- plete sequence of the four smaller ssRNAs (RNAs 2,3,4 and 5) of MSpV-Florida, USA (MSpV-Flo) and com- plete sequence of RNA3 and partial sequence of RNA1 of MSpV-Reunion (MSpV-RE) isolate infecting maize have been reported (Huiet et al., 1992; Huiet et al, 1993; Falk and Tsai, 1998; Mahmoud et al., 2007).

Sorghum, the fifth most important world cereal crop, is grown in several tropical and sub-tropical countries, mostly cultivated for food in semi-arid zones and for animal feed in developed countries. India is the third largest country cultivating sor- ghum for food (Food and Agricultural Organiza- tion, 2010). Nearly 20 viruses have been reported to naturally infect sorghum worldwide (Frederiksen and Odvody, 2010; Srinivas et al., 2010). Maize mosaic virus, Sorghum mosaic virus, Sugarcane mosaic virus, Sugarcane streak mosaic virus and an isolate of Maize stripe virus have been reported to naturally infect sorghum in India (Naidu et al., 1989; Peterschmitt et al., 1991; Srinivas et al., 2010). In Andhra Pradesh state of India, a virus isolate associated with stripe disease of sorghum was identified as MSpV-Sorg based on typical symptoms, particle morphology, transmission by delphacid vector (Peregrinus mai- dis), antigenic relationships and genome nature, composition and size (Peterschmitt et al., 1991). Subsequently, similar disease was also reported based on symptomatology, vector transmission and serology from Karnataka and Maharashtra states in India (Narayana and Muniyappa, 1995; Garud et al., 2000). The affected plants were dwarfed and had poor or no panicle formation. MSpV-Sorghum isolate in India failed to infect maize under experi- mental inoculations using P. maidis (Peterschmitt et al., 1991). In earlier studies, the isolate infecting sor- ghum in India was designated as MSpV-Sorg and the same designation is used in the present paper. Although serological relationships between vari- ous MSpV isolates were established from different countries, reports on genetic relationships among these isolates infecting maize / sorghum around the world are limited. Hence, RNA3 of four MSpV-Sorg isolates from Andhra Pradesh, India have been com- pletely sequenced to understand its genetic relation- ships with isolates of the same virus from other geo- graphic regions and also with other tenuiviruses.

Materials and methods

Collection and screening of stripe disease affected sorghum samples

Sorghum bicolor (local cultivar) leaf samples ex- hibiting either chlorotic and/or whitish interveinal streaks and/or bands were collected during the rainy season of 2009-2011 from Cuddapah (Kadapa), Kurnool and Chittoor districts of Andhra Pradesh state, India. The samples were initially screened with polyclonal antisera of MSpV-Flo and -RE isolates by direct antigen coating- enzyme-linked immuno- sorbant assay (DAC-ELISA) (Clark and Bar-Joseph, 1984). The ELISA positive samples were also con- firmed by western blotting using the same antisera (Koenig and Burgermeister, 1986).

RT-PCR, cloning and sequence analysis of RNA3

The leaf samples from sorghum plants, already confirmed for MSpV by DAC-ELISA and western blot assays, were used for the isolation of total RNA using Tri reagent (Sigma-Aldrich) according to the manufacturer's protocol. Total RNA isolated from healthy sorghum leaf samples served as healthy controls. The extracted RNA was suspended in 20 µL of RNase-free water and stored at -20°C. The primers for the amplification of MSpV-RNA3 were designed based on the RNA3 sequences of MSpV- Flo and -RE isolates deposited in the GenBank (A/C numbers M57426 and AJ969410) using the Primer3 programme (http://frodo.wi.mit.edu/primer3/). The designed primers (1st set of primers) MSpV 3-F: 5'-TATATATTACTTCTGTCCATCGAAC-3' and MSpV 3-R: 5'-ACACAAAGTCTGGGTAATCGTT-3' (Table 1) were expected to amplify [asymptotically =]2.2 kb fragment from 3' end of MSpV-RNA3. Eight µL of the isolated total RNA (from MSpV associated samples collected from Kadapa district-MSpV-Sorg-Kad1) was dena- tured at 55-60°C for 10 min and then subjected to first strand cDNA synthesis by adding 20 pmol of the MSpV 3-R primer involving M-MuLV-reverse transcriptase (Fermentas) according to the manu- facturer's protocol. The reverse transcribed cDNA was then subjected to PCR in a 25 µL reaction vol- ume involving 2.5 µL of 10 × PCR buffer, 2.5 mM MgCl2, 0.2 mM dNTP mix (Fermentas), 20 pmol of each of MSpV 3-forward and reverse primers and 1 U of Taq DNA polymerase (Fermentas). The PCR mix was subjected to thermal cycling conditions of 94°C for 5 min followed by 35 cycles of 94°C for 30 sec, 52°C for 45 sec and 72°C for 2 min, with a fi- nal extension of 72°C for 7 min. The PCR products were electrophoresed in agarose gel (Sambrook and Russell, 2001), and the amplified products ([asymptotically =]2.2 Kb) were gel extracted using Qiaquick mini gel extrac- tion kit (Qiagen) according to the manufacturer's protocol. The gel eluted PCR products were cloned into pTZ57R/T vector (Fermentas) and transformed into E.coli DH5α cells (Sambrook and Russell, 2001). The recombinant clones were confirmed by restric- tion enzyme (Eco R1 and Hind III) analysis. The confirmed positive recombinant clones were then sequenced at a commercial sequencing facility (Eurofins Biotech, India). Second set of primers (5' end-F: 5'-ACACAAAGTCCTGGGT-3' and 5' end -R-5'-CCGGACCCCTTAGTAGGC-3') (Table 1) were designed for the amplification of 5' end of the RNA3 of MSpV-Sorg isolate from Kadapa district (MSpV-Sorg-Kad1) to cover its full length sequence. The second reverse primer is an internal primer de- signed from the sequence obtained by using the first set of primers. The synthesized cDNA was again subjected to PCR thermal cycling conditions of 94°C for 5 min followed by 35 cycles of 94°C for 30 sec, 52°C for 45 sec and 72°C for 2 min with a final ex- tension of 72°C for 7 min involving 5' end primers. The amplified products ([asymptotically =]250 bp) were cloned and sequence analyzed as above. The clones were se- quenced three times to avoid any sequencing errors.

The RNA3 of MSpV-Sorg isolates from Chittoor (MspV-Sorg-Chi), Kadapa (MSpV-Sorg-Kad2) and Kurnool (MSpV-Sorg-Kur) were later amplified by RT-PCR using the third set of primers, MSpV 5énd- F: 5'-ACACAAAGTCCTGGGT-3' and MSpV RNA3- R: 5'-ACACAAAGTCTGGGTAATCGTT-3' (Table 1), employing the RT-PCR conditions as described above. This facilitated the complete amplification of RNA3 (2357 nt) and the amplified products were gel extracted, cloned, transformed and screened as de- scribed above. The respective clones were sequenced using a commercial sequencing facility. The se- quences obtained were then initially analyzed using the BLAST programme of NCBI (http://blast.ncbi. nlm.nih.gov/Blast.cgi), and further analyzed at the nucleotide and amino acid levels using MEGA ver- sion 4.0 (Tamura et al., 2007) by comparing with the MSpV maize isolates, RSV and other tenuiviruses.

Results and discussion

The virus isolates associated with stripe disease of sorghum in Andhra Pradesh, India were found to be serologically related to MSpV-Flo and -RE isolates in DAC-ELISA (A405 values range: for the buffer con- trol, 0.09-0.12; for the healthy sorghum control, 0.11- 0.16; and for infected sorghum, 0.41-1.12). In western blot analyses, the present MSpV isolate's structural protein was resolved as one major polypeptide with Mr of [asymptotically =]34 kDa (Figure 1). The first set of primers (Table 1) amplified [asymptotically =]2.2 kb fragment from 3' end of RNA3 (Figure 2a). The initial BLAST analysis of the sequence of recombinant clones having the [asymptotically =]2.2 kb insert clearly indicated that the virus associated with the stripe disease of sorghum in Andhra Pradesh, India is indeed MSpV. The 5' end of the RNA3 was also successfully amplified using the second set of designed primers (Table 1) (Figure 2b). The sequenc- es of two fragments were aligned to form complete MSpV RNA3 (2357 nucleotides in length). The later designed third set of primers, based on the gener- ated RNA3 sequence of MSpV-Sorg-Kad1 isolate (RNA3 sequence of MspV-Sorg isolate1 from Kada- pa district), were used successfully for the amplifica- tion of complete RNA3 of other MSpV-Sorg isolates collected from Chittoor, Kadapa and Kurnool dis- tricts of Andhra Pradesh state, India. The RNA3 of the four respective MSpV isolates (two from Kadapa, one from Chittoor and one from Kurnool districts) contained two open reading frames (ORFs) that were separated by a large IR. The first ORF, non-structural protein 3 (NS3) was 594 nucleotides in length, while the second ORF, structural coat protein (CP) was 951 nucleotides in length. These two ORFs were separat- ed by a large IR which is 653 nucleotides in length. The 5'- untranslated region (UTR) and 3'-UTR are, respectively, 66 and 93 nucleotides in length. These sizes are similar among the analyzed MSpV-Sorg isolates. The complete sequence (2357 nucleotides, GenBank A/C- MSpV-Sorg-Kad1: JN591726; MSpV- Sorg-Kad2: JN591724; MSpV-Sorg-Chi: JN579656; MSpV-Sorg-Kur: JN591725) of RNA3 of MSpV-Sorg isolates shared maximum identity of 82.4 and 82.6% at the nucleotide level with the RNA3 of MSpV-Flo and -RE isolates infecting maize, respectively. NS3 shared 88.2% identity at the nucleotide level with both MSpV-Flo and -RE isolates, while at the amino acid levels it has 93.9 and 94.4% identities with the respective isolates. The CP shared 87.3 and 86.6% identities with MSpV-Flo and -RE isolates at the nucleotide level, while it had 94.9 and 95.8% identi- ties at the amino acid level with the respective iso- lates (Table 2). The ORF encoding NS3 protein was present in the viral sense strand while the ORF en- coding CP was in complementary strand. Similar ambisense organization of genes was also reported in RNA3 and RNA4 of other tenuiviruses (Kakuta- ni et al., 1990; Kakutani et al., 1991; Zhu et al., 1991; Ramirez, 2008). The 5' and 3' termini of MSpV iso- lates were reported to be complementary (Mahmoud et al., 2007; Ramirez, 2008). This complementarity of 5' and 3' ends facilitated the successful amplification of complete RNA3 of MSpV-Sorg isolates.

The internal large non-coding spacer had 67.1 and 68.4% identity with the maize infecting MSpV- Flo and -RE isolates, respectively. The 5'-UTR of the MSpV-Sorg isolate shared 100% identity with the maize infecting MSpV isolates, while the 3'-UTR of the isolate had 93.5 and 92.4% identities with the MSpV-Flo and -RE isolates, respectively. The percent identities of RNA3 of MSpV-Sorg isolates from the present study with RNA3 of other tenuiviruses that have more than 50% identity at complete nucleotide level, NS3 and CP at nucleotide and amino acid lev- els, IR at nucleotide level are given in Table 2. RNA3 percent identities among the four MSpV-Sorg iso- lates from the present study were found to be more than 97%, except the MSpV-Sorg-Kunool isolate which had 89.6% identity with the other three iso- lates especially at IR (Table 2). In the phylogenetic analyses based on the complete RNA3 nucleotide sequence, the MSpV-Sorg isolates from India clus- tered along with the MSpV isolates infecting maize in Florida and Reunion (Figure 3). The same cluster- ing pattern was also observed with NS3 and CP at the amino acid levels (data not shown).

Both the NS3 and CP of the MSpV-Sorg isolates showed variations in their amino acid sequences when compared to maize infecting isolates of MSpV (Table 2). All four MSpV-Sorg isolates had 12 and nine amino acid changes in the CP and NS3 proteins, respectively when compared to their maize infecting counterparts (data not shown). Although maize is also extensively grown in India, MSpV was reported to infect only sorghum in this country. Although the plant hopper vector, P. maidis transmitting MSpV, infests both sorghum and maize, only sorghum was observed to be affected by the stripe disease. The ob- served genetic variation within the MSpV-Sorg iso- lates could be responsible for its adaptation to sor- ghum only.

The complete RNA3 sequences of the four virus isolates provided the genetic basis for confirmation of the association of MSpV with stripe disease of sorghum. This is the first complete RNA3 sequence- based study on MSpV-Sorg isolates associated with stripe disease of sorghum from Asia.

Acknowledgements

The authors thank Dr Margaret Redinbaugh, De- partment of Plant Pathology, Ohio State University and Dr Michel Peterschmitt, CIRAD, UMR BGPI, Montpellier, France for the supply of MSpV-Flo and MSpV-RU polyclonal antisera. The authors also thank the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi for financial support of a now completed re- search project.

Note: The RNA3 of MSpV-Sorg isolates were de- posited in the GenBank with accession No. JN591726; JN591724; JN579656; JN591725.

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Accepted for publication: April 16, 2013