Localization of Sweet potato chlorotic stunt virus ( SPCSV ) in synergic infection with Potyviruses in sweet potato

Among diseases reported worldwidely for sweet potato (Ipomoea batatas (L) Lam) crop, one of the most frequent is the Sweet potato virus disease (SPVD), caused by sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus (SPFMV) co-infection. In Argentina, there exists the sweet potato chlorotic dwarf (SPCD), a sweet potato disease caused by triple co-infection with SPCSV, SPFMV and sweet potato mild speckling virus (SPMSV). Both diseases cause a synergism between the potyviruses (SPFMV and SPMSV) and the crinivirus (SPCSV). Up to date, studies carried out on the interaction among these three viruses have not described their localization in the infected tissues. In single infections, virions of the crinivirus genus are limited to the phloem while potyviral virions are found in most tissues of the infected plant. The purpose of this work was to localize the heat shock protein 70 homolog (HSP70h), a movement protein for genus crinivirus, of an Argentinean SPCSV isolate in its single infection and in its double and triple co-infection with SPFMV and SPMSV. The localization was made by in situ hybridization (ISH) for electron microscopy (EM) on ultrathin sections of sweet potato cv. Morada INTA infected tissues. The results demonstrated that viral RNA coding HSP70h is restricted to phloem cells during crinivirus single infection, while it was detected outside the phloem in infections combined with the potyviruses involved in chlorotic dwarf disease. BIOCELL 2007, 31(1): 23-31 ISSN 0327 9545 PRINTED IN ARGENTINA

In 1984, Argentinean sweet potato cv Morada INTA plants, known to be tolerant to SPFMV, presented a serious viral disease named chlorotic dwarf, characterized by symptoms of chlorotic mosaic, leaf deformation and area reduction, and plant dwarfism (Di Feo and Nome, 1990).Following studies showed that there are three viruses involved in this disease: SPFMV, SPMSV and SPCSV, with a synergistic effect between SPCSV and the two potyviruses (SPFMV and SPMSV).The combined action of the three virus produced more severe symptoms on cv Morada INTA (Di Feo et al., 2000).Resent investigations in SPVD have shown that the crinivirus suppresses the RNA silencing (Kreuze et al., 2005), the concentration of potyviruses in the plant increases (Karyeija et al., 2000), therefore the synergistic effects occur.The yield reduction by SPCD may reach an 80% (Di Feo et al., 1995).
The potyvirus family does not posses one movement protein but a group of proteins related to this function (coat protein, CP; helper component protein, HC-Pro; cylindrical inclusions proteins, CI; and genome linked viral protein, VPg).Potyviral HC-Pro suppresses gene silencing and consequent plant responses, allowing an increase in viral replication (Savenkov et al., 2001).Potyviral CI are necessary for viral replication, they display RNA helicase activity and are involved in cell-to-cell movement.The conic deposits of CI may translocate the virus within the cell (already encapsulated), and the viral CP may alter the SEL (size exclusion limit) of the plasmodesms to allow the passing to the adjacent cell.HC-Pro is considered to act in long distance movement, to enter and exit the vascular system.Once in the phloem, the CP + VPg are thought to bind plant factors facilitating their distribution through the plant (Revers et al., 1999).
The protein HSP70h of the closterovirus family is related to a large family of chaperons involved in folding and transport of animal proteins.It has been observed that binding of HSP70h to the virus is required for the infectivity of the viral particles (Agronovsky et al., 1998); HSP70h may be responsible for carrying the virus from the cytoplasm to and through the plasmodesmata (Peremyslov et al., 1999).Family Closteroviridae codes two CPs: CP and minor CP (CPm).CP covers most of the viral particle, while CPm is found on one extreme of the particle.CPm is considered to be involved in cell-to-cell and long distance movements, as well as in vectors interaction (Agranovsky et al., 1995).
In the infected plants with chlorotic dwarf, the crinivirus is the only virus containing a protein with sequence homolog to HSP70, called HSP70 homolog (HSP70 h) (Yeh et al., 2000), HSP-like (Agranovsky et al., 1998), or HSP70 related protein homologue (Fauquet et al., 2005b).The sequence coding for HSP70h is highly conserved within the criniviruses and considered characteristic of the family Closteroviridae; it was, therefore, chosen as a target for this study.
The purpose of this work was to determine if the crinivirus SPCSV is localized in cells others than the phloem during synergistic infection with potyviruses SPFMV and SPMSV in chlorotic dwarf disease.

Virus isolates and detection
All experiments were performed on sweet potato plants cv Morada INTA.Plants were experimentally inoculated with the following combinations of viruses: SPMSV + SPCSV, SPFMV + SPCSV, SPMSV + SPCSV and SPCSV.
Healthy plants were propagated agamically.The virus isolates were obtained from symptomatic sweet potato plants from Cordoba (Argentina) crop fields (material involved in previous investigations, Di Feo et al., 2000), and maintained by grafting and viral transmission using the corresponding vectors, by semi-persistent transmission with whiteflies Bemisia tabaci (order Hemiptera, family Aleyrodidae) for SPCSV and by nonpersistent transmission with aphids Myzus persicae (order Hemiptera, family Aphididae) for both potyviruses.SPCSV was also isolated in Nicotiana benthamiana with whitefly transmition.

RT-PCR, Cloning and sequencing of HSP70h of SPCSV isolate
To amplify a partial sequence of the heat shock protein 70 homologue gene of SPCSV isolate from Argentina, RT-PCR was performed using specific primers (Winter et al., 1997) with total RNA purified from infected tissue.Access RT-PCR System kit (Promega Biotech, Madison WI, USA) was used.
The viral extraction was prepared by partial purification (clarified and filtrated on 25% sucrose cushion) of Nicotiana benthamiana plants infected with the SPCSV isolate.The RT-PCR product was purified with MicroSpin™ S-400 HR Columns (Amersham Pharmacia; Amersham Biosciences, AB; Uppsala; Sweden) and cloned into PCR-4-TOPO vector (Invitrogen, California, USA) following the manufactures´s instructions.Competent Escherichia coli TOP 10 cells were transformed according to Sambrook et al. (1989).Clones were analyzed with the restriction enzyme Eco RI and a clone selected was sequenced in both directions, using M13 Forward and M13 Reverse universal primers (Cornell University, USA).

Sequence accession number
The nucleotide sequence reported in this paper has been deposited in GeneBank database as accession number AY729021.

Preparation of riboprobe for SPCSV HSP70h for in situ hybridization
Once the orientation of the insert in the clone was confirmed, the plasmid was purified, linearized with PvuII (Promega, USA) and used as a template for the synthesis of riboprobe.This one, for SPCSV isolate (complementary to the HSP70h gene viral sense sequence) included the T7 promoter present in the transcription vector and was labeled with digoxigenin (DIG)-11-UTP, following provider´s specifications and protocols (Eisel et al., 2000).The evaluation of riboprobe labeling was estimated according to manufacturer´s recommendations (CDP-Star-ready to use DIG Luminescent Detection Kit, Roche Molecular Biochemicals, Mannheim, Germany).
The riboprobe reaction against healthy tissue was evaluated by dot-blot assay, using crude extracts of healthy and infected sweet potato plants with single (SPMSV) and triple co-infection (SPFMV + SPCSV + SPMSV), total RNA purified from same plants, and plasmid DNA as positive control.Plant tissues were macerated in 1/10 of extraction buffer (PBS + 0.5 ml of Tween20 + 20% PVP + sodium sulfite 1%) and centrifuged for 5 min at 5000 g.Total RNA was extracted directly from infected sweet potato tissues according the protocol of Conci et al. (1999), which was modified with the addition of 30 mg/ml polyvinylpolypyrrolidone (PVPP) to prevent the precipitation of polysaccharides and 1% sodium phosphate as antioxidant.The hybridization was made with an estimated riboprobe concentration of 100 μg/ml at 45ºC.The reaction was developed with chemiluminescent kit (CDP-Star-ready to use DIG Luminescent Detection Kit, Roche Molecular Biochemicals, Mannheim, Germany).

Virus isolates and detection
Plants with the different isolates developed the characteristic symptomatology (chlorotic mosaic, dwarfism and leaf deformation) to the given temperature, light and humidity conditions used.

RT-PCR, Cloning, sequencing and analysis of HSP70h of SPCSV isolate
The RT-PCR amplif ied product of HSP70h of SPCSV isolate showed a fragment of approximately 485 nucleotides, corresponding to the size of the Crinivirus HSP70h.
Multiple sequence alignment of these homologous proteins were carried out by using ClustalW (Fig. 2).
The analysis of the phylogenetic relationships of HSP70h from Argentinean SPCSV isolate with other crinivirus isolates showed a closer relationship with West African isolate clone 2; next, Egypt and USA isolates; and last, to East African S1EA-19b isolate.These results are consistent with the topology of the tree obtained by the maximum parsimony method using the amino acids sequence of HSP70h from Argentinean SPCSV isolate (Fig. 3).FIGURE 3. Phylogenetic tree constructed with HSP70 homologue from Argentinean SPCSV isolate and related sequences using the Parsimony method analysis (100 replicates).

Preparation of riboprobe for SPCSV HSP70h for in situ hybridization
The yield for 50 μl of labeled probe was of 250 ng/ μl.

In situ hybridization (ISH) and dot-blot assay
A positive reaction was observed in sweet potato plants infected with the Argentinean SPCSV isolate, within cells nucleus of young sieve tubes and phloem parenchyma.In sweet potatoes triply co-infected, SPFMV + SPCSV, and SPMSV + SPCSV, signal was detected in the nucleus of phloem cells and xylem parenchyma, and also extended towards mesophyll cells.Reaction was always negative in healthy plant tissues (Figs.4a, b, c, d  and e).The specific reaction was observed as small black dots, corresponding to the 5nm gold particles; bigger black dots were considered background.
Table 1 shows electron microscopy observations of in situ hybridization on ultra thin sections from healthy plants infected with SPCSV, SPMSV + SPCSV, SPFMV + SPCSV, and triply co-infected, in young, average and old leaves.
The dot-blot assay detected viral RNA on crude extracts of infected plants and total RNA purified from single infected plants (SPCSV), triply co-infected plants (SPFMV + SPCSV + SPMSV) and plasmid DNA2.
There were no hybridization signals with extraction buffer (mock) and crude extracts from healthy plants, confirming the results obtained by in situ hybridization assay (Fig. 5).
By in situ hybridization and dot-blot assays, it was demonstrated that the riboprobe for HSP70h of Argentinean SPCSV isolate detected specifically its mRNA on infected sweet potato tissues, no signal was observed when healthy tissues were tested.

Discussion
RNA silencing or post transcriptional genetic silencing (PTGS) is one of the defense mechanisms used by plants to resist viral infections; where viral genome is detected and removed by plant endonucleases (Dunoyer et al., 2004).
Infact, SPCSV induces RNAi; the exact mechanism however, is not completely elucidated yet (Kreuze et al., 2005).In chlorotic dwarf disease, there is a synergism between any of the involved potyvirus (SPFMV and SPMSV) and the crinivirus (SPCSV).The SPFMV increases its concentration in the phloem when accompanied by the crinivirus (Karyeija et al., 2000), this is not observed among both of the potyvirus (Di feo, 1994(Di feo, , 1996)).These results have been previously appreciated by the plant symptomatology.The in situ hybridization studies reported in the present work demonstrate that the crinivirus (SPCSV) is confined to the phloem in single infections, while in co-infections with the potyviruses (SPFMV and SPMSV) is found in the mesophyll to different extents.Therefore, somehow the interaction between them allows at least the subgenomic HSP70h RNA from the crinivirus to abandon the vascular tissue.
On the other hand, both potyviruses increases their concentration when accompanied by the crinivirus on sweet potato cv Morada INTA (observations by C.F. Nome, unpublished data).An attempt to explain these phenomena could be done using the previously mentioned mechanisms, suggesting that the movement protein (MP) from potyviruses allows the crinivirus to move towards tissues other than the phloem.Also it could be considered that the particles decorated by portions (Figs.1a, b and c) might be involved in this process; relocation of the crinivirus outside of the phloem may be due to the potyvirus portion, with its ability to get to and trespass the plasmodesmata.At the same time, a mechanism of gene silencing suppression by crinivirus might be acting in the multiple infection allowing the SPFMV and SPMSV increase their concentrations (observations by C.F. Nome, unpublished data).
In conclusion, our data indicates that at least one of the subgenomic SPCSV RNAs (the one coding HSP70h protein) leaves out of the phloem in presence of the potyviruses involved in chlorotic dwarf disease.

FIGURE 2 .
FIGURE 2. Multiple sequence alignment of Argentinean SPCSV isolate, West African isolate clone 2, Egypt isolate, USA isolate and East African SIEA-19b isolate-encoded proteins, constructed using the program ClustalW version 1.83.Grey regions represent conserved amino acids among the isolates.

FIGURE 4 .
FIGURE 4. nu: nucleus, c: cytoplasm, arrows: small black dots gold particles, positive reaction, arrow heads: big black dots, background and stain artifacts.a. Positive reaction within nucleus of phloem cells from sweet potato plants infected with SPCSV using HSP70h riboprobe.X 12,800.b.Positive reaction using HSP70h riboprobe within nucleus of xylem parenchyma cells of sweet potato infected with chlorotic dwarf disease.X 15,790.c.Positive reaction within nucleus of mesophyll cells of sweet potato infected with chlorotic dwarf disease.X 12,210.d.Negative reaction in phloem cells of healthy sweet potato using HSP70h riboprobe.X 67,000.e. Negative reaction of HSP70h probe with mesophyll cells of healthy sweet potato.X 9,690