Graduate School of Biotechnology, Korea University, Seoul, 136-701, Korea1
Plant Virus GenBank, Department of Horticultural Science, Seoul Womens University, Seoul, 139-774, Korea2
Division of Biological Environment, Kangwon National University, Chunchon 200-701, Korea3
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK4
Author for correspondence: Won Mok Park. Fax +82 2 923 9923. e-mail viroid{at}korea.ac.kr
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Abstract |
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In some cases, mixed infection by two viruses has been shown to overcome barriers to the cell-to-cell or long-distance movement of one of the viruses (reviewed by Atabekov & Taliansky, 1990 ; Nelson & van Bel, 1998
: Hull, 2002
). Moreover, in some mixed virus infections, there is an interaction between the two viruses, resulting in increased symptom severity and greater virus accumulation, a phenomenon referred to as synergism (reviewed by Hull, 2002
). In synergism, it is generally recognized that one virus, which is not increased in accumulation (i.e. functioning essentially as a catalyst), acts as an up-regulator of the replication and/or movement of a unrelated virus, usually increasing the intensity of symptoms induced compared with single infection by either virus alone (Rochow & Ross, 1955
; Calvert & Ghabrial, 1983
; Poolpol & Inouye, 1986a
, b
; Goldberg & Brakke 1987
; Sano & Kojima, 1989
; Vance, 1991
; Anjos et al., 1992
; Bourdin & Lecoq, 1994
; Pruss et al., 1997
). In most of the above cases of synergism, the catalytic virus is a potyvirus. Synergism between potyviruses and CMV has been described in both tobacco and cucurbit plants (Poolpol & Inouye, 1986a
, b
; Pruss et al., 1997
; Wang et al., 2002
). Zucchini squash and melon plants doubly infected with the potyvirus Zucchini yellow mosaic virus (ZYMV) and CMV have been shown to exhibit a severe synergistic pathological response and showed a strong increase in the level of accumulation of CMV positive-strand RNA and CP, with no increase in the accumulation of ZYMV (Wang et al., 2002
). This was as true for a highly virulent strain (ZYMV-AT) as for an attenuated strain (ZYMV-AG) (Wang et al., 2002
). Thus, we wanted to determine whether co-infection with ZYMV could overcome the barrier to the long-distance movement of M-CMV and whether the synergism of CMV accumulation and increased virulence also occurred in such doubly infected plants.
To determine whether ZYMV could neutralize the movement restriction of M-CMV in zucchini squash (C. pepo cv. Black Beauty), a severe isolate of ZYMV from Korea (strain A; Choi et al., 2002b ) was co-inoculated with a pseudorecombinant CMV consisting of RNAs 1 and 2 of the Fny strain of CMV and RNA 3 of the M strain of CMV. This pseudorecombinant virus has previously been shown to exhibit the same limited movement in zucchini squash as M-CMV (Shintaku & Palukaitis, 1990
; Wong et al., 1999
). The CMV RNAs were generated from infectious transcripts of cDNA clones of these RNAs (Shintaku et al., 1992
; Wong et al., 1999
) and inoculated into tobacco plants. The systemically infected tobacco plants, showing bright yellow symptoms, were tested for the presence of the virus at 10 days post-inoculation (p.i.), by either Western blot analysis or RTPCR using primers specific to the genus Cucumovirus (Choi et al., 1999
). ZYMV-A was maintained in cucumber plants (Cucumis sativus cv. Baekdadaki). Inocula were prepared by grinding equal fresh weights of young leaves from these infected plants in 50 mM phosphate buffer (pH 7) and either inoculating separately, or mixing the extracts just prior to inoculation of Carborundum-dusted squash cotyledons. The inoculated zucchini squash plants were maintained in a greenhouse at 26 °C with a photoperiod of 16 h.
ZYMV-A induced severe yellow-green mosaic and leaf malformation symptoms on zucchini squash plants 710 days p.i., while infection with M-CMV alone did not show any symptoms on the upper leaves (Fig. 1a). In contrast, zucchini squash co-infected with M-CMV and ZYMV-A showed severe systemic symptoms (Fig. 1a
). Later in the course of infection, the symptoms in these doubly infected zucchini squash plants progressed to severe vascular wilt, stunting and plant death (not shown). These same results were obtained in each of three repeated tests.
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To confirm the above conclusions, as well as to establish whether very low levels of M-CMV were present initially in the upper leaves of zucchini squash plants inoculated with M-CMV alone, RTPCR analysis was carried out. One set of primers specific to and flanking the CP gene of CMV and another primer pair specific to and flanking the sequences encoding the ZYMV CP were used for RTPCR. Gel analysis of RTPCR products obtained from nucleic acids extracted from the upper leaves of plants doubly inoculated with M-CMV plus ZYMV-A showed the expected size band for the M-CMV CP gene (Fig. 2). No M-CMV CP gene-specific RTPCR product was obtained using nucleic acids extracted from the upper leaves of plants inoculated with M-CMV alone, while the ZYMV-specific RTPCR products were present in the samples inoculated with ZYMV-A, either singly or together with M-CMV (Fig. 2
).
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To ascertain whether the systemic movement of M-CMV was dependent on mutation of the CMV CP indirectly mediated by co-infection with ZYMV, total RNAs from zucchini squash plants systemically infected with both M-CMV and ZYMV were extracted and subjected to RTPCR. The RTPCR product was analysed by sequencing, either directly or after cloning into the pGEM-T Easy vector. The sequences revealed that the CP M-CMV had not mutated (data not shown), suggesting that ZYMV facilitated the long-distance movement of M-CMV by means other than by promoting the generation of mutation in the elicitor of the resistance mechanism, the M-CMV CP.
To determine whether synergism mediated by ZYMV-A versus ZYMV-AG led to similar increases in the levels of accumulation of M-CMV in the systemically infected leaves, plant proteins were extracted and subjected to Western blot analysis. Total proteins were extracted from four leaf discs collected from the upper leaves of zucchini squash plants, as described by Choi et al. (2002a ), and were separated by SDSPAGE (Sambrook et al., 1989
). After electrophoresis and electroblotting to nitrocellulose membranes (Micron Separation Inc.), the membranes were blocked and then probed with antisera to CMV CP or ZYMV CP, as described above for tissue printing. The immunoblots showed similar levels of accumulated M-CMV CP in the upper leaves of these doubly infected plants (Fig. 2
). Although not quantitative as such, the RTPCR results on similarly infected tissues supported this conclusion (Fig. 2
).
To ascertain whether systemic movement of M-CMV had any effect on the level of accumulation of either ZYMV strain, systemically infected leaves showing yellow-mosaic symptoms (at 610 days p.i., prior to onset of plant death) were examined by Western blot analysis and ELISA for ZYMV. There was no increase in the levels of accumulation of either ZYMV strain, in singly versus doubly infected plants (Fig. 2 and data not shown). These results also indicated that CP accumulation of the two ZYMV strains was not correlated with symptom severity. Similar conclusions were drawn using melon or squash plants doubly infected with either ZYMV-A or ZYMV-AG, together with either Fny-CMV or LS-CMV; i.e. the levels of ZYMV were similar in singly vs. doubly infected plants (Wang et al., 2002
), despite differences in pathology.
The above results also indicated that ZYMV-AG could either neutralize the barrier to the long-distance movement of M-CMV, or directly facilitate such movement, as for the more severe ZYMV-A strain. Since the HC-Pro protein of potyviruses has been shown to be a factor in long-distance movement (Cronin et al., 1995 ), changes in the ZYMV-AG HC-Pro protein associated with its attenuation (Gal-On, 2000
) might have influenced its ability to interact synergistically with M-CMV. However, the above results indicated that synergistic interaction between CMV and ZYMV was not dependent on a particular genotype of ZYMV per se and that the effective long-distance movement of M-CMV was not in itself a major determinant of the extent of synergism in pathology.
Plant resistance to virus infection expressed in the form of restricted systemic movement of the virus has been described previously for various virushost systems (Dufour et al., 1989 ; Goodrick et al., 1991
; Nelson et al., 1993
; Schaad & Carrington, 1996
; Canto et al., 1997
; Derrick & Barker, 1997
; Wintermantel et al., 1997
; Kaplan et al., 1997
, 1998
; Thompson & Garcia-Arenal, 1998
; Wang et al., 1998
; Ryabov et al., 1999
). In most cases, the resistance appears to involve an inability of the virus to enter or exit the phloem, and specific cell types or locations of tissues associated with blockage of virus movement have been reported. The resistance to systemic infection by M-CMV in zucchini squash plants, which is overcome by infection with ZYMV, appears to be due to an inability of M-CMV to exit the sieve elements rather than a block in entry into the vasculature in the inoculated leaves (Haudenshield, 2001
). It is not known how the delay in cell-to-cell movement of M-CMV (Wong et al., 1999
) influences this block in systemic infection.
One possible mechanism by which ZYMV might neutralize the resistance to the systemic movement of M-CMV could be that the ZYMV-encoded HC-Pro, or one of the other ZYMV-encoded proteins involved in systemic movement, is directly able to help M-CMV move into new leaves. An alternative mechanism for systemic movement of M-CMV mediated by ZYMV could be via the ability of ZYMV to suppress the transcription of host factors involved in the formation of the barrier. The ability of HC-Pro to suppress gene silencing of reporter genes (Anandalakshmi et al., 1998 ; Brigneti et al., 1998
; Kasschau & Carrington, 1998
), as well as to neutralize a barrier against the systemic infection of Nicotiana tabacum by the potyvirus Plum pox virus (Sáenz et al., 2002
), supports a role for HC-Pro in inhibiting the induction of host defence systems. This barrier may be one that is either specific to the movement of M-CMV, or not virus-specific, but which M-CMV is unable to suppress. A barrier specific to the movement of M-CMV may be elicited by the CP of M-CMV (Wong et al., 1999
). Mutation of the CP of Y-CMV or O-CMV at amino acid 129 from serine or proline, respectively, to leucine, as is present in M-CMV, has been shown to induce a host defence response in tobacco (Suzuki et al., 1995
).
CMV has been reported to neutralize resistance against the potyvirus Pepper mottle virus (PepMoV) in pepper (Capsium annuum cv. Avelar), allowing PepMoV to invade new systemic leaves (Guerini & Murphy, 1999 ). In this case, there was no increase in accumulation of helper virus (CMV) (Guerini & Murphy, 1999
). Thus, it appears that when the movement-restricted virus is a potyvirus and CMV neutralizes resistance to the potyvirus, the potyvirus does not catalyse any increase in CMV accumulation. It will be interesting to know if this is a general or isolated phenomenon.
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References |
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Received 28 June 2002;
accepted 26 August 2002.