Department of Plant Pathology, The University of Georgia, Athens, GA 30602, USA1
Author for correspondence: Carl Deom. Fax +1 706 542 1262. e-mail deom{at}uga.edu
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CCMV-T induces an extensive systemic chlorosis in cowpea [Vigna unguiculata (L.) Walp. subsp. unguiculata California Blackeye] (Kuhn, 1964 ). Continuous propagation of CCMV-T in cowpea resulted in an attenuated variant, CCMV-M, which induces mild green mottle symptoms (Kuhn & Wyatt, 1979
). The specific infectivities of CCMV-T and CCMV-M were similar, as were their levels of replication in cowpea (Kuhn & Wyatt, 1979
). Using pseudorecombinants generated with isolated genomic RNAs from CCMV-T and CCMV-M, the determinant of systemic symptoms was mapped to RNA 3 (Kuhn & Wyatt, 1979
). In this report, we exchanged regions between biologically active cDNA clones of RNA 3 from CCMV-T and CCMV-M and localized the symptom determinant to the CP gene. Further analysis by site-directed mutagenesis identified CP amino acid residue 151 as a determinant in systemic disease symptoms in cowpea.
Cowpea plants (cv. California Blackeye) were used in all experiments. Purified viruses, in vitro transcripts from infectious viral cDNA clones or homogenates from infected cowpea leaves were inoculated onto primary leaves of 8-day-old cowpea seedlings. Virus- or mock-inoculated plants were maintained in either a growth chamber (14 h light/10 h dark cycle) at 27 °C or a greenhouse at a similar temperature. Inoculated plants were evaluated for infection by symptomology and serology (Western blot analysis or protein-A enzyme-linked immunosorbent assay, PAS-ELISA) (Edwards & Cooper, 1985 ) using polyclonal antibodies specific for the CP of CCMV-T.
RTPCR (Deom et al., 2000 ) was used to synthesize and amplify cDNA representing full-length CCMV-M RNA 3 from purified CCMV-M RNA (Kuhn & Wyatt, 1979
). The 5'-primer was 5' CGGGGTACCTAATACGACTCACTATTG-TAATCTTTACCAAACAAC 3', which contains a T7 promoter (underlined) and a unique 5'-flanking KpnI site (italics). The 3'-primer was 5' TGCTCTAGATGGTCTCCTTAGAGATCACC 3', which is complementary to the 3' end of CCMV-T RNA 3 and contains a unique 5'-flanking XbaI site (italics). The primers were based on the published sequence of CCMV-T (Allison et al., 1989
). PCR products were ligated into the KpnIXbaI sites of pUC19 (Life Technologies). Biologically active transcripts were synthesized from full-length cDNA clones of CCMV-T RNA 1, RNA 2 and RNA 3 (pCCT1, pCCT2 and pCCT3, respectively; S. Quan & C. Deom, unpublished results), CCMV-M RNA 3 (pCCM3) and from chimeric RNA 3 cDNA clones using a RiboMAX Large Scale RNA Production System-T7 (Promega).
In vitro transcripts from each of five CCMV-M RNA 3 cDNA clones were co-inoculated onto cowpea plants with in vitro transcripts from pCCT1 and pCCT2. All induced mild green mottle symptoms that were indistinguishable from those induced by CCMV-M. The clone pCCM3 was chosen for further study. The nucleotide sequence of pCCM3 was determined and compared to the sequence of pCCT3 (S. Quan & C. Deom, unpublished results). Four nucleotide differences between pCCT3 and pCCM3 were identified (Table 1). In addition, there was a deletion of seven A residues from the polyadenylation [poly(A)] tract in the intergenetic region of CCMV-M RNA 3 between the MP and CP genes. With the seven deleted A-residues in CCMV-M, the nucleotide at position 1813 in CCMV-T is equivalent to position 1806 in CCMV-M.
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The structure of native CCMV has been determined by X-ray crystallography to a resolution of 3·2 (Speir et al., 1995
). Amino acid residue 151 lies within the
GH helix, one of two helices (the second is
GDII) that make up the putative calcium-binding site that helps stabilize intercapsomere interactions along the quasi three-fold axis of the icosahedral shell. Amino acids within the
GH helix that coordinate calcium binding are Glu-148, Gln-149 and Asp-153. Not surprisingly, amino acid modifications at residue 151 might be expected to have an effect on calcium binding and, therefore, virion stability. The degree of stability between capsomeres mediated by calcium ions might play a role in symptom determination.
To determine if there is a correlation between symptoms induced by CCMV-T and CCMV-M and virus stability, a series of experiments was performed to evaluate particle stability in vitro under conditions that induce either a conformational change in virion structure (swelling) or virion dissociation. At pH<6·0 and low ionic strength (i<0·2), CCMV particles are contracted and sediment at 88S, while at pH>7·0 and low ionic strength (i<0·2) the peak of sedimentation changes to 78S as a result of swelling (Bancroft et al., 1967 ; Speir et al., 1995
). Swelling is thought to result from the removal of Ca2+ from the quasi three-fold axis of the virion (Speir et al., 1995
). Purified CCMV-T or CCMV-M was incubated in 0·1 M phosphate buffer at pH 5·0 or pH 7·5. Virus was centrifuged through 1040% linear sucrose gradients for 4 h at 25000 g (Fox et al., 1996
) and gradient profiles were monitored by absorbance at 254 nm. CCMV-T and CCMV-M were equally susceptible to swelling at pH 7·5, as indicated by identical changes in the gradient profiles of each virus, when incubation times prior to centrifugation were as short as 1 min (data not shown). Using a second approach, agarose gel electrophoresis, under conditions that result in pH-induced conformational changes (Heaton, 1992
), CCMV-T and CCMV-M were found to be equally susceptible to swelling at pH 7·5, based on the slower migration pattern of each virus relative to contracted virus particles (data not shown). Kuhn & Wyatt (1979)
previously reported that CCMV-M could swell more easily than CCMV-T in low molarity phosphate buffer (0·01 M) at pH 7·5, but we detected no difference under the same conditions.
The stability of CCMV-T and CCMV-M was also compared on sucrose gradients under conditions that either induce or do not induce particle dissociation (Fox et al., 1996 ). Virions dissociate into genomic RNA and CP at pH>7·4 and high ionic strength (i
1·0). Both CCMV-T and CCMV-M were stable under low salt (0·1 M) and low pH (pH 5·0) conditions, while both viruses dissociated under high salt (1·0 M) and high pH (pH 7·5) conditions. Taken together, the conformational and dissociation data indicate that there is no correlation between virion stability and symptoms. This conclusion is supported by research on mutants of the CP of Turnip crinkle virus, where there was no correlation detected between particle stability and symptom phenotype (Lin & Heaton, 1999
).
Although residue 151 is in the GH helix, no differences were detected in the stability of CCMV-T and CCMV-M under conditions that cause virus swelling or dissociation in vitro. We believe this indicates that Ca2+ binding is similar in CCMV-T and CCMV-M and this conclusion is in agreement with virus accumulation data. At 10 days post-inoculation (p.i.), there was no significant difference in the levels of accumulation of CCMV-T and CCMV-M in inoculated or systemic leaf tissue as determined by virus purification or ELISA. Interestingly, CCMV-M was previously shown to be as fit as CCMV-T with a strong tendency for CCMV-M to become dominant when co-inoculated with CCMV-T (Kuhn & Wyatt, 1979
). Furthermore, no significant difference was detected between the levels of viruses having substitutions at position 151 (Table 2
) and the levels of CCMV-T and CCMV-M at 10 days p.i. as determined by ELISA.
The role of viral CP in symptom development has been demonstrated at the molecular level for several viruses (Banerjee et al., 1995 ; Culver & Dawson, 1989
; Dawson et al., 1988
; Heaton et al., 1991
; Knorr & Dawson, 1988
; Lin & Heaton, 1999
; Neelman et al., 1991
; Shintaku & Palukaitis, 1992
). Significant structural changes to the
GH helix would be expected to disrupt the putative calcium-binding site that helps stabilize intercapsomere interactions. However, the similarity in stability of CCMV-T and CCMV-M and the similar accumulation levels of CCMV-T, CCMV-M and the viruses with mutations at residue 151 suggests that subtle influences by amino acid residues at position 151 likely play a role in the CPhost interactions that determine the respective symptom phenotypes.
Cowpea likely plays a host specific role in symptom induction by CCMV-T and CCMV-M, since symptoms were similar for CCMV-T and CCMV-M on other known hosts of CCMV-T, and no new hosts were identified for CCMV-M (Kuhn & Wyatt, 1979 ). This would not be surprising since CCMV-M was selected by continuous passage of CCMV-T in cowpea (Kuhn & Wyatt, 1979
).
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References |
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Received 15 October 2001;
accepted 18 December 2001.