Department of Plant Pathology, University of California, 1 Shields Avenue, Davis, CA 95616, USA
Correspondence
Bryce W. Falk
bwfalk{at}ucdavis.edu
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ABSTRACT |
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INTRODUCTION |
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Specific insect vectors transmit LIYV and several other members of the Closteroviridae from plant to plant in a non-circulative, semi-persistent manner (Karasev, 2000). Various whiteflies are vectors for LIYV and other viruses of the genus Crinivirus, while aphids are the most common vectors for BYV, CTV and viruses of the genus Closterovirus and mealy bugs appear to be vectors of at least some of the viruses in the genus Ampelovirus (Martelli et al., 2002
). As yet, the virus-encoded genetic determinants that facilitate vector-mediated transmission are not known for any virus in the Closteroviridae, thus the molecular mechanism(s) of vector transmission remains elusive. Also not known is whether vector transmission results in dissemination of defective RNAs (D-RNAs). D-RNAs are commonly associated with infections for some viruses of the family Closteroviridae, and have been well characterized, at least for CTV and LIYV (Ayllon et al., 1999
; Mawassi et al., 1995b
; Rubio et al., 2000
). One reliable approach that has greatly facilitated the study of insect vector-mediated transmission of many other plant viruses is by allowing insect vectors to acquire virus via feeding or probing in vitro through artificial membranes containing virus-infected plant sap, or purified or partially purified virions and, if needed, accessory vector transmission proteins (helper components). This approach has greatly facilitated identification of both qualitative (mutants) and quantitative determinants of virus transmission for viruses of the Potyviridae (Atreya et al., 1992
; Atreya & Pirone, 1993
; Peng et al., 1998
; Wang et al., 1996
), Caulimoviridae (Blanc et al., 1993
; Leh et al., 1999
) and Luteoviridae (Gildow, 1999
). Recently, we successfully developed an in vitro acquisition system for LIYV, the only such system so far reported for a member of the Closteroviridae. Virions alone are sufficient for acquisition and transmission, demonstrating that accessory proteins are not required. We also used antisera to neutralize Bemisia tabaci transmission of purified LIYV virions prepared from infected Chenopodium murale plants and demonstrated that the virion protein CPm is a potential determinant of whitefly transmission (Tian et al., 1999
).
Here we utilized this system to extend our knowledge of quantitative parameters affecting B. tabaci transmission of LIYV by identifying minimum concentrations of virions required for efficient transmission. In addition, we used an engineered D-RNA to demonstrate that LIYV D-RNAs can be transmitted by B. tabaci to plants and that transmission efficiency is correlated with concentration of encapsidated RNAs in the acquisition source.
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METHODS |
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Protoplast manipulation, LIYV inocula and virion purification from protoplasts.
Protoplasts were derived from cultured suspension cells of Nicotiana tabacum var. Xanthi maintained in Murashige and Skoog organics medium (Gibco-BRL) supplemented with 0·9 µM 2,4-dichlorophenoxyacetic acid, 0·4 µM kinetin, 1·5 µM thiamin hydrochloride (Passmore et al., 1993), 4·1 µM nicotinic acid and 2·4 µM pyridoxine hydrochloride. The extraction of virion RNAs and synthesis of capped transcripts of pM5gfp were done as described previously (Klaassen et al., 1994
, 1996
; Yeh et al., 2001
). Protoplasts were inoculated with virion RNAs and capped pM5gfp transcripts as described by Lindbo et al. (1993)
and Yeh et al. (2001)
. LIYV virions were purified from N. tabacum var. Xanthi protoplasts using a scaled-down version of the methods of Klaassen et al. (1994)
. Protoplasts (4x106) were harvested at 72 h post-inoculation (p.i.). Protoplasts were subjected to a brief (10 min) centrifugation (2300 g) in a Beckman SA600 rotor. Pellets were resuspended in 10 ml extraction buffer (0·1 M Tris/HCl, pH 7·4, 0·5 % w/v sodium sulfite, 0·5 % v/v 2-mercaptoethanol), stirred on ice for 2 h, with Triton X-100 added to a final concentration of 2 % (v/v), and subjected to centrifugation as previously described (Klaassen et al., 1994
). The resulting supernatant was subjected to ultracentrifugation as previously described (Klaassen et al., 1994
) and the final pellets were each resuspended overnight in 1020 µl of either 1x TE (0·01 M Tris/HCl, 1 mM EDTA, pH 7·4) or artificial diet solution (1x TE, 15 % w/v sucrose, 1 % BSA). Virions were disrupted by boiling and proteins were separated in a 12 % SDS-polyacrylamide gel (Laemmli, 1970
). Virion concentrations were estimated by densitometry (Scion Corporation) of stained gels or proteins detected by immunoblot analysis (Tian et al., 1999
) were compared with known amounts of co-electrophoresed purified virion proteins.
In vitro LIYV acquisition and whitefly transmission.
Non-viruliferous whiteflies (B. tabaci), reared on lima beans (Phaseolus limensis) (Perring et al., 1993; Tian et al., 1999
), were collected and starved for 1518 h in a room maintained at 22 °C prior to in vitro virion acquisition. To assess the effects of virion concentration on transmission efficiency, virion samples were serially diluted and 5 µl in artificial diet solution was used as the acquisition source. Following an acquisition access period (AAP) of 67 h, whiteflies were transferred onto target plants as described previously (Tian et al., 1999
). Percentage transmission was determined by the number of infected target plants over the total number of plants tested, and the results were analysed using non-linear regression analysis. To assess the effects of whitefly numbers on transmission efficiency, whiteflies were briefly anaesthetized using CO2 following in vitro acquisition and 10, 50 or 100 whiteflies were transferred onto the leaves of each target plant. Parallel plant-to-plant transmissions were performed using 1 and 10 CO2-anaesthetized whiteflies after an overnight AAP on LIYV-infected lettuce plants. Virions purified from infected protoplasts were directly resuspended in artificial diet solution and used for in vitro acquisition. Comparisons of virus transmission efficiency were made using estimates of the probability of transmission by a single whitefly (Gibbs & Gower, 1960
; Ng & Perry, 1999
).
RT-PCR, nucleotide sequence and Northern blot hybridization analyses.
Virion RNAs were extracted essentially according to Klaassen et al. (1994). Total RNA extraction was performed using TRI Reagent (MRO) according to the manufacturer's recommendations. AMV reverse transcriptase (Promega) and the oligonucleotide P26-r2 (5'-ACTATCAGTTATCGACACAACT-3'; Fig. 1a
), complementary to nucleotides 65166537 of the LIYV RNA 2 p26 coding region, were used for synthesis of the first-strand cDNA to LIYV RNA 2 or the M5gfp D-RNA (Fig. 1a
). Second-strand cDNA synthesis was primed with the oligonucleotide P26-F (5'-GACCACAGCTTTGACGACGGT-3'), corresponding to nucleotides 62356255, 64 nt upstream of the LIYV RNA 2 p26 start codon, and PCR-amplified to yield a 303 nt fragment. To detect the M5gfp D-RNA, second-strand cDNA synthesis was primed with the oligonucleotide GFP-F (5'-GATCATATGAAGCGGCACGAC-3'), corresponding to nucleotides 289309 of the green fluorescent protein (GFP)-coding sequence (Yeh et al., 2001
), and PCR-amplified to yield a 675 nt fragment. PCR-amplified products were separated on an agarose gel and visualized by ethidium bromide staining.
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For Northern hybridization analysis, RNAs were denatured with glyoxal, separated by electrophoresis in a 1 % agarose gel and transferred to Hybond-NX membrane (Amersham) as described previously (Yeh et al., 2000). Probes for Northern blot analysis were DIG-labelled transcripts from (i) pSKL16, a plasmid containing the cDNA corresponding to nucleotides 66857193 of LIYV RNA 2 (Yeh et al., 2001
) (Fig. 1a
), and (ii) AT1gfp, a plasmid containing the cDNA corresponding to nucleotides 62792 of the GFP-coding region (Fig. 1a
). Hybridization and detection procedures were as described previously (Yeh et al., 2000
).
Fluorescence and confocal laser scanning microscopy.
Fluorescence microscopy was used to visualize N. tabacum var. Xanthi cells for GFP expression at 72 h p.i. (Yeh et al., 2001). To detect GFP in whole plants, fresh tissue sections (75 µm) were prepared on a model TC-2 Sorvall microtome (DuPont) and analysed using a Leica TCS-SP laser scanning confocal microscope (Leica Microsystems). Fluorescence associated with GFP was detected using a krypton/argon laser with an excitation wavelength of 488 nm and an emission filter (500510 nm).
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RESULTS |
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We co-inoculated in vitro transcripts of the M5gfp D-RNA and LIYV virion RNAs to N. tabacum protoplasts. At 6070 h p.i., GFP fluorescence was typically observed in 812 % of inoculated cells, indicating LIYV and M5gfp D-RNA replication. Virions were then purified and used for in vitro acquisition and transmission by B. tabaci to plants of three different species (Table 3). At 2 weeks p.i., total RNAs were extracted from the upper, non-inoculated leaves of target plants, and analysis for LIYV genomic RNA 2 and the M5gfp D-RNA was performed by RT-PCR. By using primers specific for LIYV RNA 2 and specific for the GFP-coding sequence (Fig. 1a
), we identified LIYV RNA 2 and the M5gfp D-RNA in plants (data not shown). LIYV and the M5gfp D-RNA were transmitted to plants of all three species tested: L. sativa, C. bursa-pastoris and N. benthamiana (Table 3
, preparations 27). However, not all LIYV-infected plants also contained the M5gfp D-RNA. Of seven L. sativa plants analysed, all were infected by LIYV, while only 16 of 20 C. bursa-pastoris plants and 11 of 14 N. benthamiana plants were LIYV-positive (Table 3
, virion preparations 27). The M5gfp D-RNA was detected in only three of seven, 12 of 16 and four of 11 of the LIYV-infected L. sativa, C. bursa-pastoris and N. benthamiana plants, respectively (Table 3
, virion preparations 27). These data showed that an engineered D-RNA could be vector-transmitted, but its transmission was not as efficient as that of the helper virus.
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Effects of LIYV and M5gfp D-RNA concentration on whitefly transmission
Although we did not see obvious GFP expression in the above plants, because it contained a unique, non-LIYV sequence, the M5gfp D-RNA was still a good genetic marker for use in assessing D-RNA transmissibility. Our Northern hybridization and RT-PCR analyses showed that the M5gfp D-RNA was detected in leaves distal from the inoculation site; thus, it invaded infected plants systemically along with the LIYV helper virus. Therefore we assessed whether or not the M5gfp D-RNA could be stably maintained along with LIYV by whitefly transmission from plant to plant. We performed plant-to-plant transmission experiments using as source plants C. bursa-pastoris that tested positive for LIYV and the M5gfp D-RNA (Table 3, preparations 2 and 4). RT-PCR amplification of total RNAs extracted from C. bursa-pastoris target plants indicated that 63 of 69 plants were infected by LIYV, but none contained detectable levels of the M5gfp D-RNA (Table 4
, experiments 13). Northern blot analysis of total RNA extracts of these plants also showed only LIYV genomic RNA 2, not the M5gfp D-RNA (Fig. 3
, lanes 5 and 13, 6 and 14).
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DISCUSSION |
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Data accumulated so far on vector-mediated transmission of plant viruses have established that encapsidation/virion assembly is a prerequisite for transmission to occur. One of the earliest attempts to examine aphid transmission of several different viruses mechanistically showed that infectious genomic RNAs and DNAs were not aphid-transmissible, while appropriately prepared virion preparations were (Pirone & Megahed, 1966). Virions (sometimes requiring accessory helper proteins) are acquired and transmitted to plants by insect, nematode and fungal vectors of many taxonomically diverse plant viruses (Campbell, 1996
; Gray & Gildow, 2003
; MacFarlane, 2003
; Pirone & Blanc, 1996
; Rochow, 1970
). Because LIYV virions can be acquired in vitro and then transmitted to plants by B. tabaci, we were able to use specific numbers of whiteflies along with known virion concentrations and to estimate transmission efficiency at limiting thresholds for each. Although LIYV transmission was seen over a wide range of the virion concentrations tested here, the transmission frequency decreased with increasing dilutions of LIYV virions in the acquisition source. However, in all experiments here, the estimated minimum threshold virion concentration was quite consistent, between 0·01 and 0·1 ng µl1. It is remarkable, given the biological variability inherent among vector transmission experiments, that this virion concentration range was so consistent in our experiments. The transmission efficiencies using specific whitefly numbers, as reported in Table 1
, support the results from Fig. 2
and go so far as to estimate the probability for a single whitefly transmitting LIYV when specific virion concentrations are present in the acquisition source. As might be expected, and is now demonstrated by our data, whitefly numbers and virion concentration both play important roles in mediating LIYV transmission to plants.
One of our goals in elucidating the mechanism(s) associated with B. tabaci transmission of LIYV is to examine the transmission of wild-type and engineered LIYV mutants. Such analyses have been made possible in the study of several other non-mechanically transmissible plant viruses (such as the circulative, non-propagatively transmitted phloem-limited poleroviruses and luteoviruses) by combining protoplast infection and in vitro acquisition of virions by their aphid vectors (Brault et al., 1995, 2000
; Bruyere et al., 1997
; Chay et al., 1996
; Rouze-Jouan et al., 2001
; Sanger et al., 1994
). In those studies, aphids readily transmitted virus when virions in the acquisition source ranged from 10 to 100 ng µl1 and, as for LIYV, the quality and concentration (quantity) of virions in the acquisition source were important. The best way to control virion quantity and quality in the acquisition source, and then accurately to compare relative transmission efficiencies, is by using in vitro acquisition with virions of defined concentration and quality (e.g. wild-type or mutant constructs).
Although we have not demonstrated whitefly transmission of specific LIYV mutants, here we demonstrated unequivocal transmission of an engineered LIYV D-RNA. We showed that the M5gfp D-RNA was transmissible by B. tabaci to plants by using in vitro acquisition of virions purified from infected protoplasts. These protoplasts always showed high levels of M5gfp D-RNA replication, and virions purified from them also contained large amounts of the M5gfp D-RNA relative to LIYV genomic RNA 2 (Fig. 4). In contrast, although plants of three species contained the M5gfp D-RNA, it was not transmissible from these source plants to target plants unless virions were first concentrated and used for in vitro acquisition. Thus it seems that, although the M5gfp D-RNA replicated and systemically invaded plants along with the helper virus LIYV, it may not be competitive in some aspects of the infection cycle so as to ensure its subsequent transmission to new plant hosts. Most plants had higher levels of LIYV RNA 2 relative to the M5gfp D-RNA, and virions from plants analysed here contained greater amounts of LIYV RNA 2 relative to the M5gfp D-RNA (Fig. 4
). Whether this was due to competition during replication, whole-plant invasion or encapsidation or a combination of these or other critical events in maintaining the infection is not known. Similarly, engineered D-RNAs for TMV and CTV have also been transmitted to plants along with the corresponding helper viruses, and shown not always to be competitive (Knapp et al., 2001
; Yang et al., 1997
).
Although the M5gfp D-RNA was present in plants, we did not see GFP fluorescence. One possibility is that the observed reduced amount of the M5gfp D-RNA in plants versus protoplasts resulted in GFP concentrations lower than those required to allow observable discrimination of GFP fluorescence versus xylem autofluorescence. A low-level GFP fluorescence might still be detectable if the M5gfp D-RNA and the LIYV helper virus were present in tissues where autofluorescence was less abundant (e.g. the mesophyll), but because of the phloem-limited nature of LIYV this has not been possible. Still, by using the GFP-coding sequence as a marker in the M5gfp D-RNA, our study has shown that virions containing the M5gfp D-RNA could be transmitted by whiteflies, albeit only when high concentrations of the encapsidated RNA were present in the acquisition source.
This study clearly shows that the in vitro acquisition system, and knowledge of its characteristics in facilitating the delivery of LIYV and engineered RNAs to plants, should allow us to identify further LIYV-encoded determinants of transmission by B. tabaci. It remains to be seen whether in vitro acquisition will prove universally useful for other members of the Closteroviridae, as it seems to be for viruses of the Luteoviridae and of the genus Potyvirus. We have so far attempted to use in vitro acquisition for transmission of two other members of the genus Closterovirus: BYV using Myzus persicae and CTV using Aphis gossypii. So far we have been unsuccessful in transmitting these two viruses to plants after in vitro acquisition (unpublished). Thus, for the Closteroviridae, it appears that, like their virions and genomes, the molecular mechanisms facilitating vector transmission of viruses in this family may be complex and quite different for the different viruses.
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ACKNOWLEDGEMENTS |
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Received 13 April 2004;
accepted 18 May 2004.
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