Using new tools to detect and characterise plant viruses

Hao Luo

Executive summary: The overall aim of this study was to develop new methods to detect and characterise plant viruses. Generic methods for detection of virus proteins and nucleic acids were developed to detect two plant viruses, Pelargonium zonate spot virus (PZSV) and Cycas necrotic stunt virus (CNSV), neither of which were previously detected in Australia. Two new approaches, peptide mass fingerprinting (PMF) and next-generation nucleotide sequencing (NGS) were developed to detect novel or unexpected viruses without the need for previous knowledge of virus sequence or study. In this work, PZSV was found for the first time in Australia and also in a new host Cakile maritima using one dimensional electrophoresis and PMF. The second new virus in Australia, CNSV, was first described in Japan and then in New Zealand. In this work it was detected and characterised as a new strain in Australia using NGS analysis and was found in a lily plant (Lilium. longiflorum) with symptoms. Patterns of infection of a native virus Hardenbergia mosaic virus (HarMV) and the introduced virus Cucumber mosaic virus (CMV) were studied in natural and recent host plants using real-time reverse transcription polymerase chain reaction. For different virus isolates and symptoms, the virus concentration in plants varied and interaction between two co-infected viruses (such as HarMV and CMV) altered the accumulations of both viruses. Field studies were done to measure the potential impact of natural infection by HarMV on an economically-important legume crop plant, Lupinus 4 angustifolius (narrow-leafed lupin). In field studies, HarMV was spread by naturally occurring aphids, with up to 31% of the lupin plants infected. Grain yield of affected lupin plants was substantially reduced, but seed-borne infection of HarMV was not detected. Summary Peptide mass fingerprint (PMF) analysis of plant viruses A generic assay to detect and partially characterise viruses from plants was developed. Proteins extracted from virus-infected and uninfected plants were separated by one dimensional SDS polyacrylamide gel electrophoresis. Expressed coat protein bands not presented in uninfected plants were eluted after trypsin digestion and resulting peptide fragments separated according to their masses by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Resulting PMFs were compared with those present or predicted in protein databases. This assay strategy was used to identify four known viruses: the potyviruses Zucchini yellow mosaic virus (ZYMV) and Turnip mosaic virus (TuMV), an alfamovirus Alfalfa mosaic virus (AMV), and a cucumovirus (CMV). It was also used to identify a virus that manifested symptoms in wild C. maritima plants, tentatively identified as PZSV (genus Anulavirus) by its PMF, which was subsequently confirmed by Reverse transcription polymerase chain reaction (RT-PCR) and nucleotide sequencing. The detection of PZSV constitutes a first record of this virus in Australia and in this host. It is proposed that this simplified assay is a useful approach for analysis of plant samples known to harbour viruses, particularly for viruses which cannot be identified readily using antisera or nucleic acid-based assays. Although five viruses from different families and genera were identified successfully by this method, it was not a high-throughput and low cost 5 technique for sample screening, since protein extraction procedures were time-consuming, and protein identification based on PMF requires access to a high quality MALDI-TOF mass spectrometer. Nevertheless, this assay is a valuable complementary detection technique to support the identification of unexpected viruses to the species level, with further characterisation by other methods. Detection and characterisation of CNSV by NGS A lily plant (L. longiflorum), growing locally in Perth, Western Australia, with symptoms of chlorosis and streaking of leaves, was collected. Total RNA was extracted and sequenced using Illumina GA IIx technology. After assembly of reads, contigs representing the partial genome of CNSV RNA 1 and RNA2 were identified using Blastn and Blastx software. This virus was also found in bulb tissues of infected lily plants by RT-PCR and nucleotide sequencing (primers designed from the NGS sequences). This indicates that the virus can be transmitted through vegetatively propagated plant materials. Phylogenetic analysis suggested that the virus was a new strain of CNSV (family Secoviridae, genus Nepovirus). Comparison of the sequences of this virus to the CNSV (cycas strain) showed homology with identity of 88% and 96% at the nucleotide and amino acid levels, respectively, and to CNSV (gladiolus strain) of 88% and 94%, respectively; also provided in this work is a new strain of CNSV (lily strain). Another ten lily plants and nineteen cycas plants were tested for the virus by RT-PCR but none appeared to be infected. This virus has been recorded to be transmitted by seeds, nematodes and vegetative propagation, and has a very wide host range in ornamental and crop plants. This result is the 6 first record of this virus in Australia, which indicates that screening for CNSV and other nepoviruses in imported ornamental plants is essential to protect the Australian horticulture industry from incursion of new non-indigenous viruses. Natural spread of HarMV to narrow-leafed lupin Two field experiments were undertaken to study the potential threat of the Australian indigenous potyvirus, HarMV, to spread from its natural host, Hardenbergia comptoniana into narrow-leafed lupin (L. angustifolius) crops. Plants were grown in field conditions in two different years. Field plots of narrow-leafed lupin were established and interplanted with H. comptoniana plants infected with HarMV. Wild aphids were allowed to colonise the plots and spread the virus. Plants were monitored for aphids and symptoms of virus infection. Infection was confirmed by enzyme-linked immunosorbent assay (ELISA), RT-PCR and nucleotide sequencing. The first year established a pilot study, and this showed that HarMV spread naturally to 4.7% of L. angustifolius plants in the field. All infected lupin plants died within 20 days after virus symptoms became visible. In the second year a full field experiment was undertaken, and 30.7% of lupins became infected with HarMV. The majority of infected plants remained alive during this growing season, but showed symptoms of stunting, necrotic stem streaking and tip wilting. Three species of aphids were identified on plants during the experiment, including Myzus persicae (green peach aphid), Acyrthosiphon kondoi (bluegreen aphid) and Rhopalosiphon padi (oat aphid). A total of 761 seeds collected from infected plants were sown, and seedlings were tested for virus infection by ELISA. However, none of them were 7 found to be infected with HarMV, indicating that the virus was possibly not seed-borne in L. angustifolius. This aspect should be verified further by testing more seeds for HarMV from infected plants. The conclusion is that HarMV, a virus confined largely to a single native wild host, is capable of naturally extending its host range to an introduced grain legume under field conditions. Since adaption to an alternative host, as the case of HarMV invading lupin crops, is likely to be a driver of virus evolution, this pathosystem represents an ideal opportunity to study evolution of this virus in real time as it encounters new hosts, at the interface between an ancient ecosystem and a recent agroecosystem. Virus quantification using real-time quantitative PCR In this study, virus quantification by real-time quantitative PCR was used to titrate HarMV (isolate WHP-1, WHP-2 and MU-4) and CMV (Sn strain, subgroup II) expression in host L. angustifolius, H. comptoniana and Nicotiana benthamiana. A glasshouse experiment was done that showed HarMV isolate WHP-1 induced a non-necrotic (NN) response, while WHP-2 induced systemic necrosis (N) on lupin plants. Lupin cv Belara infected with WHP-2 isolate harboured a virus at a concentration of 1.34×108 copies/μl, approximately 18% higher than WHP-1 (1.13×108 copies /μl). Although typical symptoms caused by HarMV on wild H. comptoniana include: chlorosis, leaf mosaic, leaf distortion, yellow spots and blotches, the symptom severity is variable on different plants, and even different branches of the same plant show inconsistent symptoms. Their within-plant titres varied, and the highest concentration (1.75×108 copies /μl) was more than 19-fold that of the lowest (9.16×106 copies /μl). The interaction between two co-infecting viruses (HarMV and CMV) infecting N. benthamiana plants was studied. In doubly-infected N. benthamiana plants there was a strong synergistic increase in symptoms, with severe yellowing, stunting and chlorosis, and a higher overall virus titre (2.24×108 copies/μl) than for single virus infected plants (at 6.89×107 copies/μl for HarMV single infection and 4.05×107 copies/μl for CMV single infection). Compared to the single infection, CMV accumulation was enhanced (at 2.18×108 copies/μl in doubly-infected plants and 4.05×107 copies/μl in single-infected plants four weeks after inoculation), while HarMV was suppressed (at 5.66×106 copies/μl in doubly-infected plants and 6.89×107 copies/μl in single-infected plants four weeks after inoculation) in mixed infection. This study used real-time quantitative PCR to investigate the virus within-plant titre, and showed that the virus concentrations were variable in hosts depending on the virus isolate, symptoms and interaction with co-infected viruses.

Using new tools to detect and characterise plant viruses

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