Richard L. Forster

Molecular dissection of the mechanism by which potexvirus triple gene block proteins mediate cell-to-cell transport of infectious RNA

The triple gene block (TGB; consisting of proteins TGB1–3) and coat protein (CP) of white clover mosaic potexvirus (WClMV) are required for cell-to-cell movement of viral RNA. Cell-to-cell spread of WClMV mutants in which the TGB open reading frames had been mutated was rescued in transgenic plants expressing specific TGB proteins (TGBPs). This indicated that there are no requirements for the synthesis in cis of viral TGBPs. These transgenic plants provided an experimental framework to explore the roles performed by the TGBPs and CP in cell-to-cell movement of WClMV RNA. Microinjection experiments established that TGB1 functions as the WClMV cell-to-cell movement protein (MP). Furthermore, combined microinjection and dual-channel confocal laser scanning microscopy provided direct evidence that infectious transcripts of WClMV move cell to cell as a ribonucleoprotein complex, consisting of single-stranded RNA, TGB1, and CP. Movement of this ribonucleoprotein complex displayed an absolute requirement for the...

Cell-to-Cell Movement of Potexviruses: Evidence for a Ribonucleoprotein Complex Involving the Coat Protein and First Triple Gene Block Protein

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.

Presence of double-stranded RNA and virus-like particles in Botrytis cinerea

Double-stranded RNA (dsRNA) was demonstrated in mycelial extracts in 143 of 200 isolates of Botrytis cinerea from a range of hosts in New Zealand. The dsRNA profiles differed widely between isolates in the number, size and relative concentration of dsRNA species present. Virus-like particles (VLPs) were observed by electron microscopy in partly purified virus preparations in three of five dsRNA-containing isolates examined. Morphological types present included isometric particles of varying size classes (approx. 30, 35, 40 and 45 nm) and bacilliform particles (approx. 25 × 63 nm). Such particles were not observed in five isolates apparently lacking dsRNA but flexuous rods with a modal length of approx. 720 nm were present in one. Total RNA extraction of partly purified VLP preparations gave similar profiles to those obtained from mycelial dsRNA extractions of the same isolates, suggesting that the VLPs represent encapsidated dsRNA. Comparison of 12 dsRNA-containing and 12 dsRNA-free isolates indicated slight differences between the two groups in radial growth rate, virulence on bean leaves and sclerotial number but the differences were minor and the ranges overlapped.

Dark Green Islands in Plant Virus Infection are the Result of Posttranscriptional Gene Silencing

Dark green islands (DGIs) are a common symptom of plants systemically infected with a mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow, virus-infected tissue. We report here on two lines of evidence showing that DGIs are caused by posttranscriptional gene silencing (PTGS). First, transcripts of a transgene derived from the coat protein of Tamarillo mosaic potyvirus (TaMV) were reduced in DGIs relative to adjacent yellow tissues when the plants were infected with TaMV. Second, nontransgenic plants coinfected with TaMV and a heterologous virus vector carrying TaMV sequences showed reduced titers of the vector in DGIs compared with surrounding tissues. DGIs also were compared with recovered tissue at the top of transgenic plants because recovery has been shown previously to involve PTGS. Cytological analysis of the cells at the junction between recovered and infected tissue was undertaken. The interface between recovered and infected cells had very similar features to that surrounding DGIs. We conclude that DGIs and recovery are related phenomena, differing in their ability to amplify or transport the silencing signal.

'Candidatus Phytoplasma australiense' is the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases

Sequence comparisons and phylogenetic analysis of the 16S rRNA genes and the 16S/23S spacer regions of the phytoplasmas associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases revealed minimal nucleotide differences between them resulting in the formation of a monophyletic group. Therefore, along with Australian grapevine yellows, the phytoplasmas associated with Phormium yellow leaf and papaya dieback should also be considered as ‘Candidatus Phytoplasma australiense’.

Phormium Yellow Leaf Phytoplasma Is Associated with Strawberry Lethal Yellows Disease in New Zealand

A yellows disease of strawberry plants was identified in propagation beds in New Zealand. Affected plants were flatter to the ground, showed purpling of older leaves, reduced leaf size, yellowing of younger leaves, and sometimes plant death. A phytoplasma was observed in the phloem of affected plants. The 16S rRNA gene of the phytoplasma was amplified by polymerase chain reaction from symptomatic plants and from one asymptomatic plant, but not from 36 other asymptomatic plants. Nucleotide sequence analysis of the 16S rRNA gene showed that the phytoplasma is closely related or identical to the phytoplasma associated with the yellow leaf disease of New Zealand flax (Phormium tenax).

The potential of plant viral vectors and transgenic plants for subunit vaccine production.

Publisher Summary This chapter presents the case that plants can be used for the production of subunit vaccines and outlines the systems that are available for their production. The limits of plant biology are explored along with the limits of the differing expression systems that are available. Vaccination has led to a significant improvement in the health of the worlds population. The use of vaccines has reduced the spread and infection of a number of major human diseases, including measles, mumps, rubella, and tetanus. With the advent of recombinant technologies, subunit vaccines based on proteins expressed in bacteria and yeast have grown in popularity. Because subunit vaccines do not contain an infectious agent that can revert to a more virulent form or survive the inactivation process, they offer advantages over live vaccines, because they are incapable of causing disease. Interest in vaccine production in plants has also expanded rapidly. There are considerable advantages in expressing antigenic proteins in plants. Plants can be grown locally and cheaply using standard methods, thus reducing problems with distribution, transport, and storage. Several considerations must be addressed when expressing proteins in plants for the purpose of vaccination; the protein must retain the immunogenic characteristics of the original protein and be capable of inducing a protective response to the disease.

Estimating the agronomic impact of white clover mosaic virus on white clover performance in the North Island of New Zealand

Abstract The agronomic impact of white clover mosaic potexvirus (WC1MV) was determined using yield trials and a survey of virus incidence. The virus was shown to occur at high levels in white clover plants in pastures throughcut the North Island. The virus was detected in 69% of white clover plants from 42 mature pastures examined (>4 years old) and in 52% of white clover plants examined on roadsides. The virus was also detected in 15 pastures less than 4 years old, but at a lower level (18% infection). The effect of WC1MV on plant growth was determined using clonal and non‐clonal lines of the modern white clover cv. Grasslands Tahora in a replicated spaced plant field trial. Uninoculated plants were shown to out‐produce inoculated plants at each of three harvest dates over one year. Infected plants produced, in total, 36.5% less dry matter than uninoculated plants. The major effect of WC1MV infection was on stolon elongation, with lesser effects on leaf length and petiole height. These results indicate t...

Planthopper transmission of Phormium yellow leaf phytoplasma

Phonnium yellow leaf (PYL) phytoplasma was transmitted from diseased to healthy New Zealand flax (Phormium tenax) by the native planthopper, Oliarus atkinsoni (Homoptera: Cixiidae). By contrast, transmission was not effected by the introduced passionvine hopper, Scolypopa australis (Homoptera: Ricaniidae). Successful transmission of PYL phytoplasma from New Zealand flax to New Zealand flax by O. atkinsoni was demonstrated by symptomatology and by polymerase chain reaction (PCR) of the test plants using phytoplasma-specific primers to the 16s rRNA genes. When the salivary glands and the remaining body of the planthoppers used in the transmission studies were tested separately by PCR for the presence of phytoplasma, PYL phytoplasma was detected in 100% of both the salivary glands and the bodies of pre-transmission O. atkinsoni, and in 44% and 67% of the salivary glands and the bodies of post-transmission planthoppers, respectively. The phytoplasma was not detected by PCR in the whole bodies of hoppers of S. australis.

Sequence-, tissue-, and delivery-specific targeting of RNA during post-transcriptional gene silencing.

Transgenic Nicotiana benthamiana plants expressing an untranslatable version of the coat protein (CP) gene from the Tamarillo mosaic virus (TaMV) were either resistant to TaMV infection or recovered from infection. These phenotypes were the result of a post-transcriptional gene silencing (PTGS) mechanism that targeted TaMV-CP sequences for degradation. The TaMV-CP sequences were degraded when present in the wild-type TaMV potyvirus, in transgene mRNA, or in chimeric viral vectors based on White clover mosaic virus. The more efficiently targeted region was mapped to a 134-nt segment. Differences were observed in the efficiency of targeting during cell-to-cell and long-distance movement of the chimeric viruses. However, the TaMV-CP sequences do not appear to be targeted for degradation when delivered by biolistics.